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
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
• • 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
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)

Definitions

• the present invention relates to nucleic acid sequences encoding cytochrome P450 enzymes (hereinafter referred to as P450 and P450 enzymes) in Nicotiana plants and methods for using those nucleic acid sequences to alter plant phenotypes.
• P450 and P450 enzymes cytochrome P450 enzymes
• Cytochrome P450s catalyze enzymatic reactions for a diverse range of chemically dissimilar substrates that include the oxidative, peroxidative and reductive metabolism of endogenous and xenobiotic substrates.
• P450s participate in biochemical pathways that include the synthesis of plant products such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates
• Cytochrome P450s also known as P450 he e- thiolate proteins, usually act as terminal oxidases in multi- component electron transfer chains, called P450- containing monooxygenase systems. Specific reactions catalyzed include demethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S-, and 0- dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-oxide groups.
• Nicotiana plant P450 enzymes have been implicated in effecting a variety of plant metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosinolates and a host of other chemical entities.
• plant metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosinolates and a host of other chemical entities.
• P450 enzymes can impact the composition of plant metabolites in plants. For example, it has been long desired to improve the flavor and aroma of certain plants by altering its profile of selected fatty acids through breeding; however very little is known about mechanisms involved in controlling the levels of these leaf constituents.
• the down regulation of P450 enzymes associated with the modification of fatty acids may facilitate accumulation of desired fatty acids that provide more preferred leaf phenotypic qualities.
• P450 enzymes The function of P450 enzymes and their broadening roles in plant constituents is still being discovered. For instance, a special class of P450 enzymes was found to catalyze the breakdown of fatty acid into volatile C ⁇ - and C9-aldehydes and -alcohols that are major contributors of "fresh green" odor of fruits and vegetables.
• the level of other novel targeted P450s may be altered to enhance the qualities of leaf constituents by modifying lipid composition and related break down metabolites in Nicotiana leaf. Several of these constituents in leaf are affected by senescence that stimulates the maturation of leaf quality properties. Still other reports have shown that P450s enzymes are play a functional role in altering fatty acids that are involved in plant-pathogen interactions and disease resistance.
• Nornicotine is a minor alkaloid found in Nicotiana tabaccum. It is has been postulated that it is produced by the P450 mediated demethylation of nicotine followed by acylation and nitrosation at the N position thereby producing a series of N- acylnonicotines and N-nitrosonornicotines .
• N-demethylation, catalyzed by a putative P450 demethylase, is thought to be a primary source of nornicotine biosyntheses in Nicotiana . While the enzyme is believed to be microsomal, thus far a nicotine demethylase enzyme has not been successfully purified, nor have the genes involved been isolated.
• the activity of P450 enzymes is genetically controlled and also strongly influenced by 5 environment factors.
• the demethylation of nicotine in Nicotiana is thought to increase substantially when the plants reach a mature stage.
• the demethylase gene contains a transposable element that can inhibit translation of RNA when present.
• the present invention is directed to plant P450 enzymes.
• the present invention is further directed to plant P450 enzymes from Nicotiana .
• the present invention is also directed to P450 enzymes in plants whose expression is induced by ethylene and/or plant senescence.
• the present invention is yet further directed to nucleic acid sequences in plants having enzymatic activities, for example, oxygenase, demethylase and the like, or other and the use of those sequences to reduce or silence the expression of these enzymes.
• the invention also relates to P450 enzymes found in plants containing higher nornicotine levels than plants exhibiting lower nornicotine levels.
• the invention is directed to nucleic acid sequences as set forth in SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13,
• those fragments containing greater than 75% identity in nucleic acid sequence were placed into groups dependent upon their identity in a region corresponding to the first nucleic acid following the cytochrome P450 motif GXRXCX(G/A) to the stop codon.
• the representative nucleic acid groups and respective species are shown in Table I.
• the invention is directed to amino acid sequences as set forth in SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14,
• those fragments containing greater than 71% identity in amino acid sequence were placed into groups dependent upon their identity to each other in a region corresponding to the first amino acid following the cytochrome P450 motif GXRXCX(G/A) to the stop codon.
• the representative amino acid groups and respective species are shown in Table II.
• a fifth aspect of the invention is the use of nucleic acids sequences as set forth in SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 , 45, 47 , 49, 51 , 53 , 55, 57 , 59 , 61 , 63, 65, 67 , 69, 71 , 73 , 75, 77 , 79, 81, 83, 85, 87 , 89, 91, 93, 95, 97 , 99, 101, 103, 105, 107, 109, 111 , 113, 115, 117 , 119, 121, 123, 125, 127 , 129, 131 , 133, 135, 137 , 139, 141, 143, 145 and 147 .
• the reduction or elimination of P450 enzymes in Nicotiana plants may be accomplished transiently using RNA viral systems. Resulting transformed or infected plants are assessed for phenotypic changes including, but not limited to, analysis of endogenous P450 RNA transcripts, P450 expressed peptides, and concentrations of plant metabolites using techniques commonly available to one having ordinary skill in the art.
• the present invention is also directed to generation of trangenic Nicotiana lines that have altered P450 enzyme activity levels.
• these transgenic lines include nucleic acid sequences that are effective for reducing or silencing the expression of certain enzyme thus resulting in phenotypic effects within Nicotiana.
• nucleic acid sequences include SEQ. ID. Nos.
• plant cultivars including nucleic acids of the present invention in a down regulation capacity will have altered metabolite profiles relative to control plants.
• the present invention is directed to the screening of plants, more preferably Nicotiana, that contain genes that have substantial nucleic acid identity to the taught nucleic acid sequence.
• the use of the invention would be advantageous to identify and select plants that contain a nucleic acid sequence with exact or substantial identity where such plants are part of a breeding program for traditional or transgenic varieties, a mutagenesis program, or naturally occurring diverse plant populations.
• the screening of plants for substantial nucleic acid identity may be accomplished by evaluating plant nucleic acid materials using a nucleic acid probe in conjunction with nucleic acid detection protocols including, but not limited to, nucleic acid hybridization and PCR analysis.
• the nucleic acid probe may consist of the taught nucleic acid sequence or fragment thereof corresponding to SEQ ID 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 and 147.
• the present invention is directed to the identification of plant genes, more preferably Nicotiana, that share substantial amino acid identity corresponding to the taught nucleic acid sequence.
• the identification of plant genes including both cDNA and genomic clans of those cDNAs and genomic clones, preferably from Nicotiana may be accomplished by screening plant cDNA libraries using a nucleic acid probe in conjunction with nucleic acid detection protocols including, but not limited to, nucleic acid hybridization and PCR analysis.
• the nucleic acid probe may be comprised of nucleic acid sequence or fragment thereof corresponding to SEQ ID 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107 , 109, 111, 113, 115, 117 , 119, 121, 123, 125, 127 , 129, 131 , 133, 135, 137, 139, 141, 143, 145 and 147
• cDNA expression libraries that express peptides may be screened using antibodies directed to part or all of the taught amino acid sequence.
• amino acid sequences include SEQ ID 2, 4, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146 and 148.
• Figure 1 shows nucleic acid SEQ. ID. No . : 1 and amino acid SEQ. ID. No. :2.
• Figure 2 shows nucleic acid SEQ. ID. No.: 3 and amino acid SEQ. ID. No.:4.
• Figure 3 shows nucleic acid SEQ. ID. No.: 5 and amino acid SEQ. ID. No.: 6.
• Figure 4 shows nucleic acid SEQ. ID. No.: 7 and amino acid SEQ. ID. No.:8.
• Figure 5 shows nucleic acid SEQ. ID. No.: 9 and amino acid SEQ. ID. No. :10.
• Figure 6 shows nucleic acid SEQ. ID. No.:ll and amino acid SEQ. ID. No. :12.
• Figure 7 shows nucleic acid SEQ. ID. No.:13 and amino acid SEQ. ID. No.:14.
• Figure 8 shows nucleic acid SEQ. ID. No.: 15 and amino acid SEQ. ID. No. :16.
• Figure 9 shows nucleic acid SEQ. ID. No.:17 and amino acid ⁇ EQ. ID. No. :18.
• Figure 10 shows nucleic acid SEQ. ID. No.: 19 and amino acid SEQ. ID. No. :20.
• Figure 11 shows nucleic acid SEQ. ID. No.: 21 and amino acid SEQ. ID. No. :22.
• Figure 12 shows nucleic acid SEQ. ID. No.: 23 and amino acid SEQ. ID. No.:24.
• Figure 13 shows nucleic acid SEQ. ID. No.: 25 and amino acid SEQ. ID. No.:26.
• Figure 14 shows nucleic acid SEQ. ID. No.:27 and amino acid SEQ. ID. No. :28.
• Figure 15 shows nucleic acid SEQ. ID. No.: 29 and amino acid SEQ. ID. No. :30.
• Figure 16 shows nucleic acid SEQ. ID. No.: 31 and amino acid SEQ. ID. No. :32.
• Figure 17 shows nucleic acid SEQ. ID. No.: 33 and amino acid SEQ. ID. No.:34.
• Figure 18 shows nucleic acid SEQ. ID. No.:35 and amino acid SEQ. ID. No. : 36.
• Figure 19 shows nucleic acid SEQ. ID. No.: 37 and amino acid SEQ. ID. No. :38.
• Figure 20 shows nucleic acid SEQ. ID. No.: 39 and amino acid SEQ. ID. No.: 40.
• Figure 21 shows nucleic acid SEQ. ID. No.: 41 and amino acid SEQ. ID. No. :42.
• Figure 22 shows nucleic acid SEQ.* ID. No.: 43 and amino acid SEQ. ID. No. :44.
• Figure 23 shows nucleic acid SEQ. ID. No.: 45 and amino acid SEQ. ID. No.: 46.
• Figure 24 shows nucleic acid SEQ. ID. No.:47 and amino acid SEQ. ID. No. :48.
• Figure 25 shows nucleic acid SEQ. ID. No.: 49 and amino acid SEQ. ID. No.: 50.
• Figure 26 shows nucleic acid SEQ. ID. No.: 51 and amino acid SEQ. ID. No. : 52.
• Figure 27 shows nucleic acid SEQ. ID. No.: 53 and amino acid SEQ. ID. No. :54.
• Figure 28 shows nucleic acid SEQ. ID. No.: 55 and amino acid SEQ. ID. No.:56.
• Figure 29 shows nucleic acid SEQ. ID. No.: 57 and amino acid SEQ. ID. No. :58.
• Figure 30 shows nucleic acid SEQ. ID. No.:59 and amino acid SEQ. ID. No. : 60.
• Figure 31 shows nucleic acid SEQ. ID. No.: 61 and amino acid SEQ. ID. No. : 62.
• Figure 32 shows nucleic acid SEQ. ID. No.: 63 and amino acid SEQ. ID. No. :64.
• Figure 33 shows nucleic acid SEQ. ID. No.: 65 and amino acid SEQ. ID. No. :66.
• Figure 34 shows nucleic acid SEQ. ID. No.: 67 and amino acid SEQ. ID. No.: 68.
• Figure 35 shows nucleic acid SEQ. ID. No.:69 and amino acid SEQ. ID. No. :70.
• Figure 36 shows nucleic acid SEQ. ID. No.: 71 and amino acid SEQ. ID. No. :72.
• Figure 37 shows nucleic acid SEQ. ID. No.: 73 and amino acid SEQ. ID. No.: 74.
• Figure 38 shows nucleic acid SEQ. ID. No.: 75 and amino acid SEQ. ID. No. :76.
• Figure 39 shows nucleic acid SEQ. ID. No.: 77 and amino acid SEQ. ID. No. :78.
• Figure 40 shows nucleic acid SEQ. ID. No.: 79 and amino acid SEQ. ID. No. :80.
• Figure 41 shows nucleic acid SEQ. ID. No.: 81 and amino acid SEQ. ID. No. :82.
• Figure 42 shows nucleic acid SEQ. ID. No.: 83 and amino acid SEQ. ID. No.: 84.
• Figure 43 shows nucleic acid SEQ. ID. No.: 85 and amino acid SEQ. ID. No. :86.
• Figure 44 shows nucleic acid SEQ. ID. No.: 87 and amino acid SEQ. ID. No. :88.
• Figure 45 shows nucleic acid SEQ. ID. No.: 89 and amino acid SEQ. ID. No. :90.
• Figure 46 shows nucleic acid SEQ. ID. No.: 91 and amino acid SEQ. ID. No. : 92.
• Figure 47 shows nucleic acid SEQ. ID. No.: 93 and amino acid SEQ. ID. No.: 94.
• Figure 48 shows nucleic acid SEQ. ID. No.: 95 and amino acid SEQ. ID. No. :96.
• Figure 49 shows nucleic acid SEQ. ID. No.: 97 and amino acid SEQ. ID. No.: 98.
• Figure 50 shows nucleic acid SEQ. ID. No.: 99 and amino acid SEQ. ID. No. :100.
• Figure 51 shows nucleic acid SEQ. ID. No.:101 and amino acid SEQ. ID. No. :102.
• Figure 52 shows nucleic acid SEQ. ID. No.: 103 and amino acid SEQ. ID. No. :104.
• Figure 53 shows nucleic acid SEQ. ID. No.:105 and amino acid SEQ. ID. No. :106.
• Figure 54 shows nucleic acid SEQ. ID. No.:107 and amino acid SEQ. ID. No.: 108.
• Figure 55 shows nucleic acid SEQ. ID. No.:109 and amino acid SEQ. ID. No. :110.
• Figure 56 shows nucleic acid SEQ. ID. No.: Ill and amino acid SEQ. ID. No.: 112.
• Figure 57 shows nucleic acid SEQ. ID. No.: 113 and amino acid SEQ. ID. No. :114.
• Figure 58 shows nucleic acid SEQ. ID. No.: 115 and amino acid SEQ. ID. No. :116.
• Figure 59 shows nucleic acid SEQ. ID. No.: 117 and amino acid SEQ. ID. No.: 118.
• Figure 60 shows nucleic acid SEQ. ID. No.: 119 and amino acid SEQ. ID. No. :120.
• Figure 61 shows nucleic acid SEQ. ID. No.: 121 and amino acid SEQ. ID. No. :122.
• Figure 62 shows nucleic acid SEQ. ID. No.: 123 and amino acid SEQ. ID. No.: 124.
• Figure 63 shows nucleic acid SEQ. ID. No.: 125 and amino acid SEQ. ID. No.:126.
• Figure 64 shows nucleic acid SEQ. ID. No.:127 and amino acid SEQ. ID. No.: 128.
• Figure 65 shows nucleic acid SEQ. ID. No.: 129 and amino acid SEQ. ID. No. :130.
• Figure 66 shows nucleic acid SEQ. ID. No.: 131 and amino acid SEQ. ID. No.: 132.
• Figure 67 shows nucleic acid SEQ. ID. No.:133 and amino acid SEQ. ID. No. :134.
• Figure 68 shows nucleic acid SEQ. ID. No.:135 and amino acid SEQ. ID. No. :136.
• Figure 69 shows nucleic acid SEQ. ID. No.: 137 and amino acid SEQ. ID. No. :138.
• Figure 70 shows nucleic acid SEQ. ID. No.:139 and amino acid SEQ. ID. No. :140.
• Figure 71 shows nucleic acid SEQ. ID. No.:141 and amino acid SEQ. ID. No.:142.
• Figure 72 shows nucleic acid SEQ. ID. No.:143 and amino acid SEQ. ID. No.: 144.
• Figure 73 shows nucleic acid SEQ. ID. No.:145 and amino acid SEQ. ID. No.: 146.
• Figure 74 shows nucleic acid SEQ. ID. No.:147 and amino acid SEQ. ID. No.:148.
• Figure 75 shows a procedure used for cloning of cytochrome P450 cDNA fragments by PCR. SEQ. ID. Nos. 149-156 are shown.
• Figure 76 illustrates amino acid identity of group members .
• Enzymatic activity is meant to include demethylation, hydroxylation, epoxidation, N-oxidation, sulfooxidation, N-, S- , and 0- dealkylations, desulfation, deamination, and reduction of azo, nitro, and N-oxide groups.
• nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, or sense or anti-sense, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
• nucleic acid sequence includes the complementary sequence thereof.
• operable combination refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence.
• Recombinant when used with reference to a cell indicates that the cell replicates a heterologous nucleic acid, expresses said nucleic acid or expresses a peptide, heterologous peptide, or protein encoded by a heterologous nucleic acid.
• Recombinant cells can express genes or gene fragments in either the sense or antisense form that are not found within the native (non- recombinant) form, of the cell.
• Recombinant cells can also express genes that are found in the native form of the cell, but wherein the genes are modified and re- introduced into the cell by artificial means.
• a “structural gene” is that portion of a gene comprising a DNA segment encoding a protein, polypeptide or a portion thereof, and excluding the 5' sequence which drives the initiation of transcription.
• the structural gene may alternatively encode a nontranslatable product.
• the structural gene may be one which is normally found in the cell or one which is not normally found in the cell or cellular location wherein it is introduced, in which case it is termed a "heterologous gene".
• a heterologous gene may be derived in whole or in part from any source known to the art, including a bacterial genome or episome, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA.
• a structural gene may contain one or more modifications that could effect biological activity or its characteristics, the biological activity or the chemical structure of the expression product, the rate of expression or the manner of expression control. Such modifications include, but are not limited to, mutations, insertions, deletions and substitutions of one or more nucleotides.
• the structural gene may constitute an uninterrupted coding sequence or it may include one or more introns, bounded by the appropriate splice junctions.
• the structural gene may be translatable or non-translatable, including in an anti-sense orientation.
• the structural gene may be a composite of segments derived from a plurality of sources and from a plurality of gene sequences (naturally occurring or synthetic, where synthetic refers to DNA that is chemically synthesized) .
• “Derived from” is used to mean taken, obtained, received, traced, replicated or descended from a source (chemical and/or biological) .
• a derivative may be produced by chemical or biological manipulation (including, but not limited to, substitution, addition, insertion, deletion, extraction, isolation, mutation and replication) of the original source.
• “Chemically synthesized”, as related to a sequence of DNA, means that portions of the component nucleotides were assembled in vitro.
• Manual chemical synthesis of DNA may be accomplished using well established procedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983), Weissman (ed.), Praeger Publishers, New York, Chapter 1) ; automated chemical synthesis can be performed using one of a number of commercially available machines.
• Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.
• NCBI National Center for Biological Information
• substantially amino acid identity or “substantial amino acid sequence identity” as applied to amino acid sequences and as used herein denote a characteristic of a polypeptide, wherein the peptide comprises a sequence that has at least 70 percent sequence identity, preferably 80 percent amino acid sequence identity, more preferably 90 percent amino acid sequence identity, and most preferably at least 99 to 100 percent sequence identity as compared to a reference group over region corresponding to the first amino acid following the cytochrome P450 motif GXRXCX(G/A) to the stop codon of the translated peptide.
• nucleic acid identity or “substantial nucleic acid sequence identity” as applied to nucleic acid sequences and as used herein denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 75 percent sequence identity, preferably 81 percent amino acid sequence identity, more preferably at least 91 to 99 percent sequence identity, and most preferably at least 99 to 100 percent sequence identity as compared to a reference group over region corresponding to the first nucleic acid following the cytochrome P450 motif GXRXCX(G/A) to the stop codon of the translated peptide.
• stringent conditions are sequence-dependent and will be different in different circumstances.
• stringent conditions are selected to be about 5°C to about 20°C, usually about 10°C to about 15°C, lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
• Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a matched probe.
• stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60°C.
• stringent conditions will include an initial wash in 6xSSC at 42 °C followed by one or more additional washes in 0.2xSSC at a temperature of at least about 55°C, typically about 60°C and often about 65°C.
• Nucleotide sequences are also substantially identical for purposes of this invention when the polypeptides and/or proteins which they encode are substantially identical. Thus, where one nucleic acid sequence encodes essentially the same polypeptide as a second nucleic acid sequence, the two nucleic acid sequences are substantially identical, even if they would not hybridize under stringent conditions due to degeneracy permitted by the genetic code (see, Darnell et al. (1990) Molecular Cell Biology, Second Edition Scientific American Books W. H. Freeman and Company New York for an explanation of codon degeneracy and the genetic code) .
• Protein purity or homogeneity can be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon staining. For certain purposes high resolution may be needed and HPLC or a similar means for purification may be utilized.
• vector is used in reference to nucleic acid molecules that transfer DNA segment (s) into a cell.
• a vector may act to replicate DNA and may reproduce independently in a host cell.
• vector is sometimes used interchangeably with “vector.”
• expression vector refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
• Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
• Eucaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
• a nucleic acid may be inserted into plant cells, for example, by any technique such as in vivo inoculation or by any of the known in vitro tissue culture techniques to produce transformed plant cells that can be regenerated into complete plants.
• the insertion into plant cells may be by in vitro inoculation by pathogenic or non-pathogenic A. tu efaciens .
• Other such tissue culture techniques may also be employed.
• Plant tissue includes differentiated and undifferentiated tissues of plants, including, but not limited to, roots, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells in culture, such as single cells, protoplasts, embryos and callus tissue.
• the plant tissue may be in planta or in organ, tissue or cell culture.
• Plant cell as used herein includes plant cells in planta and plant cells and protoplasts in culture.
• cDNA or “complementary DNA” generally refers to ⁇ a single stranded DNA molecule with a nucleotide sequence that is complementary to an RNA molecule. cDNA is formed by the action of the enzyme reverse transcriptase on an RNA template.
• RNA was extracted from Nicotiana tissue of converter and non-converter Nicotiana lines. The extracted RNA was then used to create cDNA. Nucleic acid sequences of the present invention were then generated using two strategies.
• the poly A enriched RNA was extracted from plant tissue and cDNA was made by reverse transcription PCR.
• the single strand cDNA was then used to create P450 specific PCR populations using degenerate primers plus a oligo d(T) reverse primer.
• the primer design was based on the highly conserved motifs of P450. Examples of specific degenerate primers are set forth in Figure 1. Sequence fragments from plasmids containing appropriate size inserts were further analyzed. • These size inserts typically ranged from about 300 to about 800 nucleotides depending on which primers were used.
• a cDNA library was initially constructed.
• the cDNA in the plasmids was used to create p450 specific PCR populations using degenerate primers plus T7 primer on plasmid as reverse primer.
• sequence fragments from plasmids containing appropriate size inserts were further analyzed.
• Nicotiana plant lines known to produce high levels of nornicotine (converter) and plant lines having undetectable levels of nornicotine may be used as starting materials.
• Leaves can then be removed from plants and treated with ethylene to activate P450 enzymatic activities defined herein.
• Total RNA is extracted using techniques known in the art.
• cDNA fragments can then be generated using PCR (RT-PCR) with the oligo d(T) primer as described in Figure 1.
• the cDNA library can then be constructed more fully described in examples herein.
• the conserved region of P450 type enzymes can be used as a template for degenerate primers ( Figure 75) .
• degenerate primers P450 specific bands can be amplified by PCR. Bands indicative for P450 like enzymes can be identified by DNA sequencing.
• PCR fragments can be characterized using BLAST search, alignment or other tools to identify appropriate candidates.
• Sequence information from identified fragments can be used to develop PCR primers. These primers are used to conduct quantitative RT-PCR from the RNA' s of converter and non- converter ethylene treated plant tissue. Only appropriate sized DNA bands (300-800 bp) from converter lines or bands with higher density denoting higher expression in converter lines were used for further characterization. Large scale Southern reverse analysis were conducted to examine the differential expression for all clones obtained. In this aspect of the invention, these large scale reverse Southern assays can be conducted using labeled total cDNA' s from different tissues as a probe to hybridize with cloned DNA fragments in order to screen all cloned inserts.
• Nonradioactive Northern blotting assay was also used to characterize clones P450 fragments.
• Nucleic acid sequences identified as described above can be examined by using virus induced gene silencing technology (VIGS, Baulcombe, Current Opinions in Plant Biology, 1999, 2:109-113) .
• RNAi interfering RNA technology
• Plants may be transformed using Agrobacterium technology, see US Patent 5,177,010 to University of Toledo, 5,104,310 to Texas A&M, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, US Patents 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500 all to MaxPlanck, European Patent Applications 604662 and 627752 to Japan Nicotiana, European Patent Applications 0267159, and 0292435 and US Patent 5,231,019 all to Ciba Geigy, US Patents 5,463,174 and 4,762,785 both to Calgene, and US Patents 5,004,863 and 5,159,135 both to Agracetus.
• Agrobacterium technology see US Patent 5,177,010 to University of Toledo, 5,104,310 to Texas A&M
• transformation technology includes whiskers technology, see U.S. Patents 5,302,523 and 5,464,765 both to Zeneca. Electroporation technology has also been used to transform plants, see WO 87/06614 to Boyce Thompson Institute, 5,472,869 and 5,384,253 both to Dekalb, WO9209696 and W09321335 both to PGS . All of these transformation patents and publications are incorporated by reference.
• tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue type I and II, hypocotyl, meristem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques within the skill of an artisan.
• Foreign genetic material introduced into a plant may include a selectable marker.
• selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which code for resistance or tolerance to glyphosate; hygromycin; methotrexate; p osphinothricin (bar) ; imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorosulfuron; bromoxynil, dalapon and the like.
• reporter gene In addition to a selectable marker, it may be desirous to use a reporter gene. In some instances a reporter gene may be used without a selectable marker. Reporter genes are genes which are typically not present or expressed in the recipient organism or tissue. The reporter gene typically encodes for a protein which provide for some phenotypic change or enzymatic property. Examples of such genes are provided in K. Weising et al. Ann. Rev. Genetics, 22, 421 (1988), which is incorporated herein by reference. Preferred reporter genes include without limitation glucuronidase (GUS) gene and GFP genes.
• GUS glucuronidase
• the expression of the structural gene may be assayed by any means known to the art, and expression may be measured as mRNA transcribed, protein synthesized, or the amount of gene silencing that occurs (see U.S. Patent No. 5,583,021 which is hereby incorporated by reference) .
• Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration into whole plants (EP Appln No. 88810309.0). Procedures for transferring the introduced expression complex to commercially useful cultivars are known to those skilled in the art.
• plant tissues and whole plants can be regenerated therefrom using methods and techniques well-known in the art.
• the regenerated plants are then reproduced by conventional means and the introduced genes can be transferred to other strains and cultivars by conventional plant breeding techniques .
• Plants were seeded in pots and grown in a greenhouse for 4 weeks.
• the 4 week old seedlings were transplanted into individual pots and grown in the greenhouse for 2 months.
• the plants were watered 2 times a day with water containing 150ppm NPK fertilizer during growth.
• the expanded green leaves were detached from plants to do the ethylene treatment described below.
• Tobacco line 78379 which is a burley line released by the University of Kentucky was used as a source of plant material .
• One hundred plants were cultured as standard in the art of growing tobacco and transplanted and tagged with a distinctive number (1-100) . Fertilization and field management were conducted as recommended.
• Nicotiana line 4407 which is a burley line was used as a source of plant material. Uniform and representative plants (100) were selected and tagged. Of the 100 plants 97 were non- converters and three were converters. Plant number 56 had the least amount of conversion (1.2%) and plant number 58 had the highest level of conversion (96%) . Self-pollenated seeds and crossed seeds were made with these two plants.
• Plants from selfed-58 were segregating in about a 3:1 converter to non-converter ratio.
• the 58-33 and 58-25 were identified as homozygous converter and nonconverter plant lines, respectively.
• the stable conversion of 58-33 was confirmed by analysis of its progenies of next generation.
• Green leaves were detached from 2-3 month greenhouse grown plants and sprayed with 0.3% ethylene solution (Prep brand Ethephon (Rhone-Poulenc) ) . Each sprayed leaf was hung in a curing rack equipped with humidifier and covered with plastic. During the treatment, the sample leaves were periodically sprayed with the ethylene solution. Approximately 24-48 hour post ethylene treatment, leaves were collected for RNA extraction. Another sub-sample was taken for metabolic consituents analysis to determine the concentration of leaf metabolites and more specific constituents of interest such as a variety of alkaloids.
• alkaloids analysis could be performed as follows. Samples (0.1 g) were shaken at 150 rpm with 0.5 ml 2N NaOH, and a 5 ml extraction solution which contained quinoline as an internal standard and methyl t-butyl ether. Samples were analyzed on a HP 6890 GC equipped with a FID detector. A temperature of 250°C was used for the detector and injector. An HP column (30m-0.32nm-l *m) consisting of fused silica crosslinked with 5% phenol and 95% methyl silicon was used at a temperature gradient of 110-185 °C at 10°C per minute. The column was operated at a flow rate at 100°C at 1.7cm 3 min -1 with a split ratio of 40:1 with a 2-1 injection volume using helium as the carrier gas .
• RNA extractions Middle leaves from 2 month old greenhouse grown plants were treated with ethylene as described. The 0 and 24-48 hours samples were used for RNA extraction. In some cases, leaf samples under the senescence process were taken from the plants 10 days post flower-head removal. These samples were also used for extraction. Total RNA was isolated using Rneasy Plant Mini Kit (Qiagen, Inc., Valencia, California) following manuf cturer's protocol.
• the tissue sample was grinded under liquid nitrogen to a fine powder using a DEPC treated mortar and pestle. Approximately 100 mg of ground tissue was transferred to a sterile 1.5 ml eppendorf tube. This sample tube was placed in liquid nitrogen until all samples were collected. Then, 450 ⁇ l of Buffer RLT as provided in the kit (with the addition of ⁇ - Mercaptoethanol) was added to each individual tube. The sample was vortexed vigorously and incubated at 56° C for 3 minutes. The lysate was then, applied to the QIAshredder spin column sitting in a 2-ml collection tube, and centrifuged for 2 minutes at maximum speed. The flow through was collected and 0.5 volume of ethanol was added to the cleared lysate.
• the sample is mixed well and transferred to an Rneasy mini spin column sitting in a 2 ml collection tube.
• the sample was centrifuged for 1 minute at 10, OOOrpm.
• 700 ⁇ l of buffer RW1 was pipeted onto the Rneasy column and centrifuged for 1 minute at 10, OOOrpm.
• Buffer RPE was pipetted onto the Rneasy column in a new collection tube and centrifuged for 1 minute at 10,000 rpm. Buffer RPE was again, added to the Rneasy spin column and centrifuged for 2 minutes at maximum speed to dry the membrane.
• the memebrane was placed in a separate collection tube and centrifuged for an additional 1 minute at maximum speed.
• the contents were mixed thoroughly by pipetting and incubated for 3 minutes at 70°C on a heating block.
• the sample was then, placed at room temperature for approximately 20 minutes.
• the oligotex:mRNA complex was pelleted by centrifugation for 2 minutes at maximum speed. All but 50 ⁇ l of the supernatant was removed from the icrocentrifuge tube.
• the Sample was treated further by OBB buffer.
• the oligotex:mRNA pellet was resuspended in 400 ⁇ l of Buffer OW2 by vortexing. This mix was transferred onto a small spin column placed in a new tube and centrifuged for 1 minute at maximum speed. The spin column was transferred to a new tube and an additional 400 ⁇ l of Buffer OW2 was added to the column.
• the tube was then centrifuged for 1 minute at maximum speed.
• the spin column was transferred to a final 1.5ml microcentrifuge tube.
• the sample was eluted with 60 ul of hot (70 C) Buffer OEB.
• Poly A product was analyzed by denatured formaldehyde gels and spectrophotometric analysis.
• First strand cDNA was produced using Superscript reverse transcriptase following manufacturer' s protocol (Invitrogen, Carlsbad, California) .
• the poly A enriched RNA/oligo dT primer mix consisted of less than 5 ⁇ g of total RNA, 1 ⁇ l of lOmM dNTP mix, 1 ⁇ l of Oligo d(T) 12-18 (0.5 ⁇ g/ ⁇ l), and up to 10 ⁇ l of DEPC-treated water. Each sample was incubated at 65° C for 5 minutes, then placed on ice for at least 1 minute.
• a reaction mixture was prepared by adding each of the following components in order: 2 ⁇ l 10X RT buffer, 4 ⁇ l of 25 mM MgC12, 2 ⁇ l of 0.1 M DTT, and ' 1 ⁇ l of RNase OUT Recombinant RNase Inhibitor. An addition of 9 ⁇ l of reaction mixture was pipetted to each RNA/primer mixture and gently mixed. It was incubated at 42° C for 2 minutes and 1 ⁇ l of Super Script II RT was added to each tube. The tube was incubated for 50 minutes at 42° C. The reaction was terminated at 70° C for 15 minutes and chilled on ice.
• the sample was collected by centrifugation and 1 ⁇ l of RNase H was added to each tube and incubated for 20 minutes at 37° C.
• the second PCR was carried out with 200 pmoles of forward primer (degenerate primers as in Figure 75, SEQ. ID Nos. 149-156) and 100 pmoles reverse primer (mix of 18nt oligo d(T) followed by 1 random base) .
• Reaction conditions were 94 °C for 2 minutes and then performed 40 cycles of PCR at 94°C for 1 minute, 45° to 60°C for 2 minutes, 72°C for 3 minutes with a 72 °C extension for an extra 10 min.
• PCR fragments from Example 3 were ligated into a pGEM-T Easy Vector (Promega, Madison, Wisconsin) following manufacturer's instructions.
• the ligated product was transformed into JM109 competent cells and plated on LB media plates for blue/white selection. Colonies were selected and grown in a 96 well plate with 1.2 ml of LB media overnight at 37 °C. Frozen stock was generated for all selected colonies.
• Plasmid DNA from plates were purified using Beckman's Biomeck 2000 miniprep robotics with Wizard SV Miniprep kit (Promega) . Plasmid DNA was eluted with lOO ⁇ lwater and stored in a 96 well plate.
• Plasmids were digested by EcoRI and were analyzed using 1% agarose gel to confirm the DNA quantity and size of inserts.
• the plasmids containing a 400-600 bp insert were sequenced using an CEQ 2000 sequencer (Beckman, Fullerton, California) .
• the sequences were aligned with GenBank database by BLAST search.
• the P-450 related fragments were identified and further analyzed.
• a cDNA library was constructed by preparing total RNA from ethylene treated leaves as follows. First, total RNA was extracted from ethylene treated leaves of tobacco line 58-33 using a modified acid phenol and chloroform extraction protocol. Protocol was modified to use one gram of tissue that was ground and subsequently vortexed in 5 ml of extraction buffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; lOmM EDTA; 0.5% SDS) to which 5 ml phenol (pH5.5) and 5 ml chloroform was added. The extracted sample was centrifuged and the supernatant was saved. This extraction step was repeated 2-3 more times until the supernatant appeared clear.
• extraction buffer 100 mM Tris-HCl, pH 8.5; 200 mM NaCl; lOmM EDTA; 0.5% SDS
• RNA containing gels were transferred overnight using 20X SSC as a transfer buffer.
• poly A+ RNA was used as template to produce a cDNA library employing cDNA synthesis kit, ZAP-cDNA synthesis kit, and ZAP-cDNA Gigapack III gold cloning kit (Stratagene, La Jolla, California) .
• the method involved following the manufacture's protocol as specified.
• a quality background test of the library was completed by complementation assays using IPTG and'-X-gal, where recombinant plaques was expressed at more than 100-fold above the background reaction.
• a more quantitative analysis of the library by random PCR showed that average size of insert cDNA was approximately 1.2 kb.
• the method used a two-step PCR method as followed.
• reverse primers were designed based on the preliminary sequence information obtained from P450 fragments.
• the designed reverse primers and T3 (forward) primers were used amplify corresponding genes from the cDNA library.
• PCR reactions were subjected to agarose electrophoresis and the corresponding bands of high molecular weight were excised, purified, cloned and sequenced.
• new primers designed from 5'UTR or the start coding region of P450 as the forward primers together with the reverse primers were used in the subsequent PCR to obtain full-length P450 clones.
• the P450 fragments were generated by PCR amplification from the constructed cDNA library as described in example 3 with the exception of the reverse primer.
• the T7 primer located on the plasmid downstream of cDNA inserts was used as a reverse primer. PCR fragments were isolated, cloned and sequenced as described in Example 4.
• EXAMPLE 6 CHARACTERIZATION OF CLONED FRAGMENTS - REVERSE SOUTHERN BLOTTING ANALYSIS
• Nonradioactive large scale reverse southern blotting assay was performed on all P450 clones identified in above examples to detect the differential expression. It was observed that the level of expression among different P450 clusters was very different. Further real time detection was conducted on those with high expression.
• Nonradioactive southern blotting procedures were conducted as follows .
• Probe was produced by biotin-tail labeling a single strand cDNA derived from poly A enriched RNA generated in above step. This labeled single strand cDNA was generated by RT-PCR of the converter and nonconverter total RNA (Invitrogen) as described in example 3 with the exception of using biotinalyted oligo dT as a primer (Promega) ; These were used as a probe to hybridize with cloned DNA.
• Plasmid DNA was digested with restriction enzyme EcoRI and run on agarose gels. Gels were simultaneously dried and transferred to two nylon membranes (Biodyne B) . One membrane was hybridized with converter probe and the other with nonconverter probe. Membranes were UV-crosslinked (auto crosslink setting, 254 nm, Stratagene, Stratalinker ) before hybridization. Alternatively, the inserts were PCR amplified from each plasmid using the sequences located on both arms of p-GEM plasmid, T3 and SP6, as primers. The PCR products were analyzed by running on a 96 well Ready-to-run agarose gels. The confirmed inserts were dotted on two nylon membranes. One membrane was hybridized with converter probe and the other with nonconverter probe.
• the membranes were hybridized and washed following manufacture's instruction with the modification of washing stringency (Enzo Diagnostics, Inc, Farmingdale, NY) .
• the membranes were prehybridized with hybridization buffer (2x SSC buffered formamide, containing detergent and hybridization enhancers) at 42°C for 30 min and hybridized with lO ⁇ l denatured probe overnight at 42°C.
• the membranes then were washed in IX hybridization wash buffer 1 time at room temperature for 10 min and 4 times at 68°C for 15 min. The membranes were ready for the detection.
• RT-PCR (Gibco Kit, Carlsbad, California) was performed on the total RNA' s from non-converter (58-25) and converter (58-33) lines using primers specific to the P-450 fragments. Comparative RT-PCR was conducted as follows:
• RNA from ethylene treated converter (58-33) and nonconverter (58-25) plant leaves was extracted as described in example 2.
• Poly (A) RNA from total RNA was extracted using Qiagen kit as described in example 2.
• RT-PCR was conducted using primers specific to cloned P450 following the manufactures procedure (Invitogen) .
• the poly A enriched RNA was added to the reaction mix, along with, 25 ⁇ l of 2X Reaction Mix, l ⁇ l of lO ⁇ M Sense Primer, l ⁇ l of 10 ⁇ M Anti-sense Primer, 1 ⁇ l of RT/ Platinum taq Mix, and up to 50 ⁇ l of water.
• Reaction conditions were 50°C for 20 minutes and then 94 C for 2 min, performed 40 cycles of PCR at 94°C for 30 sec, 55° to for 30 sec, 70°C for 1 minute with a 72 °C extension for an extra 10 min.
• Ten microliters of the amplified sample were analyzed by electrophoresis using a 1% agarose gel.
• EXAMPLE 7 CHARACTERI ATION OF CLONED FRAGMENTS - NORTHERN BLOT ANALYSIS
• probe preparation the random priming method was used to prepare probes from cloned p450 DNA fragments (Random Primer DNA Biotinylation Kit, KPL) .
• the following components were mixed: 0.5 ⁇ g DNA template (boiled in a water bath for 5-10 minutes and chilled on ice before use) ; IX Random Primer Solution; IX dNTP mix; 10 units of Klenow and water was added to bring the reaction to 50 ⁇ l.
• the mixture was incubated in 37 °C for 1-4 hours.
• the reaction was stopped with 2 ⁇ l of 200 mM EDTA.
• the probe was denatured by incubating at 95 °C for 5 minutes before use.
• RNA samples were prepared from ethylene treated and non-treated fresh leaves, and senescence leaves. In some cases poly A enriched RNA was used. Approximately 15 ⁇ g total RNA or 1.8 ⁇ g mRNA (Methods of RNA and mRNA extraction are described in Example 5) was brought to equal volume with DEPC H20(5-10 ⁇ l) . The same volume loading buffer (1 x MOPS; 18.5 % Formaldehyde; 50 % Formamide; 4 % Ficoll400; Bromophenolblue) and 0.5 ⁇ l EtBr (0.5 ⁇ g / ⁇ l) were added. The samples were heated at 90 °C for 5 minutes, and chilled on ice.
• RNAs were transferred to Hybond-N+ membrane (Nylon, Amersham Pharmacia Biotech) by capillary method in 10 X SSC buffer (1.5 M NaCl; 0.15 M Na-citrate ) for 24 hours.
• Membranes with RNA samples were UV-crosslinked (auto crosslink setting, 254 nm,
• the membrane was prehybridized for 1- 4 hours at 42 °C with 5-10 ml prehybridization buffer (5 x SSC; 50 % Formamide; 5 x Denhardt ' s-solution; 1 % SDS; lOO ⁇ g/ml heat-denatured sheared non- homologous DNA) .
• Old prehybridization buffer was discarded, and new prehybridization buffer and probe were added.
• the hybridization was carried out over night at 42 °C.
• the membrane was washed for 15 minutes with 2 x SSC at room temperature, followed by a wash with 2 x SSC, 0.1 % SDS at 65 °C for 2 times, and a final wash with 0.1 x SSC, or more wash with 0.1 x SDS at 65 °C (optional) .
• AP-Streptavidin and CDP-Star were used to detect the hybridization signal ( KPL' s DNA Detector Northern blotting Kit) .
• the membrane was blocked with IX Detector Block Solution for 30 minutes at room temperature. The blocking buffer was discarded and the membrane was incubated in new IX detector Block Solution with 1:10,000 AP-SA at room temperature for 1 hour. The membrane was washed in IX Phosphatase Wash Solution for 3 times, followed by a wash with IX Phosphatase Assay Buffer for two times.
• the signal was detected with CDP-Star Chemiluminescent Substrate.
• the wet membrane was exposed to X-Ray film under saranTM wrap. The results were analyzed and recorded.
• a major focus of the invention was the discovery of novel genes that may be induced as a result of ethylene treatment or play a key role in tobacco leaf quality and constituents.
• Northern blots were useful in determining which genes were induced by ethylene treatment relative to non-induced plants. Interestingly, not all fragments were affected similarly in the converter and nonconverter.
• the cytochrome P450 fragments of interest were partially sequenced to determine their structural relatedness. This information was used to subsequently isolate and sequence full length gene clones. Functional analysis utilizing down-regulation methods was performed in whole plants with the fragments genes .
• P450 fragments were sequenced in conjunction with Northern blot analysis to determine their structural relatedness.
• the approach used utilized forward primers based either of two common P450 motifs located near the carboxyl-terminus of the P450 genes.
• the forward primers corresponded to cytochrome P450 motifs FXPERF or GRRXCP(A/G) as denoted in Figure 1.
• the reverse primers used standard primers from either the plasmid, SP6 or T7 located on both arms of pGEM plasmid, or a poly A tail. The protocol used is described below.
• Spectrophotometry was used to estimate the concentration of starting double stranded DNA following the manufacturer' s protocol (Beckman Coulter) .
• the template was diluted with water to the appropriate concentration, denatured by heating at 95° C for 2 minutes, and subsequently placed on ice.
• the sequencing reaction was prepared on ice using 0.5 to lO ⁇ l of denatured DNA template, 2 ⁇ l of 1.6 pmole of the forward primer, 8 ⁇ l of DTCS Quick Start Master Mix and the total volume brought to 20 ⁇ l with water.
• the thermocycling program consisted of 30 cycles of the follow cycle: 96° C for 20 seconds, 50° C for 20 seconds, and 60° C for 4 minutes followed by holding at 4° C.
• the sequence was stopped by adding 5 ⁇ l of stop buffer (equal volume of 3M NaOAc and lOOmM EDTA and 1 ⁇ l of 20 mg/ml glycogen) .
• the sample was precipitated with 60 ⁇ l of cold 95% ethanol and centrifuged at 6000g for 6 minutes. Ethanol was discarded. The pellet was 2 washes with 200 ⁇ l of cold 70% ethanol. After the pellet was dry, 40 ⁇ l of SLS solution was added and the pellet was resuspended. A layer of mineral oil was over laid. The sample was then, placed on the CEQ 8000 Automated Sequencer for further analysis.
• nucleic acid sequence was re-sequenced in both directions using forward primers to the
• the nucleic acid sequences of cytochrome P450 fragments were compared to each other from the coding region corresponding to the first nucleic acid after the region encoding the GRRXCP (A/G) motif through to the stop codon. This region was selected as an indicator of genetic diversity among P450 proteins. A large number of genetically distinct P450 genes, in excess of 70 genes, was observed similar to that of other plant species. Upon comparison of nucleic acid sequences, it was found that the genes could be placed into distinct sequences groups based on their sequence identity. It was found that the best unique grouping of P450 members was determined to be those sequences with 75% nucleic acid identity or greater (shown in Table I) . Reducing the percentage identity resulted in significantly larger groups.
• a preferred grouping was observed for those sequences with 81% nucleic acid identity or greater, a more preferred grouping 91% nucleic acid identity or greater, and a most preferred grouping for those sequences 99% nucleic acid identity of greater. Most of the groups contained at least two members and frequently three or more members. Others were not repeatedly discovered suggesting that approach taken was able to isolated both low and high expressing mRNA in the tissue used.
• cytochrome P450 groups Based on 75% nucleic acid identity or greater, two cytochrome P450 groups were found to contain nucleic acid sequence identity to previously tobacco cytochrome genes that genetically distinct from that within the group. Group 23, showed nucleic acid identity, within the parameters used for Table I, to prior GenBank sequences of
• Gl: 1171579 had nucleic acid identity to Group 23 members ranging 96.9% to 99.5% identity to members of Group 23 while Gl: 14423327 ranged 95.4% to 96.9% identity to this group.
• the members of Group 31 had nucleic acid identity ranging from 76.7% to 97.8% identity to the GenBank reported sequence of Gl: 14423319 (AAK62342) by Ralston et al .
• D58-BG7 (SEQ ID No.il), D58-AB1 (SEQ ID No.:3); D58-BE4 (SEQ ID No. :7)
• D35-BB7 (SEQ ID No.:23); D177-BA7 (SEQ ID No.:25) D56A- AB6 (SEQ ID No.:27); D144-AE2 (SEQ ID No.:29) 6 D56-AG11 (SEQ ID No.: 31); D179-AA1 (SEQ ID No.: 33)
• D56-AC12 (SEQ ID No.:45) 11 D58-AB9 (SEQ ID No.:47); D56-AG9 (SEQ ID No. :49); D56-
• D56A-AG10 SEQ ID No.: 71
• D58-BC5 SEQ ID No.: 73
• D58-AD12 SEQ ID No.:75
• D56-AC11 SEQ ID No.:77
• D35-39 SEQ ID No.:79
• D58- BH4 SEQ ID No.: 81
• D56-AD6 SEQ ID No.: 87
• 17 D73A-AD6
• D70A-BA11 SEQ ID No.:91
• D70A-BB5 SEQ ID No.: 93
• D70A-AB8 (SEQ ID No.: 99); D70A-BH2 (SEQ ID No. : 101); D70A-AA4 (SEQ ID No.:103)
• D70A-BA1 (SEQ ID No.: 105); D70A-BA9 (SEQ ID No. : 107); D176-BG2 (SEQ ID No.:141)
• D58-AA1 SEQ ID No.: 121
• D185-BC1 SEQ ID No. : 133
• D185-BG2 SEQ ID No.: 135
• D73-AE10 SEQ ID No.:123
• EXAMPLE 9 RELATED AMINO ACID SEQUENCE IDENTITY OF ISOLATED NUCLEIC ACID FRAGMENTS
• the amino acid sequences of nucleic acid sequences obtained for cytochrome P450 fragments from Example 8 were deduced.
• the deduced region corresponded to the amino acid immediately after the GXRXCP(A/G) sequence motif to the end of the carboxyl-terminus, or stop codon.
• sequence identity of the fragments Upon comparison of sequence identity of the fragments, a unique grouping was observed for those sequences with 70% amino acid identity or greater.
• a preferred grouping was observed for those sequences with 80% amino acid identity or greater, more preferred with 90% amino acid identity or greater, and a most preferred grouping for those sequences 99% amino acid identity of greater.
• the groups and corresponding amino acid sequences of group members are shown in Figure 2. Several of the unique nucleic acid sequences were found to have complete amino acid identity to other fragments and therefore only one member with the identical amino acid was reported.
• the amino acid identity for Group 19 of Table II corresponded to three distinct groups based on their nucleic acid sequences.
• the amino acid sequences of each group member and their identity is shown in Figure. 77.
• the amino acid differences are appropriated marked.
• At least one member of each amino acid identity group was selected for gene cloning and functional studies using plants.
• group members that are differentially affected by ethylene treatment or other biological differences as assessed by Northern and Southern analysis were selected for gene cloning and functional studies.
• peptide specific antibodies will be prepared on sequence identity and differential sequence.
• D56-AH7 (SEQ ID No.: 10); D13a-5 (SEQ ID No.: 12) 4 D56-AG10 (SEQ ID No.:14); D34-62 (SEQ ID No. :18)
• D56-AA7 (SEQ ID No.: 20); D56-AE1 (SEQ ID No.: 22); 185- BD3 (SEQ ID No. :144) 6 D35-BB7 (SEQ ID No.:24); D177-BA7 (SEQ ID No.:26);
• D56A-AB6 (SEQ ID No.:28); D144-AE2 (SEQ ID No.: 30)
• D58-AB9 (SEQ ID No.: 48); D56-AG9 (SEQ ID No.: 50); D56- AG6 (SEQ ID No.:52); D35-BG11 (SEQ ID No.:54); D35-42 (SEQ ID No.: 56); D35-BA3 (SEQ ID No.: 58); D34-57 (SEQ ID No.:60); D34-52 (SEQ ID o.:62)
• D56A-AG10 (SEQ ID No.: 72); D58-BC5 (SEQ ID No.: 74); D58-AD12 (SEQ ID No.: 76)
• D70A-AB5 (SEQ ID No.:96); D70A-AB8 (SEQ ID No.:100); D70A-BH2 (SEQ ID No.:102); D70A-AA4 (SEQ ID No.:104); D70A- BAl (SEQ ID No.:106); D70A-BA9 (SEQ ID No.:108); D176-BG2
• a cDNA library was constructed by preparing total RNA from ethylene treated leaves as follows. First, total RNA was extracted from ethylene treated leaves using a' modified acid phenol and chloroform extraction protocol. Protocol was modified to use one gram of tissue that was ground and subsequently vortexed in 5 ml of extraction buffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; lOmM EDTA; 0.5% SDS) to which 5 ml phenol (pH5.5) and 5 ml chloroform was added. The extracted sample was centrifuged and the supernatant was saved. This extraction step was repeated 2-3 more times until the supernatant appeared clear. Approximately 5 ml of chloroform was added to remove trace amounts of phenol.
• extraction buffer 100 mM Tris-HCl, pH 8.5; 200 mM NaCl; lOmM EDTA; 0.5% SDS
• RNA containing gels were transferred overnight using 20X SSC as a transfer buffer.
• poly A+ RNA was used as template to produce a cDNA library employing cDNA synthesis kit, ZAP-cDNA synthesis kit, and ZAP-cDNA Gigapack III gold cloning kit (Stratagene) .
• the method involved following the manufacture's protocol as specified.
• Approximately 8 ug of poly A+ RNA was used to construct cDNA library.
• Analysis of the primary library revealed about 2.5 x 106 - lx 107 pfu.
• a quality background test of the library was completed by a- complementation using IPTG and X-gal, where recombinant plaques was expressed at more than 100-fold above the background reaction.
• a more quantitative analysis of the library by random PCR showed that average size of insert cDNA was approximately 1.2 kb.
• the method used a two-step PCR method as followed.
• reverse primers were designed based on the preliminary sequence information obtained from p450 fragments.
• the designed reverse primers and T3 (forward) primers were used amplify corresponding genes from the cDNA library.
• PCR reactions were subjected to agarose electrophoresis and the corresponding bands of high molecular weight were excised, purified, cloned and sequenced.
• Full-length p450 genes were isolated by PCR method from constructed cDNA library. Two steps of PCR were used to clone the full-length genes. In the first step PCR, unspecific reverse primer (T3) and specific forward primer (generated from the downstream sequence of P450s) were used to clone the 5 'end of the P450s from cDNA library. PCR fragments were isolated, cloned and sequenced for designing the forward primers in next step PCR. Two specific primers were used to clone the full- length p450 clones in the second step PCR. The clones were subsequently sequenced.
• T3 unspecific reverse primer
• specific forward primer generated from the downstream sequence of P450s

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  • 2003-03-12 AP APAP/P/2004/003122A patent/AP2004003122A0/en unknown
  • 2003-03-12 JP JP2003576572A patent/JP2005531297A/ja not_active Withdrawn
  • 2003-03-12 WO PCT/US2003/007430 patent/WO2003078577A2/en not_active Ceased
  • 2003-03-12 BR BRPI0308351-9A patent/BR0308351A/pt not_active IP Right Cessation
  • 2003-03-12 CA CA002477452A patent/CA2477452A1/en not_active Abandoned
  • 2003-03-12 MX MXPA04008785A patent/MXPA04008785A/es not_active Application Discontinuation
  • 2003-03-12 AU AU2003220165A patent/AU2003220165A1/en not_active Abandoned
  • 2003-03-12 CN CNA038057476A patent/CN101166824A/zh active Pending
• 2010
  • 2010-02-01 JP JP2010019868A patent/JP2010131027A/ja active Pending

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