Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

• This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding plant defensins.
• Defensins are small, basic, cysteine-rich proteins that have broad antimicrobial activity through the formation of multimeric pores in outer or inner biological membranes that lead to membrane disruption and depolarization. They have a wide phylogenetic distribution, having been found in insects, mammals, and plants.
• Defensins have also been shown to accumulate systemically upon challenge by fungal pathogens (Manners, J. M. et al. (1998) Plant Mol Biol. 35:1071-1080; Terras, F. R. et al. (1998) Planta 206:111-124; Terras, F. R. et al. (1995) Plant Cell 7:573-588). Furthermore, transgenic tobacco that expressed constitutively a radish defensin was found to have improved resistance to infection by a fungal pathogen (Terras, F. R et al, (1995) Plant Cell 7:573-588).
• the present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 49 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:6; (b) a second nucleotide sequence encoding a polypeptide of at least 49 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2; (c) a third nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:26 and 28; (d) a fourth nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least
• the isolated polynucleotide of the claimed invention comprises a first nucleotide sequence which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21. 23, 25, and 27 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.
• this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and the complement of such nucleotide sequences.
• this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
• the present invention concerns a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention.
• the host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell.
• the present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
• the invention also relates to a process for producing a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting a compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
• the invention concerns a defensin polypeptide selected from the group consisting of: (a) a polypeptide of at least 49 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:6; (b) a polypeptide of at least 49 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2; (c) a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:26 and 28; (d) a polypeptide of at least 50 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:8; and (e) a polypeptide of at least 50 amino acids having at least 95% identity based on the Clustal method of alignment
• the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a plant defensin polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or a chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the chimeric gene into a host cell; (c) measuring the level of the plant defensin polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the plant defensin polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the plant defensin polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
• the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a plant defensin polypeptide, preferably a plant defensin polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs.l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer.
• the amplified nucleic acid fragment preferably will encode a substantial portion of a plant defensin amino acid sequence.
• this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a plant defensin polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
• this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide or a polypeptide of the present invention.
• this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or a construct of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the plant defensin polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
• Figure 1 depicts the amino acid sequence alignment between the defensin encoded by the nucleotide sequences derived from Dimorphotheca sinuata clone dms2c.pk001.d3 (SEQ ID NO:4), Picramnia pentandra clone pps.pkOOl l.a9 (SEQ ID NO:8), Parthenium argentatum Grey clones epblc.pk002.h2 (SEQ ID NO:12), epblc.pk001.hl5 (SEQ ID NO: 14), epblc.pk003.pl 4 (SEQ ID NO: 16), epblc.pk004.p22 (SEQ ID NO: 18), epblc.pk
• Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing.
• Table 1 also identifies the cDNA clones as individual ESTs ("EST"), the sequences of the entire cDNA inserts comprising the indicated cDNA clones ("FIS"), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding at a minimum the mature protein derived from an EST, FIS, a contig, or an FIS and PCR (“CGS”).
• Nucleotide SEQ ID NOs: l, 5, 9, and 25 correspond to nucleotide SEQ ID NOs: l, 5, 3, and 7, respectively, presented in U.S. Provisional Application No. 60/133039. filed May 7, 1999.
• Amino acid SEQ ID NOs:2, 6, 10, and 26 correspond to amino acid SEQ ID NOs:2, 6, 4, and 8, respectively, presented in U.S. Provisional Application No. 60/133,039, filed May 7, 1999.
• the 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. TABLE 1 Plant Defensins
• Plant Defensin Plant Defensin (Nicotiana tdrlc pk002 g7(EST) CGS 25 26 benthamiana)
• Plant Defensin Plant Defensin (Nicotiana tdrlc pk002 g7(FIS) CGS 27 28 benthamiana)
• the Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for ammo acids as defined m conformity with the IUPAC-IUBMB standards described m Nucleic Acids Res 13 3021-3030 (1985) and in the Biochemical J 219 (No 2) 345-373 (1984) 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.
• polynucleotide polynucleotide sequence
• nucleic acid sequence nucleic acid sequence
• nucleic acid fragment'Visolated 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-natural 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 at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides derived from SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27, or the complement of such sequences.
• isolated polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as and not limited to other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
• nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.
• substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence.
• substantially similar also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co- suppression technology.
• 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-a-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.
• substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell.
• a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell.
• the level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.
• antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using nucleic acid fragments that do not share 100%) sequence identity with the gene to be suppressed.
• alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide are well known in the art.
• a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
• a codon encoding another less hydrophobic residue such as glycine
• a more hydrophobic residue such as valine, leucine, or isoleucine.
• changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product.
• Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide.
• an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9. 11, 13, 15, 17, 19, 21, 23. 25, and 27 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a plant defensin polypeptide in a host cell.
• a method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell may comprise the steps of: constructing an isolated polynucleotide of the present invention or a chimeric gene of the present invention; introducing the isolated polynucleotide or the chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.
• substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.
• One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min.
• a more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60°C.
• Another preferred set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65°C.
• 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 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 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.
• nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities but typically encode a polypeptide having at least 49 or 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS.
• a "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises.
• Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer- based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).
• a sequence often or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
• gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
• oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
• a "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
• the instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
• Codon degeneracy refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting 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 sequences set forth herein.
• 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 nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment 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 a 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.
• nucleic acid fragments can be tailored for optimal gene expression based on optimization of the 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 preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
• “Native gene” refers to a gene as found in nature with its own regulatory sequences.
• Chimeric gene refers any gene that is not a native gene, comprising 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 nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA.
• 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.
• 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 may be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments.
• 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 which cause a nucleic acid fragment 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 (1989) Biochemistry of Plants 75:1 -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.
• Translation leader sequence refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence.
• the translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence.
• 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).
• "3' non-coding sequences” refers to nucleotide sequences located downstream of a coding sequence and includes 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 :611 -680.
• RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.
• the primary transcript 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 polypeptides by the cell.
• cDNA refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I.
• Sense RNA refers to an RNA transcript that includes the mRNA and 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. Patent No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, 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.
• operably linked refers to the association of two or more nucleic acid fragments so that the function of one is affected by the other.
• 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). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
• expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment 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. "Underexpression” refers to the production of a gene product in transgenic organisms at levels below that of 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. Patent No. 5,231,020, inco ⁇ orated herein by reference).
• a “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.
• 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.
• Null mutant refers to a host cell which either lacks the expression of a certain polypeptide or expresses a polypeptide which is inactive or does not have any detectable expected enzymatic function.
• “Mature protein” refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
• "Precursor protein” refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
• chloroplast transit peptide is an amino acid sequence which 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.
• a “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels ( ⁇ 99 ⁇ ) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53).
• a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
• any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 700:1627-1632).
• 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.
• isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
• a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypepti de-encoding sequence in a given host cell.
• 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.
• a promoter regulatory region e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally- regulated, or cell- or tissue-specific expression
• PCR or “polymerase chain reaction” is a technique used for the amplification of specific DNA segments (U.S. Patent Nos. 4,683,195 and 4,800,159).
• the present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 49 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:6; (b) a second nucleotide sequence encoding a polypeptide of at least 49 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2; (c) a third nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:26 and 28; (d) a fourth nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 85% identity based on the C
• the first nucleotide sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28.
• Nucleic acid fragments encoding at least a substantial portion of several plant defensins have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art.
• 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.
• 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).
• nucleic acid amplification technologies e.g., polymerase chain reaction, ligase chain reaction.
• genes encoding other plant defensin, either as cDNAs or genomic DNAs could be isolated directly by using all or a substantial 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 (Maniatis). Moreover, the entire sequence(s) can be used directly to synthesize
• DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems.
• 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.
• 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.
• 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.
• a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.
• the present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a defensin polypeptide, preferably a substantial portion of a plant defensin polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:l.
• nucleic acid fragment preferably a cDNA inserted in a cloning vector
• amplifying a nucleic acid fragment preferably a cDNA inserted in a cloning vector
• the amplified nucleic acid fragment preferably will encode a portion of a plant defensin polypeptide.
• this invention concerns viruses and host cells comprising either the chimeric genes of the invention as described herein or an isolated polynucleotide of the invention as described herein.
• Examples of host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants.
• the nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed plant defensin are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of disease (e.g., fungal) resistance and stress tolerance in those cells.
• Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development.
• the chimeric gene 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 chimeric gene may also comprise one or more introns in order to facilitate gene expression.
• Plasmid vectors comprising the instant isolated polynucleotide (or chimeric gene) may be constructed.
• the choice of plasmid vector is dependent upon the method that will be used to transform host plants. 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. ( ⁇ 9 5) EMBOJ. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 275:78-86), and thus that multiple events must 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.
• the chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:241-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Planl Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys.700:1627-1632) with or without removing targeting sequences that are already present.
• appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:241-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Planl Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys.700:1627-1632) with or without
• a chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences.
• 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.
• Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Patent Nos. 5,190,931, 5,107,065 and 5,283,323).
• An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective.
• tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.
• the present invention concerns a defensin polypeptide selected from the group consisting of: (a) a polypeptide of at least 49 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:6; (b) a polypeptide of at least 49 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:2; (c) a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:26 and 28; (d) a polypeptide of at least 50 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptid
• the instant polypeptides may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the proteins by methods well known to those skilled in the art.
• the antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts.
• Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded plant defensin.
• An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 6).
• All or a substantial portion of the polynucleotides of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes.
• the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers.
• RFLP restriction fragment length polymorphism
• Southern blots Mantonis
• the resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 7:174-181) in order to construct a genetic map.
• nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DN As of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
• nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7: 149-154).
• FISH direct fluorescence in situ hybridization
• nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 77:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 7-5:325-332), allele- specific ligation (Landegren et al. (1988) Science 247:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 75:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet.
• Loss of function mutant phenotypes may be identified for the instant cD ⁇ A clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl Acad. Sci USA 56:9402-9406; Koes et al. (1995) Proc. Nai Acad. Sci USA 2:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways.
• short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on D ⁇ As prepared from a population of plants in which Mutator transposons or some other mutation-causing D ⁇ A element has been introduced (see Bensen. supra).
• the amplification of a specific D ⁇ A fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptide.
• the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor.
• an arbitrary genomic site primer such as that for a restriction enzyme site-anchored synthetic adaptor.
• composition of cDNA Libraries Isolation and Sequencing of cDNA Clones cDNA libraries representing mRNAs from various Dimorphotheca sinuata, Picramnia pentandra, Parthenium argentatum Grey, and Nicotiana benthamiana tissues were prepared. The characteristics of the libraries are described below.
• cDNA libraries may be prepared by any one of many methods available.
• the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA).
• the Uni-ZAPTM XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript.
• the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products).
• plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or
• ESTs see Adams et al., (1991) Science 252:1651-1656).
• the resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
• SEQ ID NO: 1 the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:2.
• the sequence of a portion of the cDNA insert from clone epblc.pk002.h2 is shown in SEQ ID NO:9; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO: 10.
• the sequence of a portion of the cDNA insert from clone pps.pkOOl 1.a9 is shown in SEQ ID NO: 5; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:6.
• the sequence of a portion of the cDNA insert from clone tdrlc.pk002.g7 is shown in SEQ ID NO:25; the deduced amino acid sequence of this cDNA is shown in SEQ ID NO:26.
• BLAST scores and probabilities indicate that the instant nucleic acid fragments encode portions of plant defensin.
• the BLASTX search using the EST sequences from clones listed in Table 4 revealed similarity of the polypeptides encoded by the cDNAs to a defensin from Dahlia merckii (NCBI GenBank Identifier (GI) No. 2147320; WO 99/02038-Al; Osborn et al.
• Figure 1 presents an alignment of the amino acid sequences set forth in SEQ ID NOs:4, 8, 12, 14, 16, 18, 20, 22, 24, and 28 and the Dahlia merckii sequence (NCBI GenBank Identifier (GI) No. 2147320; SEQ ID NO:29 ).
• the data in Table 5 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:4, 8, 12, 14, 16, 18, 20, 22, 24, and 28 and the Dahlia merckii sequence (NCBI GenBank Identifier (GI) No. 2147320; SEQ ID NO:29).
• a chimeric gene comprising a cDNA encoding the instant polypeptide in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed.
• the cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be inco ⁇ orated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below.
• Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Boulevard., Manassas, VA 20110-2209), and bears accession number ATCC 97366.
• the DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment of the maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega).
• Vector and insert DNA can be ligated at 15°C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL 1 -Blue (Epicurian Coli XL-1 BlueTM; Stratagene).
• Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (SequenaseTM DNA Sequencing Kit; U.S. Biochemical).
• the resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptide, and the 10 kD zein 3' region.
• the chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132.
• the embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long.
• the embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668).
• the embryos are kept in the dark at 27°C.
• Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos.
• the embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
• the plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker.
• This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT).
• PAT phosphinothricin acetyl transferase
• the enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin.
• the pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al.
• the particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells.
• gold particles (1 ⁇ m in diameter) are coated with DNA using the following technique.
• Ten ⁇ g of plasmid DNAs are added to 50 ⁇ L of a suspension of gold particles (60 mg per mL).
• Calcium chloride 50 ⁇ L of a 2.5 M solution
• spermidine free base (20 ⁇ L of a 1.0 M solution) are added to the particles.
• the suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 ⁇ m) and the supernatant removed. The particles are resuspended in 200 ⁇ L of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 ⁇ L of ethanol. An aliquot (5 ⁇ L) of the DNA-coated gold particles can be placed in the center of a KaptonTM flying disc (Bio-Rad Labs).
• the particles are then accelerated into the corn tissue with a BiolisticTM PDS-1000/He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
• BiolisticTM PDS-1000/He Bio-Rad Instruments, Hercules CA
• the embryogenic tissue is placed on filter paper over agarose- solidified N6 medium.
• the tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter.
• the petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen.
• the air in the chamber is then evacuated to a vacuum of 28 inches of Hg.
• the macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
• tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate- supplemented medium. These calli may continue to grow when sub-cultured on the selective medium. Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 5:833-839).
• a seed-specific construct composed of the promoter and transcription terminator from the gene encoding the ⁇ subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris can be used for expression of the instant polypeptides in transformed soybean.
• the phaseolin construct includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire construct is flanked by Hind III sites.
• the cDNA fragment of this gene may be generated by polymerase chain reaction
• Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides.
• somatic embryos cotyledons. 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26°C on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.
• Soybean embryogenic suspension cultures can be maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26°C with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
• Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Patent No. 4.945,050).
• a DuPont BiolisticTM PDS1000/HE instrument helium retrofit
• a selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 373:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.
• the seed construct comprising the phaseolin 5' region, the fragment encoding the instant polypeptide and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
• Approximately 300-400 mg of a two- week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette.
• approximately 5-10 plates of tissue are normally bombarded.
• Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury.
• the tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
• Five to seven days post bombardment the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment the liquid media may be exchanged with fresh media containing 50 mg/mL hygromycin.
• This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be 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. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos. EXAMPLE 6
• the cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430.
• This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 5(5:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system.
• Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a.
• Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTGTM low melting agarose gel (FMC). Buffer and agarose contain 10 ⁇ g/mL ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELaseTM (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 ⁇ L of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA).
• the fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above.
• the vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above.
• the prepared vector pBT430 and fragment can then be ligated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL).
• Transformants can be selected on agar plates containing LB media and 100 ⁇ g/mL ampicillin. Transformants containing the gene encoding the instant polypeptide are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.
• a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximatel ⁇ 1, IPTG (isopropylthio- ⁇ -galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°.
• Cells are then harvested by centrifugation and re-suspended in 50 ⁇ L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride.
• a small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator.
• the mixture is centrifuged and the protein concentration of the supernatant determined.
• One ⁇ g of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.
• polypeptides described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 6, or expression in eukaryotic cell culture, inplanta, and using viral expression systems in suitably infected organisms or cell lines.
• the instant polypeptides may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags.
• Common fusion protein partners include glutathione S-transferase ("GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide
• the fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme.
• proteases include thrombin, enterokinase and factor Xa.
• any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.
• Purification of the instant polypeptides may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor.
• the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme.
• the instant polypeptides may be expressed as a fusion protein coupled to the C-terminus of thioredoxin.
• a (His)g peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification.
• Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B.
• a thioredoxin fusion protein may be eluted using dithiothreitol: however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include ⁇ -mercaptoethanol or other reduced thiol.
• the eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired.
• Proteolytic cleavage of the thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBondTM affinity resin or other resin.
• Crude, partially purified or purified enzyme may be utilized in assays to verify over- or underexpression of functional plant defensins in transgenic plants and transformed bacterial cells. Assays may be conducted under well known experimental conditions which permit optimal enzymatic activity. For example, assays for plant defensin are presented by Thevissen, K. et al. (1996) J. Biol Chem. 277(25 :15018-15025.

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