US 6,060,644 AGrant

Isolation and Use of Cuticular Lipid Genes

Issue Date:2000-05-09

•13 Claims

•5 Drawing Sheets

Abstract

The present invention relates to cuticular lipid genes, their gene products, and methods of use thereof, for the generation of new plant varieties having novel environmental, disease and pest resistance. In particular, certain isolated or enriched nucleic acids specifying cuticular lipid genes, or portions thereof, are disclosed.

Metadata

Assignee

Iowa State University Research Foundation, Inc.

Inventors

Patrick S. Schnable
Donald S. Robertson
Joel D. Hansen
Basil J. Nikolau
Xiaojie Xu
Yiji Xia

Application Information

Application Number:US 5811481

Filing Date:1995-12-29

Priority Date:1994-03-24

Art Unit:169

Classifications

IPC:

A01H 500C12N 504C12N 1582

Field of Search:

53643580023.6152.3;240.4;320.1;419;468205;DIG. 69;281;298

Patent Drawings (5 sheets)

Description

Technical Field of the Invention

The present invention relates to the field of genetically engineered biosynthesis of plant lipids, and specifically relates to the isolation and use of cloned cuticular lipid genes.

Background of the Invention

Plant surfaces are covered with a complex mixture of lipids, which are thought to be synthesized by the cells of the epidermal tissue, the outermost single layer of cells. In addition to functioning as a water barrier, the surface lipids of plants have been suggested to function, inter alia, in frost resistance, in plant-pathogen interactions, and to provide protection from UV irradiation. Despite these diverse and important physiological functions of the surface lipids of plants, little is known about the biochemical and molecular genetic mechanisms that regulate their biogenesis. In particular, only subsequent to the filing of the parent of this application were any of the genes that encode regulatory or enzymatic functions in the cuticular lipid biosynthetic pathway cloned, despite the earlier identification in the art of genes believed to be involved therein.

The outermost barrier of the aerial portions of plants, the cuticle, consists of a meshwork ("cutin") of cross-esterified polymerized hydroxy-fatty acids, which are embedded in a complex mixture of nonpolar lipids, commonly referred to as the cuticular waxes. Maintenance of this interface between a plant and its environment permits sequestration of the highly regulated internal biochemical processes crucial for the plant's survival and the establishment of a water barrier. See Martin and Juniper, The Cuticle of Plants (Edward Arnold Ltd., Edinburgh, UK, 1970); and Kolattukudy, Ann. Rev. Plant Physiol., 32, 539-567 (1981). Chemical analyses of the cuticle have shown that its composition is genetically controlled and is unique to each species. See Kolattukudy and Walton, Prog. Chem. Fats Other Lipids, 13, 121-175 (1973); Kolattukudy et al., in Chemistry and Biochemistry of Natural Waxes (Kolattukudy, ed., Elsevier Press, New York, N.Y., 1976), pages 289-347; Tulloch, in Chemistry and Biochemistry of Natural Waxes, supra, pages 235-287; and Kolattukudy, in The Biochemistry of Plants: A Comprehensive Treatise, Vol. 4 (Stumpf and Conn, eds., Academic Press, New York, N.Y., 1980), pages 571-645.

The biochemistry of cuticular lipid biosynthesis has been investigated by: (1) monitoring the in vivo incorporation of radioactively labeled precursors into the lipids; (2) observing the effect of inhibitors on lipid composition; and (3) determining the in vitro activity of specific enzymes involved in lipid biosynthesis. These investigations have led to the formulation of a hypothesized general scheme of lipid biosynthesis, which is depicted in FIG. 1. See Kolattukudy and Walton, supra (1973); Kolattukudy et al., supra (1976); Tulloch, supra (1980); and Kolattukudy, supra (1980).

The cuticle contains many lipid compounds, the majority of which are derivatives of fatty acids. For example, cutin is composed of fatty acid derivatives such as diols, hydroxy fatty acids, dihydroxy fatty acids and dicarboxylic fatty acids, which are polymerized by ester linkages. The resultant polymers are embedded in the wax components of the cuticle, which is composed of a mixture of hydrocarbons, including n-alkanes, branched alkanes, cyclic alkanes, alkenes, ketones, ketols, alcohols, aldehydes, diols, acids, esters, and the like.

The major components of the cuticular wax, another category of lipids, are very long-chain fatty acids ("VLCFAs") and their derivatives. VLCFAs are considered to be the products of de novo fatty acid biosynthesis, which occurs in plastids, and subsequent elongation by specific elongases that are understood to be localized on the endoplasmic reticulum membranes. Partial purification and characterization of these elongases have been reported. See, for example, Agrawal et al., Arch. Biochem. Biophys., 230, 580-589 (1984); and Agrawal and Stumpf, Arch. Biochem. Biophys., 240, 154-165 (1985). Fatty acyl aldehydes and alcohols are the products of the sequential reduction of the appropriate fatty acid. Wax ester formation probably occurs via a transferase type reaction utilizing fatty acyl-CoAs or phospholipids as the donor of a fatty acid, which is transferred to a fatty alcohol (Kolattukudy et al., supra (1976)). Indeed, a fatty acyl-CoA-fatty alcohol transacylase has been partially purified from young broccoli leaves, and more recently, a similar enzyme has been purified from jojoba seeds (Lardizabal et al., Plant Physiol. (Suppl.), 102, 93 (1993)). As stated previously, prior to the filing of the parent of this application, none of the enzymes involved in cuticular lipid biosynthesis were cloned, accordingly no readily available source of them has been available. Thus, there has existed no ability to use them to alter the cuticular lipid components of a plant or its seed oils, and no ability to use them for in vitro synthesis of the aforementioned lipids.

Maize is a well-characterized experimental plant system that has been the subject of prior investigations of cuticular lipids. The cuticular wax of wildtype maize seedlings is composed of long-chain alcohols (63%), aldehydes (20%), alkanes (1%) and esters of alcohols and long-chain fatty acids (16%). The alcohols and aldehydes are predominantly 32 carbons in length, i.e., n-dotriacontanol (99% of all the alcohols) and dotriacontaldehyde (96% of the aldehydes). The alkane fraction is mainly 31 carbons in length, i.e., hentriacontane (Bianchi et al., Maydica, 30, 179 (1985)). Developmental differences in the quantity and quality of cuticular wax in maize have been noted in that adult maize leaves, as compared to juvenile leaves, have considerably less cuticular wax, which is of a different composition.

Seventeen loci (the glossy or gl genes) have been identified in standard crossbreeding studies of maize as affecting the quantity and composition of cuticular lipids on seedling leaves (namely, glossy1, glossy2, glossy3, glossy4, glossy5, glossy6, glossy8, glossy9, glossy11, glossy14, glossy15, glossy17, glossy18, glossy19, glossy20, glossy21, glossy22), although, as further explained hereinbelow, one of these genes (i.e., glossy15) apparently encodes a developmental control of the activity of cuticular lipid genes. The glossy15 gene also affects cell shape, cell wall composition, and the presence/absence of adult-type epidermal hairs. Hence, it mediates the entire juvenile to adult transition, i.e., it does not regulate cuticular wax biosynthesis per se. See Moose et al., Plant Cell, 6, 1343-1355 (1994) and Evans et al., Development, 120, 1971-1981 (1994).

Mutant seedlings are usually identified because applied water forms droplets on their leaf surfaces (Bianchi, in Maize Breeding and Genetics (Walden, ed., John Wiley and Sons, New York, N.Y., 1978), page 533); in addition, they present a "glossy" appearance. Four of the 17 glossy loci are members of duplicate gene pairs (gl5 & gl20, and gl21 & gl22), i.e., a seedling must be homozygous mutant for both members of such pairs before it expresses the mutant phenotype. Of the 17 glossy loci, 12 have been relatively precisely mapped genetically and three more have been mapped to a chromosome or chromosome arm.

Comparisons of the composition of the waxes produced by seedlings homozygous for each of the various glossy mutants to the wax of wildtype seedlings have been used to identify putatively the biochemical steps encoded by many of the glossy genes. In view of these data, it has been suggested that cuticular wax biosynthesis occurs via two hypothesized pathways, namely ED-I and ED-II (Bianchi et al., supra). The ED-II pathway is thought to be active throughout the life of the plant, with the end product being mainly esters. The ED-I pathway is thought to be active only during the seedling stage of the plant and the end-products of this pathway are mainly alcohols, aldehydes and alkanes.

However, a comparison between the chain lengths of the alcohol moiety of the esters and the chain lengths of the free alcohols in the wax of a number of different glossy mutants suggests an alternative hypothesis that cuticular wax biosynthesis in maize is the result of four reductive systems that are juxtaposed on elongase system(s) (von Wettstein-Knowles, in The Metabolism, Structure, and Function of Plant Lipids (Stumpf et al., Elsevier/North-Holland, Amsterdam, 1987), pages 489-498). One of these reductive systems operates in young seedlings (R1) and appears to produce free alcohols and free aldehydes. The second reductase system (R2) operates in mature plants and catalyzes the esterification of the alcohol products. It has been suggested that the glossy1, glossy8 and glossy18 genes may affect a fatty acid elongation reaction that feeds the R1 reductive system. The glossy7 gene (which is allelic to glossy6), on the other hand, may affect a third reductive system (R3) that provides C28 and C30 alcohols for ester formation. And lastly, according to this interpretation, the glossy15 gene may affect a fourth reductive system (R4) that provides alcohols of even shorter chain length for ester formation. However, more recent analyses of glossy15 have established that glossy15 may not be involved in cuticular wax biosynthesis per se, but rather have an indirect or incidental role in plant cuticle formation, as in the control of the phase change between juvenile and adult leaves (Moose and Sisco, in Abstracts, 35th Annual Maize 1993; and Evans and Poethig, 1993). Mutations at glossy15 result in an early transition from the juvenile to adult stage, resulting in seedling leaves with adult-type wax. Accordingly, it appears that all of the maize glossy mutants are involved in lipid biosynthesis; all but one of these genes (i.e., glossy15) apparently provide the enzymes and, perhaps, the cofactors that mediate lipid biosynthesis. One of the mutants (i.e., glossy 15) apparently relates to the developmental control of the activity of the indicated genes, as well as that of other genes.

Another experimental plant system for the study of cuticular lipids is Arabidopsis. The chemical composition and the genetics of the cuticular lipids of Arabidopsis is less well-defined than for maize. Preliminary analyses of the lipids of Arabidopsis indicate that the composition of the Arabidopsis cuticular wax is distinct from the wax of maize (Hannoufa et al., Phytochem., 33, 851-855 (1993); and Jenks et al., Plant Physiol., 108, 396-377 (1995)). The predominant cuticular lipids of Arabidopsis are alkanes, ketones and secondary alcohols. In contrast to maize, where fatty acyl aldehydes are reduced to primary alcohols, in Arabidopsis a considerable portion of the aldehydes are decarboxylated to form alkanes, which are then further metabolized to ketones and secondary alcohols. Thus, it appears that the Arabidopsis pathway is divergent from that which occurs in maize. However, based on evolutionary considerations, it is considered likely that many of the enzymes involved in the cuticular lipid synthesis of one plant are related in sequence and function to those of another plant. It is understood that fatty acid synthesis occurs in plastids in all plants. Fatty acids of C.sub.16 -C.sub.18 content are transported from the plastid to the cytosol, where it is believed that evolutionarily conserved elongation enzymes act on the de novo fatty acids to form long-chain fatty acids, very long-chain fatty acids, and other lipid compounds. These enzymes are believed to be the same or ancestrally related between species of plants, i.e., they are likely to have related nucleic acid and amino acid sequences.

As with maize, mutations have defined loci that affect the biosynthesis and/or deposition of the cuticular waxes of Arabidopsis. Many of these 21 loci, termed the eceriferum (CER) loci, present readily identifiable phenotypes (Koornneef et al., J. Hered., 80, 118-122 (1989)); some have pleiotropic effects on fertility. Analysis of one of these mutants, at the cer2 gene, indicates that the mutant allele affects changes in the lipid classes and in the predominant carbon chain length of the acyl moieties.

Being at the boundary between the organism and its environment, it would be desirable to control the lipid content of plant cuticle, the biochemical and genetic regulatory mechanisms of which are not well understood. Doing so would facilitate the generation of new plant varieties having altered abilities to conserve water, prevent loss of extracellular components by leeching, and resisting injury from environmental effects such as wind and frost. Because the cuticle is also instrumental in mediating the interaction between plants and other organisms, such as fungi, insects, and bacteria, control of the lipid content of cuticle would also impact on the generation of resistance to these pests. Furthermore, because the nature of the cuticle greatly affects the deposition and behavior of chemicals, including pesticides, growth regulators, and foliar nutrients sprayed on plants, it would be desirable to control the lipid content of cuticles in order to optimize such content for desirable plant variants. It also would be desirable to affect the lipid content of a plant's seeds or to synthesize various plant lipids by in vitro means.

Accordingly, it is an object of the present invention to provide new materials and methods that will allow one to create plant varieties having novel environmental and disease resistances. It is a further object of the present invention to provide new plants having novel environmental and disease resistance and/or plants producing seeds having high levels of saturated or unsaturated long-chain fatty acids and lipids. It is also an object of the present invention to provide plants able to synthesize such fatty acids and lipids of variable and defined chain lengths as well as to provide methods for generating such plants. It is yet a further object of the present invention to provide the isolated genes and gene products responsible for lipid biosynthesis in plants, for in vivo and in vitro synthesis of plant lipids.

These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

Brief Summary of the Invention

New materials and methods are disclosed herein for the generation of new plant tissue varieties having novel environmental and disease resistances. Such new materials include cloned cuticular lipid genes, which are useful not only for the generation of new plant varieties that can synthesize different quantities or kinds of plant lipids, combinations thereof, but also for the generation of cuticular lipid gene product for in vitro synthesis of plant lipids.

In particular, the present invention provides certain enriched or isolated nucleic acids comprising, respectively, a nucleotide sequence that encodes a cuticular lipid gene product of a plant, as well as vectors that incorporate such sequences. The present invention further provides an enriched or isolated polypeptide comprising an amino acid sequence encoded by one of the aforementioned nucleic acids. In accordance with the present invention, organisms, seeds, and plant cells that include one of the aforementioned vectors are disclosed as well.

The present invention further provides a method for the establishment of a lipid biosynthesis variety of a plant, comprising the steps of:

(a) constructing a nucleic acid comprising a nucleotide sequence that encodes a cuticular lipid gene product;

(b) inserting the nucleic acid into plant cells; and

Brief Description of the Drawings

FIG. 1 is a diagram that presents a hypothesized general scheme of lipid biosynthesis in plants.

FIG. 2 is a physical map that indicates the relative locations of restriction endonuclease digestion sites for the glossy8-3142 8.6 kb HindIII genomic DNA fragment.

FIG. 3 is a partial genetic map of Arabidopsis as it correlates to a physical map for the region between the cer2 and 600 genetic markers. This figure further provides the relative positions of certain RFLP probes in this region and the identification of YACs that cover this region, all of which were used to accomplish a chromosome walk to the CER2 locus.

FIGS. 4A, 4B, and 4C are diagrammatic drawings of CER2-containing clones.

FIG. 5 is a hydrophobicity plot of the deduced CER2 amino acid sequence.

Description of the Preferred Embodiments

The present invention provides certain genetic sequences relating to lipid biosynthesis in plants, and further provides methods for the use of such sequences for the generation of new varieties of plants having improved resistance to disease and/or certain environmental conditions, or altered lipid content in seeds, for example. The genetic sequences disclosed herein encode at least a portion of individual genes that encode gene products that directly participate in the biosynthesis of cuticular lipid products. Such genes encode enzymes included in lipid biosynthetic pathways, for example. The present invention does not include genes that have indirect or incidental effects on the plant cuticular lipids, as in the case of glossy15 of maize, discussed hereinabove. The precise function of any of the identified genes may or may not be known, and is immaterial to the use of such genes as contemplated in the present invention. For example, one can use one of the identified genes, or portions thereof, for the purpose of altering the cuticular lipid component of a plant or of a portion of a plant by the insertion of said gene or genes into a plant using suitable means followed by screening the progeny of such a plant for variants or mutants that have the desired altered cuticular lipid component, again using suitable, conventionally available means. Such variants or mutants may incorporate new or altered or nullified gene functions as a result of methods disclosed herein.

The availability of reverse genetics systems, which are well-known in the art, makes the generation and isolation of down-regulated or null mutants feasible, given the availability of a defined nucleic acid sequence, as provided herein. One such system (the Trait Utility System for Corn, i.e., TUSC) is based on successful systems from other organisms (Ballinger et al., Proc. Natl. Acad. Sci. USA, 86, 9402-9406 (1989); Kaiser et al. (1990), Proc. Natl. Acad. Sci. USA, 87, 1686-1690; and Rushforth et al., Mol. Cell. Biol., 13, 902-910 (1993)). The central feature of the system is to identify Mu transposon insertions within a DNA sequence of interest in anticipation that at least some of these insertion alleles will be mutants. To develop the system, DNA was collected from a large population of Mutator transposon stocks that were then self-pollinated to produced F2 seed. To find Mu transposon insertions within a specific DNA sequence, the collection of DNA samples is screened via PCR using a gene-specific primer and a primer that anneals to the inverted repeats of Mu transposons. A PCR product is expected only when the template DNA comes from a plant that contains a Mu transposon insertion within the target gene. Once such a DNA sample is identified, F2 seed from the corresponding plant is screened for a transposon insertion allele. Transposon insertion mutations of the an1 gene have been obtained via the TUSC procedure (Bensen et al. (1995)). This system is applicable to other plant species, at times modified in accordance with knowledge and skills reasonably attributed to ordinary artisans.

In addition, in accordance with the present invention, long-chain fatty acids (LCFA's) and very long-chain fatty acids (VLCFA's), such as are currently obtained from the jojoba plant and sperm whale oil, may be produced. Both in vitro approaches and in vivo approaches are recited herein. For example, the various enzymes required for the appropriate lipid synthesis can be prepared from strains of host organisms, such as E. coli or S. cerevisiae, each strain of which includes the genetic information that encodes each enzyme needed for a suitable lipid biosynthetic pathway. Combining a suitable combination of such lipid biosynthetic enzymes in a reaction vessel with suitable substrates, energy source, co-factors and other ingredients known in the art will result in the in vitro production of LCFA's and VLCFA's. Alternatively, organisms can be transformed or transfected with suitable genetic material such that the quantity and/or quality of LCFA's or LVCFA's that are synthesized in vivo are altered to produce LCFA's and LVCFA's of a quantity and a quality that is desired. For example, such genetic engineering manipulation of an oil-producing plant will result in a plant or tissue culture that will produce LCFA's and/or VLCFA's of a superior sort and/or quantity, relative to what is produced by naturally-occurring oil-producing plants. By oil-producing plants, it is intended that maize, soybean, rapeseed, canola, cotton, safflower, peanut, palm, sunflower and the like are included.

In particular, the present invention provides an enriched or isolated nucleic acid, wherein the nucleic acid hybridizes under at least moderately stringent hybridization conditions to a second nucleic acid that includes a polynucleotide that encodes a cuticular lipid gene product of a plant. A cuticular gene product may be a polypeptide or an RNA molecule, and is preferably a polypeptide. The cuticular lipid gene product is involved in cuticular lipid biosynthesis, such as a biosynthetic enzyme for lipids, wherein precursor substrates are caused to react suitably to form LCFA's and VLCFA's or precursors thereof. Genes in maize that effect changes in the cuticular lipids have been named "glossy," in consequence of the appearance that typical mutations at a cuticular lipid gene in maize causes. See Schnable et al., Current Topics in Plant Physiology, 9, 196-206 (1993). Similar effects have been noted in Arabidopsis, wherein cuticular lipid gene mutants have been named "Cer," which is short for Ecerferum. As noted above, all plants have cuticular lipid genes, the isolation of which are enabled by the methods of the present invention. As illustrated hereinbelow, cloned polynucleotides that encode maize glossy gene products may be used to identify and isolate related or homologous polynucleotides in maize and other genomes.

The term "nucleic acid" refers to a polymer of DNA or RNA, i.e., a polynucleotide, which can be single- or double-stranded, and can optionally contain synthetic, nonnatural, or altered nucleotides. Any combination of such nucleotides can be incorporated into DNA or RNA polymers. The nucleic acid is "enriched" in that the concentration of the material is at least about 2, 5, 10, 100, or 1,000 times its natural concentration, for example, advantageously 0.01% by weight, preferably at least about 0.1% by weight. Enriched preparations of about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. A polynucleotide is "isolated" in that the material has been removed from its original environment, e.g., the genome of a plant, presuming that it is naturally occurring. It is also advantageous that the nucleic acids be in purified form, wherein "purified" does not mean absolute purity but rather relative purity, wherein, for example, the nucleic acids of the present invention are isolated in a laboratory vessel in a mixture of other nucleic acids, such as portions of a vector or other molecules associated with genetic engineering.

Preferably, the enriched or isolated nucleic acid of the present invention hybridizes under at least moderately stringent hybridization conditions to a second nucleic acid that includes nucleotide sequences specific to a gene selected from the group consisting of glossy1, glossy2, glossy3, glossy4, glossy6, glossy8, glossy11, glossy19, glossy25, glossy26, and cer2, or substantial portions thereof; more preferred, the nucleic acid hybridizes under the aforementioned conditions to a second nucleic acid that includes nucleotide sequences specific to a gene selected from the group consisting of glossy1, glossy2, glossy8, glossy25, glossy26, and cer2, or substantial portions thereof; yet more preferred the selected nucleotide sequence are specific to the glossy1, glossy2, glossy8, or cer2 genes; most preferred, the selected nucleotide sequences are specific to glossy1 or glossy8. Another preferred embodiment of the present invention relates to the enriched or isolated nucleic acid that hybridizes under stringent hybridization conditions to a second nucleic acid, as listed above.

Stringency of hybridization is a term of art that refers to the conditions used for a hybridization reaction, whereby complementary single strands of nucleic acid join to one another to form double-stranded nucleic acid with some degree of mismatch, the degree of which is a function of the stringency used. In particular, the stringency will depend upon the size and composition of the strands of nucleic acid that are caused to react, the degree of mismatching allowed, the desired cross reactivity, and the like. The degree of stringency can be affected by the ionic conditions employed and temperature, among others, as is well known in the art. Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989).

As used in the context of the present invention, the specified stringency of hybridization in part define the inventive nucleic acid. Accordingly, the hybridization conditions are designed suitably to be at least moderately stringent or stringent. In the former case, suitable conditions of salt, temperature, reaction mixture, and size of nucleic acid reactants are set in accordance with conventional knowledge to provide from about 45% to about 70% mismatch of the sequence of nucleotides of the nucleic acid. Preferably, moderately stringent hybridization conditions are set to provide from about 55% to about 75% mismatch; and more preferably, such conditions are set to provide from about 60% to about 70% mismatch. In the latter case, suitable conditions for hybridization are set in accordance with conventional knowledge to provide from about 10% to about 40% mismatch. Preferably, stringent hybridization conditions are set to provide from about 20% to about 40% mismatch; and more preferably, such conditions are set to provide from about 30% to about 40% mismatch. By mismatch, it is meant the degree to which non-complementary base pairs are found opposite one another in otherwise duplex nucleic acid, thereby forming bubble structures and causing the melting temperature of the duplex to be lower as compared to a 100% matched duplex of the same length and base composition.

Seventeen loci of the glossy (gl) genes of maize have been described in the technical literature (for example, see Bianchi et al., Maydica, 30, 179 (1985)), and have been further elucidated by work disclosed herein. In addition, the present invention provides two more glossy loci, namely glossy25 and glossy26, which, since filing of the parent application, have been disclosed by Schnable et al., Maydica, 39, 279-287 (1994). The glossy loci affect the quantity and/or composition of cuticular lipids on seedling leaves, and, perhaps, elsewhere on the plant cuticle. The names of the known loci are glossy1, glossy2, glossy3, glossy4, glossy5, glossy6, glossy8, glossy9, glossy11, glossy14, glossy15, glossy17, glossy18, glossy19, glossy2o, glossy21, glossy22, glossy25, and glossy26; albeit, as noted above, glossy15 apparently encodes a developmental control mechanism that provides indirect or incidental effects upon the plant cuticle, and, thus, is not included as a cuticular lipid gene as that term is used in the context of the present invention. In contrast, the other glossy genes apparently encode gene products that have direct effects upon the plant cuticle, such as would be the effect of a gene in a lipid biosynthetic pathway. Mutant seedlings are usually identified because applied water forms droplets on their leaf surfaces and a "glossy" appearance is obtained thereby.

Four of the 19 glossy loci are members of duplicate gene pairs (namely, glossy5 and glossy20, and glossy21 and glossy22). Accordingly, a seedling must be homozygous mutant for both members of such pairs before it expresses a mutant phenotype. The existence of the duplicate gene pairs suggests that the glossy genes, or a subset thereof, may represent a gene family, wherein the members are related to an ancestral locus that duplicated, diverged in sequence, followed by succeeding rounds of gene duplication and sequence divergence over evolutionary time. The importance of this observation and hypothesis is that the identification of one nucleic acid sequence specific to one of the glossy genes, which is disclosed should provide probes therefrom for the identification and isolation of other so-related glossy genes.

The present invention is also directed to a nucleic acid as described hereinabove that is flanked by regulatory sequences. Preferably, such regulatory sequences are specific to plants, fungi, and bacteria. More preferably, such regulatory sequences are specific to a plant or plants, such as one of the aforementioned oil-producing plants. As noted above, the present invention provides, inter alia, plant varieties that differ from wildtype by the inclusion of nucleic acids in accordance with the present invention. In this context, regulatory sequences of nucleic acid that direct the activity of a particular gene to occur at a point in or period of the development of the plant and/or direct the accumulation of LCFA's or VLCFA's in a particular structure of the plant, such as the seed or fruit, may be affixed upstream or downstream of the cuticular lipid gene of interest. Such regulatory DNA sequences are known to those of ordinary skill in the art.

The nucleic acid of the present invention can be isolated from any plant. As noted in the Background section above, all plants have lipids in their respective cuticles, which lipids are synthesized de novo as fatty acids in the plastids of the plant's cells, after which the fatty acids are transported to the cytosol, where chain elongation reactions are mediated by the cuticular lipid gene products disclosed herein, and, perhaps, other enzymes generically referred to as elongases, acylases, and the like. Both monocotyledons and dicotyledons may be sources of such nucleic acids as provided by the present invention.

Preferred nucleic acids of the present invention are isolated from any suitable oil-producing plant. Such plants are either monocotyledonous or dicotyledonous, including maize, soybean, rapeseed, canola, cotton, safflower, peanut, palm, and sunflower. The more preferred plants from which the cuticular lipid genes are derived are maize or canola.

The present invention also provides an enriched or isolated polynucleotide, wherein the polynucleotide hybridizes under at least moderately stringent hybridization conditions to a second polynucleotide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, and polynucleotides complementary thereto. A preferred nucleic acid according to the present invention hybridizes to the same set of second polynucleotides only under stringent hybridization conditions. A more preferred enriched or isolated nucleic acid of the present invention comprises a polynucleotide selected from the group of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, and polynucleotides complementary thereto, and polynucleotides substantially equivalent thereto. The polynucleotides identified by sequence identification number are fully described in the Examples below.

A "substantially equivalent" polynucleotide is a polynucleotide that varies from the identified polynucleotide by one or more substitutions, deletions, or additions, the effect of which does not result in an undesirable functional dissimilarity between the two polynucleotides. In other words, the polypeptide that results from the substantially equivalent polynucleotide has the activity characteristic of, for example, the glossy8 gene product, when considering polynucleotides that are substantially equivalent. A difference in sequence at the amino acid level will be understood to include amino acid differences, which range from a single amino acid substitution, deletion, or insertion to a number of amino acid substitutions, deletions, and/or insertions, wherein the resulting polypeptide is still recognizable as related to the glossy8 protein as well as those amino acid sequence differences, which result in a larger polypeptide, such as a precursor protein, a complete mature protein, and a truncated protein.

The enriched or isolated nucleic acid which comprises the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21 is most preferred. The present inventive nucleic acid may be identified for enrichment or isolation by hybridization to any subfragment of a polynucleotide of the aforementioned set of polynucleotides of at least 20 nucleotides under stringent hybridization conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989). Accordingly, this invention preferably encompasses the entire sequence of the glossy8, glossy1, glossy2, or CER2 gene and fragments thereof, which have been generated by any suitable technique, such as by restriction enzyme digestion of chromosomal or plasmid DNA, or by synthesis, and which may be either DNA or RNA.

In addition to the methods recited in Example 1 for the identification and isolation of cuticular lipid genes and related nucleic acids of the present invention, other methods may be used alternatively, such as, inter alia, chromosome walking and heterologous probe selection.

Chromosome walking, which is described in Example 6, is a particularly useful technology that can facilitate the molecular isolation of any mapped gene (Bender et al., J. Mol. Biol., 168, 17-338 (1983)) and has been found to be particularly useful with plants that have a relatively small genome size, such as that of Arabidopsis. Meyerowitz, in Methods in Arabidopsis Research, pp. 100-118 (Koncz et al., eds., World Scientific, Singapore, 1992). In addition, five YAC libraries, representing 28 genome equivalents, exist for this species. Gibson et al., in Methods in Arabidopsis Research (C. Koncz et al., eds., River Edge N.J., World Scientific, 1992), pp. 119-143. Several Arabidopsis genes have been cloned via this strategy (Yanofsky et al., Nature, 346, 35-39 (1990); Giraudat et al., Plant Cell, 4, 1251-1261 (1992); and Arondel et al., Science, 258, 1353-1354 (1992)). This technique is useful, of course, in species of larger genome size as well, such as maize.

A chromosome walk is initiated by identifying from a library of large DNA fragments the specific fragment(s) that contain sequences homologous to a restriction fragment length polymorphism (RFLP) marker that is closely linked to the target gene. Typically, the library of DNA fragments is maintained as yeast artificial chromosomes, i.e., YACs (Burke et al., Science, 236, 806-811 (1987)), although cosmids, P1 phage or .lambda. phage have been used. Single copy sequences from the termini of YACs that contain sequences homologous to a starting RFLP are then used as hybridization probes to isolate overlapping DNA fragments. This process is repeated until the entire chromosomal region, from the starting RFLP marker to beyond the target gene, has been cloned as a contiguous segment (a "contig"). Typically, the contig is oriented by mapping DNA sequences from the growing contig to the genetic/RFLP map. Similarly, the endpoint of the walk is established by demonstrating that the contig contains DNA sequences from both sides of the target gene. For both of these operations, DNA sequences from the contig must be genetically mapped. The efficiency of this mapping can be greatly increased by selecting a population of plants that have a high probability of carrying recombination breakpoints in the region defined by the contig. Such a mapping population is established by selecting plants that carry a recombination breakpoint between two visible genetic markers that flank the interval to be walked. The precision of the mapping increases proportionally with the number of genetic recombinants. The greater the precision of this mapping, the smaller the uncertainty associated with the positioning of the target gene on the contig. Once the target gene has been localized in the contig to as small an interval as the mapping population permits, the target gene is identified from the interval via its ability to complement genetically the mutant phenotype. The ability of a sequence to complement the mutant phenotype is assayed by transforming plants homozygous for a mutant allele of the target gene. Alternatively, comparisons between wildtype and mutant sequences can also identify the target gene from the interval.

Other technologies for gene isolation in Arabidopsis and other plants include genomic subtraction, and transposon and T-DNA tagging. Genomic subtraction requires the availability of strains having deletions of the target gene (Strauss and Ausubel, Proc. Natl. Acad. Sci. USA, 87, 1889-1893 (1990); and Sun et al., Plant Cell, 4, 119-128 (1992)); however, such deletions are not available for CER2, for example. A transposon tagging system in Arabidopsis has recently become available. The success in tagging and cloning a petunia gene using a heterologous maize transposon (Chuck et al., Plant Cell, 5, 371-378 (1993)) provided the direction to extend this technique to Arabidopsis, and further supports the view that this technique is amenable to tagging virtually any plant with heterologous (Dean et al., Plant J., 2, 69-81 (1992); Grevelding et al., Proc. Natl. Acad. Sci. USA, 89, 6085-6089 (1992); Swinburne et al., Plant Cell, 4, 583-595 (1992); and Fedoroff and Smith, Plant J., 3, 273-289 (1993)) and/or endogenous (Tsay et al., Science, 260, 342-344 (1993)) transposons. T-DNA tagging, another method to locate a gene, has been realized (Feldmann, Plant J., 1, 71-82 (1991)) and is in wide use (e.g., Feldmann et al., Science, 243, 1351-1354 (1989); Herman et al., Plant Cell, 11, 1051-1055 (1989); Konz et al., EMBO J., 9, 1337-1346 (1989); and Kieber et al., Cell, 72, 427-441 (1993)). Additionally, having isolated at least one cuticular lipid gene, the nucleic acid thereof can be used whole or in part (by sub-cloning fragments thereof) as a probe in heterologous systems, as exemplified in Example 4 hereof. Preferably, such a technique requires that the stringency of the selective hybridization procedure be lowered, and then slowly raised, as is well known in the art.

Although T-DNA tagging, chromosome walking or heterologous probe selection can identify a DNA fragment that putatively contains the gene of interest, in each instance these DNA fragments must be confirmed by genetic complementation or some other means, which is fully disclosed in Examples 1 and 7. Although the methods of identification of a particular gene sequence has been described herein largely with reference to maize and Arabidopsis only, it is abundantly clear to one of ordinary skill that such methods may be adapted for gene identification in other species, particularly in the context of the present invention. Moreover, having identified and isolated particular cuticular lipid genes from maize and Arabidopsis, counterpart or homologous genes in other species may be identified and isolated using the sequences disclosed herein as probes, using suitable conventional means as, for example, illustrated in Example 5. Accordingly, the identification of the cuticular lipid genes, and cloning and using thereof, is enabled hereby for any of the aforementioned oil-producing plants, as well as other plants that have lipid components in their cuticle.

The nucleic acids of the present invention may be cloned in any suitable vector and the vector as constructed with nucleic acid insert of the present invention is used to transform or transfect any suitable host. E. coli, in particular E. coli DH5.alpha., SA2821 or Y1090 is a preferred host. Suitable vectors include those designed for propagation and expansion or for expression or both. Constructs of vectors can be prepared, either circular or linear, to contain the entire cuticular lipid gene nucleotide sequence or a portion thereof ligated to a replication system functional in a microorganism host, whether prokaryotic or eukaryotic. Suitable hosts include E. coli, B. subtilis, P. aerugenosa, S. cerevisiae, and N. crassa. Replication systems may be derived from ColE1, 2 m.mu. plasmid, .lambda., SV40, bovine papilloma virus, or the like. In addition to the replication system and the inserted DNA, the construct usually will include one or more markers, which allow for selection of transformed or transfected hosts. Markers may include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. A preferred cloning vector is selected from the group consisting of pUC18, pET11d, EMBL4, NM1149, and pLZO3.

More preferably, the present invention provides expression vectors for the expression of such polypeptides. A preferred expression vector is one that comprises a nucleic acid comprising, as an insert, a nucleotide sequence that encodes a cuticular lipid gene product, preferably that of one of the aforementioned oil-producing plants. A more preferred expression vector comprises a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, a sequence complementary thereto, or a substantially equivalent sequence. The most preferred expression vector comprises a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21 and a vector as described above.

One skilled in the art will appreciate that any one of a number of expression vectors may be utilized in the context of the present invention with some degree of success, including, but not limited to, the following: pGEX2T, pATH11, pNH8A (Strategene, Inc., La Jolla, Calif.), pGL2 (Promega, Madison, Wis.), pEX2 (Boehringer Mannheim Biochemicals, Indianapolis, Ind.), and pMOSELlox (Amersham Corporation, Arlington Heights, Ill.).

Care should be taken to choose a vector that does not result in cytotoxic expression of the amino acid sequence encoded in the insert of the vector. For expression in microorganisms, the expression vector may differ from the cloning vector in having transcriptional and translational initiation and termination regulatory signal sequences and may or may not include a replication system that is functional in the expression host. The coding sequence is inserted between the initiation and termination regulatory signals so as to be under their regulatory control. Expression vectors may also include the use of regulatable promoters, e.g., temperature-sensitive or inducible by chemicals, or genes that allow for integration and amplification of the vector and insert DNA, such as tk, dhfr, metallothionein, and the like. Such controls, if incorporated into a plant, could allow for drought resistance, for example, by promoting expression of drought resistant cuticular lipids upon the advent of a triggering level of heat. Other environmental resistant varieties may promote expansion in response to water stress or UV stress, for further examples.

The vector can be used to express a dsDNA sequence, either isolated and cloned or synthesized, to obtain a precursor protein, which is subject to further manipulation by cleavage, a complete mature protein, or a fragment thereof by introducing the expression vector into an appropriate host, where the regulatory signals are functional in the host. The expression host is grown in an appropriate nutrient medium, whereby the desired polypeptide is produced and isolated from cells or from the medium, when the polypeptide is secreted. Where a host is employed in which the vector's transcriptional and translational regulatory signals are functional, then the cuticular lipid gene DNA sequence may be manipulated to provide for the expression of the desired polypeptide in proper juxtaposition to the regulatory signals. The polypeptide products can be obtained in substantially pure form, particularly free of cellular debris, which may include such contaminants as, for example, proteins, polysaccharides, lipids, nucleic acids, viruses, bacteria, fungi, and combinations thereof, using methods well known in the art.

The nucleic acids described above may be used in a wide variety of ways, depending upon their size, their natural function, the use for which they are desired, and the degree to which they can be manipulated to modify their function. For example, nucleic acids of at least about 20 bases, more usually at least 50 bases, and usually not exceeding about 1,000 bases, more usually not exceeding about 5,000 bases, may serve as probes for the detection of the presence of a cuticular lipid gene or homologous nucleic acids in an organism. Such detection can provide information relating to whether manipulation of the plant with a particular cuticular lipid gene could provide an opportunity to generate new, resistant varieties of the plant, and provide novel sources of various oils.

The method of detection involves duplex formation by annealing or hybridization of the oligonucleotide probe, either labeled or unlabeled, depending upon the nature of the detection system, with the DNA or RNA of an organism believed to produce the particular cuticular lipid gene. Usually this method of detection involves cell lysis, extraction of nucleic acids with organic solvents, precipitation of nucleic acids in an appropriately buffered medium, and isolation of the DNA or RNA. The DNA may be fragmented by mechanical shearing or restriction endonuclease digestion. The nucleic acid may then be bound to a support or may be used in solution depending upon the nature of the protocol. The Southern technique (Southern, J. Mol. Biol., 98, 503 (1975)) may be employed with denatured DNA by binding the single-stranded fragments, for example, to a nitrocellulose or nylon filter. RNA also may be blotted onto a filter (Thomas, Proc. Natl. Acad. Sci. USA, 77, 5201 (1980)). Preferably, the fragments are subjected to electrophoresis prior to binding to a support so as to enable the selection of variously sized fractions. Alternatively, the assay may be accomplished on plant cells fixed to a substrate and permeabilized using methods known in the art, whereupon the hybridization procedure can be conducted to determine if a homologous gene to a particular cuticular lipid gene exists in the plant of interest, and/or if that plant is expressing RNA that is homologous to the cuticular lipid gene.

The oligonucleotide probes may be DNA or RNA, albeit usually they are DNA. The oligonucleotide sequence may be prepared synthetically or by cloning. Suitable cloning vectors are well-known to those skilled in the art. The oligonucleotide probe may be labeled or unlabeled. A wide variety of techniques exist for labeling DNA and RNA and include radiolabeling by nick translation, tailing with terminal deoxytransferase, or the like, where the bases employed are labeled, for example, with radioactive .sup.32 P. Other labels, that may be used include fluorophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, and the like. Alternatively, instead of using a label, which provides a detectable signal by itself or in conjunction with other reactive agents, ligands can be used to which receptors bind, where the receptors are labeled, such as with the above-indicated labels, to provide detectable signals by themselves or in conjunction with other reagents (see, e.g., Leary et al., Proc. Natl. Acad. Sci. USA, 80, 4045-4049 (1983)). The oligonucleotide probes are hybridized with the denatured nucleic acid, substantially intact or fragmented, or fractions thereof, under conditions of predetermined stringency, the practicalities of which have been discussed hereinabove.

The present invention also provides an enriched or isolated polypeptide comprising an amino acid sequence of a cuticular lipid gene product from a plant, one example of which comprises the polypeptide encoded by the glossy8 gene of maize. Other such polypeptides disclosed in the context of the present invention include SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, and SEQ ID NO:23. Preferably, the enriched or isolated polypeptide comprises an amino acid sequence encoded by a nucleic acid that comprises the structural coding portion of a nucleotide sequence, such as one selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, polynucleotides complementary thereto, or polynucleotides substantially equivalent thereto. More preferably, the enriched or isolated polypeptide comprises the polypeptide selected from the group consisting of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:22, and SEQ ID NO:23.

The enriched or isolated polypeptide is "enriched" in that the concentration of the material is at least about 10, 100, 1,000 or 10,000 times its natural concentration, for example, advantageously 0.1% by weight, preferably at least about 1% by weight. Enriched preparations of about 5%, 10%, 20%, 50% and 75% or more by weight are also contemplated. A polypeptide is "isolated" in that the material has been removed from its original environment, e.g., the cytoplasm or endoplasmic reticulum of a plant cell or microbial host cell, such as E. coli or S. cerevisiae. It is also advantageous that the polypeptides of the present invention be in purified form, wherein "purified" does not mean absolute purity but rather relative purity, which is defined herein as in excess of 80% pure by weight. Such polypeptides can be combined to catalyze reactions required in the biosynthesis of valuable LCFA's and VLCFA's, as discussed herein above, using methods known in the art.

The polypeptides of the present invention are encoded by the cuticular lipid genes of a plant. Plants used in the context of the present invention that provide the genes of interest and the polypeptides encoded therein may be any suitable plant, and is preferably an oil-producing plant, as delineated hereinabove.

Most preferred polypeptides of the present invention are those that are encoded by an enriched or isolated nucleic acid comprising a polynucleotide selected from the group consisting SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, a polynucleotide complementary thereto, and a polynucleotide substantially equivalent thereto, as understood by the earlier discussion regarding the nucleic acids of the present invention.

In accordance with another aspect of the present invention, the nucleic acids disclosed herein are used in a method for the establishment of a lipid biosynthesis variety of a plant cell or of a plant, comprising the steps of: (a) constructing a sense nucleic acid or an anti-sense nucleic acid specific for a cuticular lipid gene product or a portion of the cuticular lipid gene product; (b) inserting the nucleic acid into plant cells; and (c) culturing the cells. Additionally, the inventive method further comprises the (d) culturing of the cells to provide the plant, which plant can then produce seeds. Such seeds may or may not be fertile. Accordingly, the inventive method involves plant tissue culture techniques known in the art, and may involve standard genetic and reverse genetic approaches to the generation and identification of variants or mutants having new, altered, or nullified gene functions with respect to specific genes.

Insertion of nucleic acids into plant cells is accomplished by any suitable means, including cell bombardment, i.e., attaching the DNA to metallic pellets and blasting them through the plant's cell wall (Fromm et al., Bio/Technology, 8, 833-839 (1990); and Gordon-Kamm et al., Plant Cell, 2, 603-618 (1990)), and, for the introduction of exogenous DNA to a dicotyledonous plant cell, insertion of the nucleic acid of the present invention into the Ti plasmid of Agrobacterium and adding suitable ingredients to promote transformation thereby (Horsch et al., Science, 223, 496-498 (1984); and DeBlock et al., EMBO J., 3, 1681-1689 (1984)). Other techniques are available for the introduction of exogenous DNA into a plant and/or a subset of its constituent cells, including electroporation, protoplast-mediated gene transfer, and silicon carbide crystal-mediated gene transfer. These various techniques are discussed in Genetic Engineering News, vol. 14, no. 4 (Feb. 15, 1994) pages 1, 3, and 24, and are generally known in the art.

The nucleic acid that is used in the inventive method hybridizes under at least moderately stringent hybridization conditions to a DNA that includes nucleotide sequences specific to a gene selected from the group of maize glossy genes, as disclosed above. Such DNA preferably is flanked with a regulatory sequence, such that a new plant variety, or a cell thereof, derived from the aforementioned procedure, expresses its extra or altered or nullified cuticular lipid genes in a developmentally and/or tissue-specific fashion. That is, lipid genes used in the context of the present invention may be used to add, alter, or remove a gene function that affects at least one cuticular lipid of a treated plant.

The method of the present invention is used to establish a new lipid biosynthesis plant variety of any suitable plant, which new variety is also an embodiment of the present invention. Indeed, the present invention relates to a new plant variety that is generated and isolated by the aforementioned method for the establishment of a lipid biosynthesis variety of a plant, comprising the steps as articulated above. Preferably, such a plant produces oil, such as maize, soybean, rapeseed, canola, cotton, safflower, peanut, palm, and sunflower. Most preferably, the method is used to produce new varieties of maize and canola.

The new varieties that are provided by the inventive method include environmental, disease, and pest resistance varieties. Environmental resistance relates to wind, frost, UV or drought. The connection between the lipid complement of the cuticle and such resistance is substantial, in view of the knowledge in the art that the lipid cuticular component lends strength to the overall plant, serves to prevent undue evaporation of internal liquids, as well as serves to filter UV radiation, thereby preventing undue exposure to environmental UV levels that keep rising.

Disease and insect resistance mediated by the cuticular lipids includes the blocking or retarding of fungal-, viral-, or bacterial-borne disease vectors, the most important of which are the fungal-borne rust diseases. Such disease vectors typically are opportunistic, entering the plant by way of tears or cuts in the cuticle. Obviously, altering the lipid complement of the cuticle so as to increase the strength and toughness of the cuticle necessarily reduces inadvertent tears and cuts, which, in turn, reduce opportunistic infestation by microbial pests. Similarly, strengthening and toughening the cuticle protects the plant against attack by various other pests, including insects, spiders, aphids, and the like.

Disease resistance in accordance with the present invention may also be conferred by the alteration or elimination of certain lipids that are attractive or otherwise stimulate certain microbial pathogens or opportunistic insects. The present invention provides the means for the removal or alteration of such lipid-based chemical signals.

Particularly with regard to microbe or insect pest resistance, a factor in the susceptibility to damage by such pests is the availability of water. Lipid components that foster retention of water droplets on the cuticle surface will tend to invite growth and attention by microbes and insects. Similarly, frost resistance is a function of the retention of as little surface water as possible on the plant. Accordingly, altering the lipid components of any plant provides environmental- and microbe-resistant varieties of plants.

Another aspect of the present invention relates to a method of altering the lipid content of suitable plant structures or tissues, such as seed, fruit and flower. Such an alteration can increase or decrease the normal levels, or change the kind of lipids present in a particular plant structure. Growth of plants or tissue cultures of suitable cuticular lipid gene constitution, therefore, can provide commercial sources of such lipids or combination of lipids.

The present invention also relates to any suitable organism comprising the vector comprising a nucleic acid comprising a nucleotide sequence that encodes a cuticular lipid gene product of a plant. A suitable organism can be any suitable plant, yeast, or bacteria, such as discussed hereinabove regarding suitable plants from which to isolate the cuticular lipid genes and regarding suitable hosts in which to insert the enriched or isolated nucleic acid of the present invention. Preferred organisms are oil-producing plants, as described above, and yeast, such as S. cerevisiae. A seed-bearing plant that hosts a vector/nucleic acid construct can bear seeds, which, themselves, include the construct, which seeds also constitute a preferred embodiment of the present invention.

Finally, a plant cell hosting a vector/nucleic acid construct of the present invention constitutes another preferred embodiment. Such cells can be cultured and kept as plant tissue culture cells, or certain plant hormones known in the art may be included in the culture media, thereby causing the plant tissue culture cells to differentiate and thereby form a new plant variety. Such plant culturing methods useful in the performance of this aspect of the invention are well-known in the art. Such a new plant variety may be fertile or infertile.

The following examples further illustrate the present invention, but, of course, should not be construed as in any way limiting its scope.

Example 1

This example sets forth methods used in the identification and isolation of glossy genes.

Gene tagging using naturally-occurring transposons, as described by Walbot, Ann. Rev. Plant Physiol. Plant Mol. Biol., 43, 49-82 (1992), was used to identify previously mapped glossy genes. Specifically, glossy1, glossy2, glossy3, glossy4, glossy6, glossy8, and glossy11 were targeted in the following isolation plot cross using the method of Peterson, in Maize Breeding and Genetics (D. B. Walden, ed., John Wiley & Sons, New York, N.Y., 1978), pages 601-631 (the female parent is listed first in all crosses herein):

wherein x is one of 1, 2, 3, 4, 6, 8, or 11. In the absence of mutation, progeny kernels from each cross are heterozygous for the wildtype allele (e.g., glossy1/Glossy1 or glossy8/Glossy8), and should, therefore, yield wildtype seedlings. Only if a gamete from the female (Mutator) parent carries a newly generated mutant allele (e.g., via the insertion of a Mu1 element, signified as glossy1-Mu, for example) will a glossy seedling be obtained (glossy1-Mu/glossy1 or glossy8-Mu/glossy8, for example). Data of number kernels obtained, progeny screened, and mutants identified are provided in the following table.

For isolation of glossy genes for which no existing mutant allele exists, or which are lethal as homozygotes, a random gene tagging method was used, as described by Schnable et al., Proceedings, 28th Annual Illinois Corn Breeder's School (1992), pages 24-45. The first step was to cross a standard transposon stock to a standard wildtype line of maize. Gametes produced by the transposon stock carry newly-generated transposon insertion mutants at random loci. Thus, many of the progeny from this cross will be heterozygous for new mutations, which are uncovered by self-pollinated crosses and observation of the progeny of such S1 families. Over 10,000 Mutator self-crossed progeny were screened for visible mutants, from which 42 independent glossy mutants have been recovered.

To determine the affected locus of recovered random tagged mutants, TB tests and allelism tests were conducted. TB tests locate mutants to chromosome arm by exploiting the fact that BA translocations fail to disjoin during the second meiotic division during microsporogenesis, according to Birchler, in Maize for Biological Research (W. F. Sheridan, ed., Plant Mol. Biol. Assoc., Charlottesville, N.C., 1982), pages 75-78. Allelism tests can be performed by crossing plants homozygous for the Mu1-tagged allele by separate tester lines, each of which is homozygous recessive for one of the existing standard loci. If the tagged allele is allelic to a given standard allele, all seedlings from that cross (and only that cross) will be glossy. Seedlings from all other crosses will have a wildtype phenotype. These tests have established that 23 of the 49 glossy mutants identified in the random tagging process are allelic to previously defined glossy mutants. This collection consists of 6 alleles of glossy1, 1 of glossy2, 1 of glossy3, 3 of glossy4, 13 of glossy8, 2 of glossy11, and 1 of glossy19. In addition, two of the 42 glossy mutants are not allelic to known mutants or to each other; these mutants define previously unknown genes, glossy25, and glossy26. The allelism status of 13 of the glossy mutants is still under analysis.

Using standard maize breeding techniques to analyze over 100 Mutator-induced glossy mutants, it was established that the tagged mutant glossy alleles were heritable. Cloning the various glossy-related genes, both wildtype and mutant forms thereof, was predicated on the availability of the transposon element, itself, as a molecular probe, as disclosed by Chandler and Hardeman, Adv. Genet., 30, 77-122 (1993). Mu1, one of the nine classes of Mu elements, is known to be responsible for the vast majority of mutations recovered from Mutator lines (Brown et al., Genetics, 122, 439-445 (1989)); therefore, a Mu1 sequence was used to probe for glossy genes. Mu8 was also used.

Because the Mu elements are typically present in 10-50 copies in the genomes of plants carrying an active Mutator system, a serial outcrossing program was undertaken to reduce the number of Mu elements in the glossy mutant lines. The glossy-Mu lines were crossed to lines that either lack or have low copy numbers of Mu elements in their genomes. Genomic DNA from the resulting progeny plants derived from Mutator parents was prepared and digested with HindIII, which does not cut within the Mu1 element. Southern genomic analyses of these DNAs were conducted according to Stinard et al., Plant Cell, 5, 1555-1566 (1993). Internal sequences from Mu1 were used as a probe to identify the plants having the lowest Mu1 copy numbers. These low-copy number plants were then backcrossed to the recurrent parent that either lacks Mu1 elements or has a low copy number. After three to four generations, this procedure provided lines that each carry a glossy allele and only 15-20 copies of the Mu1 element.

The next step was to utilize the low Mu copy number lines in co-segregation analyses to identify the individual Mu element inserted at the glossy locus. First, sibling plants derived from the cross of gl-Mu/Gl with gl-ref/gl-ref, with and without the glossy-Mu allele (glossy and wildtype seedlings, respectively), were obtained. DNA from each individual was digested with a restriction enzyme that does not cut within the Mu element, such as HindIII. Mu specific sequences were used to probe genomic Southern blots of these DNAs in an effort to identify a Mu-containing band that co-segregates with the glossy-Mu allele, i.e., present in all glossy seedlings, but absent in wildtype siblings.

For example, the Mu1 element co-segregated with glossy1-5048 in 93 individuals.

Genomic DNA from progeny resulting from the cross glossy1-5048/Gl1.times.Gl1/Gl1 was prepared and digested with HindIII. Genotypes of progeny from this cross were established via appropriate testcrosses. Southern analyses of these DNAs using a Mu1-specific probe revealed a 12.5 kb Mu1-containing restriction fragment that co-segregates with the glossy1-Mu allele. Accordingly, the identified fragment is present in all plants carrying the glossy1-Mu allele (i.e., glossy1-5048) and absent in all plants that do not carry this allele.

Similarly, a Mu8-containing 8.6 kb HindIII DNA fragment that co-segregates with glossy8-3142 in 88 individuals was identified.

Genomic DNA from progeny resulting from the cross glossy8-3142/Glossy8.times.Glossy8/Glossy8 was prepared and digested with HindIII. Genotypes of progeny from this cross were established via standard test-crosses. Southern blot analysis, as described above, of these DNA preparations using a Mu8-specific probe revealed an 8.6 kb Mu8-containing restriction fragment that co-segregates with the gl8-Mu allele. As above, the fragment of interest is present in all plants carrying the mutant allele, here glossy8-3142, and absent in all plants that do not carry this allele.

Genomic libraries were constructed for each of the glossy mutant lines that display a co-segregating Mu-hybridizing restriction fragment, identified hereinabove. These libraries were constructed in lambda-based cloning vehicles, such as EMBL4, Charon 33, or NM1149, using the methods of Frischauf et al., J. Mol. Biol., 170, 827-842 (1983), and Murray in The Bacteriophaqe Lambda II (Hedrix, ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1983), pages 395-432. DNA from an identified glossy line was digested with the identified restriction enzyme and size-selected to include the co-segregating, Mu-containing fragment. Once completed, these libraries were screened with a Mu probe and positive clones were identified via the procedure of Benton and Davis, Science, 196, 180-182 (1977). Following plaque purification, DNA from positive recombinant phage was isolated according to the method of Yamamoto et al., Virology, 40, 734-744 (1970).

To illustrate, the 8.6 kb HindIII DNA fragment associated with gl8-3142 was cloned by first isolating DNA from an immature unpollinated ear of a maize plant with the genotype gl8-3142/Gl8. This DNA was digested to completion with HindIII, electrophoresed through an agarose gel, and DNA fragments in the size range of 7 to 9 kb were recovered by electro-elution. Size-fractionated DNAs were ligated into the HindIII site of the replacement .lambda. vector Charon 33, packaged using commercial packaging extracts (Stratagene) and plated on E. coli strain Y1090. Plaques were lifted on nylon membranes (MSI) and hybridized using a Mu8-specific probe. Hybridizing plaques were picked and purified. Phage DNA was isolated using miniprep DNA purification from two independent isolated positive clones and the 8.6 kb HindIII maize genomic inserts were isolated. Based on restriction enzyme digestion patterns, these two clones are identical. The restriction map based on these analyses is shown in FIG. 2.

The 12.5-kb Mu1-containing DNA fragment associated with the gl1-755048 allele was cloned in the same manner with the following minor modifications. DNA isolated from maize plants carrying the gl1-755048 allele was digested with HindIII, and 12-13 kb fragments were gel-purified and cloned into the .lambda. vector DASHII (Stratagene).

Putative glossy gene DNA sequences were then isolated from the recombinant phage. Because much of the maize genome consists of repetitive sequences, isolation of near-unique DNA sequences that flank the Mu element in positive clones were subjected to "reverse Southerns," according to the techniques of Gupta et al., EMBO J., 3, 133-139 (1984). This procedure requires size fractionating restriction enzyme digested DNA from each clone on an agarose gel, and transferring the DNA, in duplicate, to membranes, as in the procedure of Southern, supra. One of the membranes was probed consecutively with Mu and lambda vector sequences to identify the cloned fragment or fragments that contain the Mu element and those that correspond to the phage vector used to construct the library. The second membrane was probed with labeled total maize genomic DNA to identify repetitive DNA fragments, as unique sequences do not give a signal in this procedure. Fragments that do not give a signal on any of the three hybridizations represent unique or near-unique sequences flanking the Mu element in the maize genome and putatively consist of the glossy locus. These fragments have been subcloned.

To verify the identity of the putative glossy clones, flanking sequences from a putative clone were used as a probe against a series of allelic transposon-induced mutants and their wildtype progenitor alleles. Flanking sequences from a correct clone would be expected to reveal polymorphisms between progenitor wildtype alleles and their corresponding mutant derivatives (as a result of the insertion of a transposon). Exceptional glossy seedlings were isolated from the cross Mutator Glossy8/Glossy8.times.glossy8-Ref/glossy8-Ref, and had the genotype glossy8-Mu/glossy8-Ref. Sibling wildtype seedlings had the genotype Glossy8/glossy8-Ref. The smaller HindIII-SacI fragment from the putative glossy8 clone (see FIG. 2) hybridized strongly to two bands in HindIII-digested DNA prepared from the glossy and wildtype seedlings. The upper band, which is common to all plants, represents the glossy8-Ref allele. The second (lower) band represents the second allele present in these seedlings (Gl8 in the case of the wildtype seedlings and glossy8-Mu in the glossy seedlings). This second band is larger in plants carrying the glossy8-Mu alleles than in plants carrying their progenitor wildtype Glossy8 allele. These alterations in DNA fragment sizes revealed by the putative glossy8 probe were coincident with mutations at glossy8, thereby demonstrating that the putative glossy8 clone represents the glossy8 gene.

Accordingly, using the procedures above, one can obtain clones of any of the glossy genes of maize and/or confirm the identity of a glossy clone. This procedure may be applied to any organism that has a defined transposon system or is amenable to a heterologous transposon system, and identifiable genetic markers.

Example 2

This example sets forth the sequence identified for the insert of a glossy8 cDNA clone, and the method by which the sequence was obtained.

A single copy isolated from the 8.6 kb gl8 genomic clone (i.e., the 3.6 kb HindIII-SacI fragment as shown in FIG. 2) was used to screen a cDNA library according to Sambrook et al., supra. This cDNA library had been prepared with RNA isolated from two-week-old seedlings of the maize inbred line B73. From a total of 800,000 plaques, 13 cDNA clones that could be grouped into three different classes were recovered. These classes of clones have inserts of 1.2 kb, 0.8 kb, and 500 bp in length, respectively. DNA isolated from these clones was double-digested with the restriction enzymes EcoRI and XhoI and then subcloned into pBSK and pBKS plasmid vectors for sequencing. The 0.8 kb cDNA was completely sequenced using an Applied Biosystems 370A Automated DNA sequencer. The partial 5' and 3' nucleotide sequences, confirmed by triplicate sequencing runs, follow:

The sequence displays the 3' poly A tail that is characteristic of cDNAs derived from mRNA. This sequence was compared to the genomic sequence of the glossy8-3142 allele. The 5' end of the cDNA matches the genomic region from the SacI site of the genomic clone (FIG. 2), close to the Mu8 insertion site. The 3' end of the cDNA matches the region of the genomic clone from the XhoI site towards the SacI site. This analysis has, therefore, established that the glossy8 gene is transcribed away from the Mu8 transposon insertion site and towards the left, as can be seen in FIG. 2.

The C-terminal portion of the gl8 protein exhibits a high degree of sequence similarity to .beta.-keto-acyl reductases, which provides suggestive evidence that the glossy8 gene product functions as a reductase in an elongation reaction (or reactions) required for the biosynthesis of cuticular lipids. Further cDNA clones are disclosed in Example 4.

Example 3

This example illustrates the endogenous expression of the glossy8 gene in maize, as assessed in a Northern blot experiment.

RNAs were isolated from two-leaf stage seedlings with the genotypes Glossy8/Glossy8 (Q60 F2), glossy8-ref/glossy8-ref and glossy8-3142/glossy8-3142, using the procedure of Dean et al., EMBO J., 4, 3055-3061 (1985). The glossy8-ref and glossy8-3142 alleles had both been backcrossed with the F1 hybrid Q60 for two generations to minimize differences in expression patterns caused by genetic background effect. The 3.6 kb HindIII-SacI fragment from the gl8-3142 genomic clone (FIG. 2) revealed a 1.4-kb gl8 transcript in Northern blots that were prepared using the method disclosed in Sambrook et al., supra. This mRNA was detected in both wildtype and mutant seedlings, but accumulated at higher levels in wildtype seedlings. In wildtype plants, the glossy8 mRNA accumulates to detectable levels at all stages of development and in all organs analyzed, including mature leaf, mature husk, mature ear, endosperm, Q60 F.sub.2 seedling root, and mature root.

Example 4

This example illustrates methods for the identification of DNA segments that crosshybridize with the glossy8 clone and portions thereof.

Leek and barley libraries were screened to isolate the respective maize gl8 homologs. Hybridization for the first screen was carried out at 50.degree. C. using an 800 bp maize gl8 partial cDNA as the probe. The first screen was washed at 50.degree. C. for 20 minutes each in solutions of 0.1% SDS and decreasing SSPE concentration (1.times., 0.5.times., 0.2.times., and 0.1.times.). Positives clones were hybridized at 70.degree. C. and washed at 70.degree. C. for 20 minutes each in solutions of 0.1% SDS and decreasing concentrations of SSPE (1.times., 0.5.times., and 0.2.times.) in subsequent purifications.

In particular, the barley homolog to maize gl8 was isolated from a sprouted barley-morex cDNA library constructed in the EcoRI site of .lambda. zap II. Phage were plated at a concentration of 40,000 pfu per plate with five plates used. The plates were grown overnight to allow lysis. The plaques were lifted to magna nylon. The filters were hybridized to a 800 bp maize gl8 partial cDNA probe. Filters were washed using standard methods and exposed for two days at -70.degree. C. and a single positive plaque was identified (M1B1). This plaque was purified by a second and third screen using standard methods. A cDNA containing plasmid was excised from purified .lambda. phage by using Stratagene's rapid excision protocol. The plasmid was isolated and retransformed into DH5.alpha.. The cDNA was sequenced on both strands using universal and reverse primers on a series of subclones of deletions from the original clone and also sequence-specific primers (CD696 and Bar1) were designed. The cDNA is 1140 bp in length with a 325 amino acid open reading frame. The cDNA appears to be full-length based on a 5' methionine and homologies to similar genes in databases.

The sequence of the barley homolog of gl8 follows:

The amino acid sequence derived from SEQ ID NO:3 follows:

The leek homolog was isolated from screening a leek epidermal cDNA library with the partial maize gl8 cDNA as the probe, using standard methods. DNA was extracted from purified plaques and digested with EcoRI and XhoI and ligated into the EcoRI/XhoI sites of pBSK. Both strands of the cDNA were sequenced using partial deletion subclones and a gene-specific primer (leek1). The leek cDNA clone is 872 bp in length.

The double-stranded sequence of the cDNA insert [SEQ ID NOS:5 and 6] and the translated first 200 amino acids [SEQ ID NO:7] for the leek glossy8 homolog follow:

Screening the GenBank database for homologous sequences to the maize glossy8 amino acid sequence also allowed identification of a related Arabidopsis sequence. The algorithm Blast identified an expressed sequence tag (EST), which is a randomly sequenced cDNA identified as Clone ID 105H4T7, GenBank ID T22476, and NCBI ID 312378. The Arabidopsis EST was obtained from the Arabidopsis Biological Resource Center DNA Stock Center. Dideoxy sequencing of both strands of purified plasmid from all three homologs was performed. Universal and reverse primers were used on the native clones and on subclones from the original clone, where convenient. Gene-specific primers were designed as necessary to complete double-stranded sequencing.

In particular, the Arabidopsis homolog 105H4T7 was sequenced using universal, reverse, and designed primers (AR612 and AR675). The clone was 896 bp long with a 253 amino acid open reading frame. Here follow the nucleotide and amino acid sequences of the Arabidopsis gl8 homolog:

Accordingly, using standard hybridization methods of cDNA libraries from other organisms and standard computer search methods of a sequence databank, related or homologous nucleic acids and polypeptides were identified with respect to maize glossy8.

Example 5

This example sets forth the DNA and amino acid sequences of the maize glossy1 gene and glossy1 homologs from other species.

A cDNA clone was isolated using the methods set forth in Example 2, and as further elucidated in Sambrook et al., supra. Specifically, a cDNA library made from mRNA isolated from seedlings of the maize inbred line B73 was screened by hybridization using as a probe a mixture of fragments A and B isolated from the 12.5-kb HindIII clone of the g11-755048 clone. Fragment A is a 600 bp BamHI/SalI fragment located approximately 800 bp from the Mu1 transposon in this clone. Fragment B is a 700 bp SacI fragment located approximately 1 kb on the other side of the Mu1 transposon. From a total of 500,000 recombinant phage that were screened, two classes of cDNA clones were isolated. One class contains a 0.8 kb cDNA insert and the other class contains a 1.6 kb insert. Restriction mapping and cross-hybridization experiments established that the 0.8 kb cDNA is a partial clone of the 1.6 kb cDNA. All subsequent investigations were performed using the longer 1.6 kb cDNA clone.

The partial 5' and 3' sequences of the cDNA insert and the translated amino acids of the maize glossy1 gene follow:

A partial homolog to maize glossy1 was identified from the GenBank database. The partial homolog (Accession No. L33792) is identified in the database as a Senecio odorus epidermal mRNA, the 3' end of which has no known function. In comparing the maize sequences recited above with the corresponding Senecio sequences recited below, the two genes can be seen to differ a great deal at their carboxy termini. The Senecio homologous glossy1 sequences follow:

Other homologous sequences were also identified via the computer database search, namely a rice-derived and an Arabidopsis-derived EST. The rice EST has been cataloged as NCBI ID 72320, EST ID R1CS2751A, Clone ID S2751, and GenBank ID D40658. The organism used was Oryza sativa, strain Nipponbase, subspecies Japonica, and the cDNA was made from mRNA isolated from 8-day old rice etiolated shoots. The nucleic acid and amino acid sequences of the rice gl1 homolog follow:

The Arabidopsis-derived EST, believed to be a glossy1 homolog based on preliminary sequencing, was obtained and sequenced in its entirety using standard methods. The nucleic acid and deduced amino acid sequences follow:

Accordingly, using standard computer search methods of a sequence databank, related or homologous nucleic acids and polypeptides, which had been previously described with respect to sequence only, were correlated to gl1. It is of interest to note that, but for the results disclosed herein, the identified homologs had no known function.

Example 6

This example sets forth the procedures used to clone the CER2 locus of Arabidopsis.

The CER2 locus was cloned using a standard method of genetic engineering called "chromosome walking," using the method of Bender et al., supra. This method requires the availability of known genetic markers of the subject organism, here Arabidopsis, which are shown in FIG. 3. As can be seen in FIG. 3, chromosome 4 has known RFLP and visible genetic markers in the region that includes CER2, which is situated between the visible markers IM and AP2 (Koorneef, in Genetic Maps (S. J. O'Brien, ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1987), pp. 742-745); estimated genetic and physical distances are recited thereon as well. In the conduct of the present work, recombinants having crossover events noted between the recited markers were identified, the numbers of which are recited in the figure. The genetic distances are estimated based on a subset of the recombinants.

As a first step in cloning the CER2 locus via chromosome walking, F3 families segregating for the genetic markers AG, IM, CER2, and AP2 were screened for genetic recombinants, as follows: A Landsberg erecta (Ler) genetic marker line that carries the recessive markers BREVIPEDICELLUS (BP), ECERIFERUM2 (CER2), and APETALA2 (AP2) (Koorneef, supra) was crossed to a Columbia stock that carries the mutant IMMUTANS (IM) (Wetzel et al., Plant J., 6, 161-175 (1994)). The resulting F1 plants were allowed to self-pollinate, and were grown at 23.degree. C., under 16 hours of daylight and 8 hours of dark (same growing conditions used for all of the plants). Individual F3 families were scored for recombinant phenotypes, using the aforementioned markers, which are readily recognized by ordinary artisans. CAPS mapping using primers based on the AGAMOUS (AG) sequence (see Konieczny et al., Plant J., 4, 403-410 (1993) for primer sequences and conditions for assay) was used in some instances to score F2 plants and thereby identify recombinants with break points between AG and CER2. The selected F2 plants were allowed to self-pollinate. Analysis of the resulting F3 families provided genotypic data for the IM locus. The cer2-2 mutant allele (Stock #CS8) was generated via EMS mutagenesis of the Ler ecotype (Koorneef et al., supra) and was obtained from the Arabidopsis Biological Resource Center at Ohio State University, Columbus, Ohio.

To more precisely map the locations of these recombination breakpoints, plants carrying these recombinant chromosomes were analyzed with several RFLP markers. Of the 92 recombinants between CER2 and AP2, 11 occurred between CER2 and m600. Based on these results, the RFLP marker m600 is approximately 2.5 centiMorgans (cM) from CER2. Assuming a value of 185 kilobases (kb) per cM for Arabidopsis chromosome 4 (Schmidt et al., Science, 270, 480-483 (1995)), this interval is estimated to be approximately 460 kb.

For the RFLP and other analyses and procedures used in the identification and elucidation of the present invention, a variety of standard nucleic acid chemical techniques were applied. For example, Arabidopsis DNA was isolated from 20-30 day old plants using a modified CTAB procedure, as disclosed in Saghai-Marroof et al. (Proc. Natl. Acad. Sci. USA, 81, 8014-8018 (1984)). Digestions with restriction endonucleases were conducted according to manufacturers' specifications. For Southern blot analysis, 0.5 .mu.g aliquots of digested genomic DNA were loaded per lane and subjected to electrophoresis through an agarose gel. RNA was isolated from the aerial parts of soil-grown adult plants, including leaves, stems, young siliques, and inflorescences, using the method of Dean et al. (EMBO J., 4, 3055-3061 (1985)). Poly A-enriched RNA was isolated using PolyATract mRNA Isolation System III.TM. (Promega, Madison, Wis.). For Northern blot analysis, 15 .mu.g RNA and/or 150 ng poly A-enriched RNA aliquots were loaded per lane and subjected to electrophoresis. Probe preparation, electrophoresis, gel blotting, and hybridizations were conducted according to standard procedures (Sambrook et al., supra).

Four clones (namely, EG24D9, EW14G12, EW9C10, and EW11E4; shown in FIG. 3) that contain sequences homologous to m600 were identified by screening a number of Arabidopsis YAC libraries, namely the EG1, EW, ABI-1, and CIC YAC libraries (Grill et al., Mol. Gen. Genet., 226, 484-490 (1991); Ward et al., Plant Mol. Biol., 14, 561-568 (1990); and Creusot et al., Plant J., 8, 763-770 (1995)), using the method of Gibson et al., supra. Yeast chromosomes were separated as described in Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc., 1994). The YAC DNA from agarose slices was purified by electroelution (Sambrook et al., supra) or using the GeneClean.TM. Kit (BIO 101, Inc., Vista, Calif.). Both ends of these YACS were subcloned via either plasmid rescue, as described in Burke et al., supra, or lambda subcloning. For lambda subcloning, the recovered YAC DNA was digested using the restriction enzyme HindIII and ligated to the lambda insertion vector NM1149 (Murray, supra) and in vitro packaged using Gigapack.RTM.II (Stratagene, La Jolla, Calif.). To isolate the subclones containing the YAC ends, the resulting lambda subclone libraries were screened using DNA fragments of pYAC4 flanking the cloning site as centromeric (i.e., the 0.5 kb HindIII/EcoRI fragment) and VRA3-containing end (i.e., the 3 kb HindITl/EcoRI fragment) specific probes, following standard procedures (Sambrook et al., supra).

The identified YACS were oriented relative to the genetic map and each other by using the subcloned YAC ends as RFLF markers to analyze the collection of genetic recombinants and in cross-hybridization experiments involving the other YACS. The YAC end closer to the CER2 locus was then used to isolate another set of YACS closer to the CER2 locus. This cycle was repeated seven times, the results of which are depicted in FIG. 3, for the blown-up area between CER2 and m600. In FIG. 3, the open circles and squares represent the left ends and right ends of the YACS, respectively. The shaded circles and squares indicate YAC ends having repetitive sequences. The dashed line of YAC clones represents a chimeric YAC clone.

YAC CIC9C5 was subcloned into the lambda insertion vector NM1149 as HindIII fragments of up to 8.5 kb in size. The resulting DNA fragments were used as RFLP markers, using the methods just discussed, to analyze recombinants. These analyses established that two of the 26 recombinants between IM and CER2 have recombination breakpoints between marker C9L (the centromeric end of YAC CIC9C5) and CER2. Similarly, one of the 11 recombinants between CER2 and m600 has a breakpoint between CER2 and marker C9-15, which lies approximately 150 kb from the centrometric end of CIC9C5. The CER2 locus is, therefore, located within an approximately 150 kb interval of YAC CIC9C5. The ends of this interval are defined by the positions of markers C9L and C9-15 on YAC CIC9C5.

To more precisely define the chromosomal region that contains the CER2 gene, DNA fragments from the interval defined by the probes C9L and C9-15 were tested for their ability to complement the cer2 mutation. A lambda genomic library prepared with DNA from wild-type Arabidopsis ecotype Landsberg erecta (Ler) (Voytas et al., Genetics, 126, 713-721 (1990)) was screened using standard procedures (Sambrook et al., supra) and the subcloned DNA fragments contained within the CIC9C5 interval. The inserts of the lambda genomic clones were subcloned into the binary vector pBI121 as overlapping fragments and used to transform Arabidopsis plants homozygous for the cer2-2 allele. Initially, the complementation test was performed using an Agrobacterium-mediated Arabidopsis root explant transformation system, described by Huang et al. (Plant Mol. Bio. Reporter, 10, 372-383 (1992)). Although transgenic plants were obtained, the resulting plants seldom initiated roots and cuticular wax deposition was noted to be strongly influenced by tissue culture conditions. Thus, the CER2 phenotype was not easily scored on the resulting regenerated plants. An in planta transformation procedure according to Bechtold et al. (C.R. Acad. Sci. Paris, Life Sciences, 316, 1194-1199 (1993)) was used to successfully generate transgenic seed at a rate of approximately one in 150 seeds.

Two small overlapping genomic fragments (pG1H and pG1RSc) isolated from the lambda genomic clone G1 (illustrated in FIG. 4A) have been shown to complement the cer2-2 mutant phenotype. Eight out of 10 transgenic plants carrying pG1H and 8 out of 9 transgenic plants carrying pG1RSc exhibited a phenotype indistinguishable from the wild type plant, as examined by the unaided eye.

One of the genomic DNA fragments capable of complementing the mutant phenotype, pG1H, was partially sequenced using standard methods, and was reported to GenBank (GenBank Accession #U40894). One end of pG1H contains the 3' half of the ATRl gene, which encodes an NADPH-cytochrome P450 reductase GenBank Accession #X66016). Adjacent to the ATR1 gene is a sequence with near-identity to the Arabidopsis EST 154C7T7 (GenBank Accession #T76511). This 1.4-kb cDNA (EST 154C7T7) was obtained from the Arabidopsis Biological Resource Center and subcloned into pbI121, such that its transcription is under the control of the CaMV 35S promoter (FIG. 4A). The resulting construct (pG1C1) was transformed into plants homozygous for the cer2-2 allele and was found to be sufficient to complement the cer2 mutant phenotype. The EST 154C7T7, therefore, includes the CER2 coding region, or a portion thereof.

Cuticular wax crystals on stems of wild-type, mutant, and transgenic plants were examined via scanning electronic microscopy (SEM). SEM examination of stem cuticular wax crystals was conducted at the Iowa State University Microscopy Facility. The samples were frozen in liquid nitrogen using EM Scope SP2000A cryo-system, coated with gold, and observed using Joel JSM-35 scanning electron microscope at 15 kv. Samples from two Ler wild type plants, one each of the cer2-2 mutant plant, the transgenic plant harboring pG1H and pG1RSc, and two transgenic plants harboring pG1C1 were examined. The SEM results demonstrated that wild-type plants produce a large number of condensed tube-shaped wax crystals. In contrast, mutant plants produce relatively few crystals. Four transgenic plants carrying a CER2 genomic fragment (the pG1H or PG1RSc constructs, FIG. 4A) or constitutively expressing the CER2 cDNA (construct pG1C1, FIG. 4A) produced wax crystals in numbers similar to those on wild-type plants. Although the crystals present on the plant carrying the pG1H construct differed somewhat in shape from those present on wild-type plants, in the remaining three instances, the shape of the wax crystals was similar to wild-type. Accordingly, the CER2 locus was identified and substantially characterized at the level of identifiable portions of the Arabidopsis genome, and such portions were shown capable of reversing the effect of a known mutation at the CER2 locus.

Example 7

This example sets forth the sequence identified for the insert of a CER2 cDNA, the corresponding genomic DNA, and a CER2-containing region of pG1H, as well as further characterization of these CER2 sequences.

The CER2 genomic clone G1 was subcloned into PBKS+ and pBKS+ (Stratagene, La Jolla, Calif.). The cDNA clone was subcloned into pGem3fz(+) (Promega, Madison, Wis.). PCR-amplified fragments were purified by electroelution (Sambrook et al., 1989) and directly sequenced. DNA sequencing was performed at the Iowa State University Nucleic Acid Facility using the double-stranded dye terminator technique on an ABI 373 Automated DNA Sequencer (Applied Biosystems, Foster City, Calif.). Both DNA strands were sequenced. Sequence comparisons and analyses were performed using the GCG sequence analysis software package (Version 8, 1994, Genetics Computer Group, Inc., Madison, Wis.) and the PSORT, TMpred, Blast, FASTA algorithms, as described previously (Nakai et al., Genomics, 14, 897-911 (1992); Hofmann et al., Biol. Chem., 374, 166 (1993); Altschul et al., J. Mol. Biol., 215, 403-410 (1990); and Pearson et al., Proc. Natl. Acad. Sci. USA, 85, 2444-2448 (1988)).

The sequence of the CER2 cDNA insert was:

The corresponding genomic clone for CER2 was found to have the following sequence.

As noted in Example 6, an Arabidopsis EST labeled 154C7T7 was also identified as having a homologous sequence to that of CER2. To provide additional evidence that this clone represents a new full-length cDNA, the region of pG1H that includes the CER2 coding region was sequenced as follows:

The DNA sequence of the 1407 nucleotide (nt) EST 154C7T7 disclosed in Example 6 contains a 134 nt 3' untranslated region that includes a putative polyadenylation signal (AATAAA) and a 24-nt poly A tail. The size of this cDNA is indistinguishable from the size of the 1.4-kb transcript detected when it is used as a probe on RNA gel blots (see below). Two putative TATA boxes (TATAAG and TATATA) that exhibit a high degree of similarity to the TATA consensus sequence (Joshi, Nucleic Acid Res., 15, 6643-6653 (1987)) were identified at positions -37 and -61 (relative to the 5' end of the cDNA), respectively. In addition, two putative CAAT boxes were identified at positions -68 and -85. In combination, these data support the view that the EST 145C7T7 cDNA is near full-length. The only sequence polymorphism between the cDNA derived from the Columbia ecotype and the genomic clone derived from the Ler ecotype is a single conservative nucleotide substitution at position +1495.

The CER2 cDNA contains an open reading frame that could encode a protein of 421 amino acid residues with a predicted molecular weight of 47 kD. No alternative open reading frames of significant length were identified. Computer-based homology searches using various derivatives of the Blast (Altschul et al., supra) and FASTA (Pearson et al., supra) algorithms have failed to reveal any significant sequence similarities between the deduced CER2 protein and entries in the nonredundant nucleotide and protein databases that have a known biochemical function. However, the deduced CER2 protein exhibits a high level of sequence similarity to that of glossy2 (gl2), a gene that plays an undefined role in cuticular wax biosynthesis in maize (GenBank Accession No. X88779, GI: 949979; SEQ ID NO: 30). The deduced CER2 protein does not contain a recognizable protein targeting signal sequence or a transmembrane domain when analyzed with the PSORT algorithm (Nakai et al., supra). However, the TMpred algorithm (Hofmann and Stoffel (1993)) predicts a putative transmembrane domain (indicated by "TM" in the hydrophobicity plot depicted in FIG. 5) between residues 147 and 172 of the deduced CER2 protein.

CER2-specific PCR primers were used to amplify the CER2-2 mutant allele and the location of respective homologies are represented in FIG. 4B. The primer sequences follow:

p1: 5'-AGGTGGACGTAATAAAGTGTG-3' [SEQ ID NO:24]

p2: 5'-GGTGGTGCCTGCTTCTTTGGTA-3' [SEQ ID NO:25]

p3: 5'-AAATCGAACCACTTCCCCACTG-3' [SEQ ID NO:26]

p4: 5'-GAGGATGATAGAACAAGATAAAGG-3' [SEQ ID NO:27]

p5: 5'-GGCATCACTACACTTACAATCCT-3' [SEQ ID NO:28]

p6: 5'-CAGTGACACCAAACAAGAACAA-3' [SEQ ID NO:29]

The paired primers: p1 and p3, p2 and p5, and p4 and p6 were used to PCR amplify the CER2-2 mutant allele. The primers were synthesized at the Iowa State University Nucleic Acid Facility using a 394 DNA/RNA Synthesizer (Applied Biosystems, Foster City, Calif.). Amplification reactions were conducted in 50 .mu.l volumes containing 50-100 ng of genomic DNA and 50 mM KCl, 10 mM Tris-HC1, pH 9.0, 0.1% Triton X-100, 1.5 mM MgCl.sub.2, 150 .mu.M dNTP, and 0.5 .mu.M primers. The reactions were overlaid with 100 .mu.l of mineral oil and denatured at 94.degree. C. for 1 minute, followed by 30 cycles of denaturation at 94.degree. C. for 45 seconds, annealing at 54.degree. C. (for the pair of p1 and p3) or 58.degree. C. (for the pairs of p2 and p5, p4 and p6) for 45 seconds and extension at 72.degree. C. for 1 to 2 minutes. The reactions were given a final extension at 72.degree. C. for 10 minutes to complete the elongation.

The entire coding region and 300 bp upstream and 100 bp downstream of the CER2-2 gene were sequenced and compared to the sequence of genomic DNA derived from wild-type progenitor (Voytas et al., Genetics, 126, 713-721 (1990)). Based upon this comparison, there is only a single difference between the CER2-2 mutant allele and its wild-type progenitor. This difference is a G to A transition mutation at position +1150 that changes a tryptophan codon to a premature stop codon, resulting in a truncated 295 amino acid peptide. The result therefore further confirms that the identified gene corresponds to the CER2 locus.

The CER2 cDNA was used to hybridize a DNA gel blot containing 500 mg of Ler genomic DNA digested by five different restriction enzymes, namely BglII, EcoRPI, EcoRV, HindIII, and XbaI. Using even relatively low stringency washes (washes in 1.times.SSC at 65.degree. C. for 40 minutes), a single-copy hybridization pattern was revealed, suggesting that CER2 is a single-copy gene. When this experiment was repeated at even lower stringency (whereby the hybridization was conducted at 45.degree. C. and the final wash was performed with 1.times.SSC at 45.degree. C.), several weakly hybridizing bonds were revealed, suggesting that some CER2-homologous sequences are present in the Arabidopsis genome.

Accordingly, the CER2-specific sequences have characteristics that are fully consistent with their being the CER2 genes.

Example 8

This example sets forth data concerning the accumulation of CER2 mRNA in wild-type and mutant plants, and spatial patterns of expression of the CEP2 gene in a plant.

The CER2 cDNA clone was used as a probe in RNA gel blotting experiments. Total RNA and poly A-enriched RNA were isolated from aerial parts of adult wild-type and mutant plants homozygous for the cer2-2 allele, as discussed in Example 6, and applied to an electrophoretic gel in 15 .mu.g and 150 ng aliquots, respectively. After the Northern blot was effected, i.e., the RNA was transferred to a solid medium from the electrophoretic gel using standard means, the blot was probed with the cDNA insert of pG1C1. These hybridization experiments revealed a single 1.4-kb mRNA in RNA isolated from a pool of leaves, stems, young siliques, and inflorescences. The amount of steady-state CER2 RNA in the cer2-2 mutant plants is approximately 5 to 10-fold lower than in wild-type plants (lanes labeled WT in FIG. 11).

Based on the phenotype associated with the cer2 mutation, it would be expected that the CER2 mRNA would accumulate in the epidermis of siliques and stems. To test this hypothesis, the 1.243 kb HindIII-BamHI fragment of pG1H that includes positions -1009 to position +234 of the CER2 gene was fused in-frame with the .beta.-glucuronidase reporter gene in the binary vector pB1101.3. The resulting construct (pCER2-GUS; FIG. 8C) was transformed into Arabidopsis Ler ecotype. Samples from six individual transgenic plants (T1) representing at least three independent transformation events were stained for GUS activity with X-Gluc.

Construction of G1H, pG1RSc, pG1C1, and pCER2-GUS, and plant transformation therewith, was accomplished using conventional means, as follows: For the subcloning of pG1H, pG1RSc, and pG1C1, the DNA fragments from the genomic clone G1 and the insert of cDNA clone were inserted into pB1121 (Clontech, Palo Alto, Calif.). To construct pCER2-GUS, the 1.4 kb HindIII/BamHI fragment of pG1H was inserted into the HindIII/BamHI cloning site of pBI101.3 (Clontech, Palo Alto, Calif.), which fragment includes the CER promoter. The resulting plasmids were introduced into Agrobacterium strain C58C1 (Koncz et al., Mol. Gen. Genet., 204, 383-396 (1986)) via freeze/thaw procedure (An et al., in Plant Molecular Biology Manual, part A3, pp. 1-9 (Gelvin and Schilperoot, eds., Vilumer Academic Publishers, Vordrecht, 1988)).

Agrobacterium-mediated root explant transformation was conducted according to the procedure of Huang et al., Plant Mol. Bio. Reporter, 10, 372-383 (1992). For the in planta transformation, an adaptation of Bechtold et al., C.R. Acad. Sci. Paris, Life Sciences, 316, 1194-1199 (1993) was used. The variant aspects with respect to the Bechtol et al. protocol were that seeds harvested from the vacuum infiltration treated plants (T.sub.0) in the same pot were bulked. The transgenic plants (T.sub.1) from the same bulk were considered to represent at least one independent transformation event.

Histochemical analysis of .beta.-glucuronidase activity in the transformed plants were performed using 5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic acid, as described by Jefferson, Plant Mol. Biol Rep., 5, 387-405 (1987). The cross-section of the stems was performed by freehand, and then subjected, along with the other structures, to the histochemical procedure.

All six transgenic plants showed similar expression patterns as described below. As expected, the pCER2-GUS chimeric gene is expressed in siliques and stems. However, the most prominent expression occurs in young siliques, and mature ovaries. Only the upper portion of stems stains for GUS activity. Hence, the localization of GUS activity is highest in regions where cells are rapidly expanding. A low level of GUS staining is also apparent in mature anthers. Detailed examination of these stained anthers revealed that only the pollen grains were stained. The expression of the CER2-GUS gene was further examined in cross-sections of GUS-stained stems. GUS activity was detected not only in the epidermal layers, but also in the vascular bundles of stems, siliques, filaments, petals and sepals. Expression of the pCER2-GUS gene was not detected in rosette leaves (before or after bolting) or in cauline leaves. In addition, no expression was detected in sepals or petals, except in the vascular bundles.

Accordingly, CER2 transcription appears correlated in general terms with the phenotype conditioned by the cer2 mutation. The analysis of transgenic plants demonstrated that the CER2-GUS gene is expressed in those portions of stems where cells are expanding, and in the developing siliques. GUS activity was also detectable in anthers, although at lower levels. Because CER2 was found to be expressed in anthers and pollen grains, it is evident that cuticular waxes have a function in pollen development or function. In contrast, the CER2 gene is not likely to be involved in the cuticular wax deposition of leaves, although it appears to be involved in such activity in the cells of the vascular bundle tissues. Thus, the CER2 promoter confers spatial and/or temporal control of expression of its controlled structural gene.

Example 9

This example illustrates the utility of probing a plant's genome for identification of related genes using a cloned cuticular lipid gene or fragment thereof.

The polynucleotides described hereinabove have been used as probes to identify the existence of related sequences within heterologous and homologous genomes. Polynucleotides encoding glossy1 and glossy8 were used as probes of maize genomic DNA, using standard methods. In particular, the autoradiogram provided herein as FIG. 12 was generated from a Southern blot of DNA isolated from different maize strains that was digested with restriction endonuclease KpnI and probed with the entire glossy1 cDNA sequence [SEQ ID NOS:10 and 11]. The source of digested DNA in the lanes is listed parenthetically after recital of the label on each lane as follows: 91g138 (i.e., gl1-mu91g138/gl1-ref); 91g139 (i.e., gl1-mu91g139/gl1-ref); 92 3508-1 (i.e., Gl1/gl1-ref); 92 3508-3 (i.e., Gl1/gl1-ref); 92 3058-4 (i.e., Gl1/gl1-ref); 92 3508-5 (i.e., Gl1/gl1-ref); 92 3508-6 (i.e., Gl1/gl1-ref); 92 3508-7 (i.e., Gl1/gl1-ref); 92 3508-8 (i.e., Gl1/gl1-ref); 92 3508-9 (i.e., Gl1/gl1-ref); 92 3508-10 (i.e., Gl1/gl1-ref); 92 1442-3 (i.e., Gl1/gl1-ref); 91g137 (i.e., Gl1-mu91g137/gl1-ref); 92 3507-3 (i.e., Gl1/gl1-ref); 92 3507-5 (i.e., Gl1/gl1-ref); and 92 3507-9 (i.e., Gl1/gl1-ref). The last lane, labeled .lambda.H3, contains .lambda. DNA digested with HindIII for size standards.

An autoradiogram was generated from a Southern blot of DNA isolated from different maize strains as well, but digested with restriction endonuclease HindIII and SacI instead, and probed with the 0.8 kb glossy8 cDNA [SEQ ID NO:1].

The probe protocol used for the Southern analysis was as described by Sambrook et al., supra. In particular, the protocol involved taking the Southern blot of the gel in which the various DNAs were separated by size, incubating the blot with labeled probe in 6.times.SSPE at 68.degree. C., washing free labeled probe in 2.times.SSPE at 68.degree. C. for 30 minutes, followed by 10 mM Tris for another 30 minutes. The autoradiogram, itself, was exposed for 2 or 3 days at -70.degree. C.

The autoradiograms clearly established that the maize glossy1 and glossy8 probes detect multiple sequences in the maize genome. Accordingly, it is believed that the present invention provides a means for the isolation of other lipid biosynthesis-involved genes, among others.

All of the references cited herein, including patents, patent applications, and technical literature, are hereby incorporated in their entireties by reference.

While this invention has been described with an emphasis upon preferred embodiments, it will be obvious to those of ordinary skill in the art that variations of the preferred embodiments may be used and that it is intended that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

What is claimed is:

1. A method of introducing a nucleic acid into a plant cell, which method comprises the steps of: (a) inserting into a plant cell (i) a sense nucleic acid, wherein said sense nucleic acid encodes a product of a cuticular lipid gene to be expressed in said plant cell, or (ii) an antisense nucleic acid, wherein said antisense nucleic acid expresses an antisense nucleic acid molecule, which is specific for mRNA or a portion thereof of a cuticular lipid gene present in and expressed in said plant cell, wherein said antisense nucleic acid molecule is of sufficient length to inhibit expression of said cuticular lipid gene, wherein said sense or antisense nucleic acid is operatively linked to one or more regulatory sequences, and wherein said cuticular lipid gene comprises a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 30, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 and a sequence which hybridizes to any of the aforementioned nucleic acid sequences under stringent conditions; (b) expressing said sense or antisense nucleic acid in said plant cell; and (c) culturing said plant cell.

2. The method of claim 1, further comprising the step of: (d) generating from the plant cell a plant.

3. The method of claim 1, wherein said plant is selected from the group consisting of maize, soybean, rapeseed, canola, cotton, safflower, peanut, palm, and sunflower.

4. The method of claim 1, wherein at least one of said regulatory sequences is developmentally specific or tissue-specific.

5. The method of claim 2, wherein said plant is selected from the group consisting of maize, soybean, rapeseed, canola, cotton, safflower, peanut, palm, and sunflower.

6. The method of claim 5, wherein said plant is maize or canola.

7. A plant cell comprising an exogenous nucleic acid selected from the group consisting of a sense nucleic acid encoding a product of a cuticular lipid gene and an antisense nucleic acid expressing an antisense nucleic acid molecule specific for mRNA or a portion thereof of an endogenous cuticular lipid gene, wherein said antisense nucleic acid molecule is of sufficient length to inhibit expression of said cuticular lipid gene, wherein said exogenous nucleic acid is operatively linked to one or more regulatory sequences and said cuticular lipid gene comprises a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 30, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 and a sequence which hybridizes to any of the aforementioned nucleic acid sequences under stringent conditions.

8. A plant comprising an exogenous nucleic acid selected from the group consisting of a sense nucleic acid encoding a product of a cuticular lipid gene and an antisense nucleic acid expressing an antisense nucleic acid molecule specific for mRNA or a portion thereof of an endogenous cuticular lipid gene, wherein said antisense nucleic acid molecule is of sufficient length to inhibit expression of said cuticular lipid gene, wherein said exogenous nucleic acid is operatively linked to one or more regulatory sequences and said cuticular lipid gene comprises a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 30, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO. 6, SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 and a sequence which hybridizes to any of the aforementioned nucleic acid sequences under stringent conditions.

9. The method of claim 1, wherein at least one of said regulatory sequences is a regulatable promoter selected from the group consisting of a temperature-sensitive promoter and a promoter that is inducible by a chemical.

10. The plant cell of claim 7, wherein at least one of said regulatory sequences is developmentally specific or tissue-specific.

11. The plant cell of claim 7, wherein at least one of said regulatory sequences is a regulatable promoter selected from the group consisting of a temperature-sensitive promoter and a promoter that is inducible by a chemical.

12. The plant of claim 8, wherein at least one of said regulatory sequences is developmentally specific or tissue-specific.

13. The plant of claim 8, wherein at least one of said regulatory sequences is a regulatable promoter selected from the group consisting of a temperature-sensitive promoter and a promoter that is inducible by a chemical.

Non-Patent Literature (30)

Lewin R., Science 237:1570 Sep. 25, 1987.
Agrawal et al. "Biosynthesis of Very Long Chain Fatty Acids in Microsomes from Epidermal Cells of Allium porrum L.," Arch. Biochem. Biophys., 230, 580-589 (1984).
Agrawal et al., "Characterization and Solubilization of an Acyl Chain Elongation System in Microsomes of Leek Epidermal Cells," Arch. Biochem. Biophys., 240, 154-165 (1985).
Bianchi, "Glossy Mutants: Level of Action and Level of Analysis," Maize Breeding and Genetics, pp. 533-550, Walden, ed., John Wiley and Sons, New York, New York (1978).
Bianchi et al., "Biosynthesis Pathways of Epicuticular Wax of Maize as Assessed by Mutation, Light, Plant Age and Inhibitor Studies," Maydica, 30, 179-198 (1985).
Brown et al., "Molecular Analysis of Multiple Mutator-Derived Alleles of the Bronze Locus of Maize," Genetics, 122, 439-445 (1989).
Gibson et al., "Chromosome walking in Arabidopis thaliana using Yeast Artificial Chromosomes," Methods in Arabidospis Research, pp. 119-123, Koncz et al., eds., World Scientific, Singapore (1992).
Grill et al., "Construction and characterization of yeast artificial chromosome library of Arabidopsis which is suitable for chromosome walking," Mol. Gen. Genet., 226, 484-490 (1991).
Hansen et al., Plant Physical, 102 (i) , 93 (Abstratct #519, "Molecular Cloning and Characterization of Plant Cuticular Wax Genes") (May 1993).
Kolattukudy et al., "The Biochemistry of Plant Cuticular Lipids," Proq. Chem. Fats Other Lipids, 13, 121-175 (1973).
Kolattukudy et al., "Biochemistry of Plant Waxes," Chemistry and Biochemistry of Natural Waxes, pp. 571-645, Kolattukudy, ed., Elsevier Press, New York, New York (1976).
Kolattukudy, "Cutin, Suberin, and Waxes," Biochem. of Plants, 4, 571-645, Stumpf et al., eds., Academic Press, New York, New York (1980).
Kolattukudy, "Structure, Biosynthesis, and Biodegradation of Cutin and Suberin," Ann. Rev. Plant Physiol., 32, 539-567 (1981).
Lardizabal et al., "Substrate Tagging, Solubilization and Partial Purification of Jojoba (Simmondsia chinensis) Embryo Fatty Acyl-Coa: Fatty Alcohol Acyl Transferase," Plant Physiol. (Suppl.) , 102, 93 (1993).
Lazo et al., "A DNA Transformation-Competent Arabidopsis Genetic Library in Agrobacterium," Bio/Technology, 9, 963-967 (1991).
Lemieux et al., "GC-MS Analysis of the Wax of Eceriferum (CER) Mutants of Arabidopsis thaliana and Isolation of CER Genes fron T-DNA Tagged Arabidopsis Mutant Lines," Plant Physiol. (Suppl.) , 99, 14 (1992).
McNevin et al., "Isolation and Characterization of Eceriferum (ecr) Mutants Induced by T-DNA Insertions in Arabidopsis Thaliana" Genome, 36 (3), 610-618 (Jun. 1983).
Meyerowitz, "Introduction to the Arabidopsis genome," Methods in Arabidopsis Research, pp. 100-118, Koncz et al., eds., World Scientific, Singapore (1992).
Moose et al, "Molecular Cloning of the glossy-15 Locus, a Cell-Specific Regulator of Epidermal Differentiation," Abstracts, 35th Annual Maize 1993, Evans et al., eds. (1993).
Schnable et al, "Genetic Approaches to Isolating Maize and Arabidopsis Genes Involved in Cuticular Wax Biosynthesis," Current Topics in Plant Physiology, 9, 196-206 (1993).
Schnable et al., "Transposon Tagging Agronomically Important Loci in Maize," Proceedings, 28th Annual Illinois Corn Breeder's School (1992).
Stinard et al., "Genetic Isolation, Cloning, and Analysis of a Mutator-Induced, Dominant Antimorph of the Maize amylose extenderl Locus," Plant Cell, 5, 1555-1566 (1993).
Tulloch, "Chemistry of Waxes of Higher Plants," Chemistry and Biochemistry of Natural Waxes, pp. 235-287, Kolattukudy, ed., Elsevier Press, New York, New York (1976).
Vogel et al., J. Cellular Biochemistry, 17A, 32 (Abstract #A327) (Jan. 9, 1993).
von Wettstein-Knowles, "Genes, Elongases and Associated Enzyme Systems in Epicuticular Wax Synthesis," The Metabolism, Structure, and Function of Plant Lipids, pp. 489-498, Stump et al., eds., Elsevier Press, North-Holland, Amsterdam (1987).
Walbot, "Strategies for Mutagenesis and Gene Cloning Using Transposon Tagging and T-DNA Insertional Mutagenesis," Annu. Rev. Plant Physiol. Plant Mol. Biol., 43, 49-82 (1992).
Ward et al., "Isolation of single-copy-sequence clones from a yeast artificial chromosome library of randomly-sheared Arabidopsis thaliana DNA," Plant Mol. Biol., 14, 561-568 (1990).
Vander Knol et al. 1988 Bio/Techniques 6: 958-976.
Vander Knol et al. 1990 Plant Molec Biol 14: 457-466.
Finnegan et al. 1994 Bio/Technology 12: 883-888.

Abstract

Description

Technical Field of the Invention

The present invention relates to the field of genetically engineered biosynthesis of plant lipids, and specifically relates to the isolation and use of cloned cuticular lipid genes.

Background of the Invention

These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

Brief Summary of the Invention

The present invention further provides a method for the establishment of a lipid biosynthesis variety of a plant, comprising the steps of:

(a) constructing a nucleic acid comprising a nucleotide sequence that encodes a cuticular lipid gene product;

(b) inserting the nucleic acid into plant cells; and

Brief Description of the Drawings

FIG. 1 is a diagram that presents a hypothesized general scheme of lipid biosynthesis in plants.

FIG. 2 is a physical map that indicates the relative locations of restriction endonuclease digestion sites for the glossy8-3142 8.6 kb HindIII genomic DNA fragment.

FIGS. 4A, 4B, and 4C are diagrammatic drawings of CER2-containing clones.

FIG. 5 is a hydrophobicity plot of the deduced CER2 amino acid sequence.

Description of the Preferred Embodiments

The following examples further illustrate the present invention, but, of course, should not be construed as in any way limiting its scope.

Example 1

This example sets forth methods used in the identification and isolation of glossy genes.

For example, the Mu1 element co-segregated with glossy1-5048 in 93 individuals.

Similarly, a Mu8-containing 8.6 kb HindIII DNA fragment that co-segregates with glossy8-3142 in 88 individuals was identified.

Example 2

This example sets forth the sequence identified for the insert of a glossy8 cDNA clone, and the method by which the sequence was obtained.

Example 3

This example illustrates the endogenous expression of the glossy8 gene in maize, as assessed in a Northern blot experiment.

Example 4

This example illustrates methods for the identification of DNA segments that crosshybridize with the glossy8 clone and portions thereof.

The sequence of the barley homolog of gl8 follows:

The amino acid sequence derived from SEQ ID NO:3 follows:

The double-stranded sequence of the cDNA insert [SEQ ID NOS:5 and 6] and the translated first 200 amino acids [SEQ ID NO:7] for the leek glossy8 homolog follow:

Example 5

This example sets forth the DNA and amino acid sequences of the maize glossy1 gene and glossy1 homologs from other species.

The partial 5' and 3' sequences of the cDNA insert and the translated amino acids of the maize glossy1 gene follow:

Example 6

This example sets forth the procedures used to clone the CER2 locus of Arabidopsis.

Example 7

The sequence of the CER2 cDNA insert was:

The corresponding genomic clone for CER2 was found to have the following sequence.

CER2-specific PCR primers were used to amplify the CER2-2 mutant allele and the location of respective homologies are represented in FIG. 4B. The primer sequences follow:

p1: 5'-AGGTGGACGTAATAAAGTGTG-3' [SEQ ID NO:24]

p2: 5'-GGTGGTGCCTGCTTCTTTGGTA-3' [SEQ ID NO:25]

p3: 5'-AAATCGAACCACTTCCCCACTG-3' [SEQ ID NO:26]

p4: 5'-GAGGATGATAGAACAAGATAAAGG-3' [SEQ ID NO:27]

p5: 5'-GGCATCACTACACTTACAATCCT-3' [SEQ ID NO:28]

p6: 5'-CAGTGACACCAAACAAGAACAA-3' [SEQ ID NO:29]

Accordingly, the CER2-specific sequences have characteristics that are fully consistent with their being the CER2 genes.

Example 8

This example sets forth data concerning the accumulation of CER2 mRNA in wild-type and mutant plants, and spatial patterns of expression of the CEP2 gene in a plant.

Example 9

This example illustrates the utility of probing a plant's genome for identification of related genes using a cloned cuticular lipid gene or fragment thereof.

All of the references cited herein, including patents, patent applications, and technical literature, are hereby incorporated in their entireties by reference.

Claims

What is claimed is:

2. The method of claim 1, further comprising the step of: (d) generating from the plant cell a plant.

3. The method of claim 1, wherein said plant is selected from the group consisting of maize, soybean, rapeseed, canola, cotton, safflower, peanut, palm, and sunflower.

4. The method of claim 1, wherein at least one of said regulatory sequences is developmentally specific or tissue-specific.

5. The method of claim 2, wherein said plant is selected from the group consisting of maize, soybean, rapeseed, canola, cotton, safflower, peanut, palm, and sunflower.

6. The method of claim 5, wherein said plant is maize or canola.

10. The plant cell of claim 7, wherein at least one of said regulatory sequences is developmentally specific or tissue-specific.

12. The plant of claim 8, wherein at least one of said regulatory sequences is developmentally specific or tissue-specific.

Non-Patent Literature (30)

Lewin R., Science 237:1570 Sep. 25, 1987.

Agrawal et al. "Biosynthesis of Very Long Chain Fatty Acids in Microsomes from Epidermal Cells of Allium porrum L.," Arch. Biochem. Biophys., 230, 580-589 (1984).

Agrawal et al., "Characterization and Solubilization of an Acyl Chain Elongation System in Microsomes of Leek Epidermal Cells," Arch. Biochem. Biophys., 240, 154-165 (1985).

Bianchi, "Glossy Mutants: Level of Action and Level of Analysis," Maize Breeding and Genetics, pp. 533-550, Walden, ed., John Wiley and Sons, New York, New York (1978).

Bianchi et al., "Biosynthesis Pathways of Epicuticular Wax of Maize as Assessed by Mutation, Light, Plant Age and Inhibitor Studies," Maydica, 30, 179-198 (1985).

Brown et al., "Molecular Analysis of Multiple Mutator-Derived Alleles of the Bronze Locus of Maize," Genetics, 122, 439-445 (1989).

Gibson et al., "Chromosome walking in Arabidopis thaliana using Yeast Artificial Chromosomes," Methods in Arabidospis Research, pp. 119-123, Koncz et al., eds., World Scientific, Singapore (1992).

Grill et al., "Construction and characterization of yeast artificial chromosome library of Arabidopsis which is suitable for chromosome walking," Mol. Gen. Genet., 226, 484-490 (1991).

Hansen et al., Plant Physical, 102 (i) , 93 (Abstratct #519, "Molecular Cloning and Characterization of Plant Cuticular Wax Genes") (May 1993).

Kolattukudy et al., "The Biochemistry of Plant Cuticular Lipids," Proq. Chem. Fats Other Lipids, 13, 121-175 (1973).

Kolattukudy et al., "Biochemistry of Plant Waxes," Chemistry and Biochemistry of Natural Waxes, pp. 571-645, Kolattukudy, ed., Elsevier Press, New York, New York (1976).

Kolattukudy, "Cutin, Suberin, and Waxes," Biochem. of Plants, 4, 571-645, Stumpf et al., eds., Academic Press, New York, New York (1980).

Kolattukudy, "Structure, Biosynthesis, and Biodegradation of Cutin and Suberin," Ann. Rev. Plant Physiol., 32, 539-567 (1981).

Lardizabal et al., "Substrate Tagging, Solubilization and Partial Purification of Jojoba (Simmondsia chinensis) Embryo Fatty Acyl-Coa: Fatty Alcohol Acyl Transferase," Plant Physiol. (Suppl.) , 102, 93 (1993).

Lazo et al., "A DNA Transformation-Competent Arabidopsis Genetic Library in Agrobacterium," Bio/Technology, 9, 963-967 (1991).

Lemieux et al., "GC-MS Analysis of the Wax of Eceriferum (CER) Mutants of Arabidopsis thaliana and Isolation of CER Genes fron T-DNA Tagged Arabidopsis Mutant Lines," Plant Physiol. (Suppl.) , 99, 14 (1992).

McNevin et al., "Isolation and Characterization of Eceriferum (ecr) Mutants Induced by T-DNA Insertions in Arabidopsis Thaliana" Genome, 36 (3), 610-618 (Jun. 1983).

Meyerowitz, "Introduction to the Arabidopsis genome," Methods in Arabidopsis Research, pp. 100-118, Koncz et al., eds., World Scientific, Singapore (1992).

Moose et al, "Molecular Cloning of the glossy-15 Locus, a Cell-Specific Regulator of Epidermal Differentiation," Abstracts, 35th Annual Maize 1993, Evans et al., eds. (1993).

Schnable et al, "Genetic Approaches to Isolating Maize and Arabidopsis Genes Involved in Cuticular Wax Biosynthesis," Current Topics in Plant Physiology, 9, 196-206 (1993).

Schnable et al., "Transposon Tagging Agronomically Important Loci in Maize," Proceedings, 28th Annual Illinois Corn Breeder's School (1992).

Stinard et al., "Genetic Isolation, Cloning, and Analysis of a Mutator-Induced, Dominant Antimorph of the Maize amylose extenderl Locus," Plant Cell, 5, 1555-1566 (1993).

Tulloch, "Chemistry of Waxes of Higher Plants," Chemistry and Biochemistry of Natural Waxes, pp. 235-287, Kolattukudy, ed., Elsevier Press, New York, New York (1976).

Vogel et al., J. Cellular Biochemistry, 17A, 32 (Abstract #A327) (Jan. 9, 1993).

von Wettstein-Knowles, "Genes, Elongases and Associated Enzyme Systems in Epicuticular Wax Synthesis," The Metabolism, Structure, and Function of Plant Lipids, pp. 489-498, Stump et al., eds., Elsevier Press, North-Holland, Amsterdam (1987).

Walbot, "Strategies for Mutagenesis and Gene Cloning Using Transposon Tagging and T-DNA Insertional Mutagenesis," Annu. Rev. Plant Physiol. Plant Mol. Biol., 43, 49-82 (1992).

Ward et al., "Isolation of single-copy-sequence clones from a yeast artificial chromosome library of randomly-sheared Arabidopsis thaliana DNA," Plant Mol. Biol., 14, 561-568 (1990).

Vander Knol et al. 1988 Bio/Techniques 6: 958-976.

Vander Knol et al. 1990 Plant Molec Biol 14: 457-466.

Finnegan et al. 1994 Bio/Technology 12: 883-888.