# Patent

## Title
Enzymes involved in triterpene synthesis

## Abstract

This invention relates to isolated polynucleotides encoding a CYP51H. The invention also relates to the construction of recombinant DNA constructs comprising all or a portion of the isolated polynucleotide of the invention, in sense or antisense orientation, operably linked to at least one regulatory sequence.

## Background
This application claims the benefit of U.S. Provisional Application No. 60/619,203, filed Oct. 15, 2004. The entire content of this application is herein incorporated by reference.


FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to polynucleotides encoding enzymes involved in the modification of β-amyrin during the biosynthesis of β-amyrin-derived triterpenes in plants and seeds. This invention also includes transgenic plants where the altered expression levels of the polynucleotides of the present invention results in altered levels or structures of β-amyrin-derived triterpenes, including saponins.
BACKGROUND OF THE INVENTION
The terpenoids, also called isoprenoids, constitute the largest family of natural products with over 22,000 individual compounds of this class having been described. The triterpenes or terpenoids (hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, polyprenols, and the like) play diverse functional roles in plants as hormones, photosynthetic pigments, electron carriers, mediators of polysaccharide assembly, and structural components of membranes. The majority of plant terpenoids are found in resins, latex, waxes, and oils.
Triterpenoids are of relevance to a variety of plant characteristics, including palatability to animals, and resistance to pathogens and predators. Triterpenes are mostly stored in plant roots as their glycosides, saponins (see Price K. R. et al, 1987, CRC Crit. Rev. Food Sci. Nutr. 26:27-133). Thus, for example, mutants of the diploid oat species, Avena strigosa, which lack the major oat root saponin, avenacin A-1 (so called saponin-deficient or “sad” mutants) have been shown to have compromised disease resistance (Papadopoulou K. et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:12923-12928). These mutants have increased susceptibility to a number of different root-infecting fungi, including Gaeumannomyces graminis var. tritici, which is normally non-pathogenic to oats. Genetic analysis suggests that increased disease susceptibility and reduced avenacin content are causally related. Furthermore, a sad mutant which produces reduced avenacin levels (around 15% of that of the wild type) gives only limited disease symptoms when inoculated with G. graminis var. tritici in comparison to other mutants which lack avenacins completely, providing a further link between avenacin content and disease resistance.
Triterpenoid saponins are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give pentacyclic triterpenoids, primarily oleanane (β-amyrin) or dammarane skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution, and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases, and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterized, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. However, the first dedicated step in saponin biosynthesis is now understood to be carried out by the oxidosqualene cyclase β-amyrin synthase, which has recently been cloned and characterized (Haralampidis K. et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98:13431-13436).
Many of the primary modifications to β-amyrin indicated in FIG. 1, which compares the structures of β-amyrin and avenacin A-1, are likely to be mediated by cytochrome P450 monooxygenases. These include oxidation at C16, C21, C30, or C23, and epoxidation at C12, C13. Besides their involvement in saponin biosynthesis, cytochrome P450 monooxygenases are involved in the biosynthesis of a multitude of other compounds, as described in (Nelson D. R., 1999, Arch. Biochem. Biophys. 369:1-10). While some single cytochrome P450 monooxygenase enzymes can metabolize multiple substrates, many of these enzymes are highly substrate specific. For example, in maize four P450s (BX2-5) sharing 45-60% amino acid identity belonging to the CYP71C family carry out successive hydroxylation events in the conversion of indole to the cyclic hydroxamic acid 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), each enzyme catalyzing predominantly only one reaction in the pathway. Available P450 structures show that the overall P450 structural fold is preserved during evolution from bacteria through plants and mammals. At the same time there are variable regions that appear to be associated with recognition and binding of structurally diverse substrates and redox partners.
The CYP51 (sterol 14α-demethylase) family is an essential enzyme in sterol biosynthesis and is the only P450 family that serves the same function in different biological kingdoms (Lepesheva G. I. et al., 2003, Biochemistry 42:9091-9101; Kelly S. L. et al., 2001, Biochem. Soc. Trans. 29:122-128). CYP51 enzymes catalyze the oxidative removal of the 14α-methyl group from lanosterol and 24-methylene-24,25-dihydrolanosterol in yeast and fungi, from obtusifoliol in plants and from 24,25-dihydrolanosterol in mammals. The products of action of sterol 14α-demethylases are Δ14,15-desaturated intermediates in ergosterol (fungi), phytosterol (plants) and cholesterol (animals) biosynthesis. The reaction includes three steps of successive conversion of the 14α-methyl group to 14α-hydroxymethyl, 14α-carboaldehyde, and 14α-formyl intermediates followed by elimination of formic acid with concomitant introduction of the Δ14,15 double bond into the sterol core. CYP51 s are targets for antifungal and cholesterol-lowering drugs.
The present invention describes polynucleotides encoding novel CYP51s, one of which modifies β-amyrin or a β-amyrin derivative. Identification of the genes encoding enzymes responsible for modification of β-amyrin or β-amyrin derivatives in a variety of crops will allow the manipulation of the same. Manipulation of the β-amyrin pathway will result in changes in the levels or structures of the saponins. A decrease in saponin production will result in an enhancement of plant resistance to pests. Foods originating from plants having an increased level of triterpenes are thought to have a cholesterol lowering effect while decreased triterpenes are believed to result in better tasting foods. Thus, transgenic plants having altered levels of triterpenes may be resistant to pests and foods prepared with seeds having altered levels or structures of saponins will have increased nutritional value or better flavor.
SUMMARY OF THE INVENTION
The instant invention relates to isolated polynucleotides encoding enzymes involved in triterpene synthesis. Specifically, this invention concerns isolated polynucleotides encoding novel cytochrome P450 monooxygenase enzymes of the CYP51 class, designated CYP51H, that modify β-amyrin or β-amyrin derivatives.
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence encoding a CYP51H polypeptide having an amino acid sequence of at least 80% sequence identity, based on the Clustal V method of alignment, when compared to SEQ ID NOs:14 and 26; or a full complement of such polynucleotide.
In a further embodiment, the instant invention is directed to an isolated polynucleotide selected from SEQ ID NOs:5, 13, 19, and 25. The invention also includes the full complement of any of these polynucleotides.
In another embodiment, the instant invention relates to a recombinant DNA construct comprising the isolated polynucleotide of the present invention operably linked to at least one regulatory sequence.
In a further embodiment, the instant invention concerns an isolated host cell comprising the recombinant DNA construct of the present invention. The host cell may be a yeast cell, bacterial cell, or a plant cell.
Compositions, including plants and plant parts, comprising the isolated polypeptide or polynucleotide of the present invention are also embodied by the present invention. The invention also includes transformed plants that arise from transformed host cells of higher plants and seeds or grains derived from such transformed plants. Such transgenic plants include those having an altered level of molecules derived from β-amyrin, or molecules with altered modifications.
The present invention also relates to a method of altering the level of expression of CYP51H polypeptide in a plant cell comprising: transforming plant tissue with a nucleic acid fragment from at least a portion of the isolated polynucleotide of the present invention, wherein the nucleic acid fragment is capable of altering expression of native CYP51H, regenerating the plant tissue into a transgenic plant, and evaluating the transgenic plant for altered level of expression of CYP51H when compared to a plant having wild type level of expression of native CYP51H.
In addition, the present invention relates to a method of producing a plant with altered levels of CYP51H comprising: transforming a plant cell with a recombinant DNA construct of the present invention; growing the transformed plant cell under conditions that promote the regeneration of a whole plant from the transformed cell; wherein the plant regenerated from the transformed cell produces an amount of CYP51H that is greater than the amount of the CYP51H that is produced in a plant that is regenerated from a plant cell of the same species as the plant that is not transformed with the recombinant DNA construct of the present invention; and optionally transforming the plant cell with a second recombinant DNA construct comprising a nucleic acid sequence encoding a polypeptide that regulates expression of at least one enzyme of the triterpene pathway; and growing the transformed plant cell under conditions that promote the regeneration of a whole plant from the transformed cell; wherein the plant regenerated from the transformed cell produces an amount of CYP51H that is greater than the amount of the CYP51H that is produced in a plant that is regenerated from a plant cell of the same species that is not transformed with the recombinant DNA construct and enzyme of the triterpene pathway of the second recombinant DNA construct.
The present invention is also directed to a method of producing a plant resistant to at least one fungus comprising: transforming a plant cell with the recombinant DNA construct of the present invention; growing the transformed plant cell under conditions that promote the regeneration of a whole plant from the transformed cell; wherein the plant regenerated from the transformed cell produces an amount of CYP51H that is greater than the amount of the CYP51H that is produced in a plant that is regenerated from a plant cell of the same species as the plant that is not transformed with the recombinant DNA construct; and optionally transforming the plant cell with a second recombinant DNA construct comprising a nucleic acid sequence encoding a polypeptide that regulates expression of at least one enzyme of the triterpene pathway; and growing the transformed plant cell under conditions that promote the regeneration of a whole plant from the transformed cell; wherein the plant regenerated from the transformed cell produces an amount of CYP51H that is greater than the amount of the CYP51H that is produced in a plant that is regenerated from a plant cell of the same species as the plant that is not transformed with the recombinant DNA construct and said enzyme of the triterpene pathway of said second recombinant DNA construct, thereby producing a plant resistant to fungi.
Also included in the invention are the grains from the transgenic plants of the invention.



BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE LISTING
The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.
The following sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
FIG. 1 depicts the structures of β-amyrin and Avenacin A-1 highlighting the multiple modifications that must take place to derive the latter from the former.



SEQ ID NO:1 is the nucleotide sequence of the hexaploid oat RFLP probe isu441.
SEQ ID NO:2 is the nucleotide sequence of primer ISU441-GSPF1 used to obtain additional 3′ end sequence of the gene cluster for avenacin biosynthesis in A. strigosa accession S75 and used for sequencing the genomic fragment encoding AsCyp51H1.
SEQ ID NO:3 is the nucleotide sequence of primer ISU441-GSPF2 used to obtain additional 3′ end sequence of the gene cluster for avenacin biosynthesis in A. strigosa accession S75.
SEQ ID NO:4 is the nucleotide sequence of primer ISU441-GSPR2 used to amplify the 5′ end of the gene cluster for avenacin biosynthesis in A. strigosa accession S75.
SEQ ID NO:5 is the nucleotide sequence of the cDNA encoding AsCyp51H1.
SEQ ID NO:6 is the nucleotide sequence of primer ISU441cF01 used in the PCR amplification of the 1639-bp cDNA containing the coding region of the AsCyp51H1 gene, and for sequencing the genomic fragment encoding AsCyp51H1 and the sad2 mutants.
SEQ ID NO:7 is the nucleotide sequence of primer ISU441cR01 used in the PCR amplification of the 1639-bp cDNA containing the coding region of the AsCyp5l H1 gene and the sad2 mutants, and used for sequencing the sad2 mutants.
SEQ ID NO:8 is the nucleotide sequence of primer ISU441gF1 used to sequence pCR®4-TOPO plasmids that might contain the 1639-bp cDNA comprising the coding region of the AsCyp51H1 gene, and used for sequencing the genomic fragment encoding AsCyp51H1 and the sad2 mutants.
SEQ ID NO:9 is the nucleotide sequence of primer ISU441cF03 used for sequencing the genomic fragment encoding AsCyp51H1.
SEQ ID NO:10 is the nucleotide sequence of primer ISU441cF04 used for sequencing the genomic fragment encoding AsCyp51H1.
SEQ ID NO:11 is the nucleotide sequence of primer ISU441gF2 used for sequencing the genomic fragment encoding AsCyp51H1.
SEQ ID NO:12 is the nucleotide sequence of primer ISU441gF4 used for sequencing the genomic fragment encoding AsCyp51H1 and the sad2 mutants.
SEQ ID NO:13 is the nucleotide sequence of the genomic fragment encoding AsCyp51H1.
SEQ ID NO:14 is the amino acid sequence of AsCyp51H1 derived from the cDNA fragment shown in SEQ ID NO:5 or the genomic fragment shown in SEQ ID NO:13.
SEQ ID NO:15 is the nucleotide sequence of primer ISU441pF01 used to amplify the sad2 mutants.
SEQ ID NO:16 is the nucleotide sequence of primer ISU441cR03 used for sequencing the sad2 mutants.
SEQ ID NO:17 is the nucleotide sequence of primer ISU441indeR used for sequencing the sad2 mutants.
SEQ ID NO:18 is the nucleotide sequence of primer ISU441gF5 used for sequencing the sad2 mutants.
SEQ ID NO:19 is the nucleotide sequence of the genomic fragment encoding AsCyp51H2.
SEQ ID NO:20 is the nucleotide sequence of primer ASCYPA2F01.
SEQ ID NO:21 is the nucleotide sequence of primer ASCYPA2R02.
SEQ ID NO:22 is the nucleotide sequence of primer ASCYPA2F03.
SEQ ID NO:23 is the nucleotide sequence of primer ASCYPA2R04.
SEQ ID NO:24 is the nucleotide sequence of primer ASCYPA2F05.
SEQ ID NO:25 is the nucleotide sequence of the cDNA fragment encoding AsCyp51H2.
SEQ ID NO:26 is the amino acid sequence of AsCyp51H2 derived from the genomic fragment shown in SEQ ID NO:19 or the cDNA fragment shown in SEQ ID NO:25.
SEQ ID NO:27 is the nucleotide sequence of the entry Vector for AsCyp51H1 comprising ATTL1-AsCyp51H1-ATTL2.
SEQ ID NO:28 is the nucleotide sequence of the entry Vector for BAS comprising ATTL3-BAS-ATTL4.
SEQ ID NO:29 is the nucleotide sequence of the section between the RB and LB of the maize recombinant DNA construct 1.
SEQ ID NO:30 is the nucleotide sequence of the section between the RB and LB of the maize recombinant DNA construct 2.
SEQ ID NO:31 is the nucleotide sequence of the section between the RB and LB of the soybean recombinant DNA construct 1.
SEQ ID NO:32 is the nucleotide sequence of the section between the RB and LB of the soybean recombinant DNA construct 2.
The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in 1984 in the Biochemical J. 219:345-373 and in 1985 in Nucleic Acids Res. 13:3021-3030 which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
DETAILED DESCRIPTION OF THE INVENTION
In the context of this disclosure, a number of terms shall be utilized. The terms “polynucleotide/isolated polynucleotide” and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-nautral or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include all or part of the isolated polynucleotide, such as for example a polynucleotide comprising the nucleotide sequence selected from the group consisting of SEQ ID NOs:5, 13, 19, and 25, or the full complement of such nucleotide sequences.
The term “isolated” polynucleotide is one that has been substantially separated or purified from other polynucleotides of the organism in which the polynucleotide naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, by conventional nucleic acid purification methods. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
The present invention is directed to isolated polynucleotides encoding CYP51Hs. While not intending to be bound by any theory or theories of operation, it is believed that these enzymes are membrane bound.
As used herein “CYP51H polynucleotides” refers to polynucleotides that encode novel cytochrome P450 monooxygenase enzymes which modify β-amyrin or a β-amyrin derivative in a reaction subsequent to that of β-amyrin synthase. “CYP51H enzymes” refer to the cytochrome P450 enzymes of the invention.
As used herein “cytochrome P450”, “P450”, “CYP450”, and “cytochrome P450 monooxygenase” are used interchangeably herein. These comprise a large number of polypeptides that are grouped into families based solely on sequence homology. Many of the primary modifications to β-amyrin indicated in FIG. 1 are likely to be mediated by cytochrome P450 monooxygenases. These include oxidation at C16, C21, C30, or C23, and epoxidation at C12, C13. Cytochrome P450 monooxygenases are also involved in the biosynthesis of a multitude of other compounds, as described in Nelson D. R., 1999, Arch. Biochem. Biophys. 369:1-10. While some single cytochrome P450 monooxygenase enzymes can metabolize multiple substrates, many of these enzymes are highly substrate specific.
Triterpenoid saponins are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give pentacyclic triterpenoids, primarily oleanane (β-amyrin) or dammarane skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution, and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases, and other enzymes.
Triterpenes, also known as triterpenoids, include and are not limited to sapinogenins and sterols.
As used herein, “substantially similar” refers to polynucleotides having nucleic acid sequences wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, that do not affect the functional properties of the polypeptide encoded by the nucleic acid sequence. “Substantially similar” also refers to polynucleotides wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid sequence to mediate alteration of gene expression by antisense or co-suppression technology among others. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting polypeptide. It is therefore understood that the invention encompasses more than the specific exemplary sequences.
Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal V method of alignment (Higgins, D. G. et al., 1992, Comput. Appl. Biosci. 8(2):189-191) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal V method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequence encoding the CYP51H proteins as set forth in SEQ ID NOs:14 and 26. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a polynucleotide for improved expression of a specific gene in a host cell, it is desirable to design the polynucleotide such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
“Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences upstream (5′ non-coding sequences), within, and downstream (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences, not necessarily in its natural location. “Chimeric or heterologous” “gene or polynucleotide” refers any gene or polynucleotide that is not native to a plant. A chimeric or heterologous gene may comprise regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.
“Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
“Promoter” refers to a polynucleotide capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements; the latter elements often referred to as enhancers.
Accordingly, an “enhancer” is a nucleotide sequence, which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg published in 1989 (Biochem. Plants 15:1-82). It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
The “translation leader sequence” or “leader” refers to a polynucleotide sequence located between the promoter of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start site. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster, 1995, Mol. Biotechnol. 3:225-236).
The “3′ non-coding region” or “terminator region” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al., 1989, Plant Cell 1:671-680.
“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a DNA that is complementary to and derived from an mRNA. The cDNA can be single-stranded or converted into the double stranded form using, for example, the Klenow fragment of DNA polymerase I. “Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
The term “operably linked” and “under the control of” refer to the association of nucleic acid fragments on a single polynucleotide so that the function of one is affected by the function of the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Similarly, a polynucleotide may be under the control of a promoter that is capable of affecting the expression of the polynucleotide. Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
The term “recombinant DNA construct” means, for example, that a recombinant nucleic acid sequence is made by an artificial combination of two otherwise separated nucleotide segments, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
The term “expression”, as used herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a polynucleotide of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference). One can also envision the use of “RNAi” related techniques to reduce the expression of the genes of the present invention. See for example U.S. Pat. No. 6,506,559. Such techniques rely on the use of constructs resulting in the accumulation of double stranded RNA with one strand complementary to the target gene to be silenced.
“Altered levels” or “altered expression” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms. Altered levels include an increase and a decrease in gene product amounts compared to normal or non-transformed organisms. Accordingly, altered includes increase, enhance, amplify, multiply, elevate, raise, and the like as well as decrease, reduce, lower, prevent, inhibit, stop, eliminate, and the like.
“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or pro-peptides present in the primary translation product has been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and pro-peptides still present. Pre- and pro-peptides may be but are not limited to intracellular localization signals.
A “signal peptide” is an amino acid sequence that is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels, M. (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel, N. (1992) Plant Phys. 100:1627-1632). A “chloroplast transit peptide” is an amino acid sequence that is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide.
“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. “Host cell” refers the cell into which transformation of the recombinant DNA construct takes place and may include a yeast cell, a bacterial cell, and a plant cell. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al., 1987, Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al., 1987, Nature (London) 327:70-73; U.S. Pat. No. 4,945,050), among others.
Expression of a chimeric CYP51H, for example, results in the production of a level of the encoded CYP51H protein in a transformed host cell that is altered as compared to the level produced in an untransformed host cell. Also, a transgenic plant, or plant part, comprising a polynucleotide of the present invention, such as for example, SEQ ID NOs:5, 13, 19, and 25, under the control of a heterologous promoter results in plants having altered levels of triterpenes. Plants may be selected from the group consisting of monocots and dicots. Monocots include and are not limited to corn, oat, rice, wheat, barley, palm, and the like. Dicots include and are not limited to Arabidopsis, soybean, oilseed Brassica, peanut, sunflower, safflower, cotton, tobacco, tomato, potato, cocoa, and the like. Plant parts include and are not limited to seeds and grains, for example.
Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs capable of introduction into and replication in a host cell. A “vector” may be such a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).
“PCR” or “polymerase chain reaction” is a technique for the synthesis of large quantities of specific DNA segments. It consists of a series of repetitive cycles (Perkin Elmer Cetus Instruments, Norwark, Conn.). Typically, the double-stranded DNA is heat denatured, the two primers complementary to the 3′ boundaries of the target segments are annealed at low temperature and then extended at an intermediate temperature. One set of these three consecutive steps is referred to as a cycle.
Oats having a sad2 mutation produce β-amyrin but produce very little or no avenacins. Eight mutations conferring a sad2 phenotype have been identified. Each of these has a lesion in the polynucleotide of the present invention that would render the polynucleotide incapable of expressing a functional mRNA encoding a functional protein. These data together with the biochemical data presented herein indicate that the non-mutated polynucleotide of the present invention encodes the enzyme AsCyp51H1 (also known in some portions of the literature as CYP51H10) responsible for a modification of β-amyrin or a β-amyrin derivative, which is not carried out in the sad2 mutants. Genomic and cDNA fragments encoding AsCyp51H1 are disclosed. Also identified is an AsCyp51H1 homolog AsCyp51H2 (also known in some portions of the literature as CYP51H11). The nucleotide sequence of AsCyp51H2 hybridizes to a probe prepared with the genomic sequence that encodes AsCyp51H1.
The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
For example, genes encoding other CYP51Hs, either as cDNAs or genomic DNAs, may be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Sambrook). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length cDNA or genomic fragments under conditions of appropriate stringency.
In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., 1989, Proc. Natl. Acad. Sci. U.S.A 86:5673-5677; Loh et al., 1989, Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin, 1989, Techniques 1:165).
Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner,1984, Adv. Immunol. 36:1-34; Sambrook).
The nucleic acid fragments of the instant invention may be used to create transgenic plants in which CYP51Hs of the present invention are present at higher levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering production of triterpenes in those cells. It is believed that overexpression of the polynucleotides of the invention, optionally in combination with polynucleotides encoding enzymes responsible for other steps in the saponin biosynthetic pathway, enhances resistance to at least one fungus. Suppression of the polynucleotides of the invention may result in legumes producing lower saponins, which in turn may improve the flavor.
A “plant resistant to at least one fungus” refers to a plant comprising a recombinant DNA construct of the present invention which when infected with a fungus is able to resist infection or to tolerate infection to a greater degree, resulting in less damage, more vigorous health and less or no loss of yield due to fungal infection relative to plants without the recombinant DNA construct of the present invention. The fungus is typically pathogenic. “Pathogenic” or “fungal pathogen” refer to a fungus that under conditions that do not include the recombinant DNA construct of the present invention, would cause disease in a plant. A transgenic plant comprising the recombinant DNA construct of the present invention is typically a plant more resistant to at least one fungus than a plant of the same species without the recombinant DNA construct of the present invention.
The embodiments of the present invention may be effective against a variety of plant fungal pathogens. Some specific fungal pathogens for the major crops include, but are not limited to, the following: Soybeans: Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var. caulivora, Sclerotium rolfsii, Cercospora kikuchii, Cercospora sojina, Colletotrichum dematium (Colletotichum truncatum), Corynespora cassiicola, Septoria glycines, Phyllosticta sojicola, Alternaria alternata, Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Glomerella glycines, Phakopsora pachyrhizi, Fusarium solani; Canola: Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassicicola, Fusarium roseum, Alternaria alternata; Alfalfa: Phoma medicaginis var. medicaginis, Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrichila medicaginis, Fusarium oxysporum, Verticillium albo-atrum, Stemphylium herbarum, Stemphylium alfalfae, Colletotrichum trifolii, Leptosphaerulina briosiana, Uromyces striatus, Sclerotinia trifoliorum, Stagonospora meliloti, Stemphylium botryosum, Leptotrochila medicaginis; Wheat: Urocystis agropyri, Alternaria aternata, Cladosporium herbarum, Fusarium avenaceum, Fusarium culmorum, Ustilago tritici, Ascochyta tritici, Cephalosporium gramineum, Collotetrichum graminicola, Erysiphe graminis f.sp. tritici, Puccinia graminis f.sp. tritici, Puccinia recondite f.sp. tritici, Puccinia striiformis, Pyrenophora tritici-repentis, Septoria nodorum, Septoria tritici, Septoria avenae, Pseudocercosporella herpotrichoides, Rhizoctonia solani, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Bipolaris sorokiniana, Claviceps purpurea, Tilletia tritici, Tilletia laevis, Ustilago tritici, Tilletia indica, Rhizoctonia solani; Sunflower: Plasmophora halstedii, Sclerotinia sclerotiorum, Septoria helianthi, Phomopsis helianthi, Alternaria helianthi, Alternaria zinniae, Botrytis cinerea, Phoma macdonaldii, Macrophomina phaseolina, Erysiphe cichoracearum, Rhizopus oryzae, Rhizopus arrhizus, Rhizopus stolonifer, Puccinia helianthi, Verticillium dahliae, Cephalosporium acremonium; Corn: Colletotrichum graminicola (Glomerella graminicola), Stenocarpella maydi (Diplodia maydis), Fusarium moniliforme var. subglutinans, Fusarium verticillioides, Gibberella zeae (Fusarium graminearum), Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus), Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella maydis, Cercospora sorghi, Ustilago maydis, Puccinia sorghi, Puccinia polysora, Macrophomina phaseolina, Penicillium oxalicum, Nigrospora oryzae, Cladosporium herbarum, Curvularia lunata, Curvularia inaequalis, Curvularia pallescens, Trichoderma viride, Claviceps sorghi, Diplodia macrospora, Sclerophthora macrospora, Sphacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Cephalosporium acremonium; Sorghum: Exserohilum turcicum, Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternata, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Colletotrichum (Glomerella) graminicola (C. sublineolum), Fusarium graminearum, Fusarium oxysporum; and the like.
Overexpression of CYP51H proteins of the instant invention may be accomplished by first constructing a recombinant DNA construct in which the coding region is operably linked to a promoter capable of directing expression of CYP51H in the desired tissues at the desired stage of development. The recombinant DNA construct may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ non-coding sequences encoding transcription termination signals may also be provided. The instant recombinant DNA construct may also comprise one or more introns in order to facilitate gene expression.
Plasmid vectors comprising the isolated polynucleotide of the invention may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host cells. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., 1985, EMBO J. 4:2411-2418; De Almeida et al., 1989, Mol. Gen. Genetics 218:78-86), and thus that multiple events may have to be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.
For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate their secretion from the cell. It is thus envisioned that the recombinant DNA constructs described above may be further supplemented by altering the coding sequence to encode appropriate intracellular targeting signals such as transit signals (Keegstra, 1989, Cell 56:247-253), signal sequences with or without endoplasmic reticulum retention signals (Chrispeels, 1991, Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel, N., 1992, Plant Phys. 100:1627-1632) with or without removing targeting signals that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future.
It may also be desirable to reduce or eliminate expression of CYP51H in plants for some applications. In order to accomplish this, a recombinant DNA construct designed for co-suppression of such enzymes can be constructed by linking a polynucleotide encoding an CYP51H to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated. Construction of chimeric nucleic acid fragments that result in the formation of hair-loop structures where portions of the polynucleotides of the invention are either the stem or the loop or the structure may also be prepared. It may also be possible to use small fragments of the nucleotides encoding CYP51H to prepare constructs that would serve as RNAi to suppress its expression. Any of the recombinant DNA constructs mentioned above may be introduced into a cell to eliminate expression of CYP51H in plants.
A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include, and are not limited to, allele-specific amplification (Kazazian, H. H. jr, 1989, J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield, V. C., et al., 1993, Genomics 16:325-332), allele-specific ligation (Landegren, U., et al., 1988, Science 241:1077-1080), nucleotide extension reactions (Sokolov, B. P., 1990, Nucleic Acid Res. 18:3671), radiation hybrid mapping (Walter, M. A. et al., 1994, Nat. Genet. 7:22-28), fluorescence in situ hybridization (FISH; Svitashev, S. K. and Somers, D. A., 2002, Plant Cell Tissue Organ Cult. 69:205-214), and Happy Mapping (Dear, P. H. and Cook, P. R., 1989, Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for all mapping methods.
While not intending to be bound by any theory or theories of operation, it is believed by those of skill in the art that altered levels of triterpenes have different effects. Increased levels of triterpenes such as avenacin in parts of the plant normally susceptible to fungal pathogen infection may endow the plant with resistance to at least some such pathogens, protecting the plants and so enhancing yield in circumstances of fungal pressure. Foods originating from plants having an increased level of triterpenes are thought to have a cholesterol lowering effect while decreased triterpenes are believed to result in better tasting foods. Accordingly, plants grown with altered levels of CYP51H may contribute to nutritious and/or better-flavored foods. Thus, also included in the invention are the grains from the transgenic plants of the invention.
The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. Examples 1-4 are actual, Examples 5-7 are prophetic. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
The disclosure of each reference set forth herein is incorporated by reference in its entirety.
EXAMPLE 1
Generation of Mutants and Biochemical Characterization of sad2 Oat Mutants
Seed of the diploid oat species Avena strigosa were mutagenized with sodium azide and M2 seed from individual M1 plants were germinated and assessed for root fluorescence as a preliminary screen to identify saponin-deficient, or sad, oat mutants. Seedlings not producing avenacins were identified by HPLC and TLC analyses of methanolic root extracts from homozygous M3 seedlings of putative mutants.
Generation of Mutants
Seed of the diploid oat species Avena strigosa (accession S75 from the Institute of Grasslands and Environmental Research, Aberystwyth, Wales, UK) was mutagenized with sodium azide essentially as described (Rines, H. W., 1985, Env. Exp. Bot., 25:7-17). Briefly, mutagenesis was performed as follows. Seeds were presoaked in an Erlenmeyer flask sealed with a rubber stopper using 0.5 ml water per seed while shaking in an orbital platform shaker at 120 cycles per minute. After presoaking for 4 hours the water was decanted. A solution of 10 mM sodium azide in 0.1 M sodium phosphate, pH 3.2 was prepared and immediately added to the seeds. After shaking, as above, for 1 hour the mutagen solution was decanted and the seeds rinsed with 5 to 6 changes of water with the last three water rinses extending over a period of 30 minutes. Rinsed seeds were drained and spread over paper in a fume hood to dry. M2 seed from individual M1 plants were germinated and assessed for root fluorescence as indicated below.
The major oat-root saponin avenacin A-1 contains N-methyl anthranilic acid and, thus, is primarily responsible for the bright blue fluorescence of young oat roots (Osbourn A. E. et al., 1994, Physiol. Mol. Plant Pathol. 45:457-467). The fact that avenacin A-1 is detectable by UV light allows root fluorescence to be used as a preliminary screen to identify saponin-deficient (sad) oat mutants. Seed of individual M2 families were germinated and assessed for root fluorescence. In the initial screens ten independent mutants with reduced fluorescence were identified after screening seedlings representing 1,289 M2 families as reported by Papadopoulou K. et al. (1999, Proc. Natl. Acad. Sci. U.S.A. 96:12923-1928). Subsequent mutant screens identified a further 40 independent avenacin-deficient mutants isolated on the basis of reduced root fluorescence.
Biochemical Characterization
Analysis of the root extracts of the original ten mutants was carried out as described (Papadopoulou K. et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:12923-1928). Briefly, M3 seeds were germinated on moist filter paper for 2 days and terminal 0.5 cm sections of the roots from 20 seedlings per line were harvested and extracted in methanol. For HPLC analysis crude methanolic root extracts from M3 seedlings were prepared in triplicate and 100 μl aliquots were analyzed directly on a Hichrom Nucleosil 5 C18 reverse phase column (4.5×250 mm) under isocratic conditions in 75% methanol (flow rate 1 ml/min) with detection at 225 nm. The four avenacins were quantified by comparison of peak areas with those of standards of known concentration. Extracts for TLC analysis were dried down, resuspended in 1 ml water and applied to SepPak C18 reverse phase cartridges (Waters, Milford, Mass.) that had been pre-conditioned with 10 ml of methanol followed by 10 ml distilled water. After elution with 75% methanol samples were dried down, resuspended in 15 μl of 100% methanol, applied to the TLC plates, and separated using chloroform:methanol:water (13:6:1; v:v:v). Avenacins A-1 and B-1 and other fluorescent components were visualized under UV illumination at 302 nm. The TLC plate was then sprayed with p-anisaldehyde/sulphuric acid/acetic acid (1:1:48, v:v:v) and baked at 130° C. for 5 min to detect all four saponins. Root extracts derived from either M3 or F3 seedlings were compared on at least seven occasions with essentially the same outcome.
HPLC analysis of crude root extracts confirmed the absence of all four avenacins in mutant #1027; and reduced levels of avenacins (approximately 15% of that of the wild type) in extracts from mutant #791 (Papadopoulou K. et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:12923-1928).
Genetic Analysis of sad Mutants
Test crosses were performed between the sad mutants and the wild type A. strigosa to determine if the saponin-deficient phenotype was due to a single mutation. Analysis of F2 generations from intermutant crosses identified at least 4 complementation groups in the initial 10 mutant lines. These loci were designated sad1 through sad4 (Papadopoulou K. et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:12923-1928). Further analysis of the original 10 mutant lines determined 4 additional loci designated sad5 through sad8 (Qi X. et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101:8233-8238). Additional loci sad9 and sad10 were identified while analyzing the additional 40 mutant lines identified later. The sad2 locus was identified as a single dominant locus defined by independent mutants #791 and #1027 (Papadopoulou K. et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96:12923-1928).
Subsequent feeding experiments using radiolabled mevalonic acid (R-[2-14C] MVA) on mutant roots indicated that sad2 mutants incorporated radioactivity into β-amyrin but either produced very small amounts of avenacins or not at all suggesting that they are blocked in a step early in the avenacin biosynthetic pathway (Trojanowska M. R. et al., 2001, Phytochemistry 56:121-129). Of the original 10 sad mutants the sad2 mutants were the only ones that accumulate β-amyrin. Screening of root extracts of the additional 40 mutant lines led to the identification of a further six candidate sad2-like mutants (mutants #283, #500, #638, #698, #1325 and #1412) on the basis of metabolite profiling experiments performed as described below.
A single seed of each line was soaked in 1% bleach for 10 minutes, rinsed three times with sterile distilled water, kept at 4° C. for approximately 24 hours, and then germinated on 1% agar at 22° C. for 6 days. Individual roots were harvested, freeze-dried, ground in liquid nitrogen, and extracted with methanol. Extracts were centrifuged, the supernatant removed and dried down prior to extraction with 100 μl CHCl3/MeOH (7:3, v/v). Extracts were then spotted onto TLC plates together with a β-amyrin standard dissolved in chloroform. The TLC plates were developed with hexane: acetone (80:20, v/v). Iodine vapor was used to detect β-amyrin and other compounds.
Using this screen a further six candidate sad2-like mutants were identified that accumulate elevated levels of β-amyrin. These results were confirmed by quantitative GC/MS analysis as described below.
TMS Ether Derivatization and GC-MS Analysis.
To 50 μl of each sample extract prepared as above from sad mutants and wild type roots 100 μl of Tri-Sil reagent and 24.48 μg of 5β-cholestan-3β-ol (TMS) was added in glass-stoppered small clear reaction vials. After swirling to dissolve the samples, the vials were heated at 60° C. for 60 minutes. Excess reagent and solvent were removed under a nitrogen stream, and normally the residue was diluted to 200 μl with HPLC grade hexane for quantitation by gas chromatography (GC) with flame ionization detection (FID).
Gas chromatography-mass spectrometry (GC-MS) was carried out on a Hewlett Packard 5973 mass selective detector coupled to a Hewlett Packard 6890 gas chromatograph with a Hewlett Packard 6890 auto injector. The column was a 30 m long DB-5MS (J & W scientific Ltd, United Kingdom) with a 0.25 mm internal diameter and a film thickness of 0.25 micron. It was held for 1 minute at 250° C., then programmed to increase at 5° C./minute to 325° C., and held for 10 minutes at 325° C. The injector was set at 250° C. and a 2 μl injection volume was used. The flow was set at 3 psi and operated in split mode with a split ratio of 10:1. The mass spectrometer source was set at 230° C. and the quadrupole at 106° C. The mass spectrometer was scanned between masses 35 and 800 in 1 second for full scan spectra after a 5 minute solvent delay. Selected ion recording masses of 498.4, 218.2, 203.2, 460.4, 370.4 and 355.3 were sequentially monitored with a dwell setting of 30 (3.64 cycles/second) between 8 and 20 minutes.
Quantitation of β-amyrin was performed using 5β-Cholestan-3β-ol (TMS) as internal standard and preparing a calibration line by analyzing a fixed amount of internal standard against a varying amount of β-amyrin. The area of the 370 ion was used for the internal standard and the area of the 218 ion used for β-amyrin. The results in Table 1, below, clearly demonstrate that sad2 mutants 791 and 1027 have much larger amounts of β-amyrin than sad1 mutants or wild type plants. Table 1 presents the quantity of β-amyrin obtained from GC-MS analyses of S75 (wt), 610 and 109 (sad1), and 791 and 1027 (sad2) roots. The results are presented as the mean β-amyrin content or μg/g of fresh freeze-dried root ± the standard deviation. Two independent extractions were done for each root except, for the sad1 mutant 610.







TABLE 1







Quantity of β-amyrin in sad1, sad2, and wt Roots










Sample
Mean β-amyrin Content







791
48.5 ± 9.0 



791
38.7 ± 3.2 



1027 
42.4 ± 5.4 



1027 
47.7 ± 4.7 



S75
2.7 ± 0.2



S75
2.0 ± 0.0



610
0.7 ± 0.0



109
0.8 ± 0.0



109
0.8 ± 0.0










These results suggest that the sad2 mutations affect a step downstream of β-amyrin synthase.
EXAMPLE 2
Isolation of the AsCypH1 Genomic and cDNA Fragments
The genomic polynucleotide fragment encoding the gene affected in sad2 mutants was isolated from A. strigosa accession S75 genomic DNA and from a library prepared from oat as follows.
The genomic polynucleotide fragment present in A. strigosa accession S75 and affected by the sad2 mutations was identified from a gene cluster identified for avenacin biosynthesis (Qi X. et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101:8233-8238). First, the hexaploid oat RFLP probe isu441 (Rayapati, P. J., et al., 1994, Theor. Appl. Genet. 89:831-837) previously mapped to the gene cluster for avenacin biosynthesis in diploid oat (Qi X. et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101:8233-8238) was used. Probe isu441 (which is a cDNA-derived probe) was sequenced by using the ABI PRISM® Big-DyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) with M13 forward and reverse primers (Qiagen Ltd) and its nucleotide sequence is shown in SEQ ID NO:1. The resulting 480-nt fragment was found to share sequence similarity with obtusifoliol 14α-demethylases, sterol biosynthetic enzymes belonging to the CYP51 family of P450s. The fragment lies in the 3′ region of the predicted P450 coding sequence and includes the polyA tail. Further sequence was obtained towards the 3′-end of the gene using the GenomeWalkerTM kit following instructions provided by the manufacturer (Clontech Ltd) and DNA from A. strigosa accession S75 as the template. Two primers, ISU441-GSPF1 and ISU441-GSPF2 were used in this experiment. The nucleotide sequences of these primers are shown in SEQ ID NOs:2 and 3, respectively.









ISU441-GSPF1:




5′-CTGACTTCTCCATTTCCCAAGCAAGA-3′
(SEQ ID NO:2)


 


ISU441-GSPF2:


5′-CTACTAGCACCTATTTGCACGGATGT-3′
(SEQ ID NO:3)






The 5′-end cDNA fragment was obtained by using GeneRacerTM Kit following instructions provided by the manufacturer (Invitrogen Ltd). Total RNA was isolated from the root tips of S75. A PCR fragment of around 1.3 kb was amplified from RACE-ready cDNA using the GeneRacerTM 5′ Primer and ISU441-GSPR2 (shown in SEQ ID NO:4), which is an isu441-specific primer.









ISU441-GSPR2:




5′-ATCCTCCTCTCTTCCAACACGAAACC-3′
(SEQ ID NO:4)






This 1.3-kb PCR fragment was cloned into PCR-Script Amp SK (+) plasmid by following the protocol for the PCR-ScriptTM Amp Electroporation-Competent cell Cloning Kit provided by the manufacturer (Stratagene Ltd). Sequencing was conducted by using the ABI PRISM® Big-DyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) with M13 forward and reverse primers (Qiagen Ltd). By merging the 5′-end and 3′-end sequences a sequence corresponding to an approximately 1790 bp fragment derived from isu441 was obtained from S75 and is shown in SEQ ID NO:5. This cDNA contains an entire open reading frame corresponding to nucleotides 103-1572. The gene corresponding to this cDNA was designated AsCyp51H1. The 1639-bp cDNA containing the coding region of this gene was amplified by PCR with primer pair ISU441cF01 and ISU441cR01 (shown in SEQ ID NOs:6 and 7, respectively), and cloned into pCR®4-TOPO plasmid (Invitrogen Ltd).









ISU441cF01




5′-CCAGTCAGGAGGATTTCAAATTCGTATTCA-3′
(SEQ ID NO:6)


 


ISU441cR01


5′-CGACGCCTTATTGTAAATAAGCCCAT-3′
(SEQ ID NO:7)






Plasmids from 8 positive clones were sequenced with M13 forward and reverse primers (Qiagen Ltd), and primer ISU441gF1 (shown in SEQ ID NO:8), respectively. A mutation-free clone was identified (pCR®4-TOPO:isu441c-7) and was used for further experiments.









ISU441gF1




5′-ACGAGGGTGAAGTCGATCTGAAACAAGAG-3′
(SEQ ID NO:8)






The genomic DNA fragment of the AsCyp51H1 gene was amplified from A. strigosa accession S75 genomic DNA by PCR using oligonucleotide primers ISU441cF01 and ISU441cR01 (mentioned above) using Expand High Fidelity PCR System (Roche Molecular Biochemicals). The 50 μl PCR reaction contained 100 ng genomic DNA, 0.2 μM forward primer, 0.2 μM reverse primer, 200 μM dNTPs, 1× reaction buffer with 1.5 mM MgCl2, and 2.6 U of Expand High Fidelity PCR System Enzyme Mix. After initial denaturation at 94° C. for 2 minutes, amplification was carried out with 35 cycles of 1) denaturation at 94° C. for 30 seconds, 2) annealing at 63° C. for 30 seconds, and 3) extension at 68° C. for 4 minutes. The amplified product was purified using a Qiagen PCR Purification Kit (Qiagen Ltd) and then used for direct sequencing with primers ISU441cF01, ISU441cF03, ISU441cF04, ISU441gF1, ISU441gF2 and ISU441-GSPF1 (the nucleotide sequences of which are shown in SEQ ID NOs:6, 9, 10, 8, 11, and 2, respectively).









ISU441cF03




5′-CAATTATATCCATCGCTGCAGTAG-3′
(SEQ ID NO:9)


 


ISU441cF04


5′-ATGTTGATCTCATTCGACAGGAAGT-3′
(SEQ ID NO:10)


 


ISU441gF2


5′-TGTCGAGGAGCAAAAGCAAATGATGAG-3′
(SEQ ID NO:11)


 


ISU441gF4:


5′-GAACAAGTGCGATGGATTATGGTA-3′
(SEQ ID NO:12)






Oligonucleotide primer ISU441gF4 (the nucleotide sequence of which is shown in SEQ ID NO:12) was designed and used for sequencing through the remaining gap to produce an entire genomic sequence encoding AsCyp51H. Comparison of the genomic DNA sequence with that obtained for the full-length cDNA identified two introns. The cDNA sequence starts at nucleotide 2881 of the genomic sequence. There is a 348-nucleotide intron at nucleotide 77 of the cDNA sequence and a 973-nucleotide intron at nucleotide 576 of the cDNA sequence.
To extend the sequence towards the 5′ end in order to obtain a possible promoter sequence a BAC library derived from A. strigosa accession S75 genomic DNA was screened with a probe generated from plasmid pCR®4-TOPO:isu441c-7. For this purpose a 1639-bp cDNA probe was generated from plasmid DNA of clone pCR®4-TOPO:isu441c-7 (containing AsCyp51H1 cDNA and described above) by PCR with the primer pair ISU441cF01 and ISU441cR01 (shown in SEQ ID NOs:6 and 7). A BAC library was constructed as described by Bakht et. al. (Plant & Animal Genome XI Conference, Jan. 11-15, 2003, San Diego, Calif., P82) and was screened with this probe. Shotgun sequencing of one of the positive clones (clone #B460D15) yielded a further 2882 bp of sequence upstream from 5′-end of the full-length cDNA. This region was defined as putative promoter sequence of AsCyp51H1 gene. The 5992 bp genomic sequence of the AsCyp51H gene is shown in SEQ ID NO:13. The amino acid sequence of the enzyme encoded by this gene has 490 amino acids and are shown in SEQ ID NO:14.
EXAMPLE 3
Cloning and Sequencing of AsCyp51H1 Alleles From Different sad2 Mutants
The sad2 mutants #791 and #1027 accumulate β-amyrin and so were considered likely to be blocked in a cytochrome P450-mediated step early in the pathway. Previous genetic analysis (Qi X. et al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101:8233-8238) indicated that Sad2 is closely linked to Sad1. This gene, Sad1, has been designated AsbAS1 and has been previously cloned, characterized, and demonstrated to encode β-amyrin synthase, the enzyme that catalyzes the first committed step in avenacin biosynthesis (Haralampidis K. et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98:13431-13436). Sad2 co-segregates with Sad1 in a population of 2040 F2 individuals (Qi X. at al., 2004, Proc. Natl. Acad. Sci. U.S.A. 101:8233-8238). Several BAC clones that contained both AsCyp51H1 and AsbAS1 were identified by hybridization of the BAC colony filters with the cDNA probes from the two genes. Analysis of the sequence of one of these clones (clone #B460D15) indicated that AsCyp51H1 was within 100 kb of AsbAS1. AsCyp51H1 had been predicted to encode a cytochrome P450 enzyme and was known to be genetically linked to Sad1 therefore it was a candidate for Sad2. This was addressed by sequencing the AsCyp51H1 gene in the two original sad2 mutants (#791 and #1027) and in the new sad2-like mutants identified by metabolite profiling.
Genomic DNA from S75, the confirmed sad2 mutants #791, #1027 and the six candidate sad2-like mutants #283, #500, #638, #698, #1325 and #1412 were amplified by PCR with the primer pair ISU441pF01 (shown in SEQ ID NO:15) and ISU441cR01 (shown in SEQ ID NO:7).











ISU441pF01:





5′-CGTGGCTTTTTTCCATTTCTCC-3′
(SEQ ID NO:15)






The PCR products were purified using Qiagen PCR Purification Kit (Qiagen Ltd) and used for direct sequencing with primers ISU441pF01, ISU441cR03, ISU441indeR, isu441gF5, ISU441gF4, ISU441gF1, ISU441gF2, and ISU441cR01 (shown in SEQ ID NOs:15, 16, 17, 18, 12, 8, 11, and 7, respectively).









ISU441cR03:




5′-GAGATCAATTCCTGTCACCACC-3′
(SEQ ID NO:16)


 


ISU441indeR:


5′-GCACACTAACATTTTCTATATCGTTTC A-3′
(SEQ ID NO:17)


 


ISU441gF5:


5′-TACTATGTGAATATAAGTAATGTT-3′
(SEQ ID NO:18)






Sequencing was carried out using the ABI PRISM® Big-DyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Point mutations were found in all the sad2 and sad2-like mutants. In the original sad2 mutants #791 and #1027 and in five of the six sad2-like mutants these mutations were found to be in the coding region of the AsCyp51H1 gene and are predicted to cause amino acid substitutions as follows. In mutant #1412 nucleotide 338 was thymine instead of cytosine resulting in amino acid 113 being changed from threonine to isoleucine. In mutant #1027 nucleotide 371 was thymine instead of cytosine to resulting in amino acid 124 being changed from alanine to valine. In mutant #698 nucleotide 1670 was adenine instead of guanine resulting in amino acid 233 being changed from alanine to threonine. In mutant #1325 nucleotide 1866 was thymine instead of cytosine resulting in amino acid 298 being changed from serine to phenylalanine. In mutant #638 nucleotide 1922 was adenine instead of guanine resulting in amino acid 317 being changed from glutamic acid to lysine. In mutant #283 nucleotide 2277 was adenine instead of guanine resulting in amino acid 435 being changed from glycine to aspartic acid. In mutant #791 nucleotide 2360 was thymine instead of cytosine resulting in amino acid 463 being changed from proline to serine. In mutant #500 the mutation was at the exon-intron boundary having adenine at nucleotide 475 instead of guanine resulting in a longer exon.
One would expect that mutations causing amino acid substitutions would not effect transcription, but mutations that disrupt splicing might result in an unstable message. Northern blot analysis of transcripts from the sad2 mutants was consistent with this. Mutant #500 lacks AsCyp51H1 transcript while the other mutants still possess transcripts corresponding to AsCyp51H1.
In summary, multiple independent alleles of the sad2 mutant were isolated. All accumulate β-amyrin and either lack or produce reduced levels of avenacins. Each mutant has a copy of the AsCyp51H1 gene containing a molecular lesion that would be expected to encode a non-functional enzyme or an unstable transcript. Taken together these data indicate that Sad2 is synonymous with AsCyp51H1, which encodes an enzyme catalyzing a step subsequent to that carried out by β-amyrin synthase in the biosynthetic pathway for avenacins.
EXAMPLE 4
Cloning of AsCyp51H2
Other P450s that may be involved in the modification of β-amyrin may be found by sequencing DNA that hybridizes with probes prepared from AsCyp51H1. For this purpose a BAC clone that showed a positive reaction when hybridizing with AsCyp51H1 cDNA as a probe was sequenced and analyzed as follows.
Shotgun sequencing analysis of a BAC clone (clone# B286H18) which showed a positive reaction when using AsCyp51H1 cDNA as probe revealed some fragments with sequence similarity to AsCyp51H1 (74% sequence identity at the nucleic acid level). Comparison of the genomic AsCyp51H1 sequence with the newly obtained BAC sequences enabled the identification of a putative homologous gene. This putative homologous gene contains a 3-kb promoter region, three exons, and two introns, was designated AsCyp51H2, and its nucleotide sequence is shown in SEQ ID NO:19.
The tissue distribution of AsCyp51H2 was analyzed by PCR amplification of total RNA isolated from the root tips, shoots, old leaves, and flowers of S75. RT-PCR amplification using primer pair ASCYPA2F01 and ASCYPA2R02 (shown in SEQ ID NOs:20 and 21, respectively) revealed that AsCyp51H2 only expresses in oat flowers.











ASCYPA2F01





5′-CAGTTAGCGTCATGTTGTTCTC-3′
(SEQ ID NO:20)



 



ASCYPA2R02



5′GAACACGCTAAAGGCTTGCAT-3′
(SEQ ID NO:21)






The cDNA fragment containing the coding sequence for AsCyp51H2 was obtained by PCR amplification of total RNA with primer pair ASCYPA2F03 and ASCYPA2R04 (shown in SEQ ID NOs:22 and 23, respectively).











ASCYPA2F03





5′-GCTTCCCTGAGAACTACACCATGG-3′
(SEQ ID NO:22)



 



ASCYPA2R04



5′-ATCAACCACACCTTCTTCCTCC-3′
(SEQ ID NO:23)






The amplified PCR fragment was cloned into pCR®4-TOPO (Invitrogen Ltd). Plasmids from 7 positive clones were sequenced with M13 forward and reverse primers (Qiagen Ltd), and primer ASCYPA2F05 (shown in SEQ ID NO:24), respectively.











ASCYPA2F05





5′-AGCATACCCGCTTCATCGTTG-3′
(SEQ ID NO:24)






Sequencing was carried out using the ABI PRISM® Big-DyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). A mutation-free clone was identified and designated pCR®4-TOPO:AsCypA2. The nucleotide sequence of the cDNA insert in this clone is shown in SEQ ID NO:25. The deduced amino acid sequence of nucleotides 18 through 1487 of SEQ ID NO:25 are shown in SEQ ID NO:26. Nucleotides 1488-1490 represent a stop codon.
EXAMPLE 5
Recombinant DNA Constructs to Express AsCyp51H1 in Other Species
Following are examples of recombinant DNA constructs that can be used to express AsCyp51H1 in monocot or dicot species, either alone or in combination with another gene from the same biosynthetic pathway, using corn and soybean as examples. Constitutive promoters are used, and a person skilled in the art will appreciate that, depending on the target pathogen or other considerations, targeted promoters such as those of the examples described earlier in this text may be equally or even more efficacious or preferable due to special end uses of the plant material. Depending on the species and the enzymatic activities present in that species, other genes from the biosynthetic pathways might be included to increase expression levels.
In the examples below the following abbreviations for nucleic acid fragments comprising the different components are used:
“RB” and “LB” correspond to the right and left borders of the T-DNA.
“CAMV35S ENH” is the enhancer region of the cauliflower mosaic virus 35S promoter, which increases the level of expression of promoters to which it is attached (Benfey P. N., et al., 1990, EMBO J. 9:1685-1696).
“UBI PRO” is the promoter of the maize ubiquitin gene, as described in (Christensen et al., 1992, Plant Mol. Biol. 18:675-689).
“UBI 5′UTR” is the 5′ leader region of the same maize ubiquitin gene.
“UBI INTRON1” is the intron of the same ubiquitin gene. Inclusion of this intron has been shown to increase expression levels.
“ATTR1” is a recombination site as described in the GatewayTM cloning system manual (Invitrogen, Carlsbad, Calif., USA).
“CCDB” is a bacterial negative selectable marker described in the GatewayTM cloning system manual.
“ATTR2” is a recombination site as described in the GatewayTM cloning system manual.
“PINII” is the transcription termination gene from the potato protease inhibitor II gene.
“CAMV35SPRO” is the promoter of the cauliflower mosaic virus 35S gene, a constitutive promoter commonly used in plants (Odell J. T. et al., 1985, Nature 313:810-812).
“ADH1 INTRON1” is the intron of the maize ADH1 gene. Inclusion of this intron has been shown to increase expression levels (Luehrsen K. R. and Walbot V., 1991, Mol. Gen. Genet. 225:81-93).
“BAR” is an herbicide resistance gene commonly used as a selectable marker in corn transformation.
“SCP1” is a synthetic constitutive promoter for use in plants and is described in U.S. Pat. 6,072,050.
“OMEGA 5′ UTR” is the 5′ leader region of a tobacco mosaic virus gene, whose use has been shown to enhance translation levels (Gallie et al., 1989, in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256).
“BAS” is the coding sequence for the β-amyrin synthase gene (Haralampidis K. et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98:13431-13436).
“SPC1” is a coding sequence for a polypeptide that provides resistance to the antibiotic spectinomycin, allowing bacterial selection Svab, Z. and Maliga, P., 1991, Mol. Gen. Genet. 228:316-319.
“ColE1 ORI” is a DNA origin of replication functional in E. coli. 
Constructs for the Expression of Saponin Biosynthetic Genes in Maize
Fragments containing the open reading frames of AsCyp51H1 and BAS are obtained respectively from clones described in earlier examples and as described in (Haralampidis K. et al., 2001, Proc. Natl. Acad. Sci. U.S.A. 98:13431-13436). In each case PCR amplification is carried out with primers that result in the open reading frames being flanked by restriction sites allowing their cloning into the BamHI and EcoRV restriction sites of modified Gateway Entry Vectors (Invitrogen, Carlsbad, Calif., USA). BamHI and EcoRV are added to AsCyp51H1, while BamHI and HpaI are added to BAS (EcoRV and HpaI both leave blunt ends). Other restriction sites could be used so long as they do not cut the genes internally. After ligation, the two resulting “entry vectors” consist of ATTL1-AsCyp51H1-ATTL2 and ATTL3-BAS-ATTL4, and both contain kanamycin resistance for bacterial selection. The sequences of the resulting polynucleotides are shown in SEQ ID NOs:27 and 28. ATTL1, 2, 3, and 4 are recombination sites provided in the Invitrogen Gateway cloning system (Carlsbad, Calif., USA).
Maize Recombinant DNA Construct 1: E35S-UBI-AsCYP51H1-PINII
This construct can be used to express the AsCYP51H1 gene alone in corn. The AsCYP51H1 entry vector is used in a Gateway LR reaction with a Gateway modified Agrobacterium transformation vector backbone modified from pSB1 (Komari, T. et al., 1996, Plant J. 10:165-174) by the addition of the following components at the cos site: RB-CAMV35S ENH-UBI PRO-UBI 5′UTR-UBI INTRON1-ATTR1-CCDB-ATTR2-PINII+CAMV35S ENH-CAMV35S PRO-ADH1 INTRON1-BAR-PINII−LB-SPC-ColE1 ORI. In this Gateway reaction, ATTL1 and ATTL2 recombine with ATTR1 and ATTR2, thereby transferring the AsCYP51H1 gene into the destination vector, replacing CCDB, which is toxic to E. coli, and allowing screening for successful clones as described in the Gateway manual (Invitrogen, Carlsbad, Calif., USA). This resulting construct contains a T-DNA which will be transferred into the plant genome and contains RB-CAMV35S ENH-UBI PRO-UBI 5′UTR-UBI INTRON1-ATTB1-AsCYP51H1-ATTB2-PINII+CAMV35S ENH-CAMV35S PRO-ADH1 INTRON1-BAR-PINII−LB. The nucleotide sequence of the region between the RB and LB is shown in SEQ ID NO:29 and the reminder of the vector is described in Kormai, T. et al., op cit., with the exception of the SPC and ColE1 components. This construct is electroporated into LBA4404 Agrobacterium tumefaciens cells and used in transformation experiments such as those described in Example 6 below.
Maize Recombinant DNA Construct 2: E35S-UBI-AsCYP51H1-PINII+UBI-BAS−PINII
This construct allows the simultaneous expression of the AsCYP51H1 and BAS. The AsCYP51H1 and BAS entry vectors are used together in a Gateway LR reaction with a Gateway modified Agrobacterium transformation vector backbone modified from pSB1 (Komari, T. et al., 1996, Plant J. 10:165-174) by the addition of the following components at the cos site: RB-CAMV35S ENH-UBI PRO-UBI 5′UTR-UBI INTRON1-ATTR1-CCDB-ATTR2-PINII+CAMV35S ENH-UBI PRO-UBI 5′UTR-UBI INTRON1-ATTR3-CCDB-ATTR4-PINII+CAMV35S ENH-CAMV35S PRO-ADH1 INTRON1-BAR-PINII−LB-SPC-ColE1 ORI. In this Gateway reaction, ATTL1 and ATTL2 recombine with ATTR1 and ATTR2, while ATTL3 and ATTL4 recombine with ATTR3 and ATTR4. As a result both, AsCYP51H1 and BAS, are transferred in to replace the CCDB genes, which allows screening for successful recombination as noted earlier. The final construct, thus, contains as T-DNA which will be transferred into the plant genome RB-CAMV35S ENH-UBI PRO-UBI 5′UTR-UBI INTRON1-ATTB1-AsCYP51H1-ATTB2-PINII+CAMV35S ENH-UBI PRO-UBI 5′UTR-UBI INTRON1-ATTB3-BAS-ATTB4-PINII+CAMV35S ENH-CAMV35S PRO-ADH1 INTRON1-BAR-PINII−LB. The nucleotide sequence of the fragment corresponding to the region between RB and LB is shown in SEQ ID NO:30 and the reminder of the vector is described in Kormai, T. et al., op cit., with the exception of the SPC and ColE1 components. These constructs may be electroporated into LBA4404 Agrobacterium tumefaciens cells and used in transformation experiments such as those described in Example 6 below.
Constructs for the Expression of Saponin Biosynthetic Genes in Soybean
To prepare the two recombinant DNA constructs described below for the expression of saponin biosynthetic genes in soybean the following steps are done first. The AsCyp51H1 and BAS open reading frames are obtained by PCR amplification as described above for the maize constructs, except that in assembling constructs for expression in soybean, different restriction endonuclease sites are built into the PCR primers such that NcoI and BamHI will flank the AsCyp51H1 open reading frame coding sequence while XbaI and XmaI will flank that of BAS.
Soybean Recombinant DNA Construct 1: SCP1-O′-AsCyp51H1-PINII
This construct can be used to express the AsCYP51H1 gene alone in dicots. After ligating a polynucleotide comprising the open reading frame of AsCyp51H1 into a vector containing SCP1-O′-NcoI-BamHI-PINII, the plasmid is linearized for bombardment by cutting with the restriction enzymes NruI and Eco47III and extracting the desired band of DNA from a gel. This process also removes the nucleotides encoding ampicillin resistance used for bacterial selection. This insert contains SCP1 PRO-OMEGA 5′UTR-AsCyp51H1-PINII and its nucleotide sequence is shown in SEQ ID NO:31. This fragment is used for soybean transformation as described in Example 7 below.
Soybean Recombinant DNA Construct 2:
SCP1-O′-AsCyp51H1-PINII+SUP-O′-BAS-PINII
This construct allows simultaneous expression of AsCYP51H1 and BAS in dicots. A polynucleotide comprising the open reading frame of BAS will be ligated into a vector containing SUP PRO-OMEGA 5′UTR, the XbaI and XmaI restriction sites, and PINII. This resulting cassette will then be ligated into the Soybean Recombinant DNA Fragment 1 plasmid constructed above, using the restriction enzymes BlpI and Eco47III. Prior to its use in bombardment of cells this final plasmid will be linearized by digestion with the flanking restriction enzyme NruI and the desired DNA band will be isolated after separation by gel electrophoresis. This process also removes the polynucleotide encoding ampicillin resistance which is used for bacterial selection. The isolated insert contains SCP1 PRO-OMEGA 5′UTR-AsCyp51H1-PINII+SUP PRO-OMEGA 5′UTR-BAS-PINII. The nucleotide sequence of this insert is shown in SEQ ID NO:32. This fragment may be used for soybean transformation as described in Example 7 below.
EXAMPLE 6
Agrobacterium-mediated Transformation of Maize and Regeneration of Transgenic Plants
The recombinant DNA constructs prepared in Example 5 above may be used to prepare transgenic maize plants as follows.
Maize may be transformed with any of the polynucleotide constructs described in Example 5 using the method of Zhao(U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the polynucleotide construct to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). The immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period an optional “resting” step is performed. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). The immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). The callus is then regenerated into plants (step 5: the regeneration step), and calli grown on selective medium are cultured on solid medium to regenerate the plants.
EXAMPLE 7
Transformation Of Somatic Soybean Embryo Cultures and Regeneration Of Soybean Plants
Transformation of soybean with the polynucleotide constructs of Example 5 may be accomplished using the following soybean transformation procedures.
The following stock solutions and media are used for transformation and regeneration of soybean plants:
Stock Solutions (per Liter)
100× Sulfate Stock: 37.0 g MgSO4.7H2O, 1.69 g MnSO4.H2O, 0.86 g ZnSO4.7H2O, 0.0025 g CuSO4.5H2O.
100× Halides Stock: 30.0 g CaCl2.2H2O, 0.083 g Kl, 0.0025 g CoCl2.6H2O,
100× P, B, Mo Stock: 18.5 g KH2PO4, 0.62 g H3BO3, 0.025 g Na2MoO4.2H2O
100× Fe EDTA Stock: 3.724 g Na2EDTA, 2.784 g FeSO4.7H2O.
2,4-D Stock: 10 mg/mL.
1000× Vitamin B5 Stock: 10.0 g myo-inositol, 0.10 g nicotinic acid, 0.10 g pyridoxine HCl, 1 g thiamine.
Media (per Liter)
SB196: 1 ml B5 vitamin stock, 1 mL 2,4-D stock, 10 ml of each of the remaining above stock solutions, 0.463 g (NH4)2 SO4, 2.83 g KNO3, 1 g asparagine, 10 g sucrose, pH 5.7.
SB103: 1 package Murashige & Skoog salts mixture, 1 ml B5 vitamin stock, 750 mg MgCl2 hexahydrate, 60 g maltose, 2 g gelrite, pH 5.7.
SB166: SB103 supplemented with 5 g per liter activated charcoal.
SB71-4: Gamborg's B5 salts (Gibco-BRL catalog No. 21153-028), 1 ml B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH 5.7.
Soybean embryogenic suspension cultures are maintained in 35 ml liquid medium (SB196) on a rotary shaker (150 rpm) at 28° C. with fluorescent lights providing a 16 hour day/8 hour night cycle. Cultures are subcultured every 2 weeks by inoculating approximately 35 mg of tissue into 35 ml of fresh liquid media.
Soybean embryogenic suspension cultures are transformed by the method of particle gun bombardment (see Klein et al.,1987, Nature 327:70-73) using a DuPont Biolistic PDS1000/He instrument.
In particle gun bombardment procedures it is possible to use either purified entire plasmid DNA or DNA constructs containing only the recombinant DNA expression cassette(s) of interest. For every eight bombardment transformations, 30 μl of suspension is prepared containing 1 to 90 picograms (pg) of DNA construct per base pair of DNA fragment. The recombinant DNA plasmid or construct used to express the antifungal gene is on a separate recombinant DNA plasmid or construct from the selectable marker gene. All recombinant DNA plasmids or constructs are co-precipitated onto gold particles as follows. The DNAs in suspension are added to 50 μl of a 20 to 60 mg/ml 0.6 μm gold particle suspension and then combined with 50 μl 2.5 M CaCl2 and 20 μl 0.1 M spermidine. The mixture is pulse vortexed 5 times, centrifuged in a microfuge for 10 seconds, and the supernatant removed. The DNA-coated particles are then washed once with 150 μl of 100% ethanol, pulse vortexed, centrifuged in a microfuge again, and resuspended in 85 μl of anhydrous ethanol. Five μl of the DNA-coated gold particles are then loaded on each macrocarrier disk.
Approximately 150 to 250 mg of two-week-old soybean embryogenic suspension culture is placed in an empty 60 mm×15 mm petri plate and the residual liquid is removed from the tissue using a pipette. The tissue is placed about 3.5 inches away from the retaining screen and each plate of tissue is bombarded once. Membrane rupture pressure is set at 650 psi and the chamber is evacuated to −28 inches of Hg. Eighteen plates are bombarded, and, following bombardment, the tissue from each plate is divided between two flasks, placed back into liquid media, and cultured as described above.
Seven days after bombardment, the liquid medium is exchanged with fresh SB196 medium supplemented with 50 mg/ml hygromycin or 100 ng/ml chlorsulfuron, depending on the selectable marker gene used in transformation. The selective medium is refreshed weekly or biweekly. Seven weeks post-bombardment, green, transformed tissue is observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally-propagated, transformed embryogenic suspension cultures. Thus, each new line is treated as independent transformation event. These suspensions can then be maintained as suspensions of embryos clustered in an immature developmental stage through subculture or can be regenerated into whole plants by maturation and germination of individual somatic embryos.
Transformed embryogenic clusters are removed from liquid culture and placed on solid agar medium (SB166) containing no hormones or antibiotics for one week. Embryos are cultured at 26° C. with mixed fluorescent and incandescent lights on a 16-hour day 8-hour night schedule. After one week, the cultures are then transferred to SB103 medium and maintained in the same growth conditions for 3 additional weeks. Prior to transfer from liquid culture to solid medium, tissue from selected lines is assayed by PCR or Southern analysis for the presence of the antifungal gene.
Somatic embryos become suitable for germination after 4 weeks and are then removed from the maturation medium and dried in empty petri dishes for 1 to 5 days. The dried embryos are then planted in SB71-4 medium where they are allowed to germinate under the same light and germination conditions described above. Germinated embryos are transferred to sterile soil and grown to maturity.
Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
The disclosure of each reference set forth above is incorporated herein by reference in its entirety.

## Claims

1. An isolated polynucleotide comprising:
a) a nucleotide sequence encoding a Cyp51H enzyme having an amino acid sequence that is at least 95% identical, based on the Clustal V method of alignment with pairwise alignment default parameters of KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5, when compared to SEQ ID NO:14; or
b) a nucleotide sequence comprising the full complement of (a).

2. The polynucleotide of claim 1, wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:14.

3. The polynucleotide of claim 1 wherein the nucleotide sequence comprises one of SEQ ID NO:5 or SEQ ID NO:13.

4. A vector comprising the polynucleotide of claim 1.

5. A recombinant DNA construct comprising the isolated polynucleotide of claim 1 operably linked to at least one regulatory sequence.

6. A method for transforming a cell, comprising transforming a cell with the recombinant DNA construct of claim 5.

7. A transformed cell comprising the recombinant DNA construct of claim 5.

8. A method for producing a transgenic plant comprising transforming a plant cell with the recombinant DNA construct of claim 5 and regenerating a transgenic plant from the transformed plant cell.

9. A transgenic plant comprising the recombinant DNA construct of claim 5.

10. A seed comprising the recombinant DNA construct of claim 5.

11. An isolated host cell comprising the recombinant DNA construct of claim 5.

12. The host cell of claim 10 wherein said host cell is selected from a yeast cell, a bacterial cell, and a plant cell.

13. A transgenic plant comprising a polynucleotide of claim 1 operably linked to a heterologous promoter, said plant having an altered level of a triterpene.

14. The plant of claim 13 wherein said triterpene is a saponin derived from β-amyrin and said level is increased.

15. The plant of claim 13 wherein said triterpene is a saponin derived from β-amyrin and said level is decreased.

16. The plant of claim 13 wherein said plant is selected from the group consisting of a monocot and a dicot.

17. The plant of claim 16 wherein said monocot is selected from the group consisting of wheat, oat, rice, and corn.

18. The plant of claim 16 wherein said dicot is soybean.