ZA200602939B - Cloning of cytochrome P450 genes from nicotiana - Google Patents

Cloning of cytochrome P450 genes from nicotiana Download PDF

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ZA200602939B
ZA200602939B ZA200602939A ZA200602939A ZA200602939B ZA 200602939 B ZA200602939 B ZA 200602939B ZA 200602939 A ZA200602939 A ZA 200602939A ZA 200602939 A ZA200602939 A ZA 200602939A ZA 200602939 B ZA200602939 B ZA 200602939B
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South Africa
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seq
plant
nucleic acid
tobacco
amino acid
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ZA200602939A
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Xu Dongmei
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Us Smokeless Tobacco Co
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Description

W~ 0 2005/038018 POC T/US2004/034218
CLONING OF CYTOCHROME P45) GENES FROM NIC:OTIANA
The present invention relates to nuczleic acid sequences encoding cytochrome p450 enzymes (ereinafter referred to as p450 and p450 erazymes) in Nicot_iana plants and methods for using those nucleic acid sexquences to alter plant phenotypes.
BACKGROUND
Cytochrome p450s catal=yze enzymatic re=actions for a diverse range of chemically dissimilar subsstrates that include the oxidative, pe roxidative and. reductive . metabolism of endogenous and xenobiotic subs=strates. In
ME plants, pd50s participate in. biochemical paathways that include the synthesis of plant product-s such as phenylpropanoids, alkaloid_s, terpenoidss, lipids, cyanogenic glycosides, and gliacosinolates (Ch_appel, Annu.
Rev. Plant Physiol. Plant Mol. Biol. 198, £49:311-343). cytochrome p450s, also known as p450 heae=me-thiolate proteins, usually act as teerminal oxidase s in multi- } component electron transfeer chains, called p450- containing monooxygenase sy stems. Specif@ c reactions catalyzed include demet=hylation, hy -droxylation, epoxidation, N-oxidation, su lfooxidation, N—, S-, and O- 5 dealkylations, desulfation, c3eamination, and reduction of” azo, nitro, and N-oxide growaps.
The diverse role of Nicotiana plant p45:0 enzymes has been implicated in effec ting a variet-y of plant metabol ites such as vphenylpropano-ids, alkaloids, terpeno.ids, lipids, cyanogenic glycoside=s, glucosinolates and a host of other chemical entities. puring recent years, -it is becoming apparent that some= p450 enzymes can impact the composition of plant metabeolites in plants.
For example, it has been long desire=d to improve the flavor and aroma of certain plants by altering its profile= of selected fatty acids through breeding; however very 1 ittle is known about mechanii sms involved in control ling the levels of these leaf c=onstituents. The down reegulation of p450 enzymes assiociated with the modification of fatty acids may facili tate accumulation of desi_red fatty acids that provide more preferred leaf phenotypic qualities. The function off p450 enzymes and their Ioroadening roles in plant constituents is still being AMiscovered. For instance, a speecial class of p450 enzymess was found to catalyze the break—down of fatty acid into vo=latile C6- and C9-aldehydes and -—alcohols that are major contributors of “fresh green’ o-dor of fruits and vegetal>les. The level of other novel t-—argeted p450s may be alte=red to enhance the qualities of leaf constituents by mod-ifying lipid composition and r elated break down metabol. ites in Nicotiana leaf. Seeveral of these constitzuents in leaf are affected bys senescence that stimulates the maturation of leaf quuality properties.
Still other reports have shown that -p450s enzymes are play a functional role in altering fat—ty acids that are involveed in plant-pathogen interact.ions and disease resistance.
In otheer instances, p450 enzymes have been suggested to be invol-ved in alkaloid biosynthesis.
Nornicotine =is a minor allkaloid found in Nicotiaana tabaceum.
It has been postul ated that it is produce=d by the p450 mediateed i demethylati.on of nicotine followed by acylation amd nitrosatiora at the N position ther—eby producing a seri es of N-acylronicotines and N-nitr—osonornicotines.
N- demethylation, catalyzed by a putative p450 demethylas e, is thought= to be a primary ssource of nornicoti ne
) biosynthesess in Nicotiana.
While the enzyme is believ—ed to be microsomal, thus far a nicot—ine demethylase enzyme has not be en successfully purifie=d, mor have the gemmes involved beeen isolated.
i Furtheermore, it is hypothesi:=zed but not proven tlnat the activi ty of p450 enzymes is genetically controll ed and also s%rongly influenced by eravironment factors.
Eor example, t-he demethylation of ni cotime in Nicotiana is thought to increase substantially when the plants reach
) a mature stage.
Furthermore, it =is hypothesized yet rot proven that the demethylase gene acontains a transposakole element thaat can inhibit translation of RNA when presert.
i The 1.arge multiplicity of p&l50 enzyme forms, the=ir differing structure and function ave made their resea—xch on Nicotiecana p450 enzymes very dif ficult before fthe enclosed imvention.
In addition, cloning of p450 enzymmes has been hampered at least i-m part because theese membrane-localized proteins are typically present in low abundance and often unstable= to purification. Hence, a- need exists for the identiffication of p450 enzymes in. plants and the nucleic acid sequences associated witlma those p450 enzymes. In parti.cular, only a few cytochrome p450 proteins have been resported in Nicotiana. The inventions described hereirm entail the discovery of & substantial number of cytochrome p450 fragments that’ correspond to several groups of p450 species based omn their sequence identity.
SUMEMARY
The present invention. is directed to plant pi5=( enzymes. The present invemtion is further directed t=o plant p450 enzymes from Nicotiana. The present inventicon is also directed to p45 0 enzymes in plants whose expression is induced by ethylene and/or plarat senescence. The present— invention is yet further directed to nucleic acid sequences in plants havimg enzymatic activities, for ecxample, being categorized a=as oxygenase, demethylase and the like, or other and the usse of those sequences to reduc e or silence the expression or over-expression of these -enzymes. The invention al so relates to p450 enzymes fou nd in plants containing high er nornicotine levels tharma plants exhibiting lowwer nornicotine levels.
In one aspect, the in=vention is directed to nucleic acid sequences as set fort h in SEQ. ID. Nos. 1, 3, 5, 1,
9, 11, 13, 15, 17, 1S, 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, 25, 77, 79, 81, 83, 85, 87, 89, 91, 9s, 97, 99, 101, 103, 105, 107, 109, 111, 113 , 115, 117, - 119, 121, 123, 125, 127, 129, 131, 133, 135 _, 137, 139, 143, 145, 147, 149, 151, 153, 155, 157, 159 , 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181 , 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203 , 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225 , 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247 , 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269 , 271, 273, 275, 277, 279, 281, 2 83, 285, 287, 289, 291, =293, 295 and 297.
In a second related aspect, 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 fi rst nucleic acid following the c-ytochrome pd450 motif GXRXCX(G/A) to the stop codon. The representative nucleic acid groups and respective speci es are shown in Table I..
In a third asprect, the invention is directed to amino acid sequences as set forth in SEQ. ID . Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, S56, 58, 60, 62, 64, 66, 868, 70, 72, 74, 76, 718, 80, 82, 84, 86, 88, 90, 92, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 144, 146, 148, 150, 152, 154, 156, 158s, 160, 162,
164, 166, 168, 170, 172, 1.74, 176, 178, 180, 182, 1184, 186, 188, 190, 192, 194, 1.86, 198, 200, 202, 204, 2206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, -228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, - 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, =284, 286, 288, 290, 292, 294, 296 and 298.
In a fourth related aspect, those fragments containing greater than 71% identity in amino acid sequence were placed into groups dependent upon t. heir identity to each other in a region corresponding to= the first amino acid followirng the cytochrome p450 mmotif
GXRXCX (G/A) to the stop cocdon. The representative amino acid groups and respective species are shown in Table II.
In a fifth aspect, the invention is directead to amino acid sequences of ful l length genes as set fort=h in
SEQ. ID. Nos. 150, 152, 1 54, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, A476, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, A498, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 2220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 2242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 28 6, 288, 290, 292, 294, 296s and 298.
In a sixth related aspect, those full length genes containing 85% or greater identity in amino acid sequ ence were placed into groups despendent upon the identity to each other. The representative amino acid groups and respe=ctive species are shown ira Table III.
In a seventh aspect, the invention is directed to amineo acid sequences of the fraagments set forth in SEQ.
ID. Mos. 299-357.
In the eighth related aspect, those fragments cont.aining 90% or greater ident=ity in amino acid sequence were placed into groups dependesnt upon their identity to each. other in a region corwxesponding to the first cytoechrome p450 domain, UXXRXXZ, to the third cytochrome doma_in, GXRXO, where U is E or X, X is any amino acid and 7 is. R, T, S or M. The representative amino acid groups respsective species shown in Ta ble IV.
In a ninth related asspect, the reduction or elimination or over-expression of p450 enzymes im
Nicootiana plants may be accomplished transiently using
RNA viral systems.
Resulting transformed eor infected plants are asse=ssed for phenotypic chamges including, but not= limfi ted to, analysis of endogemous p450 RNA transcripts, pd50 expressed peptides, and. concentrations of plant met=abolites using techniques commonly available to one : : havi.ng ordinary skill in the art.
In a tenth important aspe=ct, the present inventior is aalso directed to generaticon of trangenic Nicotiana lines that have altexed p450 enzyme activity lewrels. In accordance with the invention, these transgenic lines include nucleic aci d sequences that are effecative for reducing or silencing or increasing the expreassion of certain enzyme thus resulting in phenotypic effects within Nicotiana. such nucleic acid sequencess include
SEQ. ID. Nes. 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, s3, 5, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 , 77, 79, 81, 83, 85, 87, 89, 91, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137. 139, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161. 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183 _ 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249 , 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293 295 and 297.
In a very important eleventh aspect of the invention, plant cwmltivars including nucleic ac-1ids of the present invention in a down regulation capac=ity using either full lengtka genes or fragments thereofS or in an over-expression capacity using full length cyenes will have altered metabolite profiles relative t.o control plants.
In a twelfth aspect of the inventieon, plant cultivars includin g nucleic acid of the present. invention using either full Mength genes or fragments thereof in modifying the bioswnthesis or breakdown of metabolites derived from the plant or external to the plantcs, will have use in toleramting certain exogenous chemi#.cals or plant pests. Such rucleic acid sequences include= 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, 95, 97, 99, 101, 103, 105, 107, 1.09, 111, 113, 115, 117, 119 , 121, 123, 125, 127, 129, 1231, 133, 135, 137, 139, 143 , 145, 147, 149, 151, 153, 1 55, 157, 159, 161, 163, 165 , 167, 169, 171, 173, 175, 1°77, 179, 181, 183, 185, 187 , 189, 191, 193, 195, 197, 1 99, 201, 203, 205, 207, 209 , 211, 213, 215, 217, 219, 2 21, 223, 225, 227, 229, 231 , 233, 235, 237, 239, 241, 2 43, 245, 247, 249, 251, 253 , 255, 257, 259, 261, 263, 2 65, 267, 269, 271, 273, 275 , 277, 279, 281, 283, 285, 2 87, 289, 291, 293, 295 and 2297.
In a thirteermth aspect, the present invemmtionn is directed to the s creening of plants, more pr—eferably
Nicotiana, that c:ontain genes that have sulmstantial nucleic acid idemntity to the taught nucle=ic acid sequence. The use =of the invention would be advaantageous to identify and sel ect plants that contain a nucleic acid sequence with exac:t or substantial identity wh_ere such plants are part of a breeding program for tradit=ional or transgenic varietie=s, a mutagenesis program, or mmaturally occurring diverse plant populations. The scre-ening of plants for substammtial nucleic acid identity— may be asccomplished by evaluating plarat nucleic acid materi als u.sing a nucleic acid probe in conjunction with nucl.eic a-cid detection protocols including, but not limited to, n-ucleic acid hybridization and EXR analysis. The nucl.eic a cid probe may consist of the taught nucleic amcid s equence or fragment thereof corresponding to SEQ ID 1, 3,5, 7,9, 11, 13, 15, 17, 19. 21, 23, 25, 27, 28, 31, 3 3, 35, 37, 39, 41, 43, 45, 47. 49, 51, 53, 55, 57, 59, 6 1, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 8 9, 91, 95, 97, 99, 101, 103, 105, 107, 109, 111, A113, 1.15, 117, 119, 121, 123, 125, 127, 129, 131, 133, A 35, 1.37, 139, 143, 145, 147, 149, 151, 153, 155, 157, M59, 1.61, 163, 165, 167, 169, 171, 173, 175, 177, 179, Mel, 1.83, 185, 187, 189, 191, 193, 195, 197, 199, 201, =203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 2227, 229, 231, 233, 235, 237, 239, 241, 243, 245, =47, 2=49, 251, 253, 255, 257, 259, 261, 263, 265, 267, =269, >=71, 273, 275, 277, 279, 281, 283, 285, 287, 289, =291, 2293, 295 and 297.
In a fourteenth aspect, the present inventiorm is :
Airected to the identification of plant genes, more preferably Nicotiana, that shaxe substantial amino acid i_dentity corresponding to the taught nucleic acid sequence. The identification of plant genes including
I>oth cDNA and genomic clones, those cDNAs and genomic c=lones, more preferably fxom Nicotiana may be accomplished by screening plarat cDNA libraries usirag a mucleic acid probe in conjurection with nucleic &:acid detection protocols including, but not limited to,
nucleic acid hybridization and PCR analysis. The nucleic acid probe may be comprised o=f nucleic acid sequence Or fragment thereof corresponding to SEQ ID 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 23, 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, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131i, 133, 135, 137, 139, 143, 145 and 147.
In an alterative fifteemmth aspect, cDNA expression libraries that express pepti des may be screened using antibodies directed to part er all of the taught amino acid sequence. Such amino aczd sequences include SEQ ID 2, 4, 8, 9, 10, 12, 14, 16, 1.8, 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, #4, 76, 78, 80, 82, 84, B6, 88, 90, 92, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124 , 126, 128, 130, 132, 134, 136, 138, 140, 144, 146, 148.
In a sixteenth important aspect, the present invention is also directed to generation of transgenic
Nicotiana lines that have ove r-expression of p450 enzyme activity levels. In accordance with the invention, these transgenic lines include all nucleic acid sequences encoding the amino acid sequ.ences of full length genes that are effective for increasing the expression of certain enzyme thus resulting in phenotypic effects within Nicotiana. Such amino acid sequences include SEQ.
TD. 150, 152, 154, 156, 158, 160, 162, 164, 166, 1 €8, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 1 90, 192, 194, 196, 198, 200, 202. 204, 206, 208, 210, 2 12, 214, 216, 218, 220, 222, 224. 226, 228, 230, 232, 2 34,
Ss 236, 238, 240, 242, 244, 246. 248, 250, 252, 254, 2 56, 258, 260, 262, 264, 266, 268. 270, 272, 274, 276, 2-778, 280, 282, 284, 286, 288, 290, 292, 294, 296 and 298.
A tobacco product is allso provided that inclumdes tobacco leaf (lamina and/or sftem) having reduced amorants of nornicotine. The tobacco product includes tobacco (tobacco leaf including lamin a and/or stem) from a pB. ant that includes the sequences described herein or where genes encoding tobacco speci fic nitrosamines have Ioeen i5 eliminated or suppressed. The elimination or suppresssion : of genes encoding tobacco speicific nitrosamines ig effective for reducing tobacco specific nitrosaminess in the tobacco products from about 5 to about 10%, in another aspect from about 10 to 20%, in another aspoect about 20 to 30%, and in anotlnier aspect greate than 30%, as compared to tobacco produc=ts made from tobacco plants where gnees coding for tobacco specific nitrosamines Ihave not been eliminated or suppressed. As used herein, the tobacco product may include= cigarettes, cigars, wipe tobacco, snuff chewing tobacceo, products blended with. the tobacco product, and mixturess thereof.
BRIEF DESCRIPTI®@N OF DRAWINGS
Figure 1 shows nucleic a-cid SEQ. ID. No.:1 and amnino acid SEQ. ID. No.:2.
Figure 2 shows nucleic acid SEQ. ID. No.:3 and amino aci-d SEQ. ID. No.:4.
Figure 3 shows nucleic acid SEQ. ID. No.:5 and amino aci d SEQ. ID. No.:6.
S Figure 4 shows nucleic acid SEQ. ID. No. :7 and amino aci.d SEQ. ID. No.:8.
Figure 5 shows nucleic acid SEQ. ID. No. :9 and amino aci.d SEQ. ID. No.:10.
Figure 6 shows nucleic acid S EQ. ID. No.:11 and ami_mo acid SEQ. ID. No.:12.
Figure 7 shows nucleic acid SEQ. ID. No.:13 and ami_no acid SEQ. ID. No.:14.
Figure 8 shows nucleic acid SSEQ. ID. No.:15 and ami_no acid SEQ. ID. No.:16.
Figure 9 shows nucleic acid SEQ. ID. No.:17 and amino acid SEQ. ID. No.:18.
Figure 10 shows nucleic acid SSEQ. ID. No.:19 and ami_no .acid SEQ. ID. No.:20.
Figure 11 shows nucleic acid SEQ. ID. No.:21 and ami_no acid SEQ. ID. No.:22.
Figure 12 shows nucleic acid SEQ. ID. No.:23 and ami_no acid SEQ. ID. No.:24.
Figure 13 shows nucleic acid SSEQ. ID. No.:25 and ami_no acid SEQ. ID. No.:26.
Figure 14 shows nucleic acid SEQ. ID. No.:27 and ami_no acid SEQ. ID. No.:28.
Figure 15 shows nucleic acid SSEQ. ID. No.:29 and ami _no acid SEQ. ID. No.:30.
Figure 16 shows nucleic acid SEQ. ID. No.:31 and ami_no acid SEQ. ID. No.:32.
Figure= 17 shows nucleic acid SSEQ. 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. = Figures 19 shows nucleic acid SSEQ. ID. No.:37 and amino acid SEQ. ID. No.:38.
Figur-e 20 shows nucleic acid =SEQ. ID. No.:39 and amino acid. SEQ. ID. No.:40.
Figur e 21 shows nucleic acid SEQ. ID. No.:41 and 1@ amino acid. SEQ. ID. No.:42.
Figqur—e 22 shows nucleic acid SEQ. ID. No.:43 and amino aci& SEQ. ID. No.:44.
Figur—e 23 shows nucleic acid SEQ. ID. No.:45 and amino acid SEQ. ID. No.:46.
Figur—e 24 shows nucleic acid SEQ. ID. No. :47 and amino acid SEQ. ID. No.:48.
Figumre 25 shows nucleic acid SEQ. ID. No.:49 and amino acicl SEQ. ID. No.:50.
Figume 26 shows nucleic acid SEQ. ID. No.:51 and amino acicd SEQ. ID. No.:52.
Figu—xe 27 shows nucleic acid SEQ. ID. No.:53 and amino acied SEQ. ID. No,:54.
Figu—re 28 shows nucleic acid SEQ. ID. No.:55 and. amino aci 4d SEQ. ID.- No.:56.
Figu re 29 shows nucleic acid SEQ. ID. No.:57 and. amino aci d SEQ. ID. No.:58.
Figu.re 30 shows nucleic acid SEQ. ID. No.:59 andl amino aci._d SEQ. ID. No. :60.
Figmre 31 shows nucleic acid. SEQ. ID. No.:61 an amino aci.d 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 anda amino acid SEQ. IID. No.:68.
Figure 35 shows nucleic acid SEQ. ID No. :69 and amino acid SEQ. IX, No.:70.
Figure 36 shows nucleic acid SEQ. ID No.:71 and amino acid SEQ. IM. No.:72.
Figure 37 skiows nucleic acid SEQ. ID . No.:73 ancl amino acid SEQ. ID. No.:74.
Figure 38 shows nucleic acid SEQ. ID . No.:75 aned amino acid SEQ. ID. No.:76.
Figure 39 shows nucleic acid SEQ. ID . No.:77 an d amino acid SEQ. I'D. No.:78.
Figure 40 shows nucleic acid SEQ. IDs. No.:79 an_d amino acid SEQ. ID. No.:80.
Figure 41 shows nucleic acid SEQ. ID®. No.:81 an=d amino acid SEQ. ID. No.:82.
Figure 42 shows nucleic acid SEQ. IID. No.:83 armd amino acid SEQ. XD. No.:84.
Figure 43 shows nucleic acid SEQ. IID. No.:85 arad amino acid SEQ. XD. No.:86.
Figure 44 shows nucleic acid SEQ. IID. No.:87 arad amino acid SEQ. ID. No.:88.
Figure 45 shows nucleic acid SEQ. IID. No.:89 ard . amino acid SEQ. ID. No.:90.
Figure 46 shows nucleic acid SEQ. IID. No.:81 ard amino acid SEQ. ID. No.:92.
Figure 48 shows nwicleic acid SEQ. ID. No.:95 and amino acid SEQ. ID. No. :96.
Figure 49 shows nmucleic acid SEQ. ID. No.:97 aned - amino acid SEQ. ID. No. :98.
Figure 50 shows n-ucleic acid SEQ. ID. No.:99 and amino acid SEQ. ID. No. :100.
Figure 51 shows nwmicleic acid SEQ. ID. No.:101 and amino acid SEQ. ID. No..:102.
Figure 52 shows nwcleic acid SEQ. ID. No.:103 am=d amino acid SEQ. ID. No-_ :104.
Figure 53 shows nucleic acid SEQ. ID. No.:105 amd amino acid SEQ. ID. No. :106. ' Figure 54 shows nucleic acid SEQ. ID. No.:107 arad amino acid SEQ. ID. No .:108.
Figure 55 shows n ucleic acid SEQ. ID. No.:109 arad amino acid SEQ. ID. No .:110.
Figure 56 shows nucleic acid SEQ. ID. No.:111 ard amino acid SEQ. ID. No .:112.
Figure 57 shows nucleic acid SEQ. ID. No.:113 ard amino acid SEQ. ID. No .:114.
Figure 58 shows nucleic acid SEQ.. ID. No.:115 ard amino acid SEQ. ID. No .:116.
Figure 59 shows mrucleic acid SEQ. ID. No.:117 ard amino acid SEQ. ID. No .:118.
Figure 60 shows mmucleic acid SEQ. ID. No.:119 ard amino acid SEQ. ID. No .:120. :
Figure 61 shows mucleic acid SEQ. ID. No.:121 amd amino acid SEQ. ID. No .:122.
Figure 62 shows rmucleic acid SEQ. ID. No.:123 amd amino acid SEQ. ID. No .:124.
’
Figure 63 shows nucleic acid =SEQ. ID. No.:125 anda amino acid. SEQ. ID. No.:126.
Figur-e 64 shows nucleic acid SEQ. ID. No.:127 anda - 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 ancl amino aci& SEQ. ID. No.:132.
Figur-e 67 shows nucleic acid SEQ. ID. No.:133 and amino aci& SEQ. ID. No.:134.
Figure 68 shows nucleic acid SEQ. ID. No.:135 ancl amino acicl SEQ. ID. No.:136.
Figure 69 shows nucleic acid SEQ. ID. No.:137 ancl amino acid SEQ. ID. No.:138.
Figur-e 70 shows nucleic acid SEQ. ID. No.:139 and amino acidcl SEQ. ID. No.:140.
Figur-e 72 shows nucleic acid SEQ. ID. No.:143 and amino acid SEQ. ID. No.:144.
Figur—e 73 shows nucleic acid SEQ. ID. No.:145 aned amino acid SEQ. ID. No.:146.
Figure 74 shows nucleic acid SEQ. ID. No.:147 aned amino acicl SEQ. ID. No.:148.
Figure 75 shows nucleic acid SEQ. ID No.: 149 aned amino acid& SEQ. ID. No.: 150.
Figur-e 76 shows nucleic acid SEQ. ID No.: 151 aned amino acid SEQ. ID. No.: 152.
Figur-e 77 shows nucleic acid SEQ. ID No.: 1533 an«d amino acid SEQ. ID. No.: 154.
Figur-e 78 shows nucleic acid SEQ. ID No.: 155 aned amino acid SEQ. ID. No.: 156.
Figure 79 shows nucleic acid SEQ. ID No.: 157 and amino acid SEQ. ID. No.: 158.
Figure 80 showss nucleic acid SEQ. ID No.: 159 and amino acid SEQ. ID. No.: 160.
Figure 81 showss nucleic acid SEQ. ID No.: 161. and amino acid SEQ. ID. No.: 162.
Figure 82 showss nucleic acid SEQ. ID No.: 163% and amino acid SEQ. ID. No.: 164.
Figure 83 showss nucleic acid SEQ. ID No.: 165 and amino acid SEQ. ID. DNo.: 166.
Figure 84 showss nucleic acid SEQ. ID No.: 16a and amino acid SEQ. ID. No.: 168.
Figure 85 showss nucleic acid SEQ. ID No.: 163 and amino acid SEQ. ID. No.: 170. , Figure 86 shows=s nucleic acid SEQ. ID No.: 177 and amino acid SEQ. ID. No.: 172.
Figure 87 shows=s nucleic acid SEQ. ID No.: 1733 and amino acid SEQ. ID. No.: 174.
Figure 88 shows nucleic acid SEQ. ID No.: 175 an amino acid SE3Q. ID, No.: 176.
Figure £39 shows nucleic acid SEQ. ID No.: 177 and amino acid SEQ. ID. No.: 178.
Figure 90 shows nucleic acid SEQ. ID No.: 179 and amino acid SE=Q. ID. No.: 180.
Figure 1 shows nucleic acid SEQ. ID No. : 181 aned amino acid SEQ. ID. No.: 1B2.
Figure %2 shows nucleic acid SEQ. ID No.: 183 an-d amino acid SEEQ. ID. No.: 184.
Figure 93 shows nucleic acid SEQ. ID No.: 185 amd amino acid SEQ. ID. No.: 186.
Figure 94 shows nucleic acid SEQ. ID No.: 187 amd amino acid SEQ. ID. No.: 188.
Figure 95 shows nucleic acid SEQ. ID No.: 189 armad amino acid S EQ. ID. No.: 190. . Figure 96 shows nucleic acid SEQ. ID No.: 191 ard amino acid S-EQ. ID. No.: 192.
Figure 97 shows nucleic acid SEQ. ID No.: 193 armd . amino acid SEQ. ID. No.: 194.
Figure 98 shows nucleic acid SEQ. ID No.: 195 ard amino acid SSEQ. ID. No.: 196. :
Figure 99 shows nucleic acid SEQ.. ID No.: 197 amd amino acid SEQ. ID. No.: 188.
Figure 100 shows nucleic acid SEQ . ID No.: 199 amnd amino acid SEQ. ID. No.: 200.
Figure 101 shows nucleic acid SEQ . ID No.: 201 amd amino acid SEQ. ID. No.: 202.
Figure 102 ghows nucleic acid SEQ . ID No.: 203 amd amino acid SEQ. ID. No.: 204.
VIVO 2005/038018 PCT/US2004/034218
Figure 103 shows nucleic acid SEQ. ID No.: 205 and amino acid SEQ. ID. No.: 206.
Figure 104 shows nucleic aci.d SEQ. ID No.: 207 and amino acid SEQ. ID. No.: 208.
Figure 105 shows nucleic ac3d SEQ. ID No.: 209 and amino acid SEQ. ID. No.: 210.
Figure 106 shows nucleic acZid SEQ. ID No.: 211 and amino acid SEQ. ID. No.: 212,
Figure 107 shows nucleic acZid SEQ. ID No.: 213 and amino acid SEQ. ID. No.: 214.
Figure 108 shows nucleic ac=id SEQ. ID No.: 215 and amino acid SEQ. ID. No.: 216.
Figure 109 shows nucleic ac*id SEQ. ID No.: 217 and amino acid SEQ. ID. No.: 218.
Figure 110 shows nucleic ac=id SEQ. ID No.: 219 and amino acid SEQ. ID. No.: 220.
Figure 111 shows nucleic acid SEQ. ID No.: 221 and amino acid SEQ. ID. No.: 222.
Figure 112 shows nucleic acid SEQ. ID No.: 223 and amino acid SEQ. ID. No.: 224.
Figure 113 shows nucleic acid SEQ. ID No.: 225 and amino acid SEQ. ID. No.: 226.
Figure 114 shows nucleic acid SEQ. ID No.: 227 and amino acid SEQ. ID. No.: 228.
Figure 115 shows nucleic aciid SEQ. ID No.: 229 and amino acid SEQ. ID. No.: 230.
Figure 116 shows nucleic acid SEQ. ID No.: 231 and amino acid SEQ. ID. No.: 232.
Figure 117 shows nucleic aci-d SEQ. ID No.: 233 and amino acid SEQ. ID. No.: 234.
Figure 118 shows rxucleic acid SEQ. ID No.: 2235 and amino acid SEQ. ID. No. : 236.
Figure 119 shows raucleic acid SEQ. ID No.: 237 and amino acid SEQ. ID. No. : 238. -
Figure 120 shows raucleic acid SEQ. ID No.: .239 and amino acid SEQ. ID. No. : 240.
Figure 121 shows raucleic acid SEQ. ID No.: 241 and amino acid SEQ. ID. No. : 242.
Figure 122 shows raucleic acid SEQ. ID No.: 243 and amino acid SEQ. ID. No. : 244.
Figure 123 shows raucleic acid SEQ. ID No.: 245 and ‘amino acid SEQ. ID. No. : 246.
Figure 124 shows raucleic acid SEQ. ID No.: 247 and amino acid SEQ. ID. No. : 248.
Figure 125 shows raucleic acid SEQ. ID No.: 249 and amino acid SEQ. ID. No. : 250.
Figure 126 shows raucleic acid SEQ. ID No.: 251 and amino acid SEQ. ID. No. : 252.
Figure 127 shows raucleic acid SEQ. ID No.: 253 and amino acid SEQ. ID. No. : 254.
Figure 128 shows raucleic acid SEQ. ID No.: 255 and amino acid SEQ. ID. No. : 256. "Figure 129 shows raucleic acid SEQ. ID No.: 257 and amino acid SEQ. ID. No. : 258, :
Figure 130 shows mniucleic acid SEQ. ID No.: _259 and amino acid SEQ. ID. No. : 260.
Figure 131 shows nucleic acid SEQ. ID No.: 261 and amino acid SEQ. ID. No. : 262.
Figure 132 shows nucleic acid SEQ. ID No.: =263 and amino acid SEQ. ID. No. : 264.
Figure 133 shows nucleic acid SSEQ. ID No.: 265 an.d armino acid SEQ. ID. No.: 266.
Figure 134 shows nucleic acid SEQ. ID No.: 267 armd amnino acid SEQ. ID. No.: 268. -
Figure 135 shows nucleic acid :SEQ. ID No.: 269 arad amino acid SEQ. ID. No.: 270.
Figure 136 shows nucleic acid SEQ. ID No.: 271 ard amino acid SEQ. ID. No.: 272.
Figure 137 shows nucleic acid SEQ. ID No.: 273 ard amino acid SEQ. ID. No.: 274.
Figure 138 shows nucleic acid SEQ. ID No.: 275 ard amino acid SEQ. ID. No.: 276,
Figure 139 shows nucleic acid SEQ. ID No.: 277 amd aamino acid SEQ. ID. No.: 278.
Figure 140 shows nuclei¢ acid SEQ. ID No.: 279 amd amino acid SEQ. ID. No.: 280.
Figure 141 shows nucleic acid SEQ. ID No.: 281 amd anino acid SEQ. ID. No.: 282.
Figure 142 shows nucleic acid SEQ. ID No.: 283 amd amino acid SEQ. ID. No.: 284.
Pigure 143 shows nucleic acid SEQ. ID No.: 285 a.nd amino acid SEQ. ID. No.: 286.
Figure 144 shows nucleic acid SEQ. ID No.: 287 a.nd amino acid SEQ. ID. No.: 288.
Figure 145 shows nucleic acid SEQ. ID No.: 289 a_nd &mino acid SEQ. ID. No.: 290.
Figure 146 shows nucleic acid SEQ. ID No.: 291 a.nd amino acid SEQ. ID. No.: 292.
Figure 147 shows nucleic acid SEQ. ID No.: 293 aand amino acid SEQ. ID. No.: 294.
Figuire 148 shows nucleic acid SEQ. ID No.: 295 and amino aci.d SEQ. ID. No.: 296.
Figure 149 shows nucleic acid SEQ. ID No.: 297 and amino aci.d SEQ. ID. No.: 298. -
Figurare 151 shows a comparison of Sequence Groups.
Figurare 152 illustrates alionment of £ull length clones.
Figiare 153 shows a procedure usecl for cloning of cytochromne p450 cDNA fragments by PCR
DETAILED DESCRIPTION
DEFINITIONS E
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Single-ton et al. (1994)
Dictionasmry of Microbiology and Molecular Biology, second edition, John Wiley and Sons (New York) provides one of skill with a general dictionary of many of the terms used in this invention. All patents and publ ications referred to herein are incorporated by refererice herein. For purposes of the present invention, the following terms are defimned below. “En=zymatic activity” is meamt to include demethylation, hydroxylation, epoxidat=ion, N-oxidation, sulfooxiedation, N-, S-, and O- dealkylations, desulfat=ion, deamination, and reduction of azo, nitro, and N-ox=ide groups. The term "nucleic acid" refers to a deoxyribeonucleotide or ribonucleotide poolymer in either single- or double-stranded £orm, or sense or ant i-sense, and unless otherwise limited , encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucl._eotides.
Unless otherwise indicated, a particular nucleeic acid sequence includes the comp lementary Sequence thereof.
The terms ‘operably linked”, “in operable combination”, and “in operab le order” refer to fumnctional linkage between a nuclei< acid expression control } sequence (such as a promote, signal sequence, or array of transcription factor bonding sites) and = second nucleic acid sequence, whexein the expression control sequence affects transcription and/or translaticon of the nucleic acid corresponding to the second sequerace.
The term "recombinant" when used with reference to a cell indicates that the cell replicates a hete=rologous nucleic acid, expresses said nucleic acid or exroresses a peptide, heterologous pepti de, or protein enco-ded by a heterologous nucleic acid. Recombinant cells car express genes or gene fragments in either the sense or Aantisense form that are not found within the native (non- recombinant) form of the cel 1. Recombinant cells can also express genes that are fourad in the native for-m of the cell, but wherein the genes are modified and re- introduced into the cell by artificial means.
A “structural gene” Ds that portion of a gene comprising a DNA segment encoding a protein, pol _ypeptide or a portion thereof, and execluding the 5' seguerace which drives the initi ation of transcription. The structural gene may alternatively encode a nontranslatable product.
The structural geene may be one which is normally £=ound in the cell or one which is not normally found in t—he cell or cellular locamtion wherein it is introduced, i-n which case it is terme-d a “heterologous gene”. A heter—ologous gene may be derived in whole or in part from any.” source known to the art, including a bacterial gemome or episome, eukaryoetic, nuclear or plasmid DNA, cDNA, viral
DNA or chemicall-y synthesized DNA. A structural cjene may contain one or more modifications that could. effect biological act ivity or its characteristic s, the biological acti-vity or the chemical structure of the expression prodiact, the rate of expression or the= manner of expression control. Such modifications incluade, but are not limited to, mutations, insertions, delet—ions and substitutions off one or more nucleotides. The stmructural gene may constitute an uninterrupted coding sequaence or : it may include one or more introns, bounded by the appropriate spli_ce junctions. The structural gen:e may be translatable or non-translatable, including in =n anti- sense orientation. The structural gene ma—y 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 chemicallly synthesized). “Derived f rom” is used to mean taken, okotained, received, traced, replicated or descended from a source {chemical and/osx biological). A derivative may be
V0 2005/038018 PCTAUS2004/034218 produced by chemical or biological manirpulation (including, but not limited to, substitution, a-ddition, insertion, deletion, extraction, isolation, mutamtion and replication) of the oxiginal source. “Chemically synthesized”, as related to a sequence of DNA, means that portions of the component nucleotides were assembled in vitro. Manual chemical synt-hesis of
DNA may be accomplished using well established pr—-ocedures (Caruthers, Methodology of DNA and RNA Secjuencing, (1983), Weissman (ed.), Praeger Publishers, NJew York,
Chapter 1); automated chemical synthesis can be oerformed using one of a number of commercially available rmachines.
Optimal alignment of sequences for comparisson 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 similari_ty 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 TFAST2 in the
Wisconsin Genetics Software Package, Genetics Computer
Group, 575 Science Dr ., Madison, Wis.), or by irmspection. :
The NCBI Basic I.ocal Alignment Search Toc>1 (BLAST) (Altschul et al., 1990) is available fromn several sources, including the National Center for BE3iological
Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysiss programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at htp://www.ncbi .nlm.nih.gov/BLAST/. A description of how to determine sequence identity using : this pro gram is available at http: //www.ncbi .nlm.nih.gov/BLAST/blast help.html.
The terms "substantial amino acid identity® or "substantial am-ino acid sequence identity" as applied to amino acid secuences and as used herein denote a characteristic of a polypeptide, wherein the peptide comprises a sequence that has at least 70 percent sequence ident-Dty, preferably 80 percent amino acid. sequence identi ty, more preferably 90 percent amino acid sequence identi ty, and most preferably at least 99 to 100 percent sequence identity as compared to a references group over regi on corresponding to the first amino acid following the cytochrome p450 motif GXRXCX (G/A) to the= stop codon of the translated peptide.
The terms "substantial nucleic acid identity" ox~ ssubstantial nucleic acid sequence identity" as applied to nucleic acid sequences and as used herein denote am characteristic of a polynucleotide sequence, wherein the= polynucleotide comprises a sequence that has at least 75 percent sequemce identity, preferably 81 percent sequence identity, more preferably at least 91 percent— sequence identi ty, and most preferably at least 99 to 100m percent sequence identity as compared to a reference= group over region corresponding to the first nucleic acid following the cytoclarome p450 motif
GXRXCX(G/A) to the stop codon of the tramnslated peptide.
Anothe—r indication that nucleotide sequences are substantially identical is if two molectales hybridize to each other under stringent condit_ions. Stringent conditions -are sequence-dependent and wzill be different in differesnt circumstances. Generally, 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 poimt (Tm) for the specific sequ.ence at a defined ionic stren-gth and pH. The Tm is the temperature (under defined iondc strength and pH) at which 50% of the target sequence hybridizes to a matched pmobe. Typically, stringent conditions will be those im which the salt concentrati on is about 0.02 molar at pH 7 and the temperature is at least about 60°C. For instance in a standard Seouthern hybridization proce=dure, stringent conditions will include an initial wash in 6xSSC at 42 °C followed by- one or more additional waslnes in 0.2xSSC at a temperatiare of at least about 55°C, typically about 60°C and of ten about 65°C.
Nucleo tide sequences are alse substantially identical for purposes of this inveantion when the polypeptide s and/or proteins which #they encode are substantial ly identical. Thus, where one nucleic acid sequence emcodes essentially the same polypeptide as a second nucleic acid sequence, the t-wo nucleic acid sequences ame substantially identical, e=ven if they would not hybridize under stringent conditions due to degeneracy permitted by the genetic code (see, Da-xnell et al. (1990) Molecular Cell Biology, Second Edition
Scientific Amemrican Books W. H. Freeman and Compoany New b York for an e=xplanation of codon degeneracy and the genetic code). Protein purity or homogeneity can be indicated by a- number of means well known in t=he art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualization upon stainin.g. For certain purposees high resolution may be needed =and HPLC or a similar mesans for purification may be utilized.
As used herein, the term "vector" is ~—used in reference to mucleic acid molecules that transfer DNA segment (s) int=o a cell. A vector may act to resplicate
DNA and may reproduce independently in a host ce 11. The term "vehicle" is sometimes used interchangealoly with "vector." The term "expression vector" as usecl herein refers to a rec ombinant DNA molecule containing a desired coding sequenc e and appropriate nucleic acid se=quences necessary for the expression of the operably- linked coding sequence= in a particular host organism. Nucleic acid sequences necessary for expression in proBkaryotes usually includes a promoter, an operator (optiona=al), and a ribosome b inding site, often along witli other sequences. FEuecaryotic cells are known to utilize promoters, enhamncers, and termination and polyadernylation signals.
For the purpose of reg enerating complete genetic-ally engineered plants with reoots, a nucleic acid maw” be inserted into plant cells, for example, by any techn ique such as 4n vivo inoculation or by any of the knowan in vitro tissue culture techmiques to produce transfo rmed plant cells that can be regenerated into complete pla nts.
Thus, for example, the ins ertion into plant cells mamy be by in vitro inoculation by pathogenic or non-pathogenic
A. tumefaciens. Other sucha tissue culture techniquess may also be employed. "Plant tissue” ircludes differentiated and : undifferentiated tissues of plants, including, but not limited to, roots, shoots, leaves, pollen, seeds, t—=umor tissue and various forms of cells in culture, suc’h as single cells, protoplasts, embryos and callus tissue _ The plant tissue may be in plamta or in organ, tissue or cell culture. “Plant cell” as used herein includes plant cell_s in planta and plant cells and protoplasts in cultcure. “cDNA” or “complementary DNA” generally refers to a single stranded DNA molectile with a nucleotide sequmence that is complementary to am RNA molecule. cDNA is fosrmed by the action of the enzyrmne reverse transcriptase osn an
RNA template.
STRATEGIES FOR OBTAINING NUCLEIC ACID SEQUENCES
Tn accordance with the present invention. RNA was extracted from Nic otiana tissue of converter and non- converter Nicotianea lines. The extracted RNA was then used to create cDNA. Nucleic acid sequence=s of the present invention were then generated ussing two strategies.
In the first strategy. the poly A enriche=d RNA was extracted from plant tissue and cDNA was made IDy reverse transcription PCR. The single strand cDNA was then used to create p450 specific PCR populations using Segenerate primers plus a oligo d(T) reverse primer. The primer design was based or the highly conserved motife=s of p450.
Examples of specifzic degenerate primers are se~t forth in
Figure 1. Sequence fragments from plasmids containing appropriate size inserts were further analyze=d. These : size inserts typically ranged from about 300 toes about 800 nucleotides depending on which primers were uased.
In a second sstrategy, a cDNA library was initially constructed. The «DNA in the plasmids was used to create p450 specific PCR populations using degeneraste primers plus T7 primer on plasmid as reverse primer. As in the first strategy, sequence fragments from plasmids containing appropriate size inserts were further analyzed.
Nicotiana plant lines known to produce h_igh levels of nornicotine «converter) and plant lin _.es having undete.ctable levels of nornico®tine may be used as starting materials. - L eaves can then be removed fZrom plants and treated ] with esthylene to activate p450 enzymatic activities define -d herein. Total RNA is extr—acted using techniques known in the art. cDNA fragments can then be generated using PCR (RT-PCR) with the oligo d(T) primer as descri’ed in Figure 153. The <¢DNOA library can then be constructed more fully described in examples herein.
The conserved region of p45:0 type enzymes can be used a.s a template for degeneratee primers (Figure 75).
Using degenerate primers, p450 :specific bands can be amplif died by PCR. Bands indicativ-e for p450 like enzymes can be identified by DNA sequenci ng. PCR fragments can be cha—racterized using BLAST sear-ch, alignment or other tools —to identify appropriate can.didates.
Seequence information from id.entified fragments can be use=d to develop PCR primerss. These primers in combination of plasmid primers in cDNA library were used to clomne full length p450 genes. Large-scale Southern reverse= analysis was conducteed to examine the differe=ntial expression for all fr—agment clones obtained and in some cases full length clommes. In this aspect of ) the inwrention, these large-scale r-everse Southern assays can bez conducted using labeleci total cDNA’s from different tisssues as a probe to hybridiz=ze with cloned DNA fragments in order to screen all clone.d inserts. - Nonradieactive and radioactive (P32) Northern blotting assays were also used to chaaracterize clones p450 fragmen ts and full length clones.
Peptide specific antibodies we=re made against several full -length clones by deriving their amino acid sequence amd selecting peptide reegions that were antigenic and unique relative to othezx clones. Rabbit antibodies were made to synthetic peptides conjugated to a carrier protein. Western blotting analyses or other immunologica.l methods were performed on plant tissue using these antibodies.
Nucleic acid sequences identified .as described above can be examined by using virus induce=d gene silencing technology ( VIGS, Baulcombe, Current «Opinions in Plant
Biology, 199 9, 2:109-113).
Peptide specific antibodies were made for several full-length =xclones by deriving their ammino acid sequence and selectirg peptide regions that were potentially antigenic ard were unique relative to other clones.
Rabbit anti bodies were made to s ynthetic petides conjugated to a carrier protein. Western blotting analyses wer e pexfomed using these ant=ibodies.
In another aspect o f£f the invention, interfering RNAa technology (RNAi) is used to further characterize= cytochrome p450 enzymati c¢ activities in Nicotiana plants of the present invention . The following references which describe this technolog-y are incorporated by reference herein, Smith et al., Na.ture, 2000, 407:319-320; Fire et= al., Nature, 1998, 391:3-06- 311; Waterhouse et al., PNAS. 1998, 95:13959-13964; S talberg et al., Plant Molecular—
Biology, 1993, 23:671- &83; Baulcombe, Current Opinions in Plant Biology, 1999, 2:109-113; and Brigneti et al. -
EMBO Journal, 1998, 17 (22):6739-6746. Plants may be transformed using RNAi techniques, antisense techniques . or a variety of other methods described.
Several techniques exist for introducing foreigm genetic material into plant cells, and for obtainincy plants that stably main_tain and express the introduce gene. Such techniques include acceleration of genetic material coated onto microparticles directly into cellss (US Patents 4,945,050 to Cornell and 5,141,131° to
DowElanco) . Plants may be transformed using
Agrobacterium technologwy, see US Patent 5,177,010 to
University of Toledo, 5 ,104,310 to Texas A&M, Europear
Patent Application 0131624B1, European Patent:
Applications 120516, 15941881, European Patent
Applications 120516, 159 418Bl1 and 176,112 to Schilperoot
US Patents 5,149,645, 5, 469,976, 5,464,763 and 4,940,833 and 4,693,976 to Schilperoot, European Patentc
Applications 116718, 290799, 320500 all to MaxPlanck.
European Patent Applicat-ions 604662 and 627752 to Japarm
Nicootiana, European Patent Applications 02671.59, and 02592435 and US Patent 5,231,019 all to Ciba Ge2igy, US
Pa®ents 5,463,174 and 4. 762,785 both to Calgene , and US
Patents 5,004,863 and 5, 159,135 both to Agracetuss. Other transformation technologyy includes whiskers techmology,
See U.S. Patents 5,302,523 and 5,464,765 both to Zeneca.
Eleectroporation techno logy has also been mumised to transform plants, see WO 87/06614 to Boyce "Thompson
Institute, 5,472,869 &and 5,384,253 both to Dekalb,
WO0™209696 and WO093213335 both to PGS. All of these : transformation patents and publications are inco—xporated by reference. In addit-ion to numerous technoloegies for transforming plants, whe type of tissue wIhich is . comtacted with the fore® gn genes may vary as wel.l. Such tisssue would include but would not be lim ited to emlbryogenic tissue, callus tissue type I «and II, hympoocotyl, meristem, arad the like. Almost all plant ti=ssues may be transformed during dedifferentiati_on using apporopriate techniques within the skill of an a-rtisan.
Foreign genetic mat erial introduced into a pelant may ineclude a selectable marker. The preference for a paxticular marker is at the discretion of the aartisan, bu any of the following selectable markers may be used along with any other gerae not listed herein whieh could furaction as a selectable marker. Such selectable markers include but are not= limited to aminogZlycoside phosphotransferase gene of transposon Tn5 (Aph I-T) which encodes resistance to thes antibiotics kanamycin, reomycin 0 ancl G418, as well as those genes which code for
PCT/US2004/034218 resistance or tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bar); immidazolinones, sulfonylureaas and triazolopyrimidine herbic-ides, such as chlorosulfur-on; bromoxynil, dalapon and tlhme like. hl
In addition to a selectable marker—, it may be desirous to use a reporter gene. In somee instances a reporter geme may be used without a selec=table marker.
Reporter gernes are genes which are typically not present or expressecd in the recipient organism or tissue. The reporter ge=ne typically encodes for a protein which provide for some phenotypic change or enzymsatic property.
Examples of such genes are provided in K. WVeising et al.
Ann. Rev. Ge=netics, 22, 421 (1988), which i=s incorporated herein by re-eference. Preferred reporter genes include without limitation glucuronidase (GUS) «gene and GFP genes.
Once introduced into the plant tissue, the expression of the structural gene may be amssayed by any means known to the art, and expression may koe measured as mRNA transcribed, protein synthesized, or the amount of gene silenecing that occurs (see U.S. Patent No. 5,583,021 wihich is hereby incorporated b—vy reference). 15 Techniques are known for the in vitro cul ture of plant tissue, and in a number of cases, for rege=meration into whole plants (EP Appln No. 88810309.0). Perocedures for transferringgy the introduced expression complex to commercially useful cultivars are known to those skilled 0 in the art.
Once plant cells expressing the desire=ad level of p450 enzyme are obtained, plant tissues and whole plants can be regenerat.ed therefrom using methods an& techniques well-known in tke art. The regenerated planstts are then reproduced by conventional means and the introduced genes can be transferred to other strains and cwaltivars by conventional plant breeding techniques.
The following examples illustrate methods for carrying out the invention and should be under—stood to be illustrative of, but not limiting upon, the s cope of the } invention which is defined in the appended claims.
EXAMPLES
EXAMPLE T: DEVESIOPMENT OF PLANT TISSUE AN D ETHYLENE
TREATMENT
Plant Growth
Plants were seeded in pots and grown in a greenhouse for 4 weeks. The 4 week 0ld seedlings were tr—ansplanted into individual poots and grown in the greenh-ouse for 2 months. The plan ts were watered 2 times a day with water containing 150pp»m NPK fertilizer during gr—owth. The expanded green leaves were detached from plantss to do the ethylene treatmerat described below.
Cell Line 78379 10
Tobacco line 78372, which is a burley tobacco lire released by the Univercsity of Kentucky was used as a source of plant mater-ial. One hundred plants we=e cultured as standard im the art of growing tobacco arad - transplanted and tagged with a distinctive number (Z1- 100). Fertilization and field management were conductesd as recommended.
Three quarters of the 100 plants converted between 20 and 100% of the nico tine to nornicotine. One quarteer of the 100 plants converted less than 5% of the nicotikne to nornicotine. Plant rumber 87 had the least conversicon (2%) while plant number— 21 had 100% conversion. Plants converting less than 3% were classified as nor- : converters. Self-polli nated seed of plant number 87 ard plant number 21, as welll as crossed (21 x 87 and 87 x 271) geeds were made to study genetic and phenotypic differences. Plants fr-om selfed 21 were converters, arid 99% of selfs from 87 we=xre non-converters. The other MA % of the plants from 87 showed low conversion (5-15%».
Plants from reciprocal crosses were all converters.
0 34218
WO =2005/038018 PCT/US2004/ 11 Line 44
Nicotiana line 4407, which is a burley line was us ed . &=s a source of plant material. Uniform and m-epresentative plants (100) weare selected and tagged. Of the 100 plants 97 were non- converters and three we-re converters. Plant number 5& had the least amount of conversion (1.2%) and plant number 58 had the highe=st devel of conversion (96%). Self-pollenated seeds a-nd crossed seeds were made with these two plants.
Plants from selfed-58 seegregated with 3:1 convert_er #0 non-converter ratio. Plants 58-33 and 58-25, we=rxe
Sdentified as homozygous converter and nonconverter plaant
Tlines, respectively. The stadle conversion of 58-33 wwas —onfirmed by analysis of its progenies of ne=xt cyeneration.
Cell Line PBLBO1
PBLBOl is a burley line cdeveloped by ProfiGen, In.c. and was used as a source of plant material. The converter plant was selected from foundation seeds of
BEBLBO1.
ESthvlene Treatment Proceduress
Green leaves were detachead from 2-3 month greenhouse ogrown plants and sprayed wi-th 0.3% ethylene soluti on
C Prep brand Ethephon (Rhone-Pcoulenc)). Each sprayed le af 39 o was hung i_n a curing rack equipped with humidifier and covered wi.th plastic. During the treat-ment, the sample leaves we=re periodically sprayed wilh the ethylene solution. Approximately 24-48 hour post ethylene treatment . leaves were collected for RNA extraction.
Another suab-sample was taken for metaloolic constituent analysis to determine the concent ration of leaf metabolitess and more specific constitiaents of interest such as a variety of alkaloids.
As ar example, alkaloids analysis could be performed as followss. Samples (0.1 g) were shakean at 150 rpm with 0.5 ml 2M NaOH, and a 5 ml extraction solution which contained quinoline as an internal stanciard and methyl t- butyl etloer. Samples were analyzed on a HP 6890 GC equipped wvith a FID detector. A temperature of 250°C was used for the detector and injector. A.n HP columm (30m- 0.32nm-1'n) 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 100°C wit h a flow rate of 1.7cm’min—! with a split ratio of 40:1 with a 2-1 injectiorm volume using helium as the carrier gas.
EXAMPLE 2= : RNA ISOLATION
For RNA extractions, middle leave s from 2 month old greenhousse grown plants were treated with ethylene as describe. The 0 and 24-48 hours samples were used for
RNA extreaction. In some cases, leaf samples under the senescence proceags were taken from the plants 10 days post flower-headk removal. These samples were &also used ‘for extraction. Motal RNA was isolated using Rne asy Plant
Mini RKit® (Qiagem, Inc., Valencia, California) following manufacturer's p rotocol.
The tissue sample was ground under liquid nitrogen to a fine powder using a DEPC treated mortar aned pestle.
Approximately 1 00 milligrams of ground tis sue were transferred to &a sterile 1.5 ml eppendorf tuloe. This sample tube was placed in liquid nitrogen umntil all samples were col.lected. Then, 450u-1 of Buffer RLT as provided in the kit (with the addi®ion of
Mercaptoethanol) was added to each individual t—ube. The sample was vortexed vigorously and incubated at 56° C for 3 minutes. Tihe lysate was then, applied to the
QIAshredder™ spin column sitting in a 2-ml ccollection tube, and centri fuged for 2 minutes at maxim=am speed.’
The flow through was collected and 0.5 volume o—f ethanol was added to thee cleared lysate. The sample is mixed well and transferred to an Rneasy® mini spi nm column sitting in a 2 ml collection tube. The sa.mple was centrifuged for 1 minute at 10, 000rpm. Next, 700pl of buffer RWl1l was pipetted onto the Rneasy® co lum and centrifuged for 1 minute at 10,000rpm. Buffer RPE was pipetted onto tlme Rneasy® column in a new collection tube and centrifvaged for 1 minute at 10,000 rpm. Buffer
RPE was again, a:added to the Rneasy® spin column and ]
certrifuged for 2 minutes at maximum speed to dry the mermbrane. To eliminate any ethanol carry over, t he mervnbrane was placed in a separate collection tube a.nd cermtrifuged for an additional 1 minute at maximum spee=d.
The= Rneasy® column was transferred into a new 1.5 ml collection tube, and 40 pl of Rnase-free water wvaas pimoetted directly onto the Rneasy® membrane. This firmal elwmite tube was centrifuged £or 1 minute at 10, 000xrrom.
Quality and quantity of total RNA was analyzed by dematured formaldehyde gel arad spectrophotometer.
Poly (A)RNA was isolated using Oligotex™ poly A+ FRNA puxification kit (Qiagen Inc.) following manufacture's pr=otocol., About 200 pg total RNA in 250 pl maximum vo.dume was used. 2A volume of 250nl of Buffer OBB and 15 nl of Oligotex™ suspension was added to the 250 pl of to-tal RNA. The contents were mixed thoroughly by pigpetting and incubated for 3 minutes at 70°C on a he.ating block. The sample was then, placed at room temperature for approximately 20 minutes. Whe ol _dgotex:mRNA complex was pel leted by centrifugation Sor 2 minutes at maximum speed. All but 50 nul of t=he supernatant was removed from the microcentrifuge tuloe.
Thee sample was treated fuxther by OBB buffer. he ol:dgotex:mRNA pellet was resuspended in 400 ul of Buffer
OW= by vortexing. This mix was transferred onto a small spin column placed in a new tube and centrifuged for— 1 mimiute at maximum speed. The spin column was transferr—ed to a new tube and an additional 400 pl of Buffer OW2 wazag added to the column. The tube was then centrifuged or 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 OZEB.
Poly A product was analyzed by denatured formaldeh_yde © gels and spectrophotometric analysis.
EXAMPLE 3: REVERSE TRANSCRIMPTION-PCR
First strand cDNA was poroduced using SuperScripet reverse transcriptase followwing manufacturer’s protomcol (Invitrogen, Carlsbad, California). The poly A+ enriched RNA/oligo dT prime—r mix consisted of less t—han 5 ng of total RNA, 1 pl of I10mM ANTP mix, 1 nl of Ol igo d(T),;.0s (0.5pg/nl), and up to 10 ul of DEPC-treated water. Each sample was inc—ubated at 65°C for 5 minutes, then placed on ice for at least 1 minute. A reaction mixture was prepar ed by adding each of the following components in order: 2 pl 10X RT buffer, 14 nl of 25 mM MgCl2, 2pl of O .1 M DTT, and 1 nul of RNase
OUT Recombinant RNase Inhilwitor. An addition of 9 yal of reaction mixture was pirmetted to each RNA/primer mixture and gently mixed. It was incubated at 42°'C for 2 minutes and 1 pl of Super— Script II™ RT was added to each tube. The tube was irmcubated for 50 minutes alk 42°C. The reaction was terminated at 70° C for 15 minutes and chilled on ice. The sample was collected by centrifugation and 1 pl of RNase H was added to eesach tube and incubated for 20 minutes at 37°C. The seceond
PCR was carried out with 2030 pmoles of forward primeer
(degenerate primexs as in Figure 75, SEQ.ID Nos -~ 149- 156) and 100 pmoles reverse primer (mix of 18nt oligo d(T) followed by A random base).
Reaction conditions were 94°C for 2 minutess 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 wikth a 72°C extension for an extra 10 min.
Ten microliters of the amplified sample werce analyzed by electrophoresis using a 1% agarose cel. The correct size fragments were purified from agarose gel.
EXAMPLE 4: GENERATION OF PCR _FRAGMENT POPULATIONS
PCR fragments from Example 3 were ligated Ento a pGEM-T® Easy Vector (Promega, Madison, Wisconsim) following manufacturer's instructions. The ligated product was transformed into JM109 competent cel ls and plated on LB media plates for blue/white selection.
Colonies were seleacted and grown in a 96 well pl ate 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 Bi.omeck 2000 miniprep robotics with Wizard SV Miniprep® kit (Promega). Plasmid DNA was eluted with 100plwat-er and stored in a 96 wel 1 plate. Plasmids were digeste-d by
EcoR1l and were analyzed using 1% ag arose gel to confirm the DNA quantity and size of insert s. The plasmids containing a 400-600 bp insert were: sequenced using an
CEZQ 2000 sequencer (Beckman, Fuller—ton, California).
S The sequences were aligned with Gen-Bank database by
BLLAST search. The p450 related fra_gments were iclentified and further analyzed. Al ternatively, p450 fmagments were isolated from substrTaction libraries.
TEhese fragments were also analyzed as described above.
EXAMPLE 5: CONSTRUCTION OF CDNA LI_BRARY
A cDNA library was constructed by preparing total
RENA from ethylene treated leaves ass follows. First, teotal RNA was extracted from ethylesne treated leaves of toobacco line 58-33 using a modified acid phenol and chloroform extraction protocol. Pr—otocol was modified tao use one gram of tissue that was ground and swabsequently vortexed in 5 ml of extraction buffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10M EDTA; 0.5% SDS) teo which 5 ml phenol (pH5.5) and 5 ml chloroform was aclded. The extracted sample was centrifuged and the smipernatant was saved. This extraction step was 14] re=peated 2-3 more times until the ssupernatant appeared . clear. Approximately 5 ml of chlor—oform was added to re=move trace amounts of phenol. RNJA was precipitated fmrom the combined supernatant fract—ions by adding a 3- fold volume of ETOH and 1/10 volume of 3M NaOAc (pH5.2) 10 arid storing at -20¢C for 1 hour. pmafter transferring to a Corex cglass container the RNA fraction was centrifucyed at 9,000 RPM for 45 minutes at 49C. The pellet was washed with 70% ethanol and spun for 5 minutes =t 9,000 RPM at 42°C. After dryi ng the pellet, the pellexted RNA was dissolved in 0.5 ml. RNase free water. ~The pelleted RNA was dissolved in 0.5 ml RNase free wateer. The quality and quantity of total RNA was analyzed by denatured formaldehyde gel and spectrop-hotometer, respectively.
The- resultant total RNA was isolated for poly A+
RNA using an 0ligo(dT) cellulose protocol (Invitrogen) and Microcentrifuge spin columns (Invitxogen) by the followirmg protocol. Approximately twenty mg of total
RNA was subjected to twice purification to obtain high quality poly A+ RNA. Poly A+ RNA produ-ct was analyzed by performing denatured formaldehyde ge 1 and subsequent
RT-PCR of known full-length genes to en sure high quality of mRNA. ' Next, poly A+ RNA was used as tempwlate to produce a cDNA “library employing cDNA synthesiss kit, ZAP-cDNA® synthesis kit, and ZAP-cDNA® Gigapack® III gold cloning kit (Stratagene, La Jolla, Cali fornia). The method —involved following the manufactwuare's protocol as specified. Approximately 8 ug of poly A+ RNA was used to construct cDNA library. Analysis off the primary library revealed about 2.5 x 10° - 1x 1 0’ pfu. A quality background test of the library was completed by complementation assays using IPTG arad X-gal, where recombinant plaques was expressed at- more than 100-fold above the background reaction.
A more quantitative analysis off the library by random PCR showed that average size of insert cDNA was approximately 1.2 kb. The method ussed a two-step PCR method as followed. For the first =step, reverse primers were designed based on the poreliminary sequence= information obtained from p450 fragments. The designed reverse primers and T3 (forward) primers were used amplify corresponding genes from th-e cDNA library. PCRR reactions were subjected to agarose electrophoresis anc the corresponding bands of high mol ecular weight were excised, purified, cloned and sequenced. In the secon step, new primers designed from 5'U TR or the start coding region of p450 as the forwar-d primers together with the reverse primers (designed from 3'UTR of p450) were used in the subsequent PCR to obtain full-length p450 clones.
The p450 fragments were genera_ted by PCR amplification from the constructed cDNA library as described in Example 3 with the exczeption of the reverse primer. The T7 primer locat-ed on the plasmid downstream of cDNA inserts (see Figyure 75) was used as a reverse primer. PCR fragments we=re isolated, cloned and sequenced as described in Examgole 4.
Full-length p450 genes were issolated by PCR methocd from constructed cDNA library. Gene specific reverse primers (demsigned from the downstream sequence of p450 fragments) and a forward primer (T3 on library plasmid) - were used t—o clone the full length genees. PCR fragments wrere isolated, cloned and sexquenced. If necessary, second step PCR was applied . In the second step, new f=orward primers designed froam 5'UTR of cloned p450s toget=her with the reverse primer s designed from 3'UTR of pAs0 clones were used in the subsequent PCR 10m reactions t—o obtain full-length p450 c lones. The clones were subsequently sequenced.
EXAMPLE 6: CHARACTERIZATION OF CLONED FRAGMENTS —
REVERSE SOUJTHERN BLOTTING ANALYSIS
Nonracilioactive large scale reversse southern blotting asssays were performed on all p450 clones identified in above examples to detect the differential expression . It was observed that the 1 evel of expression among different p450 cluste=rs was very different. Further real time detection was conducted on those w=ith high expression.
Nonracdioactive Southern blotting procedures were conducted as follows. 1) Teotal RNA was extracted from ethylene treated and nontreated converter (58-33) and rmonconverter (58- 25) leaves using the Qiagen Rnaeasy ki. t as described in
Example 2.
2) Probe was produced by bioti_n-tail labeling a : single stxr-and cDNA derived from poly A+ enriched RNA - generated in above step. This label ed single strand cDNA was cgenerated by RT-PCR of the converter and nonconver-ter total RNA (Invitrogen) as described in
Example 3 with the exception of using biotinalyted oligo AT as a primer (Promega). The se were used as a probe to hybridize with cloned DNA. 10 . 3) plasmid DNA was digested wazith restriction enzyme EctoR1l and run on agarose gel _s. Gels were simultaneously dried and transferread to two nylon membraness (Biodyne B®). One membrane was hybridized with consserter probe and the other with nonconverter probe. Membranes were UV-crosslinke=d (auto crosslink setting, 254 nm, Stratagene, Strat=alinker) before hybridization.
Alt ernatively, the inserts we:xe PCR amplified fmrom each pla smid using the sequences 1 ocated on both armss of p-GEM: plasmid, T3 and SP6, as perimers. The PCR products: were analyzed by running on a 96 well Ready-— to-run agarose gels. The confirmed inserts were dott ed on two raylon membranes. One membramne was hybridized with coraverter probe and the other— with nonconvertexr— probe. 4) The membranes were hybridized and washed 30e followirg manufacture’s instruction with the
WO» 2005/038018 PCT/US2004/0-34218 modification of washing stringency (Enzo MaxSence™ kit,
Enzo Diagnostics, Inc, Farmingdale, MY). The membrar-ies were prehybridized with hybridizatior: buffer (2x ssc buffered formamide, containing detergent and hybridization enhancers) at 42°C for 30 min and hybridized with 10ul denatured probe overnight at 42°sC.
The membranes then were washed in 1X hybridization wash : buffer 1 time at room temperature foxx 10 min and 4 times at 68°C for 15 min. The membranes were ready for the detection. oo 5) The washed membranes were de tected by alkali me phosphatase labeling followed by NBT- /BCIP colometric: detection as described in manufactur-e’s detection procedure (Enzo Diagnostics, Inc.). The membranes we=re blocked for one hour at room tempera ture with 1x blocking solution, washed 3 times wi th 1X detection reagents for 10 min, washed 2 times with 1x predevelopment reaction buffer for 5. min and then developed the blots in developing soslution for 30-45= min until the dots appear. All reage=nts were provided by manufacture (Enzo Diagnostics, Imc). In Addition, large scale reverse Southern assay wwas also performe=d using KPL southern hybridization andl detection kit™ following manfacturer’s instruction CKPL, Gaithersbur—g,
Maryland).
EXAMPLE 7; eCHARACTERIZATION OF CLONES - NORTHERN BLOT
ANALYSIS
. Altern. ative to Southern Blot analysis, some membranes wwere hybridized and detected ass described in the examples of Northern blotting assays. Northern
Hybridization was used to detect mRNA dif~ferentially expressed in Nicotiana as follows.
A random priming method was used to prepare probes from cloned p450 (Megaprime™ DNA Labellimng Systems,
Amersham Biosciences).
The feollowing components were mixed : 25ng denatured IDNA template; 4ul of each unlabeled dTTP, dGTP and deCTP; 5ul of reaction buffer; P-*-labelled dATP and 2-ul of Klenow I; and H,0, to br—ing the reaction t-o 50pl. The mixture was incubeated in 37°C for 1-4 ho-urs, then stopped with 2pl of 0.5 M EDTA.
The probe —was denatured by incubating at= 95°C for 5 minutes be fore use.
RNA s.amples were prepared from ethylene treated and non-tr-eated fresh leaves of several pairs of tobacco li_nes. In some cases poly A+ ermriched RNA was used. Apperoximately 15ug total RNA or 1 .8ug mRNA (methods of RNA and mRNA extraction as Clescribed in
Example 5) were brought to equal volume with DEPC H,0 (5~10 nl). The same volume of loading kouffer (1 x
MOPS; 18.5 % Formaldehyde; 50 % Formamicle; 4 $%
Ficoll400; Bromophenolblue) and 0.5 ul EtBr (0.5 ug/ul) were added. The samples were subsequently Senatured in preparation for separation of the RNA by electrophoresis . - }
S
Samples were subjected to electrophoressis on a formaldehyde gel (1 $ Agarose, 1 x MOPS, 0. 6M
Formaldehyde) with 1XMOP buffer (0.4 M
Morpholinopropanesulfonic acid; 0.1 M Na-ac etate-3 x
H20; 10 mM EDTA; adjust to pH 7.2 with NaOH:=). RNA was transferred to a Hybond-N+ membrane (Nylon, Amexsham
Pharmacia Biotech) by capillary method in 1.0 X ssc buffer (1.5 M WaCl; 0.15 M Na-citrate) for 24 hours. 1= Membranes with RNA samples were UvV-crosslirmaked (auto crosslink sett ing, 254 nm, Stratagene, Straatalinker) before hybridi zation.
The menmbrane was prehybridized for 1-4 hours at 42°C with 5-10 ml prehybridization buffer (C5 x SSC; 50 % Formamide; 5 x Denhardt's-solution; 1 % =SDS; 100ug/ml heat-denatured sheared non- homologous DNA ). 01d prehybridizati.on buffer was discarded, and new prehybridization buffer and probe were add-ed. The hybridization was carried out over night a t 42°C. The membrane was washed for 15 minutes with 2 .x SSC at room temperature, Followed by a wash with 2 x S.8C. 0
A major focus of the inventior 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. As illustrated in the table= below, Northern blots and reverse Southern Blot were useful in determining which genes wwere induced by ethylene treatment relative to non—induced plants.
Interestingly, not all fragments weere affected similarly in the converter and nonsconverter. The cytochrome p450 fragments of inters=est were partially sequenced to determine their structural relatedness.
This information was used to subseequently isolate ani characterize full length gene clon es of interest.
Indumced mRNA Expressiom
Fragments Efthylene Treatment i D56-AC7 (SEQ ID No: 35) t+ + 1
D56-AG11 (SEQ ID No: 31)
D56-AC12 (SEQ TD No: 45)
D70A-AB5 (SEQ ID No:
D73-AC9 (SEQ ID No: 43
D70A-AA12 (SEQ ID No:
D73A-AG3 ID No: 129)
D34-52 (SEO ID No: 61)
D56-AG6 (SEQ ID No: 51)
Northern analysis was perform ed using full lengt—h clones on tobacco tissue obtained from converter and nonconverter burley lines that wer-e induced by ethylesne treatment. The purpose was to identify t hose full length clones that showed elevated expres-sion in ethylene i_nduced converter lines relative= to ethylene induced converter lines relative to ethyl ene induced nonconvert—er burley lines. By so doing, the functiona® ity relationship of full lengtln clones may be determined by comparing biochemical diffearences in leaf constituerts between converter and noncoraverter lines.
As shown =n table below, six clones showead L significartly higher expression, as denoted by ++ and +++, in converter ethylene treated tissue than that of nonconver®er treated tissue, denoted by «. All of these clomes showed little or no expression in converter and nonconverter lines that wemre not ethylene treated.
Clones 0
EXAMPLE 8: IMMFUNODETECTION OF pd50S ENCODED BY THE CLONED
GENES
Peptide r-egions corresponding to 20-22 ammino acids in length from three p450 clones were selected feor 1) having lower or no homology to other clones and 2) having good hydrophilicity and anti genicity. The amino acid ssequences of the peptide regions selected from the respective pd450 clones are listed below~. The synthesized peptides were conjugated with KHL and. then injected into rabbites.
Antisera were collected 2 and 4 weeks after the 4*™ injection (Alpha Diagno stic Intl. Inc. San Antonio, TX).
D234-AD1 DIDGSKSKLVKAHIRKIDEILG
D90a-BB3 RDAFREKETFDEN_DVEELNY
DB9-AB1 FKNNGDEDRHF SQ KLGDLADKY
Antisera were exam-ined for crossreactivity tos target proteins from tobacco pllant tissue by Western Blot- analysis.
Crude protein extracts wwere obtained from ethylene treated {0 to 40 hours) middle leaves of converter and non. converter lines. Protein concentmations of the extracts wer e determined using RC DC MProtein Assay Kit (BIO-RAD) following the manufacturer‘s protocol. =20
Two micrograms of protein were loaded onto easch lane and the proteins separated on 10% - 20% gradient geels using the Ilmaemmli SDS-PAGE sysstem. The proteins were transferred from gels to PROTRAN® Nitrocellulose Transfer Membmranes (Schleicher & Schuell) wryith the Trans-Blot® Semi-Dwy cell (BIO-RAD). Target p450 proteins were detected and visualized with the ECL Advance™ Western Blotting Detection
Kit (Amersham Bioscience=s). Primary antibodies agaainst the synthetic-KLH conjugates. were made in rabbits. Secondary antibody against rabbit IgG, coupled with peroxidasse, was purchased from Sigma. Both. primary and secondary antibodi es: were used at 1:1000 dilutions. Antibodies showed strong reactivity to a single band. on the Western Blots indicatin-g that the antisera were mono-specific to the target peptide of interest. Antisera were al so crossreactive with synthetic peptides conjuated to KLH.
EXAMPLE 9: NUCLEIC ACID IDEENTITY AND STRUCTURE RELATEDNESSS
OF_ISOLATED NUCLEIC ACID FR AGMENTS
Over 100 cloned p450 f£ ragments were sequenced in conjunction with Northern belot analysis to determine their~ structural relatedness. Th.e approach used utilized forward primers based either of two common p450 motifs located nea x the carboxyl-terminus of th.e p450 genes. The forward primers corresponded to cyt ochrome p450 motifs FXPERF or
GRRXCP(A/G) as denoted in F igure 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 decuble stranded DNA following th-e manuf acturer’s protocol (Beckman Coulter). The template was diluted with water to the ampopropriate concentration, denatured by heating at 95° CC for 2 minutes, and subsequently placed on ice. The sequencing reaction was prepared on ice using 0.5 too 10ul of denatured DNA template, 2 pl of 1.6 pmole of the fomward primer, 8 pl of DTCS Quick start Master Mix and the totczal volume brought to 20 nl with water. The thermocycling program consisted of 390 cycles of the follow cycle: 96° C for 20 seconds, 50° C fox 20 seconds, and 60° CC for 4 minutes followed by hol«ding at 4°
Cc. -
The sequence was stopped by adding 5 pl of stop buffer (equal volume of 3M NaOAc and 100mM EDTA and 1 nl of 20 ng/ml glycogen). The sample was precipitated wi-th 60 nl of cold 95% ethanol and centrifuged at 6000g for 6 minutes.
Ethanol was disca rded. The pellet was 2 washes with 200 nl of cold 70% ethanol. After the pellet was dry, 40 ul of SLS solution wags added and the pellet was resuspended. A layer of mineral oil wa.s over laid. The sample was t=hen, placed on the CEQ 8000 A.utomated Sequencer for further analysis.
In order to verify nucleic acid sequences, nucleic acid sequence was re-ssequenced in both directions usi® ng forward primers to the FXIPERF or GRRXCP(A/G) region of t=he p450 gene or reverse primers to either the plasmid or polis A tail. All sequencing was pesrformed at least twice in both directions.
L
The nucleic acid sequences of cytochrome p#&50 fragments were compared to each other from the coding region corresponding to the first nucleic acid after tke region encoding the GRRXCP(A/G) motif through to the stop codon.
This region was sselected as an indicator of genetic diversity among pp450 proteins. A large number of genetically distinct p450 genes, in excess of 70 genes, were observed, similarc to that of other plant speciess. Upon comparison of nucleic acid sequences, it was fouand that the genes could be placed into distinct seqLaences groups based on their sequence identity. It was fournd that the best unique growvaping of p450 members was determined to be those sequences with 75% nucleic acid identitw or greater {shown in Table I) . Reducing the percentage iclentity resulted in significant=ly larger groups. A preferre=d grouping was observed for those sequences with 81% nwacleic acid identity or greater, a more preferred grouping 971% nucleic acid identity oxr— greater, and a most preferred grouping for those sequences 99% nucleic acid identity of «greater. Most of the groups contained at least two members amd 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 mused.
Based on 75% nucleic acid identity or greater, two cytochrome 450 groups were found to comntain nucleic acid sequence identity to previously tobacco cytochrome genes that genet® cally distinct from that witBhin the group. Group 23, showed nucleic acid identity, withimn the parameters used for Table IT, to prior GenBank sequences of GI:1171579 (CAA64635) and GI:14423327 (or AAK62346]) by Czernic et al and Ralstora et al, respectively. GI:11771579 had nucleic acid identi ty to Group 23 members rangirng 96.9% to 99.5% identity to members of Group 23 while GIX:14423327 ranged 95.4% to 96.9% identity to this group. The members of Group 31 had nucl_eic acid identity ranging freom 76.7% to 97.8% identity to the GenBank reported sequenc—e of GI:14423319 (AAK62342) by Ralston et al. None of tlie other p450 identity gr-oups of Table 1 contained par-ameter identity, as
PCT/MTIS2004/034218 used in Tabl._e 1, to Nicotiana p450s awenes reported boy
Ralston et zal, Czernic et al., Wang =t al or LaRosa and
Smigocki.
As shown in Figure 76, consensuss sequence with appropriate nucleic acid degenerate orobes could be derived for group tc» preferentially identify and isolate additional members of each group from Nicotiana plants.
TT 59 le TI: icoti A450 Nucleic Aci d Sequence Ideratit
CSroups - CSROUP FRAGMENTS 21 DS58-BG7 (SEQ ID No.:1), D58-ABL. (SEQ ID No.:3D; D58-BE4 (SEQ ID No.:7) =2 D56-AH7 (SEQ ID No.:9); Dl3a-5 (SEQ ID No.:11D 3 D56-AG1l0 (SEQ ID No.:13); D35-3 3 (SEQ ID No.:715);
FD34-62 (SEQ ID No.:17) <4 D56-AA7 (SEQ ID No.:19); D56-AE=l (SEQ ID No.:=21); 7185-BD3 (SEQ ID No. :143) [= D35-BB7 (SEQ ID No.:23); D177-B-A7 (SEQ ID No. =25);
ID56A-AB6 (SEQ ID No.:27); D144-AE2 ( SEQ ID No.:29) se D56-AG1l1 (SEQ ID No.:31); D179-_AAl (SEQ ID No. :33) 4 D56-AC7 (SEQ ID No.:35); D144-2D1 (SEQ ID No. =37) : 8 D144-AB5 (SEQ ID No.:39) b= D181-AB5 (SEQ ID No.:41l); D73-Ae«c9 (SEQ ID No. :: 43) . 10 D56-AC12 (SEQ ID No. :45) 1.1 D58-AB9 (SEQ ID No.:47); D56-AGS™ (SEQ ID No.:4-9);
ID56-AG6 (SEQ ID No.:51); D35-BGll (SEQ ID No.:53); D35-42
C SEQ ID No. :55); D35~-BA3 (SEQ ID No .:57); D34-57" (SEQ ID
NHo.:59); D34-52 (SEQ ID No.:61); D34-25 (SEQ ID® No.:63) 1.2 DS6-AD10 (SEQ ID No.:65) 13 56-8Al11l (SEQ ID No.:67) 14 D177-BDS (SEQ ID No.:69); D177-E3D7 (SEQ ID No. :83)
PCT/US2004/034218 15 DS56A-AG10 (SEQ ID No.:71) ; D58-BCS (SEQ ID No.:73);
E58-AD12 (SEQ ID No.:75) : 216 D56~AC11 (SEQ ID No.:77); D35-39 (SEQ ID No.:79);
ID58-BH4 (SEQ ID No.:81); D56-AD6 (SEQ ID No.:87) 1.7 D73A-aD6 (SEQ ID No.:89); D70A-BAall (SEQ ID No.:91) 1.8 D70A-ABS5 (SEQ ID No.:95); D70A-AAS8 (SEQ ID No.:97) 1.9 D70A-AB8 (SEQ ID No.:99); D70A-BH2 (SEQ ID No.:101);
D»70A-AA4 (SEQ ID No. :103) 20 D70A-BAl (SEQ ID No. +105) » D70A-BA9 (SEQ ID No.:107) 2.1 D70A-BD4 (SEQ ID No.:109) 22 DI181-AC5 (SEQ ID No.:111); D144-AH1 (SEQ ID No.:113);
D34-65 (SEQ ID No. :115) 23 D35-BG2 (SEQ ID No.:117) 24 D73A-AH7 (SEQ ID No.:119) 25 D58-AAl (SEQ ID No.:121); ID185-BCl (SEQ ID No.:133);
D1 85-BG2 (SEQ ID No. :135) 26 D73-AE10 (SEQ ID No.:123) 27 D56-AC12 (SEQ ID No.:125) 28 D177-BF7 (SEQ ID No.:127); D185-BEl (SEQ ID No.:137);
D1E35-BD2 (SEQ ID No.:139) 29 D73A-AG3 (SEQ ID No.:129)
D70A-AA12 (SEQ ID No.:131); D176-BF2 (SEQ ID No. :85) 31 D176-BC3 (SEQ ID No.:145) 32 D176-BB3 (SEQ ID No.: 1.47)
TT 6L
33 D186—2aH4 (SEQ ID No.:5)
EXAM-PLE 10: RELATED AMINO ACID SEQUENCE IDENTITY €OF
ISOLATED NUCLEIC ACID FRAGMENTS
The amino acid sequen.ces of nucleic acid sequ ences obta ined for cytochrome p4 50 fragments from Exampl e 8 were deduced. The deduced region corresponded to the amin .© acid immediately after the GXRXCP(A/G) seque=nce moti f to the end of the carboxyl-terminus, or stope codo-ni. Upon comparison of sequence identity of time fragments, a unique grouping was observed for thosse sequ_ences with 70% amino acid identity or greater. a pref erred grouping was obsserved for those sequence=s witha 80% amino acid identi ty or greater, more preferred witlhm 90% amino acid identi ty or greater, and a mosst prefS erred grouping for those sequences 99% amino acid idem tity of greater. The groups and corresponding amirmo acid sequences of group members are shown im
Figmzre 2. Several of the unique nucleic acid sequences were= found to have complet-e amino acid identity to ] othe=r fragments and therefore only one member witla the idemtical amino acid was reported. 5 The amino acid identity for Group 19 of Table II corr—esponded to three distinct groups based on their - nucl.eic acid sequences. he amino acid sequences of eacm 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. In addition, group members that are differentially affected by ethylene treatment or other biological differencess as assessed by Northern and Southern analysis were selected for gene cloning and functional studies. To assist in gene cloning, expression studies and whole plant evaluations, peptide specific antibodies will be prepared on sequence identity and differential se=quence.
Table II: Nicot] 450 Amino Acid Sequence I-denti Groups
GROUP FRAGMENTS
1 D58-B@7 (SE2Q ID No.:2), DS58-AB1 (SEQ ID MNJo.:4) 2 D58-BE4 (SEEQ ID No. :8) 3 D56-AH7 (SEQ ID No.:10); Dl3a-5 (SEQ ID XJo.:12) 4 D56-AGL_0 (SEQ ID
No.:14); D34-62 (SEQ ID No.:18) 5 D56-AA7 (SEEQ ID No.:20); D56-AEl (SEQ ID No.:22); 185-
BD3 (SEQ ID No. = 144) 6 D35-BB7 (SE=Q ID No.:24); D177-BA7 (SEQ IID No.:26);
DS56A-AB6 (SEQ IID No.:28); D144-AE2 (SEQ ID No :30) 7 D56-AG11l (SSEQ ID No.:32) ; D179-AAl (SEQ ID No.:34) 8 D56-AC7 (SE2Q ID No.:36); D144-AD1 (SEQ IID No. :38) 9 D144-AB5 (SSEQ ID No. :40) 10 D181-AB5 (SSEQ ID No.:42); D73-Ac9 (SEQ IID No.:44) 11 D56-AC12 (SSEQ ID No. :46) 12 D58-AB9 (SEEQ ID No.:48); D56-AGY9 (SEQ ID No.:50); DS6-
AG6 (SEQ ID No. =52); D35-BGl11 (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 No.:62) 13 D56AD10 (SESQ ID No.:66) 5 14 56-AAll (SEEQ ID No.:68)
15 D177-BD5 (SEQ ID No.:70); D177-BD7 (SEQ ID MNo.:84) 16 D56A-AGLO (SEQ ID No.:72); D58-BCS5 (SEQ ID WNo.:74):
D58-AD12 (SEQ ID No. :76) 17 DS6-ACll (SEQ ID No.:78); D56-AD6 (SEQ ID N o.:88) 18 D73A-AD6 (SEQ ID No.90:) 19 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) 20 D70A-BD4 (SEQ I'D No.:110) 21 D181-ACS5 (SEQ ID No.:112); D144-AH1 (SEQ ID® No.:114);
D34-65 (SEQ ID No.:116) 22 D35-BG2 (SEQ ID No.:118) 23 D73A-AH7 (SEQ I'D No.:120) 24 D58-AA1 (SEQ ID No.:122); D185-BCl (SEQ ID No.:134);
D185-BG2 (SEQ ID No. :136) 25 D73-AEl10 (SEQ ID No.:124) 26 DS56-ACl2 (SEQ XD No.:126) 27 D177-BF7 (SEQ ID No.:128); 185-BD2 (SEQ ID No.:140) 28 D73A-AG3 (SEQ XD No.:130) 29 D70A-AAl12 (SEQ ID No.:132); D176-BF2 (SEQ ID No.:86)
D176-BC3 (SEQ ID No.:146) 31 D176-BB3 (SEQ ID No.:148) 32 D186-RH4 (SEQ ID No.:6)
EXAMPLE 11: RELATED AMINO ACID SEQUENCE IDENTITY OF FULL
LENGTH CLONES
The nucleic acid sequences of full length Nicotiana genes cloned in Example 5 were= deduced for their— entire ~ amino acid sequence. Cytochrcome pd450 genes were= identified by the presence of three conse=rved p450 domain mwotifs, which corresponded to UXXRXXZ, PXRF=XF or GXRXC at the carboxyl- terminus where U is E or K, X is any amino acid and Z is P,
T, S or M. It was also noted that two of the cl ones appeared nearly complete but “Lacked the appropri ate stop codon, D130-AAl and D101-BA2, however but both ¢ ontained all three p450 cytochrome domains . All p450 genes were characterized for amino acid # dentity using a BL AST program comparing their full length sequences to each other and to known tobacco genes. The procyram used the NCBI special
BLAST tool (Align two sequences (bl2seq), attp: //www.ncbi.nlm.nih.gov/bl _ast/bl2seg/bl2 htm 1). Two sequences were aligned under EBLASTN without filteer for nucleic acid sequences and BLMSTP for amino acid sequences.
Based on their percentage amirmo acid identity, each sequence wwas grouped into identity growmps where the group=ing «contained members that shared at least 85% ident—ity with &nother member. A preferred grouping was observead for those
Sequences with 90% amino acid identity or greatem, a more preferred grouping had 95% ami mo acid identity ox greater, &nd a most preferred grouping “had those sequencess 99% amino acid identity or greater. Usimg these criteria, 25 unique groups were identified and are depicted in Table III. oo 66 .
Within t=he parameters used for Table IIT- for amino acid identity, thr-ee groups were found to contain greater than 85% or greate=r identity to known tobacco gene=s. Members of
Group 5 had rap to 96% amino acid identity for- full length sequences to prior GenBank sequences of GI:14-423327 (or
AAK62346) by Ralston et al. Group 23 had up to 93% amino acid identity to,GI:14423328 (or ARK62347) by- Ralston et al. and Group 24 had 92% identity to GI:14423318 (or AAK62343) by Ralston et= al.
Table III: Amnino Acid Sequence Tdentitv Groupes of Full
Length Nicoti ana p450 Genes 1 D208-ADP (SEQ. ID. No. 224); D120-AH4 (S EQ. ID. No. 180); DI1_21-AA8 (SEQ. ID. No. 182), D122-_aAF1l0 (SEQ. ID.
No. 184) ; D103-aH3 (SEQ. ID. No. 222); D 208-AC8 (SEQ.
ID. No. 218); D-235-ABI (SEQ. ID. No. 24 6) 2 D244-AD4. (SEQ. ID. No. 250); D244-AB6 (SEQ. ID. No. 274) ; Dw285-AAB; D285-AB9; D268-AE2 (SEQ . ID. No. 270) ’ 3 D100A-AC*3 (SEQ. ID. No. 168); D1OGCA-BE2 4 D205-BE9 (SEQ. ID. No. 276); D205-~-BGS (SEQ. ID. No. ] 202); D2 05-aH4 (SEQ. ID. No. 294) 5 D259-ABS (SEQ. ID, No. 260) ; D257-AE4 (SEQ. ID. No. 268); D1-47-AD3 (SEQ. ID. No. 154) 6 D249-AE8 (SEQ. ID. No. 256); D-248-AA6 (SSEQ. ID. No. 254) 7 D233-AG7 (SEQ. ID. No. 266; D224-BDl11 (SEQ. ID. No. 240); DAE"10 8 D105-AD6 (SEQ. ID. No. 172); D215-AB5 (SE=Q. ID. No. 0 220); D1335-AEl1 (SEQ. ID. No. 190)
9 D8TA-AF3 (SEQ. ID. No. 2216), D210-BD4 (SEQ. ID. No. 10 D89-AB1 (SEQ. ID. No. 1 50); Dg 9-AD2 (SEQ. ID. Mo. 152); 163-AGl1 (SEQ. ID. No. 198); 163-AF12 (SEQ.- ID No. 196) 11 D267-AF10 (SEQ. ID. No. 296); DI96-AC2 (SEQ. ID . No. 160); D96-AB6 (SEQ. ID- No. 158); D207-AA5 (SEQ. ID.
No. 204); D207-AB4 (SEQ. ID. No. 206); D207-AC 4 (SEQ.
ID. No. 208) 12 D98-2AG1 (SEQ. ID. No. -164); D98-AAL (SEQ. Ip. No. 162) 13 D209-AA12 (SEQ. ID. No . 212); D209-AA11l; D209—AH10 (SEQ. ID. No. 214); D2 09-AH12 (SEQ. ID. No. 232);
D90a-BB3 (SEQ. ID. No. 154) 14 D129-AD10 (SEQ. ID. NSo. 188); D104A-AES (SEQ. ID. No. 170) 15 D228-AHS (SEQ. ID. No. 244); D228-AD7 (seg. ID. No. 241), D250-AC11 (SEQ~ ID. No. 258); D247-AH1 (SEQ.
ID. No. 252) 16 D128-AB7 (SEQ. ID. No . 186) ; D243-AA2 (SEQ. ID. No. 248); D125-AFll (SEQ . ID. No. 228) 17 D284-AHS5 (SEQ. ID. Neo. 298); D110-AF12 (SEQ®. ID. No. 176) 18 D221~-BB8 (SEQ. ID. No . 234) 19 D222-BH4 (SEQ. ID. No. 236) 20 D134-AE11l (SEQ. ID. Mo. 230) 21 DL09-AH8 (SEQ. ID. N¥o. 174) 22 D136-AF4 (SEQ. ID. No. 278) 23 p237-AD1 (SEQ. ID. No. 226) 24 D112-AAS5 (SEQ. ID. NJo. 178) 25 D283-AC1 (SEQ. ID. No. 272)
The full length genes were furthear grouped based on the highly conversed amino acid homology Ibetween UXXRXXZ p450 domain and GXRXC p450 domain near the end the carboxyl- terminus. As shown in Figure 3, indiwridual clones were aligned for their sequence homology beatween the conserved domains relative to each other and placed in distinct identity groups. In several cases, although the nucleic acid sequence of the clone was unique,. the amino acid sequence for the region was identical. The preferred grouping was observed for those sequerices with 90% amino acid identity or greater, a more prefearred group had 95% amino acid identity or greater, and a most preferred grouping had those sequences 99% amino acid identity of greater. The final grouping was similar to that based on the percent identity for the entire armino acid sequence of : the clones except for Group 17 (of Talole III) which was divided into two distinct groups.
Within the parameters used for armino acid identity in
Table IV, three groups were found to contain 90% or greater identity to known tobacco genes. Memloers of Group 5 had up to 93.4% amino acid identity for full length sequences to prior GenBank sequences of GI:14423326 (AAKE62346) by Ralston et al. Group 23 had up to 91.8% amino acid identity to
GI:14423328 (or AAK62347) by Ralston et al. and Group 24 had 98.8% identity to GI:14423318 (or AAK&2342) by Ralston et al.
PCT/US20804/034218
Tables TV: Amino Acid Sequence Identity Groups of Regioras betwesen Congerved Domains of Ni«cotiana p450 Genes 11 D208-AD9 (SEQ. ID. No. 224); D120-AH4 (SEQ. ID. Ne. 180); D121-AA8 (SEQ. ID. No. 182), D122-AF10 (SEQ . ID.
No. 184); D103-AH3 (SEQ. ID. No. 222); D208-AC8 (=SEQ.
ID. No. 218); D-235-ABI (SEQ. ID. No. 246) 2 D244-2AD4 (SEQ. ID. No. 250); D244-aB6 (SEQ. ID. Neo. 274) ; D285-AA8; D285-AB9; D268-AE2 (SEQ. ID. No. 270) 3 D100A-AC3 (SEQ. ID. No. 16 8); D100A-BE2 4 D205-BES (SEQ. ID. No. 276); D205-BG9 (SEQ. ID. Neo. 202) ; D205-AH4 (SEQ. ID. No. 294) 5 D259-AB9 (SEQ. ID. No. 260) ; D257-AE4 (SEQ. ID. No. 268); D147-AD3 (SEQ. ID. No. 194) 6 D249-AE8 (SEQ. ID. No. 256); D-248-AA6 (SEQ. ID. No. 254) 7 D233-AG7 (SEQ. ID. No. 266 ; D224-BD1l (SEQ. ID. Neo. 240); DAF10 8 D105-AD6 (SEQ. ID. No. 172); D215-AB5 (SEQ. ID. Neo. 220) ; D135-AEl1 (SEQ. ID. No. 190) 9 D87A-AF3 (SEQ. ID. No. 216), D210-BD4 (SEQ. ID. Neo. 262) 10 D89-AB1 (SEQ. ID. No. 150) ; D89-AD2 (SEQ. ID. No. 152); 163-AG11 (SEQ. ID. No. 198); 163-AF12 (SEQ. ID. Neo. 196) 11 D267-AF10 (SEQ. ID. No. 29 6); D96-AC2 (SEQ. ID. Neo. 160); D96-AB6 (SEQ. ID. No . 158); D207-AA5 (SEQ. =ID.
No. 204); D207-AB4 (SEQ. ID. No. 206); D207-AC4 (S=SEQ.
ID. No. 208) 12 D98~AGl (SEQ. ID. No. 164) ; D98~AAl (SEQ. ID. No. 162)
N 70
1.3 D209-AA12 (SEQ. ID. No. 212); ID20S-AAll; D209-AH10 (SEQ. ID. No. 214); D209-AH12 (SEQ. ID. No. 232):
D90a-BB3 (SEQ. ID. No. 154) nd D129-AD10 (SEQ. ID. No.- 188): D104A-AE8 (SEQ. ID. No.. 170) ns D228-AH8 (SEQ. ID. No. 244); EWD228-aD7 (SEQ. ID. No. 241), D250-AC1l1 (SEQ. ID. No. 258); D247-aH1 (SEQ.
ID. No. 252) a6 D128-AB7 (SEQ. ID. No. 186) ; ID243-AA2 (SEQ. ID. No. 248); D125-AF11 (SEQ. ID. No. 228) a7 D284-AHS (SEQ. ID. No. 298); D110-AFl2 (SEQ. ID. No. 176) 18 D221-BB8 (SEQ. ID. No. 234) a9 D222-BH4 (SEQ. ID. No. 236) =20 D134-AE11 (SEQ. ID. No. 230) =21 D109~-AH8 (SEQ. ID. No. 174) =22 D136-AF4 (SEQ. ID. No. 278) =23 D237-AD1 (SEQ. ID. No. 226) 24 D112-AAS (SEQ. ID. No. 178) =25 D283-AC1l (SEQ. ID. No. 272) =26 D110-AF12 (SEQ. ID. No. 176)
IEXAMPLE 12: NICOTIANA CYTOCHROME P4 50 CLONES LACKING ONE OR ™0ORE OF THE TOBACCO CYTOCHROME Pp450Q SPECIFIC DOMATNS
Four clones had high nucleic a cid homology, ranging 90% #0 99% nucleic acid homology, to otTher tobacco cytochrome genes reported in Table III. The f-our clones included D136—
AAD5, D138-AD12, D243-AB3 and D250-A«Cll. However, due to a rucleotide frameshift these genes d.id not contain one or more of three C -terminus cytochrome p45® domains and were excluded from i dentity groups presented in Table IIT or
Table IV.
The amino acid identity of one cloene, D95-~-AG1l, did not= contain the thi_.rd domain, GXRXC, used t=o0 group p450 tobacco genes in Table III or Table IV. The numcleic acid homology of this clone ad low homology to other— tobacco cytochrome genes. This cM.one represents a novel =and different group of cytochrome p45) genes in Nicotiana.
EXAMPLE 13: USHT OF NICOTIANA CYTOCHROMES P450 FRAGMENTS AND
CLONES IN ALTEERED REGULARTION OF TOBACCO PROPERTIES
The use o—f tobacco p450 nucleic aacid fragments or who le genes are usef-ul in identifying and seTlecting those plants that have alte:xred tobacco phenotypes o=r tobacco constituen ts and, more impo xtantly, altered metabol.ites. Transgenic tobacco plants are generated by a vari-ety of transformation systems that i-mcorporate nucleic acid fragments or full length genes, selected from those repo rted herein, in orientations fF or either down-regulation, for example anti— sense orientat.ion, or over-expression for example, sense orienation. F—or over-expression to fu ll length genes, any nucleic acid ss=equence that encodes the= entire or a functional par—t or amino acide sequenc=e of the full-lengtina genes describe=d in this invention are desired that are effective for increasing the expressicon of a certain enzyme and thus resul_ting in phenotypic effect within Nicotiana.
Nicotiana line=s that are homozygous li_nes are obtained through a series o¥ backcrossing and assessed for phen» _otypic changes including, but not limited to, analysis of endogenous pd50 RNA, transcripts, p450 expressed pepti des and concentrations of plant metabolites using techniqu-.es commonly avaiable to one having ordinary skill in the art.
The changes exhibited in the tobacco plans provide information on the functional role of the selected gene of interest or are of a utility as a preffered Nicotiana plant species.
ExMPLE 14. IDENTIFICATION OF GENES INDUCED IN ETHVLENE T=REATED
CONVERTER LINES
High density oligonucleotide array technology, Affymetrix
GeneChip® (Affymet-rix Inc., Santa Clara, CA) array, waas used for quantitative and highly parallel measurements o—E£ gene expression. In using this technology, nucleic acid arrays were fabricated by direct synthesis of oligonucleotides on a solid surface. This solid-phase chemistry is able to poxoduce arrays containing hundreds of thousands of oligonucl _eotide probes packed at extremely high densities on a chip referred to as GeneChip®. Thousands of genes can be simultaroeously screened from a simgle hybridization. Each gene is tyroically represented by a set of 11-25 pairs of probes dependirmg upon size. The probes are designed to maximize sensit-ivity, specificity, and reproducibility, allowing conssistent discrimination bet-ween specific and background signal.s, and between closely related target sequerces.
Affymetrix GeneChip hybridization experiments invol=wve the followeing steps: design and produ ction of arrays, prepar-ation of £liaorescently labeled target from RNA isolated from the piolocyical specimens, hybridizat=ion of the labeled targget to the Ge=neChip, screening the array , and analysis of the secanned image and generation of gene exp.xession profiles.
A. Dessigning and Custom making Affymetrix GeneChip =A GeneChip CustomExpress AcRvantage Array was custom made Toy Affymetrix Inc. (Santa Clara, CA). Chip size was 18 microom and array format was 100—2187 that can accommodate 528 p xobe sets (11, 628 probes) — Except for GenBank deriv—ed nucleic acid sequences, all sequences were se ected from our previously identified tobacco clones and all probes were custom designed. A total of 400 tobacco genes ONC fragments were selected to be ircluded on the GeneChip. The seque=nces of oligonucleotides seelected were based on wunique regions of the 3’ end of the geme. The selected nucleic acid sequences consisted of 56 full length p450 genes and 71 p450 fragments that were cloned from tobacco, describ ed in (patent applications) . Other t obacco sequences inclu-ded 270 tobacco ESTs which were generat ed from suppression subtmraction library using Clont.ech SSH kit (BD Bioscie=nces,
Palo Alto, CA). Among these gen_es, some oligonucleoti de seque=nces were selected from cytochrome P450 genes li_sted in
GenB=ank. Up to 25 probes were msed for each full lenagth gene and 11 probes for each fragment=. A reduced number off probes were used for some clones due t=o the lack of unique, high quality probes. Appropriate control sequencess were also included on the @GeneChip®. -The probe Arrays were 25-mer oligonuclecotides that were directly synthessized onto a glass wafer by a combination of semiconductor-baased photolithography and soli.d phase chemical synthessis technologies. Each array contained up to 100,000 differermt oligonucleotide probes. Sirace oligonucleotide probes are synthesized in known locations on the array, the hybridization patterns and sicgmal intensities can be interpretzed in terms of gene identity and relative expression levels by the Affymetrix Microarraay Suite® software. Each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotic3e. The perfect match probe has a sequence exactly compliment—ary to the particular gene and thus measures the expresssion of the gene. The mismatch probe differs from the per—fect match probe by a single base substitution at the ceanter base position, which disturbs the binding of the t—arget gene transcript. The mismatch produces a nonspeci. fic hybridization si.gnal or background signal thaat was compared to the signal measured for the perfect match oligonucleotide ~
B. Sample preroaration
Hybridizati.on experiments were conduc:=ted by Genome
Explorations, Irac. (Memphis, TN). The RNA ssamples used in hybridization comsisted of six pairs of nonconv-erter/converter issogenic lines that were induced by ethylene treatment s.
Sarples included one pair of 4407-25/4407-33 non-treated bur ly teobacco samples, three pairse of ethylene treated 4407-25/440 7- _ 373 samples, one pair of ethylene treated dark tobacco NL
M.=dole/181 and one pair oX ethylene treated burly varie=ty
PrBLB01/178. Ethylene treatment was as described in Example 1.
Total RNA was extracted from above mentioned ethylene t reated and non-treated leaves using a modified acid pheraocl a nd chloroform extraction p rotocol. Protocol was modified to w.se one gram of tissue that was ground and subsequent=1ly ~~ortexed in 5 ml of extraction buffer (100 mM Tris-HCl, pH 8 .5; 200 mM NaCl; 10mM EDTA; 0.5% SDS) to which 5 ml pheriol ( pH5.5) and 5 ml chloroform was added. The extracted sample vras centrifuged and the supernatant was saved. This e=xtraction step was repeated 2-3 more times until whe ssupernatant appeared clear Approximately 5 ml of chlorofeorm was added to remove tras=ce amounts of phenol. RNA wwas precipitated from the combimed supernatant fractions by add-ng = 3-fold volume of ETOH and 1/10 volume of 3M NaOAc (pHS5 .2) =and storing at -202C for 1 hour. After transferring to a
Clorex glass container the RNA fraction was centrifuged at © ,000 RPM for 45 minutes aft 49°C. The pellet was washed with 7 0% ethanol and spun for 5 mminutes at 9,000 RPM at 42C. Af ter 5 Slrying the pellet, the pel. leted RNA was dissolved in 0.5 ml
FNase free water. The pel leted RNA was dissolved in 0.5 ml
FINase free water. The quallity and quantity of total RNA -was analyzed by denatured formamldehyde gel and spectrophotomet er, r—espectively. The total RENA samples with 3-5ng/ul were s ent 0 t—o Genome explorations, ines. to do the hybridization.
C. Hybridization, detection and data output
The preparatiom of labeled cRNA material wa Ss performed as follows. First and ssecond strand cDNA were synth-esized from 5- 15 ng of total RNA wising the SuperScript Double—Stranded cDNA
Synthesis Kit (Gibced Life Technologies) and oligeo-dT24-T7 (5'-
GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG €6G-3') primer according to the manufacturer's instructions.
The cRNA was concurrently synthesized ana labeled with biotinylated UTP ard CTP by in vitro transcrip-tion using the 77 promoter coupled double stranded cDNA as teamplate and the
T7 RNA Transcript. Labeling Kit (ENZO Diagrmostics Inc.).
Briefly, double st randed cDNA synthesized frorm the previous steps were washed twice with 70% ethanol and ressuspended in 22 pl Rnase-free H20. The cDNA was incubated with 4 nl of 10X each Reaction Buffer, Biotin Labeled Ribonucleotides, DIT,
Rnase Inhibitor Misc and 2 pl 20X 17 RNA Polymerase for 5 hr at 37¢<C. The labeleed cRNA was separated from unincorporated ribonucleotides by passing through a CHROMA SS PIN-100 column (Clontech) and pre cipitated at -202C for 1 hr to overnight.
Oligonucleoti de array hybridization and analysis were 15+ performed as follows. The cRNA pellet was ressuspended in 10 pl Rnase-free H20 and 10.0 ng was fragmented bxy heat and ion- mediated hydrolys=is at 952C for 35 mins in 200 mM Tris- acetate, pH 8.1, 500 mM KOAC, 150 mM MgOAc. The fragmented
CRNA was hybridized for 16hr at 45°C to HG_U95Aav2 co oligonucleotide ar-rays (Affymetrix) containing ~12,500 full
Dength annotated genes toegether with additional probe se=ts designed to represent EST sequences. Arrays were washed at 258C with 6 X SSPE (0.9M NamCl, 60 nMNaH2PO4, 6 mM EDTA + 0.01% qween 20) followed by a sstringent wash at 502C with 100 mM ] MES, 0.1M [Na+], 0.01% Tweeen 20. The arrays were stained w3ith phycoerythrein conjugated streptavidin (Molecular Probes) and the fluorescence intensities were determined using a la ser confocal scanner (Hewlett —Packard) . The scanned images w e€re analyzed using Microarr ay software (Affymetrix). Sarmple 10 loading and variations in staining were standardized by scaling the average of Whe fluorescent intensities of all genes on an array to consstant target intensity (250) for all arrays used. Data Analyzsis was conducted using Microaxray
Suite 5.0 (Affymetrix) foellowing user guidelines. The sicmal 15 intensity for each germe was calculated as the average intensity difference, represented by [Z(PM - MM) / (numbem: of probe pairs)l, where PIM and MM denote perfect-match and mismatch probes. 20 D. Data Analysis and res ults
Twelve sets of ybridizations were successful. as evidenced by the Expresssion Report generated using detection instruments from Genome “Explorations. The main parametems on the report included Noi_se, Scale factor, background, t—otal probe sets, number and p ercentage of present and absent porobe sets, signal intensity o f housekeeping controls. The dat-a was subsequently analyzed a.nd presented using software GCOS in combination of other Moicrosoft software. Signal compazrison ) between treatment pairss was analyzed. Overall data fomr all respective probes corresponding to gern=es and fragments of each different treatment including replicaations were compiled and compiled expression data such as call of the changes. and signal log 2 ratio changes were analyzed.
A typical application of GeneCh=-ip technology is fimding genes thats are differentially expresssed in different tis-sues.
In the p-xesent application, geneti ¢ expression variaations caused by- ethylene treatment were determined for pai xs of converter and nonconverter tobacco 1i_nes that included a 4407- 25/74407-3 3 burley variety, PRLB01/17S3 burley variety, and a NL
Madole/181 dark variety. These ana» yses detected only those genes whose expression is signif icantly altered dmue to biological variation. These analysess employed the Fold echange (signal ratio) as a major criterion t—o identify induced -genes.
Other paxxameters, such as signal &Entensity, present/ absent call, wewxe also taken into consideration.
After analyzing the data for expression differemces in 0 convertex and nonconverter pairs of samples for approxiamately
A00 genes, the results based on the signal intensities showed that only two genes, D121-AA8, and [D120-AH4 and one fragment,
D35-BG1L, ‘that is partial fragment of D121-AA83, had reproducible induction in ethylene treated converter— lines 5 wersus ron-converter lines. To il lustrate the diffe—rential expression of these genes, the data was represermted as follows - As shown in Table V, the signal of a gerne in a convertesr line, for example, burley= tobacco variety, 4-407-33, was determined as ratio to the signal of a related ) nonconvearter isogenic line, 440 7-25. Without ethylene treatment, tke ratio of converter to nonconwerter signals for all genes apoproached 1.00. Upon ethylerme treatment, two genes, D121-7ZAA8 and D120-AH4, were induced Zin converter lines relative to non-converter line as deteermined by three independent =analyses using isogenic burley 1.ines. These genes have very hicgh homology to each other, approximately 99.8% or } greater nucle=ic acid sequence homology. As depicted in Table
V, their rela tive hybridization signals in cconverter varieties ranged from .approximately 2 to 12 fold hiegher in converter lines than tBhe signals in their non-conver ter counterparts.
In comparison, two actin-like control clones, internal controls, wemxre found not to be induced iro converter lines based on the-ir normalized ratios. In addi_tion, a fragment {(D35-BG11l), —whose sequence in coding region is entirely contained in both D121-AA8 and D120-AH4 genes, was highly induced in thae same samples of paired isogemmic converter and nonconverter lines. Another isogenic pair of burley tobacco varieties, PB-ILB0l and 178, was shown to havee the same genes,
D121-AA8 and D120-AH4, induced in converteer samples under ethylene induection. Furthermore, D121-22A8 amd D120-AH4 genes were preferen:tially induced in converter 1 ines of isogenic dark tobacco pairs, NL Madole and 181, de-monstrating that ethylene induection of these genes in convert—er lines was not limited to bumrley tobacco varieties. In all. cases, the D35- '5 BGll fragment= was the most highly induceed in converter relative to nconconverter paired lines. 80 i
Table V: Comparison of Clone Induction in Ethvyle reated
Converter and Non-Converter Lines
Ethylene -
No Ethylene Treated] Treated Buriey| Ethylene Treated] Ethylene Treated EthZylene
Clones JTreatment} Burey Exp 1 p 2 Burley Exp 3 Burley Exp 4 _ Treated Dark 33:25 33:25 EtNo* J] 33:25 EtNo | 33:25 EtNo [ 33:25 EtNo 181:N LEtNo = Ratio | Ratio Ratio § Ratio Ratio | Ratio Ratio | Ratio Ratio Ratios Ratio induced
D121-AA3 1.03 2.20 2.14 13.25 12.90 6.31 5.15 12.56 12.19 17.0688 16.60
D120-AH4 1.44 2.74 1.80 18.33 12.74 4.13 2.87 10.87 7.55 11.768 8.17
Actin-Like | 0.67 0.57 (5) 1.18 1.17 0.99 088 0.74 086 073 1.20 1.02
Actin-Like | 0.86 0.79 3 1.09 1.23 7.12 0.89 0.81 1.18 0.11 1,02 0.93 *~-normalized Ratio.
EXAMPLE 15: ETHYLENE INDUCTION OF MICROSOMAL NICOTINE
DEMETHYLASE IN TOBACCO CONVERTER LINES
Biochemical analyses of demethylase enzymatic activity in microsomal enriched fractions of ethylene treated and non- treated pairs of converter and non-converter tobacco lines were performed as follows. 15 A. Preparation of Microsomes
Microsomes were isolated at 4°C. Tobacco leaves were extracted in a buffer consisting of 50 mM N- (2-hydrooxyethyl) piperazine-N’- (2-ethaneswlfonic acid) (HEPES), pH 7.5, 3 mM 0 DL-Dithiothreitol (DTT) and Protease Inhibitor Cocktail (Roche) at 1 tablet/50 mnl. The crude extract was filtered through four~ layers of cheesecloth to remove undisrupteci tissue, and the filtrate was centrifuged —for 20 min at 20,000
X g to removes cellular debris. The superratant was subjectecd to ultracent rifugation at 100,000 x g for 60 min and the resultant pesllet contained the microsomal fraction. The microsomal fr-action was suspended in the extraction buffer ancl applied to am ultracentrifugation step wloere a discontinuous sucrose gradient of 0.5 M sucrose in the extraction buffer wass used. The purified microsomes were resuspended in the extraction kmuffer supplemented with 10% (w/v) glycerol ass cryoprotectarat. Microsomal preparations were stored in a liquid nitrocgen freezer until use.
B. Protein Concentration Determination
Microsormal proteins were precipitated with 102%
Trichlorocacet=ic Acid (TCA) (w/v) in acetone, and the proteirm concentratiors of microsomes were determined using RC DCC
Protein Asseay Kit (BIO-RAD) following the manufacturer's protocol. 3) Nicotine Demethylase Activity Asssay
DL-Nicot=ine (Pyrrolidine-2-%C) was okotained from Moravels
Biochemicals and had a specific activity of 54 mCi/mmol —
Chlorpromazirae (CPZ) and oxidized cytochrome c {cyt. C), bot
P450 inhibitors, were purchased from Sigrma. Reduced form of nicotinamide adenine dinucleotide phosph.ate (NADPH) is the typical eleactron donor for cytochroene P450 via the
NADPH: cytochrome P450 reductase. NADPH was omitted for control_
incubation. Routine enzyme assay consisted of microsomal proteins (around 2 mg/ml), 6 md NADPH, 55 pM C label-ed nicotine. The concentration of CPZ and Cyt. C, when used, wvas 1 mM and 100 pM, respectively. T-he reaction was carried -at 25°C for 1 hour and was stopped with addition of 300 pul methanol to each 25 pl reaction mixture. After spinning, 20 pul of the methanol extract was sepawxated with a reverse-phasse
High Performance Liquid Chromatography (HPLC) system (Agilent=) using an Inertsil ODS-3 3p (150 x 4.6 mm) column from Variarm.
The isocratic mobile phase was the mixture of methanol and 5 0 mM potassium phosphate buffer, pH 6.25, with ratio of 60:4 0 (v/v) and the flow rate was 1 ml/mira. The nornicotine peak, as=s determined by comparison with authentic non-labelecd nornicotine, was collected and subjected to 2900 tri-carl>o
Liquid Scintillation Counter (L&SC) (Perkin Elmer) for=— quantification. The activity of ndcotine dJdemethylase iss calculated based on the production ef !¥C labeled nornicotine- over 1 hour incubation.
Samples were obtained from pa.irs of Burley converter (line 4407-33) and non-converter (lime 4407-25) tobacco lines that were ethylene treated or not. All untreated samples did not have any detectable microsomal nicotine demethylase activity. In contrast, microsomal samples obtained from 15 ethylene treated converter lines were found to contain significant levels of nicotine demesthylase activity. The nicotine demethylase activity was slyown to be inhibited by
P450 specific inhibitors demonstrating the demethylase activity was consistent to a P450 microsomal derived enzyme.
WeQ 2005/038018 PCT/US2004/0 34218
A tyroical set of enzyme assay results obtained for the burlexy convearter tobacco line is shown in thes Table VI. In contrast=, sampl_e derived from ethylene treated nonconverter tobacco did not contain any nicotine demethylase activity. These resulizs demorastrated that nicotine demethylamse activity was induced upon treatment with ethylene in convemxter lines but not in the corresponding isogenic nonconverter line. Similar result—s were obtained for an isogenic dark toloacco variety pair, where microsomal nicotine demethylase activity was induced i.n convearter lines and not detectable in nonconverter paire=d liness. Together these experimemts demonstrated thamt microssomal nicotine demethylase activity is induced upcon ethyl ene treatment in converter lin es while not in paire=d isoge=nic nonconverter lines. Thos € genes that are P45.0 derived genes and are preferentially induced in converte=r lines. relative to paired non-converter lines are candidat.e genes to encode the nicotine demethyl ase enzyme.
Table VI: DEMETHYIASE ACTIVITY TIN MICROSOMES OF ETHYLENE
INDUCED BURLEY CONVERTER AND NON CONVERTER LINES
1 mM chlor- with 100 uM [NADPH promazine vtochrome C
Conver—ter 8.3 + 0.4 0.01 + 0.01 0.2 0.2 0.4 + 0.4
EN HE SE SC protein protein protein protei-m
Converter _|wot petected [not petectect |wot Detected [vot De tected
Conver-ter Not Detected | Not Detectecd | Not Detected | Not De tected
E_XAMPLE 16: FUNCTIONAL IDENTIFI-CATION OF D121-AAB AS NICOTINE
D. EMETHYLASE
The function of the candidate clone (D121-AA 8), was ceonfirmed as the coding gene for nicotine demethyl ase, by assaying enzyme activity of hetzerologously expressed P450 in yeast cells. 1, Construction of Yeast: Expression Vector
The putative protein-codirag sequence of the P45 -encoding cIONA (12128), was cloned into the yeast expression v-ector : pW eDP60. Appropriate BamHI and Mfel sites (underlined.) were irmtroduced via PCR primers cont aining these sequences either upostream of the translation sta xt coden (ATG) or downstream of thee stop coden (TAA). The Mfel eon the amplified PCR product is co-mpatible with the EcoRI site en the vector. The prirmers used to amplify the 121AA8 cDNA were 5’ —
TASGCTACCCCGATCCATGCTTTCTCCCATAGAAGCC-3 and 57-
CTCCATCACAATTGTTAGTGATGETGATGGTGATGCGATCCTCTATARAGCTC AGE TGCCAGGC - 3‘ .. A segment of sequence coding nine extra amino acidls at the C- ter—minus of the protein, includi.ng six histidines, was incorporated into the reverse primer. This facilitates the exporession of 6 X His tagged P45 0 upon induction. PCR products wer—e ligated into pYeDP60 vector after enzyme digestioms in the sen . se orientation with reference to the GAL10-CYCl prormoter.
Corastructs were verified by enzyme rest=rictions and DNA secyuencing. 2. Yeast Transformation _
The WAT1l1l yeast line, modified t © express Arabidopsis=s
NAD PH-cytochrome P450 reductase ATR1l, weas transformed with the corstruct pYeDP60-P450 cDNA plasmids. Fifty micro-liter otf
WAT 11 yeast cell suspension was mixed wwith ~1 ug plasmid DN& in a cuvette with 0.2-cm electrode gap=. One pulse at 2.0 kW wass applied by an Eppendorf electroporat—or (Model 2510). Cellss wer—e spread onto SGI plates (5 g/L bac=tocasamino acids, 6.77 g/I= yeast nitrogen base without amino eacids, 20 g/L glucose. 40 mg/L DL-tryptophan, 20 g/L agar) . Transformants were= con_firmed by PCR analysis performed directly on randomly sel ected colonies. 3. P450 Expression in Transsformed Yeast Cells
Single yeast colonies were used too inoculate 30 ml, SGEC med ia (5 g/L bactocasamino acids, 6.7 g~’L yeast nitrogen base without amino acids, 20 g/L glucose, 4 0 mg/L DL-tryptophan): and grown at 30 °C for about 24 hours . An aliquot of thiss culture was diluted 1:50 into 1000 mL oof YPGE media (10 g/L veasst extract, 20 g/L bacto peptone, 5 ¢/L glucose, 30 ml/I. ethanol) and grown until glucose was ccompletely consumed ass 15 ind=icated by the colorimetric change of a Diastix urinalysiss reacyent strip (Bayer, Elkhart, IN). Induction of cloned P450+ was initiated by adding DL-galactose to a final concentration_ of 2%. The cultures were grown for an additional 20 hours preparation.
WAT11 yeast cells expressing pY¥eDP60-CYP71D20 (a P450 catalyzing the hydroxylation of 5-epi-aristeolochene and 1- deoxycapsidio 1 in Nicotiana tabacum) were use as control for the P450 expr ession and enzyme activity assays. 4. In Vivo Enzyme Assay
The nicotine demethylase activity in the transformed yeast cells wesre assayed by feeding of yeast culture with DL-
Nicotine (Pyrr-olidine-2-%C). To 75 pl of the gaalactose induced culture Cc lalbeled nicotine (54 mCi/mmol) was added to a final concentration of 55 uM. The assay culture wast incubated with shaking in 14 ml polypropylene tubes for 6 hours and was extracted with 900 pl methanol. After spinnireg, 20 pl of the methanol extract was separated with an rp-HPLC and the nornicotine fraction was quantitated by LSC.
The control culture of WAT1l (pYeDP60-CY P71D20) did not convert nicotine to nornicotine, showing that the WAT11l yeast strain does no t contain endogenous enzyme acti vities that can catalyze the s¥tep of nicotine bioconversion to mornicotine. In contrast, veast expressing 121AA8 gene prodvaced detectable amount of mnormicotine, indicating the nicoti_ne demethylase activity of th is P450 enzyme. 5. Yeas t Microsome Preparation
After in duction by galactose for 20 hours, yeasst cells were collecte=d by centrifugation and washed twice with TES-M buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6 M sorb-tol, 10 mM 2-mercap toethanol). The pellet was resusperaded in extraction buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6 M sorbitol, 2 mM 2-mercaptoethanol, 1% bovine serumm album,
Protease Inhi.bitor Cocktail (Roche) at 1 tablet/50 ml J). Cells were then brorken with glass beads (0.5 mm in diameter, Sigma).
Cell extract was centrifuged for 20 min at 20,000 == g to remove cellular debris. The supernatant was subje cted to ultracentrifuagation at 100,000 x g for 60 min and the resultant pellet contained the microsomal fracticon. The microsomal fr-action was suspended in TEG-M buffer (50 rvM Tris-
HCl, pH 7.5, 1 mM EDTA, 20% glycerol and 1.5 mM 2- mercaptoetharmol) at protein concentration of 1 mg/mL.
Microsomal preparations were stored in a liquid ri trogen freezer until. use. 6. Enzyme Activity Assay in Yeast Mic=rosomal
Preparati_ons
Nicotine demethylase activity assays with yeast microsomal preeparations were performed in the same way as with microsomal preparations from tobacco leaves (EXAMPLE 15) except that the protein concentrations were constamt at 1 mg/mL.
Microsomal preparations from control yeast cells expressing CY P71D20 did not have any detectable microsomal nicotine demet=hylase activity. In contrast, microsomal samples obtained from yeast cells expressing 121AA8 geme showed significant levels of nicotine demethylase activity. The nicotine demethylase activity had requirement for NADPH and was shown to be i-nhibited by P450 specific irahibitors, consistent to the P4%50 being investigated. A typical set of enzyme assay results obtained for the yeast cells iss shown in the Table VII.
Table VII: DEMETHYLASE ACTIVITY IN MICROSOMESS OF YEAST
CELLS EXPRESSING 121AA8 AND CONTROL P450
Sample Microsomes |Microsomes + | Microsomes Microsomes - 1 mM chlor- + with 100 NADPH promazine BM cytochrome CC
D121-AAS8 10.8 = A..2* |1.4 ££ 1.3 2.4 ££ 0.7 0.4 = 0.1 pkat / mg pkat / mg pkat / mg kat / mg protein protein protein protein
Control (CYP71D20) | Not Not Not Not Detected
Detected Detected Detected ’ *--Average results of 3 replicates.
Together these experiments demonstrated that tlme cloned full length gene D121—AA8 encodes cytochrome P450 protein that catalyzes the conversion of nicotine to nornicotSne when expressed in yeast.
Numerous modifications and variations in practice of the invention are expected to occur to those skilled in %he art upon consideration of the foregoing detailed description oof the invention.
Censequently”, such modifications and var—iations are intended to be included within the scope of the folleowing claims.

Claims (42)

WHAT IS CLAIMED IS:
1. An isolated nucleic acid molecule from Nicotiamma, wherein said nucleic acid mo lecule is SEQ. ID. No.: 181 Ss
2. An isolated nucleic acid molecule from Nicotiara, wherein said nucleic acid mo lecule has at least 81% sequaence identity to SEQ. ID. No.: 181.
3. An isolated nucleic acid molecule from Nicotiamna, wherein said nucleic acid molecule has at least 91% sequence identity to SEQ. ID. No.: 181.
4. An isolated protein from Nicotiana, wherein said protein comprises SEQ. ID. No.: 182.
5. An isolated proteim from Nicotiana, wherein samid protein has at least 80 percent sequence identity to SEQ. ID. NKJjo.: 182.
6. An isolated proteim from Nicotiana, wherein saaid protein has at least 90 percent sequence identity to SEQ. ID. MNJo.: 182.
7. A transgenic plant, wherein said transgenic pl_ant comprises the nucleic acid molecule of claim 1, 2 or 3 _ 2:5
8. The transgenic plant of Claim 7, wherein said plant is a tobacco plant.
9. A method of producing a transgenic plant, wheerein said method comprises the steps of: 10 (i) operably linking said nucleic acid molecule eof claim 1,
2 or 3 with a promoter functiconal in said plarmt to create a plant transformational vector; (ii) transforming said plant with =said plant - transformational vector of st ep (i): (iii) selecting a plant cell transf ormed with said transformation vector; and (iv) regenerating a transformatiora plant from said. transformed plant cell.
10. The method of claim 9, wherein the plant has reduced levels of nornicotine.
11. The method of Claim 9, where in said nucleic =acid molecule is in an antisense orientatio-nm.
12. The method of Claim 9, wherein said nucleic acid molecule ig in a sense orientation.
13. The method of Claim 9, wherein said nucleic acid molecule is in a RNA interference orie=ntation.
14. The method of Claim 9, whereein said nucleic acid mvolecule is expressed as a double str anded RNA molecule.
15. The method of Claim 9, wher-ein said transgemic plant is a tobacco plant.
16. A method of selecting a plant containing a nucleic acid molecule, wherein said plant is analyzed for the pressence of a nucleic acid sequence of claim 1, 2 or 3.
17. Tie method of selecting a plant of Claim 16, wherein said plant fs analyzed by DNA hybridization. -
18. Thhe method of selecting a plant of Claim 17, wherein said DNA hy®oridi zation is Southern blot amalysis.
19. The method of selecting a plant. of claim 17, wherein said DNA hy-bridization is Northern blot analysis.
20. The method of selecting a plant= of Claim 16, wherein said plant is analyzed by PCR detection.
21. "Whe method of Claim 16, whereim said plant is a tobacco plant.
22. = method of increasing or decreasing nornicotine levels in a plant , wherein said method comprise s the steps of: (i) opoerably linking said nucleic a«cid molecule of claim 1, 2 or 3 with a promoter functional in sai _d plant to create a plant transforma tional vector; (ii) transforming said plant with s aid plant transformational vector of step (i); (iii) selecting a plant cell transfosrmed with said transformamtion vector; and (iv) regenerating a transformation plant from said transformed plant cell.
23. The method of Claim 22, wherezin said nucleic acid molecule fis in an antisense orientation .
24. Tkae method of Claim 22, wherein said nucleic acid molecule is in a sense orientation.
25. Time method of Claim 22, wherein said nucleic ac-id molecule is in a RNA interference orientat ion.
26. Th e method of Claim 22, wherein .said nucleic ac: did molecule is -expressed as a double stranded RNA molecule.
27. The= method of Claim 22, wherein said transgenic plant is a tobacco plant.
28. A t=obacco product having reduced amounts of norrm.icotine levels, the t=obacco product comprising tobacco frém a rlan:t of claim 7.
29. The= tobacco product of claim 27 wherein the tobacco product is se lected from the group consistimg of cigarettess, cigars, pipe tobacco, snuff, chewing tobacco, products blernded with the tobaecco product, and mixtures thereof.
30. The tobacco product of claim 28 wlierein the level_s of nornicotine ar—e reduced from about 5 to abowmt 10%.
31. The tobacco product of claim 28 wh-erein the level s of nornicotine ar-e reduced from about 10 to abo-ut 20%.
32. The tobacco product of claim 28 wheerein the levelss of nornicotine ar e€ reduced from about 20 to abomit 30%. 94 AMENNDED SHEET: S APRIL 2(07
33. The tobacce product of claim 28 wherein the levels of nornicotine are reduced more than about 30%.
34. A tobacco 1 eaf having reduced amounts o_f nornicotine levels, the tobacco 1 eaf comprising tobacco leaf from a plant of claim 7.
35. The tobacco leaf of claim 30 wherein a t=obacco product is formed frorm the tobacco leaf, the tobacco product selected from the group consisting of cigarettes, cigars, pipe tobacco, snuff, chewirhg tobacco, products blended with the tobacco product, and rnixtures thereof.
36. A method of isolating a gene from a plamt using the isolated nucleic acid of claim 1, 2 or 3.
37. A method of producing a transgenic plant according to claim 9 substantially as herein described with ref erence to any one of the illustrative Examples.
38. A method of selecting a plant containing a nucleic acid molecule according to claim 16 substantially &s herein described with referen ce to any one cf the illustrative Examples.
33. A method of dncreasing or decreasing norriicotine levels in a plant accoxding to claim 22 substantial ly as herein described with reference to any one of the illustrative Examples. 95 AMENDED S HEET: 5 APRIL 2007
40. A tobacco product according to claim 28 substantially as herein described witth reference to any one of the illustrative Examples.
41. A tobacco leaf according to claim 34 substantially &s herein described with reference to any one of the illustrative Examples.
42. A method of dsolating a gene from a plant according to claim 36 substantially as herein described witsh reference to any one of the illustrative Examples. 96 AMEND®ED SHEET: 5 APRIL_ 2007
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