CN1294267C - 携带新黄素裂解酶基因的转基因植物 - Google Patents
携带新黄素裂解酶基因的转基因植物 Download PDFInfo
- Publication number
- CN1294267C CN1294267C CNB011116285A CN01111628A CN1294267C CN 1294267 C CN1294267 C CN 1294267C CN B011116285 A CNB011116285 A CN B011116285A CN 01111628 A CN01111628 A CN 01111628A CN 1294267 C CN1294267 C CN 1294267C
- Authority
- CN
- China
- Prior art keywords
- leu
- val
- gly
- ala
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- PVNVIBOWBAPFOE-UHFFFAOYSA-N Dinoxanthin Natural products CC1(O)CC(OC(=O)C)CC(C)(C)C1=C=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC1(C(CC(O)C2)(C)C)C2(C)O1 PVNVIBOWBAPFOE-UHFFFAOYSA-N 0.000 title claims abstract description 79
- OWAAYLVMANNJOG-OAKWGMHJSA-N neoxanthin Natural products CC(=C/C=C(C)/C=C/C=C(C)/C=C=C1C(C)(C)CC(O)CC1(C)O)C=CC=C(/C)C=CC23OC2(C)CC(O)CC3(C)C OWAAYLVMANNJOG-OAKWGMHJSA-N 0.000 title claims abstract description 79
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 33
- PGYAYSRVSAJXTE-CLONMANBSA-N all-trans-neoxanthin Chemical compound C(\[C@]12[C@@](O1)(C)C[C@@H](O)CC2(C)C)=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(\C)/C=C/C=C(\C)C=C=C1C(C)(C)C[C@H](O)C[C@@]1(C)O PGYAYSRVSAJXTE-CLONMANBSA-N 0.000 title claims abstract 7
- 108090000856 Lyases Proteins 0.000 title description 45
- 238000000034 method Methods 0.000 claims abstract description 73
- 241000196324 Embryophyta Species 0.000 claims description 225
- 108090000623 proteins and genes Proteins 0.000 claims description 202
- 150000001413 amino acids Chemical class 0.000 claims description 51
- 235000018102 proteins Nutrition 0.000 claims description 44
- 102000004169 proteins and genes Human genes 0.000 claims description 44
- 230000002101 lytic effect Effects 0.000 claims description 34
- 239000002773 nucleotide Substances 0.000 claims description 31
- 125000003729 nucleotide group Chemical group 0.000 claims description 31
- 235000001014 amino acid Nutrition 0.000 claims description 29
- 241000219194 Arabidopsis Species 0.000 claims description 24
- 238000009396 hybridization Methods 0.000 claims description 20
- 235000013311 vegetables Nutrition 0.000 claims description 15
- 230000008034 disappearance Effects 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 108090000790 Enzymes Proteins 0.000 abstract description 11
- 102000004190 Enzymes Human genes 0.000 abstract description 10
- 238000003776 cleavage reaction Methods 0.000 abstract 2
- 230000003247 decreasing effect Effects 0.000 abstract 2
- 230000007017 scission Effects 0.000 abstract 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 871
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 804
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 623
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 591
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 573
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 542
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 536
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 462
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 439
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 300
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 273
- 108020004414 DNA Proteins 0.000 description 133
- 102220369447 c.1352G>A Human genes 0.000 description 88
- 230000014509 gene expression Effects 0.000 description 84
- 239000002299 complementary DNA Substances 0.000 description 61
- 241000282326 Felis catus Species 0.000 description 60
- 230000035882 stress Effects 0.000 description 60
- 210000004027 cell Anatomy 0.000 description 51
- PGYAYSRVSAJXTE-FTLOKQSXSA-N 9'-cis-neoxanthin Chemical compound C(\[C@]12[C@@](O1)(C)C[C@@H](O)CC2(C)C)=C/C(/C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)C=C=C1C(C)(C)C[C@H](O)C[C@@]1(C)O PGYAYSRVSAJXTE-FTLOKQSXSA-N 0.000 description 49
- 102220369445 c.668T>C Human genes 0.000 description 44
- 230000002411 adverse Effects 0.000 description 42
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 41
- 102220023258 rs387907548 Human genes 0.000 description 34
- 102000004317 Lyases Human genes 0.000 description 29
- 241000219977 Vigna Species 0.000 description 28
- 229940024606 amino acid Drugs 0.000 description 28
- 239000000523 sample Substances 0.000 description 28
- 238000003752 polymerase chain reaction Methods 0.000 description 27
- 238000004458 analytical method Methods 0.000 description 24
- 239000012634 fragment Substances 0.000 description 24
- 102220023256 rs387907547 Human genes 0.000 description 24
- 108090000994 Catalytic RNA Proteins 0.000 description 23
- 102000053642 Catalytic RNA Human genes 0.000 description 23
- 230000000694 effects Effects 0.000 description 23
- 108091092562 ribozyme Proteins 0.000 description 23
- 102220023257 rs387907546 Human genes 0.000 description 22
- 230000000692 anti-sense effect Effects 0.000 description 19
- 230000005764 inhibitory process Effects 0.000 description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 17
- 230000018044 dehydration Effects 0.000 description 16
- 238000006297 dehydration reaction Methods 0.000 description 16
- 102220004457 rs11567847 Human genes 0.000 description 16
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 15
- 240000003768 Solanum lycopersicum Species 0.000 description 15
- 240000008042 Zea mays Species 0.000 description 15
- 230000008859 change Effects 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 15
- 230000002068 genetic effect Effects 0.000 description 14
- 150000003839 salts Chemical class 0.000 description 14
- 239000002689 soil Substances 0.000 description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 239000000284 extract Substances 0.000 description 12
- 230000012010 growth Effects 0.000 description 12
- ZTALKMXOHWQNIA-TVBSHJCBSA-N 2-cis,4-trans-xanthoxin Chemical compound C([C@H](O)C1)C(C)(C)[C@@]2(/C=C/C(/C)=C\C=O)[C@]1(C)O2 ZTALKMXOHWQNIA-TVBSHJCBSA-N 0.000 description 11
- ZTALKMXOHWQNIA-UHFFFAOYSA-N Xanthoxin Natural products C1C(O)CC(C)(C)C2(C=CC(C)=CC=O)C1(C)O2 ZTALKMXOHWQNIA-UHFFFAOYSA-N 0.000 description 11
- 101001128925 Zea mays 9-cis-epoxycarotenoid dioxygenase 1, chloroplastic Proteins 0.000 description 11
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 11
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 11
- 238000009825 accumulation Methods 0.000 description 11
- 235000005822 corn Nutrition 0.000 description 11
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 239000012528 membrane Substances 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 239000004677 Nylon Substances 0.000 description 10
- 239000002253 acid Substances 0.000 description 10
- 102220369446 c.1274G>A Human genes 0.000 description 10
- 230000001939 inductive effect Effects 0.000 description 10
- 229920001778 nylon Polymers 0.000 description 10
- 238000012545 processing Methods 0.000 description 10
- 241000588724 Escherichia coli Species 0.000 description 9
- 229920002684 Sepharose Polymers 0.000 description 9
- 239000004213 Violaxanthin Substances 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 102000039446 nucleic acids Human genes 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 150000007523 nucleic acids Chemical class 0.000 description 9
- 101001128917 Arabidopsis thaliana Probable 9-cis-epoxycarotenoid dioxygenase NCED5, chloroplastic Proteins 0.000 description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 8
- 235000010726 Vigna sinensis Nutrition 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 8
- 108020004999 messenger RNA Proteins 0.000 description 8
- 238000010369 molecular cloning Methods 0.000 description 8
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 210000002706 plastid Anatomy 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 101001128924 Solanum lycopersicum 9-cis-epoxycarotenoid dioxygenase NCED1, chloroplastic Proteins 0.000 description 7
- 238000013459 approach Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 6
- 230000006353 environmental stress Effects 0.000 description 6
- 238000013467 fragmentation Methods 0.000 description 6
- 238000006062 fragmentation reaction Methods 0.000 description 6
- 230000001976 improved effect Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 230000009182 swimming Effects 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 108020004491 Antisense DNA Proteins 0.000 description 5
- 108091092584 GDNA Proteins 0.000 description 5
- 244000061176 Nicotiana tabacum Species 0.000 description 5
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 5
- 238000000636 Northern blotting Methods 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 239000003816 antisense DNA Substances 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 238000005520 cutting process Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 230000006872 improvement Effects 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 230000037039 plant physiology Effects 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000013519 translation Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 108020005544 Antisense RNA Proteins 0.000 description 4
- 241000701489 Cauliflower mosaic virus Species 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 102000005720 Glutathione transferase Human genes 0.000 description 4
- 108010070675 Glutathione transferase Proteins 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 4
- 235000007244 Zea mays Nutrition 0.000 description 4
- 150000001299 aldehydes Chemical class 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000003184 complementary RNA Substances 0.000 description 4
- 238000006073 displacement reaction Methods 0.000 description 4
- 230000024346 drought recovery Effects 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 108020001507 fusion proteins Proteins 0.000 description 4
- 102000037865 fusion proteins Human genes 0.000 description 4
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000003375 plant hormone Substances 0.000 description 4
- 238000009331 sowing Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- JKQXZKUSFCKOGQ-JLGXGRJMSA-N (3R,3'R)-beta,beta-carotene-3,3'-diol Chemical compound C([C@H](O)CC=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C[C@@H](O)CC1(C)C JKQXZKUSFCKOGQ-JLGXGRJMSA-N 0.000 description 3
- 101001128921 Arabidopsis thaliana 9-cis-epoxycarotenoid dioxygenase NCED2, chloroplastic Proteins 0.000 description 3
- 101000910770 Arabidopsis thaliana Probable carotenoid cleavage dioxygenase 4, chloroplastic Proteins 0.000 description 3
- 241000972773 Aulopiformes Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 238000007399 DNA isolation Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 101150066516 GST gene Proteins 0.000 description 3
- JKQXZKUSFCKOGQ-LQFQNGICSA-N Z-zeaxanthin Natural products C([C@H](O)CC=1C)C(C)(C)C=1C=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC1=C(C)C[C@@H](O)CC1(C)C JKQXZKUSFCKOGQ-LQFQNGICSA-N 0.000 description 3
- QOPRSMDTRDMBNK-RNUUUQFGSA-N Zeaxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCC(O)C1(C)C)C=CC=C(/C)C=CC2=C(C)CC(O)CC2(C)C QOPRSMDTRDMBNK-RNUUUQFGSA-N 0.000 description 3
- JKQXZKUSFCKOGQ-LOFNIBRQSA-N all-trans-Zeaxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2=C(C)CC(O)CC2(C)C JKQXZKUSFCKOGQ-LOFNIBRQSA-N 0.000 description 3
- 230000003570 biosynthesizing effect Effects 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 210000003763 chloroplast Anatomy 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 241001233957 eudicotyledons Species 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000004817 gas chromatography Methods 0.000 description 3
- 239000003862 glucocorticoid Substances 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- -1 polyoxyethylene Polymers 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000001850 reproductive effect Effects 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 235000019515 salmon Nutrition 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- KBPHJBAIARWVSC-XQIHNALSSA-N trans-lutein Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CC(O)CC1(C)C)C=CC=C(/C)C=CC2C(=CC(O)CC2(C)C)C KBPHJBAIARWVSC-XQIHNALSSA-N 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- 235000010930 zeaxanthin Nutrition 0.000 description 3
- 239000001775 zeaxanthin Substances 0.000 description 3
- 229940043269 zeaxanthin Drugs 0.000 description 3
- 108020005065 3' Flanking Region Proteins 0.000 description 2
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 2
- 241000219195 Arabidopsis thaliana Species 0.000 description 2
- 241000490494 Arabis Species 0.000 description 2
- 238000009631 Broth culture Methods 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- YXHKONLOYHBTNS-UHFFFAOYSA-N Diazomethane Chemical compound C=[N+]=[N-] YXHKONLOYHBTNS-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 2
- 241000209510 Liliopsida Species 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 108020005120 Plant DNA Proteins 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 108700025695 Suppressor Genes Proteins 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 244000042314 Vigna unguiculata Species 0.000 description 2
- 235000010722 Vigna unguiculata Nutrition 0.000 description 2
- SZCBXWMUOPQSOX-LOFNIBRQSA-N Violaxanthin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C12OC1(C)CC(O)CC2(C)C)C=CC=C(/C)C=CC34OC3(C)CC(O)CC4(C)C SZCBXWMUOPQSOX-LOFNIBRQSA-N 0.000 description 2
- 101150117319 aba2 gene Proteins 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 235000021466 carotenoid Nutrition 0.000 description 2
- 150000001747 carotenoids Chemical class 0.000 description 2
- BQFCCCIRTOLPEF-UHFFFAOYSA-N chembl1976978 Chemical compound CC1=CC=CC=C1N=NC1=C(O)C=CC2=CC=CC=C12 BQFCCCIRTOLPEF-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 210000002249 digestive system Anatomy 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 239000006916 nutrient agar Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000032696 parturition Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000001902 propagating effect Effects 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 238000012882 sequential analysis Methods 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 238000005728 strengthening Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 235000019245 violaxanthin Nutrition 0.000 description 2
- SZCBXWMUOPQSOX-PSXNNQPNSA-N violaxanthin Chemical compound C(\[C@@]12[C@](O1)(C)C[C@H](O)CC2(C)C)=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(\C)/C=C/C=C(\C)/C=C/[C@]1(C(C[C@@H](O)C2)(C)C)[C@]2(C)O1 SZCBXWMUOPQSOX-PSXNNQPNSA-N 0.000 description 2
- 210000004885 white matter Anatomy 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- KKAJSJJFBSOMGS-UHFFFAOYSA-N 3,6-diamino-10-methylacridinium chloride Chemical compound [Cl-].C1=C(N)C=C2[N+](C)=C(C=C(N)C=C3)C3=CC2=C1 KKAJSJJFBSOMGS-UHFFFAOYSA-N 0.000 description 1
- 108020005029 5' Flanking Region Proteins 0.000 description 1
- 101150108171 ABA4 gene Proteins 0.000 description 1
- 241000242764 Aequorea victoria Species 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 101100439902 Arabidopsis thaliana CLE19 gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 101100494448 Caenorhabditis elegans cab-1 gene Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 235000004035 Cryptotaenia japonica Nutrition 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 102100039556 Galectin-4 Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 108090000079 Glucocorticoid Receptors Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 108090001102 Hammerhead ribozyme Proteins 0.000 description 1
- 108010068250 Herpes Simplex Virus Protein Vmw65 Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241001480167 Lotus japonicus Species 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 241000723638 Nepovirus Species 0.000 description 1
- 101000598243 Nicotiana tabacum Probable aquaporin TIP-type RB7-18C Proteins 0.000 description 1
- 101000655028 Nicotiana tabacum Probable aquaporin TIP-type RB7-5A Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102000005877 Peptide Initiation Factors Human genes 0.000 description 1
- 108010044843 Peptide Initiation Factors Proteins 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000714474 Rous sarcoma virus Species 0.000 description 1
- 108020005543 Satellite RNA Proteins 0.000 description 1
- 241000242583 Scyphozoa Species 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 244000300264 Spinacia oleracea Species 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102000007641 Trefoil Factors Human genes 0.000 description 1
- 235000015724 Trifolium pratense Nutrition 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 101150045754 aba3 gene Proteins 0.000 description 1
- HLVPIMVSSMJFPS-UHFFFAOYSA-N abscisic acid beta-D-glucopyranosyl ester Natural products O1C(CO)C(O)C(O)C(O)C1OC(=O)C=C(C)C=CC1(O)C(C)=CC(=O)CC1(C)C HLVPIMVSSMJFPS-UHFFFAOYSA-N 0.000 description 1
- 238000003811 acetone extraction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000516 activation analysis Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000036978 cell physiology Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000008641 drought stress Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006735 epoxidation reaction Methods 0.000 description 1
- 239000002038 ethyl acetate fraction Substances 0.000 description 1
- OAYLNYINCPYISS-UHFFFAOYSA-N ethyl acetate;hexane Chemical compound CCCCCC.CCOC(C)=O OAYLNYINCPYISS-UHFFFAOYSA-N 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108010083391 glycinin Proteins 0.000 description 1
- 239000002044 hexane fraction Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 239000002085 irritant Substances 0.000 description 1
- 231100000021 irritant Toxicity 0.000 description 1
- XUWPJKDMEZSVTP-LTYMHZPRSA-N kalafungina Chemical compound O=C1C2=C(O)C=CC=C2C(=O)C2=C1[C@@H](C)O[C@H]1[C@@H]2OC(=O)C1 XUWPJKDMEZSVTP-LTYMHZPRSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- PGYAYSRVSAJXTE-UHFFFAOYSA-N neoxanthene Natural products O1C(CC(O)CC2(C)C)(C)C12C=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=C=C1C(C)(C)CC(O)CC1(C)O PGYAYSRVSAJXTE-UHFFFAOYSA-N 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000010451 perlite Substances 0.000 description 1
- 235000019362 perlite Nutrition 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 210000002729 polyribosome Anatomy 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 239000011814 protection agent Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000012744 reinforcing agent Substances 0.000 description 1
- 230000008521 reorganization Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000000979 retarding effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000011122 softwood Substances 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 210000001324 spliceosome Anatomy 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000010396 two-hybrid screening Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 241001478887 unidentified soil bacteria Species 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 238000001429 visible spectrum Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Nutrition Science (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明提供了一种编码用于改进植物逆境耐性的新黄素裂解酶的DNA，一种通过将所述DNA引进植物来增强植物逆境耐性的方法，和一种其中引进了新黄素裂解酶基因的转基因植物。本发明还提供了一种用于降低植物逆境耐性的DNA，一种通过将所述DNA引进植物来降低植物逆境耐性的方法，和一种其中引进了所述DNA的转基因植物。本发明能够产生一种其中逆境耐性增强或降低的植物。
Description
本发明领域
本发明涉及一种用于增强或降低植物的逆境耐性的DNA，和一种该DNA的转基因植物。
本发明背景
由于不能自由移动，所以植物必须调节它们本身来适应各种逆境，例如，干旱、土壤中的盐和低温。在这些不利的环境因素中，干旱被认为对植物的生长影响最严重。为了在干旱的条件下生存，一些植物在进化过程中获得了一种生理学和/或形态学的特性，而其它许多植物也具有一种应答干旱逆境的机制来保护它们自己。植物中这些对于水短缺的应答和对于干旱环境的适应是由包括干旱条件下基因表达发生改变在内的各种生理变化引起的(Shinozaki，K和Yamaguchi-Shinozaki，K.，植物生理学(Plant physiol.)，115：327-334，1997；Shinozaki，K.和Yamaguchi-Shinozaki，K.，“针对干旱逆境的分子应答(Molecular responses to drought stress)”在Shinozaki，K和Yamaguchi-Shinozaki(编辑)的“高等植物中针对冷、热和盐等不利环境的分子应答(Molecular responses to cold，drought，heat and salt stress in higherplants)”中，R.G.LANDES company，Austin，Texas，USA，pp.11-28，1999)。例如，在拟南芥属(拟南芥)中，已知干旱信息是通过脱落酸(ABA)依赖途径和脱落酸(ABA)非依赖途径进行传递来调控与干旱耐受性有关的基因表达。这些基因产物被认为具有调控例如渗透保护剂如蔗糖和脯氨酸的积累、蛋白质的半衰期、逆境信号传导途径和转录的功能(Bray，E.A.，Trends inPlant科学，2：48-54，1997；Bohnert，H.J.等，植物细胞，7：1099-1111，1995；Ingram，J.和Bartels，D.，植物生理学和植物分子生物学年鉴(Annu.Rev.Plant Physiol.Plant Mol.Biol.)，47：377-403，1996；Shinozaki，K和Yamaguchi-Shinozaki，K.，植物生理学，115：327-334，1997；Shinozaki，K.和Yamaguchi-Shinozaki，K.，“针对干旱逆境的分子应答”在Shinozaki和Yamaguchi-Shinozaki(编辑)的高等植物中针对冷干旱、热和盐等不利环境的分子应答，R.G.LANDES company，Austin，Texas，USA，pp.11-28，1999)。
C40途径被认为是高等植物中的ABA的生物合成途径。该C40途径，亦称作类胡萝卜素途径，是通过玉米黄质的环氧化作用，合成堇菜黄素、新黄素、黄氧素、ABA醛(ABA aldehyde)，然后得到ABA(Zeevaart，J.A.D.和Creelman R.A.，39：439-473，1998)。这一生物合成途径是通过对ABA生物合成的突变体进行生理学研究和分析后提出的。例如，从烟草(Nicotianatabacum)中分离的突变体aba2在催化玉米黄质发生环氧化反应的玉米黄质环氧化酶的基因(aba2)上具有一个突变(Marin E等，EMBOJ.，15：2331-2342，1996)。从玉米分离的突变体vp14在催化由新黄素向黄氧素转化的新黄素裂解酶的基因(vp14)上具有一个突变(Tan，B.C.等，美国国家科学院院报，94：12235-12240，1997)。从拟南芥属植物中分离出了在催化由黄氧素转化为ABA醛的反应的酶中发生了突变的突变体aba3和氧化ABA醛生成ABA的反应中所涉及的突变体aba4(Schwartz，S.H.等，植物生理学，114：161-166，1997；Leon-Kloosterziel，K.M.等，植物学杂志(Plant J.)，10：655-661，1996)。
已知新黄素裂解酶基因(vp14)中发生了突变的玉米表现出易失水分和易枯萎的特性。然而，还不清楚使用新黄素裂解酶基因是否是可以改善植物的逆境耐性。
发明简述
本发明的目的是提供一种编码用于改进植物逆境耐性的新黄素裂解酶的DNA，一种通过将所述DNA引进植物来提高植物逆境耐性的方法，和一种其中引进了新黄素裂解酶基因的转基因植物。本发明的另一个目的是提供一种用于降低植物逆境耐性的DNA，一种通过将所述DNA引进植物来降低植物逆境耐性的方法，和一种其中引进了所述DNA的转基因植物。植物逆境耐性的改进，例如，在植物培育中非常有用。
通过示差筛选制备于豇豆植物(Vigna unguiculata)的cDNA文库，该豇豆植物经脱水处理10小时后表现出很强的干旱耐受性，本发明人分离到对应于应答干旱处理有关的基因的cDNA克隆(CPRD65)。预测CPRD65cDNA能编码被认为与脱落酸(ABA)生物合成有关的新黄素裂解酶。施加于8天龄(8-day-old)豇豆植物的干旱逆境强烈地诱导了ABA的积累和CPRD65的表达，表明CPRD65基因的密切相关性，尤其在应答干旱逆境方面。采用GST-CPRD65融合蛋白对酶活性的测定证实了CPRD65具有将9-顺式-新黄素裂解为黄氧素的活性。这些结果表明，CPRD65基因编码新黄素裂解酶并且其产物在干旱逆境下生物合成内源ABA的过程中起着关键性的作用。
尤其，通过以分离于豇豆植物的CPRD65基因的cDNA为探针从源自于拟南芥属植物的cDNA文库中筛选新黄素裂解酶基因，本发明人分离到了一种新型基因(AtNCED3)。另外，鉴定了从拟南芥属植物得到的与这些基因具有高度同源性的四种序列(AtNCED1，2，4和5)。这些基因在大肠杆菌(E.coli)中的表达和新黄素裂解活性的分析表明AtNCED1，3和5具有与CPRD65相同的的新黄素裂解酶活性。
本发明人采用AtNCED3，一种新黄素裂解酶基因，首次制备出一种拟南芥属的转基因植物。AtNCED3基因被连接到一个载体中的35S启动子的下游，并且通过真空渗入法将载体引进拟南芥属，所述载体用于以有义(一种过表达类型)或反义(一种表达抑制类型)方向将基因引进植物细胞(pBE2113N)。对所制备的转基因植物的干旱耐受性进行的评价表明过表达植物与它们的亲本株系比较，其逆境耐性有了显著的提高。相反，引进了反义的表达-抑制株系，其逆境耐受性降低了(图15和16)。以这种方式，本发明人发现，实际上引进了新黄素裂解酶基因的转基因植物显著地增强了逆境耐性并且通过降低所述基因的表达显著地降低逆境耐性来完成本发明。
特别是，本发明涉及一种编码用于改进植物逆境耐性的新黄素裂解酶的DNA，一种通过将所述DNA引进植物来提高植物逆境耐性的方法，和引进了新黄素裂解酶基因的转基因植物，还涉及一种用于降低植物逆境耐性的DNA，一种通过将所述DNA引进植物来降低植物逆境耐性的方法，和引进了所述DNA的转基因植物，更具体地说，本发明提供了：
(1)一种分离到的DNA，该DNA编码具有用于改进植物逆境耐性的新黄素裂解活性的蛋白质，
(2)一种分离的用于降低植物逆境耐性的DNA，其中所述DNA选自以下一组DNA：
(a)一种编码反义RNA的DNA，所述反义RNA与具有新黄素裂解活性的蛋白质的编码基因转录物互补；
(b)一种编码具有核酶活性的RNA的DNA，所述核酶裂解具有新黄素裂解活性的蛋白质的编码基因转录物；和
(c)一种编码RNA的DNA，所述RNA在植物细胞中通过共抑制效应抑制编码具有新黄素裂解活性的蛋白质的基因表达，
(3)(1)或(2)的DNA，其中具有新黄素裂解活性的蛋白选自下列一组蛋白质：
(a)具有SEQ ID NOS：2(AtNCED1)、6(AtNCED3)、10(AtNCED5)、12(CPRD65)、14(VP14)或16(LeNCED1)所示氨基酸序列的蛋白质，
(b)具有SEQ ID NOS：2(AtNCED1)、6(AtNCED3)、10(AtNCED5)、12(CPRD65)、14(VP14)或16(LeNCED1)所示序列中的一个或多个氨基酸发生了置换、缺失、添加，和/或插入的氨基酸序列的蛋白质，和
(c)在严格条件下由与具有SEQ ID NOS：1(AtNCED1)、5(AtNCED3)、9(AtNCED5)、11(CPRD65)、13(VP14)或15(LeNCED1)所示核苷酸序列的DNA杂交的DNA编码的蛋白质，
(4)(1)至(3)中的任意一种DNA，其中具有新黄素裂解活性的所述蛋白质源自于拟南芥属植物，
(5)一种携带(1)至(4)中任意一项的DNA的转化植物细胞，
(6)含有(5)中所述的转化植物细胞的转基因植物，
(7)(6)中所述的转基因植物的子代或克隆的转基因植物，
(8)(6)或(7)中所述的转基因植物，其中具有新黄素裂解活性的蛋白质的编码基因表达与其野生型相比被增强或降低了，
(9)(6)至(8)中的任意一种转基因植物，其中脱落酸的量与其野生型相比增加或减少了，
(10)(6)至(9)中任意一种转基因植物，其中与其野生型相比逆境耐性被提高或降低了，
(11)一种(6)至(10)中的任意一种转基因植物的繁殖材料，
(12)含有(1)至(4)中的任意一种DNA的载体，
(13)一种用于生产(6)至(10)中的任意一种转基因植物的方法，包括步骤：将(1)至(4)中的任意一种DNA引进植物细胞中并由所述植物细胞再生植物，
(14)一种用于增强或降低植物的逆境耐性的方法，包括(1)至(4)中的任意一种DNA在植物细胞中的表达，
(15)(1)中所述的DNA用于改进植物逆境耐性的用途，和
(16)(2)中所述的DNA用于降低植物逆境耐性的用途。
本发明中，“逆境耐性”是指抵抗环境逆境的耐性，例如，干旱逆境耐性、盐逆境耐性、低温逆境耐性、空气污染逆境耐性、对于低氧条件的耐性、病原体耐性、药物如那些农用化学品耐性等。已知利用ABA进行外源处理能改进多种植物抵抗这些逆境的耐性(参考Takahashi，N.和Masuda，Y.(编辑)，“植物激素手册(Plant Hormone Handbook)(The Last)，”Baifukan，日本，pp.78-160；以及其中引用的参考文献)。
附图简述
图1表示经脱水或再水化作用的CPRD65基因表达的Northern印迹分析。由进行了0、1、2、4、6、8、10和12小时的脱水或脱水10小时后又进行了0、1、2、5、10和24小时的再水化作用的生长了8天(8-day-old)的豇豆植物制备总RNA。在每一电泳泳道上加上10μg的总RNA。将所述RNA在1％的琼脂糖凝胶中分级分离后，再印迹到尼龙膜上，并采用CPRD65克隆的[32P]-标记的cDNA插入片段进行探测。
图2表示CPRD65，VP14(玉米中的新黄素裂解酶，Schwartz，S.H.等，科学，276：1872-1874，1997)和LeNCED1蛋白，番茄(Lycopersicon esculentum)中的新黄素裂解酶，Burbidge，A.等，实验植物学杂志47：2111-2112，1997；Burbidge，A.等，植物杂志17：427-431，1999)的推测氨基酸序列的比较。虚线条表示为了优化排列顺序所引进的缺口。封闭框表示相同的氨基酸。阴影区域表示相似的氨基酸。
图3表示对源自于豇豆植物2246栽种品种的基因组DNA进行的SOUTHERN印迹分析。用EcoRI(E)、HindIII(H)和XbaI(X)消化基因组DNA(每泳道10μg)，在1％的琼脂糖凝胶中分级分离后，再转移到尼龙膜上使该膜与CPRD65 cDNA的[32P]-标记的片段进行杂交。“A”和“B”代表杂交条件的不同严格程度(参考实施例)。DNA片段的大小标记以kbp表示。
图4(A)表示通过高盐度(NaCl)、高温(加热)、低温(致冷)和使用脱落酸(ABA)对CPRD65基因进行诱导的Northern印迹分析。在指定时间处理后从豇豆植物中分离总RNA。每一泳道加上10μg的总RNA。每一泳道上端的数字表示处理持续的时间(小时)。
图4(B)表示没有经过或经过10小时脱水处理的CPRD65基因的Northern印迹分析。将从豇豆2246栽种品种的叶子(L)、茎(S)和根(R)中分离的总RNA各10μg加到每一泳道上。将所述RNA在1％的琼脂糖凝胶中分级分离后，再印迹到尼龙膜上，并采用CPRD65的[32P]-标记的cDNA插入片段进行探测。
图5表示GST(A)或GST-CPRD65重组蛋白(B)的类胡萝卜素代谢产物的HPLC峰图。反应混合物含有作为底物的顺式-新黄素。cN；顺式-新黄素，C25；C25-产物。
图6表示原生质体中的CPRD65N-sGFP嵌合蛋白质的质体导向。利用聚乙二醇(PGE)将携带有35S-sGFP(A、C、E)或35S-CPRD65N-sGFP嵌合构建体(B、D、F)的构建体转染到拟南芥的原生质体中。通过光学显微镜检(A，B)或带有绿色(E、F)或红色(C、D)滤光片的荧光显微镜检观察转染的原生质体。E和F表示GFP的定位，C和D表示叶绿体。
图7表示脱水期间ABA的积累与CPRD65的基因表达之间的关系。滞留在图1尼龙膜上的放射性如下所示定量和作图。ABA的定量方法如实施例所述。误差线表示标准误差。实验重复三次。
图8表示器官分离后脱水处理期间豇豆植物的叶(L)、茎(S)和根(R)中的内源ABA的积累。ABA的定量方法与实施例7中所述的相同(图7)。
图9表示AtNCED3和CPRD65的推测氨基酸序列之间的比较。虚线条表示为了优化排列顺序所引进的缺口。封闭框表示相同的氨基酸。阴影区域表示相似的氨基酸。
图10表示AtNCED1、2、3、4和5的氨基酸序列的排列顺序。虚线条表示为了优化排列顺序所引进的缺口。封闭框表示相同的氨基酸。阴影区域表示相似的氨基酸。
图11表示测定AtNCED1、2、3、4、5和CPRD65的氨基酸序列之间关系的种系发生(phylogenic)分析的结果，以及数据库中与它们有关的序列。LeNCED1(登记号Z97215)和VP14(登记号U95953)分别表示从番茄和玉米中分离的蛋白。
图12表示AtNCED基因在每一种逆境中的表达。
图13表示AtNCED3基因在AtNCED3-转化体中的表达。上面和下面的图分别表示AtNCED3基因在进行干旱逆境处理前和干旱逆境处理后的植物中的表达。表达有义AtNCED3基因(过表达)(A和B)的植物和表达反义AtNCED3基因(表达抑制)(C和D)的植物均使用两例植株。
图14表示AtNCED3-转化体中的内源ABA量。与野生型植物相比，表达有义AtNCED3基因(过表达)(A和B)的植物中的内源ABA量增大，而表达反义AtNCED3基因(表达抑制)(C和D)的植物中的内源ABA量减小。
图15表示新黄素裂解酶转基因植物的干旱耐性的测试结果，表明结束灌溉后14天的植物和叶子中的相对含水量。
图16表示新黄素裂解酶转基因植物的干旱耐性的测试结果，表明结束灌溉后17天的植物。
发明详述
本发明涉及一种分离的DNA，该DNA编码具有用于改进逆境耐性的新黄素裂解活性的蛋白。已知新黄素裂解酶是一种与ABA生物合成有关的酶，然而，还没有证实在对植物的生长没有产生严重的影响条件下植物中引进编码这种酶的DNA是否确实导致了ABA的积累并改进了抵抗逆境的耐受性。
采用ABA进行的外源处理引起，例如，许多植物的生长抑制。已知在种籽中ABA也导致生长抑制(发芽的抑制)(参考Takahashi，N.和Masuda，Y.(编)，“植物激素手册(The Last)，”Baifukan，日本，78-160页；以及其中引用的参考文献)。ABA含量的增加给植物带来了各种损伤。还没有报导通过外源基因造成ABA产生过量是否能导致逆境耐性的获得。采用ABA进行外源处理的传统实验方法需要高浓度的处理，其强烈地抑制了生长并妨碍了对耐性的精确测量。而且，外源性处理的实验没有确定出保证正常生长和获得耐性的合适ABA浓度。通过获得ABA生物合成基因和采用该基因来制备转基因植物，本发明人首次证实了植物的逆境耐性可以得到改进。
一种“分离DNA”是这样一种DNA，其结构不同于任何天然DNA结构或包括三个以上独立基因的天然基因组DNA片段的结构。因此该术语包括，例如，(a)一种具有天然存在于生物体基因组中的部分天然基因组DNA分子的序列的DNA；(b)结合到载体或原核或真核的基因组DNA上以致于所得分子不同于任何天然载体或基因组DNA的DNA；(c)分离的分子如cDNA、基因组片段、通过多聚酶链反应(PCR)制备的片段或限制性酶切片段；和(d)为杂交基因一部分的重组核苷酸序列，即编码融合蛋白的基因。特别是，除了这一定义以外，由不同的(i)DNA分子、(ii)转染细胞或(iii)细胞克隆，例如，源自于DNA文库如cDNA或基因组DNA文库的那些克隆所组成的混合物中存在的DNA分子。
作为用于增强逆境耐性的分离DNA，任何基因只要其编码具有新黄素裂解活性的蛋白，都能被使用。例如，玉米的VP14(Zea mays)(Schwartz，S.H.等，科学，276：1872-1874，1997；Tan，B.V.等，美国国家科学院院报，94：12235-12240，1997)(cDNA：SEQ ID NO：13，蛋白质：SEQ ID NO：14)，番茄(Lycopersicon esculentum)的LeNCED1(Burbidge，A.等，实验植物学杂志(J.Exp.Bot.)，47：2111-2112，1997；Burbidge，A.等，植物学杂志(Plant J.)17：427-431，1999)(cDNA：SEQ ID NO：15，蛋白质：SEQ ID NO：16)，将它们分离出来作为新黄素裂解酶基因。这些基因对改进逆境耐性有用并能用于本发明。另外，可以方便地使用编码AtNCED1(SEQ ID NO：2)、AtNCED3(SEQID NO：6)、AtNCED5(SEQ ID NO：10)和CPRD65(SEQ ID NO：12)的DNA(分别是SEQ ID Nos：1、5、9和11)。尤其是，编码SEQ ID NO：18所示的新黄素裂解酶的DNA(cDNA：SEQ ID NO：17，蛋白质：SEQ ID NO：18)(Neill，S.J.等，实验植物学杂志49：1893-1894，1998，登记号AJ005813)也可用于本发明。编码具有新黄素裂解活性的蛋白质的DNA还可用作增强逆境耐性的试剂(逆境耐性增强剂)。本发明还提供了编码具有新黄素裂解活性的蛋白质的DNA用于增强逆境耐性的用途。
本发明的编码具有新黄素裂解活性的蛋白质的DNA包括基因组DNA、cDNA和化学合成DNA。基因组DNA和cDNA通过本领域技术人员公知的方法来制备。基因组DNA可通过，例如，按照常规方法从植物中提取基因组DNA来制备，包括制备基因组文库(其中，作为载体，例如使用质粒、噬菌体、粘粒和BAC)，以本发明所述DNA(例如，SEQ ID Nos：1，5，9，11，13，15等)作为探针来进行菌落杂交或噬菌斑杂交。或者，也可通过采用特异于本发明DNA(例如，SEQ ID Nos：1，5，9，11，13，15等)的引物进行PCR反应来制备基因组DNA。根据从植物中提取出来的mRNA合成cDNA来制备cDNA，将该cDNA插入到载体如λ噬菌体上来制备和形成cDNA文库，并以与上述相同的方式进行菌落杂交或噬菌斑杂交，或进行PCR反应。本发明的DNA不仅包括SEQ ID Nos：1，5，9，11，13和15的DNA序列，而且还包括一种DNA，该DNA含有以编码每种蛋白质的氨基酸的密码简并性可选择的核苷酸序列。
本发明的DNA还包括，例如，编码一种蛋白质的DNA，该蛋白质含有SEQ ID Nos：2，6，10，12，14或16中的一个或多个氨基酸发生了置换、丢失、增加、和/或插入的氨基酸序列，并且该蛋白质具有新黄素裂解活性。因此，本发明的DNA包括SEQ ID Nos：1，5，9，11，13或15的突变体、衍生物、等位基因、变异体和同系物，一种从天然植物中衍生的基因。
由本发明的DNA编码的修饰蛋白含有的氨基酸序列至少70％(例如，80％、90％、95％或99％)与SEQ ID Nos：2，6，10，12，14或16相同。如本文使用的，两个氨基酸序列或两个核苷酸的“同一性百分率”是利用在Karlin和Altschul(美国国家科学院院报90：5873-5877，1993)中修正的Karlin和Altschul算法(美国国家科学院院报，87：2264-2268，1990)来确定的。这一算法被编入Altschul等的NBLAST和XBLAST程序(分子生物学杂志215：403-410，1990)中。利用NBLAST程序，比数＝100，字长＝12来进行BLAST核苷酸的搜索。利用XBLAST程序，比数＝50，字长＝3来进行BLAST蛋白的搜索。当两个序列之间存在缺口时，按照Altschul等中的描述使用缺口BLAST(核酸研究25：3389-3402，1997)。使用BLAST和缺口BLAST程序(例如，XBLAST和NBLAST)时，使用了每个程序的缺省参数。见http：//www.ncbi.nlm.nih.gov。
SEQ ID Nos：2，6，10，12，14或16中一个或多个氨基酸被置换了的修饰蛋白优选地通过至少一个保守氨基酸的置换来制备。“保守氨基酸的置换”是指下组中与同组另一氨基酸有化学相似性侧链的氨基酸残基的置换。具有相似侧链的氨基酸残基组在本技术领域中已被定义。这些组包括具有碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)，具有酸性侧链的氨基酸(例如，天冬氨酸、谷氨酸)，具有不带电极性侧链的氨基酸(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)，具有非极性侧链的氨基酸(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)，具有β-枝链侧链的氨基酸(例如，苏氨酸、缬氨酸、异亮氨酸)和具有芳族侧链的氨基酸(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
制备这种编码具有修饰氨基酸序列的蛋白质的DNA的方法实例是本技术领域的技术人员熟知的，即利用PCR进行的体外诱变(Izawa，T.，“体外PCR诱变”在Shimamoto，K.和Sasaki，T.(主管s)，细胞技术，增刊，植物细胞技术系列VII，植物PCR实验方案，新版，151-158页，Shujunsha，日本)。在人工修饰的情况下，蛋白质中的氨基酸修饰通常在200个氨基酸的范围内，优选地在100个氨基酸的范围内，更优选地在50个氨基酸的范围内，进一步更优选地在10个氨基酸的范围内。由于核苷酸编码序列的修饰而导致的蛋白质的氨基酸修饰可以自然发生。编码野生型新霉素裂解酶的氨基酸序列中有一或多个氨基酸发生置换、丢失、加入、和/或插入，这样的编码DNA也包括在本发明中，只要其编码具有新黄素裂解活性的蛋白质。本发明的DNA还包括简并变异体，该简并变异体的核苷酸序列中的一个突变不导致蛋白质的氨基酸发生突变。
给定的DNA是否编码新黄素裂解酶可通过在大肠杆菌中表达该DNA来制备重组蛋白并按照以下实施例5，以顺式-新黄素为底物检测裂解活性来确定。
在编码已知新黄素裂解酶的DNA基础上，可分离到一种新型新黄素裂解酶基因。本技术领域的技术人员熟知的用于实现这一目的的方法实例是利用杂交技术的方法(Maniatis，T等，1982，分子克隆：实验室指南，冷泉港，纽约，冷泉港实验室出版社)和聚合酶链反应(PCR)技术的方法(Nakayama，H.，细胞技术，增刊，生物学实验举例(Biological ExperimentIllustrated)，卷3，新版，Shujunsha，1998)。特别是，本技术领域的技术人员可通过以已知新黄素裂解酶基因的核苷酸序列(例如，SEQ ID Nos：1，5，9，11，13，15等)或其部分序列为探针，并以与这些序列特异杂交的寡聚核苷酸为引物来从任何植物中常规地分离编码新黄素裂解酶基因的DNA。本发明中用于改进逆境耐性的DNA也包括能够通过这种杂交技术或PCR技术分离的编码新黄素裂解酶的DNA。
按照例如参考文献中的方法(Sambrook，J.等，分子克隆：实验室指南，第二版，冷泉港实验室出版社，冷泉港，NY，1989)，以随机引物法制备的[32P]-标记的DNA为探针，在严格条件下进行杂交。将SOUTHERN印迹到尼龙膜上并与[32P]-标记的片段，在例如含有30％，优选50％的甲酰胺、6XSSC、5X Denhardt溶液和100μg/ml变性鲑精DNA的杂交溶液中，于37℃，优选42℃下进行杂交。在严格条件下，例如在1X SSC、0.1％SDS(室温)中洗涤15分钟、两次，优选(更严格地)在0.5X SSC、0.5％SDS(37℃)中洗涤15分钟、两次，更优选(更严格地)在0.1X SSC、0.1％SDS(60℃)中洗涤15分钟、两次，并进行放射自显影。“严格条件下的杂交”指在上述条件下利用碱基配对与固相参照核苷酸的特异性和非共价平衡结合。
为了使用这些DNA来制备逆境耐性得到改进的转基因植物，将所述DNA插入到合适的载体，并将该载体导入植物细胞，然后由转化的植物细胞再生为转基因植物。
任何能在植物细胞中表达插入基因的载体都可用作转化该细胞的载体。例如，可使用含有在植物细胞中稳定表达基因所需之启动子(例如，花椰菜花叶病毒的35S启动子)的载体和含有经外源刺激能激活的启动子的载体。或者，通过采用特异于植物组织的启动子诱导目的基因的表达，可专为对逆境高度敏感的组织提供逆境耐性。例如，利用特异性表达于种子中的基因的启动子，如菜豆的β-菜豆碱基因启动子(Bustos等，欧洲分子生物学组织杂志，10：1469-1479，1991)和大豆的大豆球蛋白基因启动子(Lelievre等，植物生理学，98：387-391，1992)，特异性表达于叶子中的基因的启动子，如豌豆的RbcS基因的启动子，(Lam和Chua，科学，248：471-474，1990)和小麦的Cab1基因的启动子(Gotorn等，植物志3：509-518，1993)，特异性表达于根中的基因的启动子，例如番茄的TobRB7基因的启动子(Yamamoto等，植物细胞，3：371-382，1991)等，可在组织中特异性表达目的基因。或者，可以使用具有由外源刺激物诱导激活的启动子的载体。一个响应环境逆境如干旱、盐或低温的启动子的实例是rd29A基因的启动子(Yamaguchi-Shinozaki，K.和Shinozaki，K.，植物细胞，6：251-264，1994)。本发明还优选地使用通过环境逆境如干旱和高盐浓度激活的启动子。这些启动子实例是下列基因的启动子：拟南芥属AtNCED3基因、豇豆CPRD65基因等。尤其是，通过使用由药物诱导的表达系统，目的基因可以在合适的时间和合适的组织中表达。由类固醇激素(糖皮质激素)诱导的表达系统的实例是利用GVG基因(GAL4，VP16，糖皮质激素受体)的诱导系统(Aoyama T.和Chua，N.H.，植物杂志，11：605-12，1997)。
载体可被插入到任何植物细胞中，例如，拟南芥(Arabidopsis thaliana)，水稻(Oriza sativa)，烟草(Nicotiana tabacum)，番茄(Lycopersicon esculentum)，土豆(Solanum tuberosum)，玉米(Zea mays)，鸟的脚三叶(birds’foottrefoil)(Lotus japonicus)等。也可使用其它的作物或树。植物可以是针叶树，阔叶树，双子叶植物，单子叶植物等。本文所用的“植物细胞”包括不同形式的植物细胞，例如，悬浮培养细胞，原生质体，叶子切片，愈伤组织等。
为了将载体引入植物细胞中，采用了本技术领域的技术人员已知的方法，例如，聚乙二醇方法，电穿孔方法，农杆菌属方法，真空渗入方法，粒子枪方法等。可采用本领域技术人员已知的方法根据植物细胞的类型从转基因植物细胞再生出植物。例如，水稻，拟南芥属等的转化体能够按照“Simamoto，K.，Okada K.(主管)，细胞技术，增刊，植物细胞技术系列4，模型植物实验方案，Shujunsha，Japan.”中描述的方法来制备。
一旦获得了其基因组中引进了本发明的DNA的转基因植物，可通过该植物的有性或无性繁殖得到子代。或者，由植物、它的子代或克隆得到繁殖材料(例如，种籽、果实、接穗、块茎、有块茎的根、茎干、愈伤组织、原生质体等)，并且可以由它们大量制备植物。本发明包括其中引进了本发明的DNA的植物细胞，含有该细胞的植物，该植物的子代或克隆，以及该植物、它的子代和克隆的繁殖材料。
与相应野生型植物比较，以这种方式制备的转化植物具有增加的ABA含量。或者，与相应野生型植物比较，以这种方式制备的转化植物具有改进的逆境耐性。逆境耐性可通过已知的方法进行比较。例如，如下面实施例所描述的，植物生长在逆境条件下，如干旱、高盐、低温或炎热的条件下，并且比较个体的生长情况。例如，通过测定表观、植物大小或组织如叶子、茎和根的大小、重量(湿重或干重)、颜色、相对生长速率、光合活性等作为指标来进行比较。逆境耐性可在植物的至少一种组织中得到增强。植物中的ABA含量通过例如免疫分析、薄层层析(TLC)、气相色谱(GC)和HPLC(参考Takahashi，N.和Masuda，Y.(eds)，“植物激素手册(最后一版)，”Baifukan，日本，pp.1-21；以及其中引用的参考文献)来测定。例如，如实施例7所描述的，以标记的ABA作为内标，通过HPLC和GC/MS可最终可靠地定量初制的纯化级分。
通过本发明，一种有价值的作物能够生长在受环境逆境影响的地区，例如，干旱地区、寒冷地区或盐浓度高的地区。另外，本发明可使除作物以外的植物在植物栽培环境中。
本发明还涉及一种DNA，该DNA能够降低具有新黄素裂解活性的蛋白质的编码基因的表达水平，所述蛋白质用于降低逆境耐性。所述DNA也可用作降低逆境耐性的试剂(逆境耐性降低试剂)。本发明还提供了一种DNA的用途，该DNA能够降低具有新黄素裂解活性的蛋白质的编码基因的表达水平，所述蛋白质用于降低逆境耐性。
通过施用于杂草等，降低了逆境耐性的植物有利于从环境中除掉杂草等。例如，制备能够引起逆境耐性降低的植物来改善土地等。对于再生能力强的植物，例如杂草，在其由化学试剂(例如，糖皮质激素等)诱导的启动子的下游引进一个抑制新黄素裂解酶基因表达(例如，以反义方向)的构建体。这一转化的植物在没有使用化学试剂的条件下就能正常生长。将这种转化种子种植几年，喷洒糖皮质激素以便通过特异性降低杂草的逆境耐性而迅速除去杂草，并种植一种作物可改善干旱的土地。就作物而言，可种植能过表达新黄素裂解酶的转化作物(该植物中以有义方向引进了此酶的DNA)。
本发明首先成功地制备了一种转化植物，通过采用表达新黄素裂解酶基因的反义RNA的基因构建体，该转化植物中新黄素裂解酶的表达被人为地抑制了。与其野生型比较，该植物在非灌溉条件下容易枯萎，表明其逆境耐性降低(图15和16)。以这种方式，本发明建立了一种人工抑制具有新黄素裂解活性的蛋白质的编码基因表达并因此成功地降低植物逆境耐性的方法。
本发明中，为了降低植物逆境耐性，可降低具有新黄素裂解活性之蛋白质的编码基因的表达。本文中基因“表达”包括基因的转录和转录物的翻译。表达的抑制包括表达的完全终止。为了抑制具有新黄素裂解活性的蛋白质的编码基因的转录和翻译，可通过抑制编码该基因的DNA、其转录控制区域、或其转录物来抑制该基因的表达。
任何植物可被用于本发明，因此可使用各种植物。例如，可以使用拟南芥属等。具有新黄素裂解活性的蛋白质的编码基因并可作为抑制表达的靶子的实例是玉米的VP14(Zea mays)，(Schwartz，S.H.等，科学276：1872-1874，1997；Tan，B.V.等，美国国家科学院院报94：12235-12240，1997)(cDNA：SEQ ID NO：13，蛋白质：SEQ ID NO：14)，番茄的LeNCED1(Lycopersicon esculentum)(Burbidge，A.等，实验植物学杂志47：2111-2112，1997；Burbidge，A.等，植物杂志17：427-431，1999)(cDNA：SEQ ID NO：15，蛋白质：SEQ ID NO：16)，AtNCED1(cDNA：SEQ ID NO：1，蛋白质：SEQ IDNO：2)，AtNCED 3(cDNA：SEQ ID NO：5，蛋白质：SEQ ID NO：6)，和/或拟南芥属的AtNCED 5(cDNA：SEQ ID NO：9，蛋白质：SEQ ID NO：10)，豇豆的CPRD65(cDNA：SEQ ID NO：11，蛋白质：SEQ ID NO：12)等。来自其它植物的同源基因也可以是一靶子。通过例如上文描述的杂交方法等可以鉴别和/或分离其它植物的同源基因。为了降低本发明中给定的植物的逆境耐性，可利用另一植物品种的基因或基因序列信息(例如，上述基因)，通过已知方法例如基因沉默和反义方法来抑制所需植物的靶基因的表达。因此，不需要分离也不需要鉴别目的植物的靶基因。
通过将抑制基因表达的DNA插入到合适的载体上，将该载体引入植物细胞中，由得到的转化细胞再生转基因植物能够抑制本发明中具有新黄素裂解活性的蛋白质的编码基因的表达。可以使用任何启动子，例如上述用于改进逆境耐性的启动子。例如，使用表达诱导型启动子只需在一种特定条件下就能降低逆境耐性。
作为抑制植物的特定内源基因表达的方法，本技术领域的技术人员最常使用的方法是反义技术。
反义方法是一种人工基因表达抑制方法，该方法中由靶mRNA与指定基因所转录的RNA的互补DNA分子(反义核酸)形成双链以抑制表达。1960年至1970年间建立了反义法抑制基因表达的方法，在1978年，Zamecnik等利用反义寡聚体成功地抑制了小鸡劳斯肉瘤病毒的复制和反转录酶的活性(Zamecnik，P.C.和Stephenson，M.L.，美国国家科学院院报，75：280-284，1978)。
在用于引入反义DNA的方法中，将反义寡聚体直接引入细胞中，或通过将靶基因的反义DNA连结到表达载体上进行转化。下面给出的实施例对后一方法做出了说明。具体是，将具有新黄素裂解活性的蛋白质的编码基因的cDNA以反义方向连接到花椰菜花叶病毒的35S启动子下游以便将载体引进植物细胞。采用过渡基因表达方法，Ecker等通过显示植物中以电穿孔方法引进的反义RNA的反义效应首次说明了植物细胞的反义效应(Ecker，J.R.和Davis，R.W.，美国国家科学院院报，83：5372，1986)。此后，报道了烟草和矮牵牛植物中反义RNA的表达对靶基因表达的抑制(van derKrol，A.R.等，自然，333：866，1988)。目前，反义方法被作为抑制植物中靶基因表达的好方法。用反义核酸抑制靶基因表达的模式包括通过三链的形成对转录起始产生的抑制，通过杂合体的形成对转录产生的抑制，所述杂合体含有由RNA聚合酶局部作用产生一个开环结构的位点，通过发生了合成的RNA杂合体的形成对剪接产生的抑制，通过与剪接体形成位点形成杂合体对剪接产生的抑制，通过与mRNA形成杂合体对由胞核到胞质的转移产生的抑制，通过与带帽位点或Poly(A)加入位点形成杂合体对剪接产生的抑制，通过与翻译起始因子结合位点形成杂合体对翻译的起始产生的抑制，通过与起始密码子侧翼的核糖体结合位点形成杂合体对翻译产生的抑制，通过与mRNA编码区域或多核糖体结合位点形成杂合体对肽链延伸产生的抑制，通过与核酸和蛋白之间的相互作用位点形成杂合体对基因表达的抑制等。这些抑制了转录、剪接或翻译过程，从而抑制了靶基因的表达(Hirajima和Inoue，“生物化学新实验2，核酸IV，基因的复制和表达”，日本生物化学会(编)，Tokyo-Kagakudojin，pp.319-347，1993)。
反义DNA的序列优选地是待转化的植物中与编码具有新黄素裂解活性的蛋白质的内源基因的转录物或其部分互补的序列，但是只要能够有效抑制基因的表达，但没有必要完全互补。转录RNA与靶基因的转录物优选地具有90％或更高的互补性，最优选地具有95％或更高的互补性。为了有效抑制使用反义序列的靶基因的表达，反义DNA的长度至少是15个或更多的核苷酸，优选地是100或更多的核苷酸，，更优选地是500或更多的核苷酸。通常，所使用的反义DNA短于5kb，优选地短于2.5kb。
采用编码核酶的DNA还能够抑制内源基因的表达。最近，对采用编码核酶的DNA来抑制基因的表达进行了研究。核酶是一种具有催化体内反应活性的RNA。核酶具有各种活性。针对核酶作为一种切割RNA的酶的研究使得设计一种用于在特异位点切割RNA的核酶成为可能。核酶包括I组内含子，有400或更多个核苷酸的大分子如包含在RnaseP中的M1RNA，和那些被称作锤头型和发夹型的核酶，其含有40个核苷酸长的活性结构域(Koizumi，M.，和Otsuka，E.，蛋白质，核酸和酶，35：2191，1990)。
例如，锤头型核酶的自切割结构域消化G13U14C15中的C15的3’位点。U14与第9位A之间碱基对的形成被认为对该活性非常重要，如果第15位核苷酸是A或U或C，那么也能被消化(Koizumi，M.等，FEBS Lett.，228：225，1988)。如果核酶的底物结合位点被涉设计成能与靶位点侧翼的RNA序列互补，那么就产生了一种能够识别靶RNA中的UC，UU，或UA序列的限制酶样核酶(Koizumi，M.等，FEBS Lett.，239：285，1988；Koizumi，M.，Otuska E.，蛋白质，核酸和酶，35：2191，1990；Koizumi，M.等，核酸研究，17：7059，1989)。例如，拟南芥属的AtNCED3基因的编码区存在上百个这样的位点。例如在烟草环斑病毒中的卫星RNA的负链中发现了发夹型核酶(Buzayan，J.M.，自然，323：349，1986)。也显示该核酶能够被设计用来特异地切割靶RNA(Kikuchi，Y.和Sasaki，N.，核酸研究，19：6751，1992；Kikuchi，H.，化学和生物学，30：112，1992)。
为了能在植物细胞中进行转录，将用于切割靶而设计的核酶与启动子例如花椰菜花叶病毒的35S启动子和转录终止序列连接。在转录RNA的5’端和3’端加入额外序列，就可以消除核酶的活性。在这种情况下，为了只从含有核酶的转录RNA上准确切除核酶部分，可以放置另一种在核酶部分的5’端和3’端进行顺式剪切的剪切核酶(Taira，K.等，蛋白质工程，3：733，1990；Dzianott，A.M.和Bujarski，J.J.，美国国家科学院院报.，86：4823，1989；Grosshans，C.A.，和Cech，R.T.，核酸研究，19：3875，1991；Taira，K.等，核酸研究，19：5125，1991)。另外，为了提高效率，将这一结构单元串联排列以切割靶基因中的多个位点(Yuyama，N.等，Biochem.Biophys.Res.Commun.，186：1271，1992)。可通过采用这类核酶来切割本发明的靶基因转录物，从而抑制其表达。优选特异地切割靶基因的转录物的核酶。可通过采用这类核酶来特异切割本发明的靶基因转录物以抑制其表达。
通过引进含有与靶基因序列相同或相似的序列的DNA而产生的共抑制也可抑制内源基因的表达。“共抑制”是指这样一种现象，其中通过转化将与内源靶基因序列相同或相似的基因引进植物来同时抑制引进的外源基因和内源靶基因的表达。还不清楚共抑制的详细机制，然而，在植物中却能经常观察到共抑制。(Curr.Biol.，7：R793，1997；Curr.Biol.，6：810，1996)。例如，通过制备含有具有新黄素裂解活性的蛋白质的编码基因或相似序列的载体DNA，用该载体转化目的植物，并以其中具有新黄素裂解活性的蛋白质的编码基因的表达降低这一特性对获得的植物中进行筛选以便获得其中具有新黄素裂解活性的蛋白质的编码基因的表达受到共抑制的植物。
尽管用于共抑制的基因不需要与靶基因完全相同，但是通常应具有至少70％或更高，优选地80％或更高，更优选地90％或更高的同一性。Genetyx(Software Development)，一种基因信息处理软件能被用于测定同一性或互补性。这一程序采用了Lipman-Pearson方法(Lipman，D.J.和Pearson，W.R.，科学，227：1435-1441，1985)。这个方法首先比较序列数据，并计算具有高度同源性的序列之间的同一性，其中考虑了序列的缺失(GAP)。
通过采用用于本发明的上述抑制基因表达的DNA可以制备逆境耐性降低了的转化植物。具体是，通过将该DNA插入到合适的载体上，将该载体引入植物细胞中，由得到的转化细胞再生转基因植物。作为使用的载体，只要插入的基因能在植物细胞中表达，任何载体都可以以与上述增加逆境耐性时相同的方式来加以使用。任何植物细胞可以用来插入载体。植物可以是针叶树、阔叶树、双子叶植物(dicot)、单子叶植物等。本文引用的“植物细胞”包括各种形式的植物细胞，例如悬浮培养细胞、原生质体、叶子切片、愈伤组织等。
可通过采用本技术领域技术人员已知的方法，依据植物细胞的类型，以与改进逆境耐性时相同的方式将该载体引入植物细胞中并由转化细胞再生植物。一旦获得了其基因组中引进了本发明的DNA的转基因植物，通过有性或无性繁殖可由植物得到子代。或者，由植物、它的子代或克隆得到繁殖材料(例如，种籽、果实、接穗、块茎、有块茎的根、茎干、愈伤组织、原生质体等)，并且可以由它们大量制备植物。本发明涉及其中引进了本发明的DNA的植物细胞，含有该细胞的植物，植物的子代或克隆，以及植物、它的子代和克隆的繁殖材料。
与野生型植物比较，以这种方式制备的转化体植物具有降低的ABA含量。或者，与野生型植物比较，该转化体植物具有降低的逆境耐性。本发明可用于，例如杂草的有效清除。另外，采用上述的诱导型启动子，本发明的转化体植物可用于改善土质等。
本发明能够制备一种逆境耐性提高或降低的植物。逆境耐性改进的植物能够在至今植物不能生长的劣质土壤中生长。降低的逆境耐性可用于杂草而将杂草清除。本发明的方法可用于农业以扩大培育区域和提高作物产率。
将本文引用的任何专利、专利申请和公开出版物引入作为参考。
下面借助实施例来详细说明本发明，但并不是用于限制本发明。这些实施例中采用的实验条件如下所示。
豇豆的生长
将豇豆种籽(Vigna unguiculata IT84S-2246-4)种植在盆中并在光周期为16小时的温室(除了自然光外，当光照不充足时补充人工光照)中生长8天，温度为25℃，浇水适量。
脱水处理
为了进行脱水处理，将植物小心地从盆中拔出以免造成损伤，称重，并用3MM Whatman滤纸在室温和暗光(300lux)下大约60％的湿度下进行脱水。作为对照，将植物从盆中拔出后立刻转种在水份充足的土壤中，其中该土壤保持在与脱水处理组相同的条件下。
DNA的序列分析
利用PI-100型自动质粒分离系统(Automatic Plasmid Isolation SystemModel PI-100)(KURABO)制备质粒DNA模板，并利用373A型DNA测序仪(ABI)进行测序。利用GeneWorks软件系统(IntelliGenetics，Inc.)，Sequencher 3.0(Hitachi软件)，和威斯康星大学基因计算机组(GCG)程序来分析核苷酸序列和氨基酸序列。
实施例1：对应于由脱水诱导的基因的cDNA克隆的分离
采用聚腺苷酸化(Poly(A)+)RNA构建cDNA文库，该聚腺苷酸化RNA是从按下述脱水逆境作用10小时后的8天龄的豇豆植物中分离出来的。
收获完整的植物，轻轻洗掉根上的土并用3MM Whatman滤纸在室温和暗光下大约60％的湿度下进行脱水。从通过上述方法脱水处理后的植物中制备全长RNA(Nagy，F.等，“转基因植物中基因表达的分析”在Gelvin和Schilperroort(编)，“植物分子生物学手册B4.，”Kluwer Academic Publishers，Dordrecht，1-29页，1988)。按照参考文献(Sambrook，J.等，“分子克隆：实验室手册，第二版，冷泉港实验室出版社，冷泉港，纽约，1989)，将全长RNA两次通过寡脱氧胸苷酸(Oligo-dT)纤维素柱以便制备聚腺苷酸化RNA。大约2％的加样到柱上的RNA作为聚腺苷酸化RNA级分收集。利用cDNA Synthesis System Plus(Amersham Pharmacia Biotech)由聚腺苷酸化RNA合成双链cDNA。利用cDNA Cloning System(Amersham PharmaciaBiotech)由该cDNA构建cDNA文库。
采用由分离于未经逆境作用的豇豆植物的聚腺苷酸化RNA制备的cDNA和分离于经脱水逆境作用10小时的植物的聚腺苷酸化RNA制备的cDNA对cDNA文库进行示差筛选。按照参考文献(Sambrook，J.等，“分子克隆：实验室手册，第二版，冷泉港实验室出版社，冷泉港，纽约，1989)，通过噬菌斑杂交来从cDNA文库中筛选1×104个噬菌斑。
结果，从脱水10小时的豇豆植物中获得了与[32P]-标记的cDNA有较强杂交信号的噬菌斑。噬菌体克隆子的质粒区域进行体内切除后被用来转化大肠杆菌的细胞。利用限制性酶切图谱和该cDNA片段的边缘序列对获得的质粒cDNA片段进行分析。根据这些分析，将cDNA分组，并鉴别了称作CPRD(能应答脱水处理的豇豆)65的cDNA克隆。
通过RNA印记杂交分析经脱水诱导的对应于CPRD65克隆的基因的表达。从土壤中拔出8天龄的植物并进行不同时间的脱水处理，最长脱水时间为12个小时。作为对照，从土壤中拔出相似的豇豆植物并立刻转种到水份充足的土壤中。然后从脱水的植物和对照植物中分离出用于RNA印记杂交的全长RNA。
根据Nagy等的方法分离全长RNA(Nagy，F.等，“转基因植物中基因表达的分析”在Gelvin和Schilperoort(编)，“植物分子生物学手册B4.，”Kluwer Academic Plublishers，Dordrecht，1-29页，1988)，在含甲醛的1％的琼脂糖凝胶中进行分离，并转移到尼龙膜上(Sambrook，J.等，“分子克隆：实验室手册，第二版，冷泉港实验室出版社，冷泉港，纽约，1989)。42℃下，在50％的甲酰胺、5X SSC、25mM的磷酸钠缓冲液(PH6.5)、10X Denhardt溶液和250μg/ml的变性鲑精DNA中将尼龙膜与[32P]-标记的CPRD65 cDNA片段进行杂交。膜在60℃下用0.1X SSC、O.1％SDS中洗涤15分钟、两次，并进行放射自显影。
图1表示对应于经脱水处理的CPRD65基因的表达的诱导过程。通过脱水逆境作用，CPRD65的表达水平明显提高。观察到开始脱水处理后的2个小时内积累了对应于CPRD65的mRNA。
脱水10小时的豇豆植物发生枯萎。转移到水分充足的土壤(再水化处理)后的4个小时内，这些枯萎的植物得到恢复。再水化后，CPRD65 mRNA的含量降低了(图1)。CPRD65基因对干旱逆境表现出典型且明显的反应，即通过脱水诱导转录和通过再水化降低其含量。这些事实表明CPRD65基因与干旱耐性有关。
实施例2：CPRD65 cDNA的序列分析
由于实施例1中分离到的CPRD65 cDNA片段可能不是全长，所以为了分离到全长cDNA(SEQ ID NO：11)，还要以一种部分CPRD65 cDNA为探针再次筛选同一cDNA文库。由全长cDNA克隆(SEQ ID NO：12)编码的氨基酸序列如图2表示。全长CPRD65 cDNA由2432个碱基对组成，包括由125个碱基对组成的5’-侧翼区和由486个碱基对组成的3’-侧翼区。在3’-侧翼区发现了一个聚腺苷酸化的共同序列(AATAAA)。这个序列具有一个编码612个氨基酸多肽的开放读框，推测该假定蛋白的分子量为67.6kDa。如图2所示，将推测的CPRD65蛋白的氨基酸序列与蛋白数据库进行比较，结果表明与玉米(Zea mays)的VP14(61％)(Schwartz，S.H.，科学，276：1872-1874，1997)和番茄(Lycopersicon esculentum)的新黄素裂解酶(69％)(Burbidge，A.等，实验植物学杂志47：2111-2112，1997；Burbidge，A.等，植物杂志，17：427-431，1999)具有很高的同源性。就像VP14蛋白一样，推测的CPRD65在其N-末端区域似乎含有一个转运多肽。CPRD65、VP14和番茄的新黄素裂解酶的N-末端序列具有较低的序列相似性，但却具有结构相似性。
实施例3：CPRD65基因的基因组SOUTHERN印迹分析
为了分析与豇豆植物的CPRD65有关的基因，在两个严格的条件下进行基因组SOUTHERN印迹杂交(图3)。
按照参考文献的方法进行基因组SOUTHERN印迹分析(Sambrook，J.等，“分子克隆：实验室手册，第二版，冷泉港实验室出版社，冷泉港，纽约，1989)。用限制酶消化10μg的基因组DNA，在1％的琼脂糖凝胶中进行分离，并印迹到尼龙膜上。在42℃条件下，滤膜与[32P]-标记的片段在30％的甲酰胺、6X SSC、5X Denhardt溶液和100μg/ml的变性鲑精DNA中进行杂交。在60℃条件下，用0.1X SSC、0.1％SDS将膜洗涤两次，各15分钟(B)或在37℃条件下，0.5X SSC、0.5％SDS洗涤两次，各15分钟(A)，并进行放射自显影。
通过测定其核苷酸序列，CPRD65 cDNA没有EcoRI和XbaI的内在限制酶切位点，而有HindIII的两个侧翼内在限制酶切位点。以CPRD65 cDNA作为探针，测出EcoRI和XbaI消化液中有一条杂交带和HindIII消化液中有两条杂交带。在上述严格的条件(A)下测出另外的一些模糊的杂交带。这些结果表明CPRD65基因与有关基因组成一个小基因家族。
实施例4：CPRD65基因的RNA印迹分析
分析各种环境逆境对CPRD65基因表达的影响。对于高盐浓度、ABA和水处理，以与脱水处理相同的方式将植物从土壤中拔出，并分别在含有250mM NaCl，100μM ABA和去离子水的溶液中通过含水培养进行生长。对于加热和冷处理，将盆中的植物分别转移到40℃和4℃的培养箱中。植物的各种逆境处理进行0、1、2、5、10和24小时。处理后，立刻用液氮冷冻被处理的植物，并分离RNA用于RNA印迹分析。
结果发现，这一基因的表达在高盐条件下受到很强的诱导，但不受冷或热逆境的诱导(图4A)。对于经ABA处理或水处理的情况，没有检测到CPRD65基因受到诱导。
为了确定干旱逆境下CPRD65基因表达的组织特异性，在正常或干旱条件下对从叶、茎、或根提取的全长RNA进行了RNA印迹杂交(图4B)。通过干旱处理，CPRD65转录物在茎和叶中受到强烈诱导，但在根中受到的诱导较弱。
实施例5：细菌表达的CPRD65蛋白的酶活性
CPRD65基因的推测氨基酸序列与由玉米VP14基因编码的新黄素裂解酶的氨基酸序列具有高度同源性(图2)。为了检验CPRD65基因是否编码新黄素裂解酶，分析在大肠杆菌中表达的重组CPRD65蛋白的生化特性。通过PCR扩增CPRD65编码区的DNA片段并利用pGEX4T-1(Pharmacia)将该片段与GST基因融合到同一读码框中来构建如下所述的嵌合质粒pGST-CPRD65。
采用引物5’-ATTGAATTCATGCCTTCAGCTTCAAAC-3’(SEQ IDNO：19)和5’-ATTGGATCCCAAAAGCTACACGCTGGTCCCC-3’(SEQ IDNO：20)，通过PCR扩增编码CPRD65蛋白的DNA。将PCR片段插入到pBluescript II SK+载体中的EcoRV位点上。证实插入的PCR片段的序列以测定通过PCR是否在核苷酸序列中发生了突变。利用限制酶(EcoRI和XhoI)，从pBluescript II SK+载体的核苷酸序列中没有发生任何突变的PCR片段作为DNA片段分离出，并将该片段插入到pGEX4T-1(Amersham PharmaciaBiotech)中的EcoRI至XhoI位点之间来构建pGST-CPRD65。用pGST-CPRD65或pGEX4T-1转化大肠杆菌菌株JM109的细胞并在37℃下于L肉汤培养基中进行培养。当D600达到大约0.5时，加入异丙基β-D-硫代半乳糖吡喃糖苷(IPTG)，在17℃下继续培养12小时。收集细胞，洗涤并悬浮在提取缓冲液(10mM Tris-HCl(PH8.0)、5mM MgCl2，5％的甘油、0.1mM苯甲基磺酰氟(PMSF)和0.1mM二硫苏糖醇(DTT))中。按照GST基因融合系统的说明书手册(Amersham Pharmacia Biotech)纯化融合蛋白并用凝血酶消化。采用蛋白分析试剂盒(Bio-Rad)测定蛋白的浓度。
GST-CPRD65融合蛋白以与上述相同的方式在大肠杆菌中过表达，并利用谷胱甘肽-Sepharose 4B从细胞粗提物中纯化。对于该纯化的GST-CPRD65重组蛋白是否能消化顺-新黄素、反-堇菜黄素和顺-堇菜黄素来产生黄氧素进行了鉴定。
分析新黄素裂解酶活性的方法已被描述过了(Schwartz，S.H.等，科学，276：1872-1874，1997)。从菠菜叶中制备顺-新黄素和反-堇菜黄素。从桔子皮中制备顺-堇菜黄素。反应混合物(100μl)含有100mM双-Tris(PH6.7)、0.05％的Triton X-100、10mM抗坏血酸、5mM FeSO4和蛋白样品。反应在室温下进行1小时。加入1ml水后，用正己烷(1ml×2)萃取反应混合物后再用乙酸乙酯(1ml×2)萃取。浓缩正己烷级分并用Nucleosil 5 C18柱(150mm长，8mm内径(i.d.))进行HPLC分析。以1.5ml/min的流速使用溶剂A(85％的乙醇)和溶剂B(氯仿和甲醇，1∶1)之间的线性梯度对柱进行洗脱。在25分钟内溶剂B的浓度从10％增加到50％，并在50％处保持5分钟。用紫外光检测仪在440nm处检测洗脱物的吸光度。在Nucleosil 5 C18柱(150mm长，8mm内径(i.d.))上，采用HPLC提纯乙酸乙酯级分。以1.5ml/min的流速采用50％的含水甲醇对柱进行洗脱，并用紫外光检测仪在260nm处检测洗脱物的吸光度。收集预测的黄氧素级分并进行GC-MS分析。每一步骤中，样品尽可能避光。
GC-MS分析如下进行。配有5890气相色谱(Hewlett Packard)的AUTOMASS质谱仪(Nippon Denshi)用于本分析。分析条件如下：电离，EI 70eV；柱，DB-5(15m长；0.25mmi.d.；0.25μm膜厚；J&W Scientific)；载体气体，He(1ml min-1)；进样温度，250℃；转线温度，250℃；和初始加热温度80℃。进样1分钟后开始，加热温度以30℃min-1的速率升高到200℃，接着以5℃min-1的速率进一步升高到230℃。
如图5所示，通过HPLC分析，在含有GST-CPRD65蛋白和顺-新黄素的反应混合物中检测到预测的C25-产品和黄氧素。通过GC-MS分析证实了黄氧素的存在，分析过程中观察到了黄氧素的离子特性。离子及其相对强度是：m/z 250(4)，168(32)，149(77)，107(61)，和95(100)。由反-堇菜黄素没有生成黄氧素和C25-产物(没有显示数据)。这些结果不受凝血酶处理的影响，该凝血酶将GST-CPRD65重组蛋白分解成GST和CPRD65蛋白。
实施例6：由拟南芥属植物制备的原生质体中作为转运肽的CPRD65蛋白的N末端区域分析
CPRD65蛋白的N-末端区域具有与叶绿体导向有关的转运肽的典型结构特征。CPRD65蛋白的这一结构特征表明成熟的CPRD65蛋白定位在含有叶绿体的质体上。为了分析N-末端区域作为转运肽的作用，构建了编码CPRD65蛋白的N-末端区域(1-148)的嵌合基因35S::CPRD65N-sGFP，该嵌合基因位于CaMV 35S启动子和水母Aequorea victoria的合成绿开花期(synthetic green florescent)蛋白(sGFP)基因之间(Chiu，W.，等，现代生物学(Curr.Biol.)，6：325-330，1996)。
采用引物5’-ATATATCTAGAATGCCTTCATCAGCTTCAAACACTTGG-3’(SEQ ID NO：21)和5’-ATATAGGATCCCTCCGGCACCGGCGCGAAGTTCCCG-3’(SEQ ID NO：22)，通过PCR扩增对应于CPRD65蛋白N-末端肽(1至148个氨基酸)的DNA。将PCR片段插入到pBluescript II SK+载体中并证实通过PCR没有引起序列发生突变。将该DNA片段插入到35S-启动子与过渡表达载体的sGFP基因之间的位点上(Chiu，W.，等，现代生物学，6：325-330，1996)。按照上文中描述的方法进行制备、DNA转染和拟南芥属原生质体的培养(Abel，S.和Theologis，A.，植物杂志，5：421-427，1994)。
通过DNA-转染方法将35S::CPRD65-sGFP融合构建体及其对照构建体(35S::sGFP)引进由拟南芥属制备的原生质体中(Abel，S.和Theologis，A.，植物杂志，5：421-427，1994)。转化2-4天后，通过荧光显微术观察原生质体。如图6所示，当在原生质体中过渡表达35S::sGFP构建体时，荧光定位不在质体中。另外，当引进35S::sGFP构建体时，荧光定位不在质体中，而主要是定位在细胞质中。这些结果表明，CPRD65蛋白的N-末端区域作为转运肽起着将CPRD65蛋白导向到质体的作用。预测CPRD65蛋白定位在质体中，在质体中发挥着产生ABA的作用。
实施例7：通过脱水逆境作用在8天龄的豇豆植物中的ABA积累
通过脱水条件测定8天龄的豇豆植物中内源ABA含量的积累。
将样品在液氮中进行均质并用含水甲醇(20-80％)提取两次。加入[2H3]ABA之后，浓缩提取物，并进行标准溶剂分馏过程以便得到酸性-乙酸乙酯可溶级分。通过以前报道的方法(Wijayanti，L.，等，Biosci.Biotech.Biochem.，59：1533-1535，1995)，采用结合洗脱柱体(C18和DEA，Varian)进行纯化。然后采用Senshu PakODS-2101-N柱(100mm长，6mm的内径，SenshuScientific Co.)对从没有干燥的植物中纯化的样品进行HPLC分析。分析条件与前面所报道的相同(Wijayanti，L.，等，Biosci.Biotech.Biochem.，59：1533-1535，1995)。采用醚性重氮甲烷将如此纯化的样品甲基化并进行GC-SIM分析。
如图7所示，ABA在脱水后2小时内开始积累。ABA在经10小时脱水的植物中的含量比未经逆境的对照植物中的高140倍。CPRD65 mRNA积累时间比ABA mRNA积累时间要早。
在叶和茎中CPRD65基因的表达受干旱逆境的强烈诱导，而在根中受到很小的诱导作用(图4B)。检测了在干旱逆境作用下CPRD65基因的表达与内源ABA的积累之间的关系。将8天龄的豇豆植物分割成叶、茎和根，然后进行脱水。分别测定这些器官在脱水处理前或后的内源ABA含量。如图8所示，通过干旱逆境作用，叶和茎中积累了大量的内源ABA，而根中积累得很少。干旱逆境作用下的ABA积累的组织特异模式与图4B和8中所示的CPRD65基因的表达模式一致。
实施例8：豇豆叶中的叶黄素的分析
分析豇豆叶中的叶黄素以便发现与CPRD65蛋白有关的物质。
样品采用丙酮提取两次，浓缩提取物，溶解在80％的甲醇(1ml)中，并加样到结合洗脱C18柱上。用另外的4ml、80％的甲醇洗柱，并用5ml的甲醇-水-氯仿(71∶9∶20)洗脱叶黄素。浓缩洗脱物并采用Nucleosil 5 C18柱和Senshu Pak Silica-2251-S柱(250mm长，6mmd内径)进行HPLC分析。ODS-HPLC采用的条件与上述相同。对于Silica-HPLC，使用的流速为1.5ml/分钟，溶剂B浓度的线性梯度是在30分钟内从10％到100％，其中溶剂A是乙酸乙酯-正己烷(1∶1)，溶剂B是乙酸乙酯。利用叶黄素的可见光谱和紫外光谱数据鉴定叶黄素。
通过可见光和紫外光分光光度分析，检测到豇豆叶中反-新黄素、反-堇菜黄素和顺-新黄素为主要叶黄素，顺-堇菜黄素为少量成分(没有显示数据)。在正常生长条件和干旱条件下，反-新黄素、反-堇菜黄素和顺-新黄素的内源含量没有明显的区别。
如上所示，豇豆干旱-诱导型CPRD65基因编码新黄素裂解酶，其产品定位于质体中。在干旱和高盐条件下，CPRD65基因主要在叶和茎中被强烈诱导。观察到在干旱条件下，叶和茎中ABA的大量积累，这表明一种类似于CPRD65基因的表达模式。这些结果有力地说明了CPRD65蛋白主要是一种与干旱逆境作用下豇豆植物中ABA的生物合成有关的酶。
实施例9：编码新黄素裂解酶基因的同系物的拟南芥属cDNA克隆的分离
以CPRD65为探针，筛选拟南芥属的cDNA文库(Abe H.等，植物细胞，9；1859-1868，1997)。结果，获得了许多具有强杂交信号的噬菌斑。从这些噬菌斑中分离出噬菌体克隆，用限制酶切出cDNA区域以便插入到pBluescrip SK+中并转化大肠杆菌。通过DNA序列分析，将这些克隆归为一组。将其命名为AtNCED3。该AtNCED3显示出与编码新黄素裂解酶的CPRD65具有高度的同源性(图9)。AtNCED3 cDNA的核苷酸序列和AtNCED3蛋白的氨基酸序列分别由SEQ ID NO：5和6表示。
实施例10：AtNCED1、2、4和5的分离，种系发生树(phylogenic tree)的分析
利用CPRD65和AtNCED3的核苷酸序列搜索DNA数据库，鉴定出具有高度同源性的四个序列(登记号AL021713，AL021687，AJ005813，AB028621)。
为了分离与AL021713序列具有高度同源性的基因，以gDNA为模板和以5’-CCCGGGATCCCTCAAGCCTCTCTATACCG-3’(SEQ ID NO：23)和5’-CCCGGGATCCTTTATACGGATTCTGAGGGAG-3’(SEQ ID NO：24)为引物，通过PCR法扩增靶基因片段。以该片段为探针，从gDNA文库(Clontech)中分离含有靶基因的克隆。以5’-CCCGGGATCCCTCAAGCCTCTCTATACCG-3’(SEQ ID NO：23)和5’-CCCGGGATCCTTTATACGGATTCTGAGGGAG-3’(SEQ ID NO：24)为引物，再次通过PCR方法扩增该基因，并将该基因克隆到pBluescript II SK+(Stratagene)中EcoRV位点上来测定核苷酸序列。该基因被命名为AtNCED1。AtNCED1 cDNA的核苷酸序列和AtNCED1蛋白的氨基酸序列分别由SEQ ID NO：1和2表示。
为了分离与AL021687序列具有高度同源性的基因，以gDNA为模板和以5’-ATTGAATTCATGGACTCTGTTTCTTCTTCTTCC-3’(SEQ ID NO：25)和5’-ATTGAATTCTTAAAGCTTATTAAGGTCACTTTCC-3’(SEQ ID NO：26)为引物，通过PCR法扩增靶基因片段。以该片段为探针，从gDNA文库(Clontech)中分离含有该靶基因的克隆。以5’-ATTGAATTCATGGACTCTGTTTCTTCTTCTTCC-3’(SEQ ID NO：25)和5’-ATTGAATTCTTAAAGCTTATTAAGGTCACTTTCC-3’(SEQ ID NO：26)为引物，再次通过PCR方法扩增基因，并将该基因克隆到pBluescript II SK+(Stratagene)中EcoRV位点上来测定核苷酸序列。该基因被命名为AtNCED2。AtNCED2 cDNA的核苷酸序列和AtNCED2蛋白的氨基酸序列分别由SEQ ID NO：3和4表示。
为了分离与AJ005813序列具有高度同源性的基因，以gDNA为模板和以5’-AAGAATTCATGGCGGAGAAACTCAGTGATGGCAGC-3’(SEQ IDNO：27)和5’-AAAAGAATTCGGCTTATATAAGAGTTTGTTCCTG G-3’(SEQID NO：28)为引物，通过PCR扩增靶基因片段。以该片段为探针，从cDNA文库(Clontech)中分离含有靶基因的克隆。以5′-AAGAATTCATGGCGGAGAAACTCAGTGATGGCAGC-3’(SEQ ID NO：27)和5’-AAAAGAATTCGGCTTATATAAGAGTTTGTTCCTGG-3’(SEQ ID NO：28)为引物，再次通过PCR方法扩增基因，并将该基因克隆到pBluescript II SK+(Stratagene)中EcoRV位点上来测定核苷酸序列。该基因被命名为AtNCED4。AtNCED4 cDNA的核苷酸序列和AtNCED4蛋白的氨基酸序列分别由SEQ ID NO：7和8表示。
为了分离与AB028621序列具有高度同源性的基因，从P1克隆MUJ8中分离DNA。以5’-CGGGATCCATGCAACACTCTCTTCGTTCTGATCTTCTTC-3’(SEQ ID NO：29)和5’-CGGGATCCTCAGAAAACTTGTTCCTTCAACTGATTCTCGC-3’(SEQ ID NO：30)为引物，通过PCR方法扩增基因，并将该基因克隆到pBluescript II SK+(Stratagene)中EcoRV位点上来测定核苷酸序列。该基因被命名为AtNCED5。AtNCED5 cDNA的核苷酸序列和AtNCED5蛋白的氨基酸序列分别由SEQ ID NO：9和10表示。
图10显示了AtNCED1至5的氨基酸序列的对比排列。为了检测由每一序列推导出的氨基酸序列与数据库中的序列之间的关系，进行种系发生树分析(图11)。利用基因分析软件GeneWorks(Intelligenetics，Inc.)构建种系发生树。公式采用UPGMA(由Nei，M.撰写的，并由Gojo，H.和Saito，N.，Baifukan翻译的：Unweighted Pair Group Method with ArithmeticMean：Molecular Evolutionary genetics，252-256页，Japan)。
实施例11：AtNCED基因的RNA印迹分析
通过RNA印迹分析来分析各种环境逆境对每一鉴定的AtNCED基因表达的影响。
在琼脂平板上生长三周的植物被用于进行每种逆境处理。对于脱水逆境，从琼脂培养基中拔出植物，并在滤纸上风干(相对湿度为50％)。对于盐逆境、ABA处理和作为对照的水处理，从琼脂培养基中拔出植物，并放入分别含有250Mm NaCl溶液，100μM ABA溶液和蒸馏水的培养皿中，以便只将根浸入，盖上盖于室温下保持一段时间。对于冷和热逆境，将琼脂平板分别放置在4℃和40℃的恒温箱中保持一段时间。
在液氮中磨碎经上述各种逆境处理的植物，提取全长RNA(Nagy F，KaySA和Chua N-H(1988)转基因植物中基因表达的分析。在Gelvin和Schilperoort(编)，植物分子生物学手册B4.Kluwer Academic Publishers，Dordrechrt，1-29页)，并取每一样品20μg点样到1％琼脂糖凝胶的每一泳道上进行电泳。电泳后将RNA从凝胶印迹到尼龙膜上，以[32P]-标记的cDNA为探针进行RNA印迹分析(Sambrook，J.，Fritsch EF和Maniatis T(1989)“分子克隆：实验室手册”第二版，冷泉港实验室出版社，冷泉港，纽约)。
结果发现AtNCED3基因表达受到干旱、高盐浓度和冷条件的强烈诱导。加热条件不诱导表达。另外，对于ABA或水处理，没有观察到AtNCED3基因表达受到诱导(图12)。
实施例12：细菌表达的AtNCED蛋白的酶特性
推导的AtNCED3基因的氨基酸序列与编码新黄素裂解酶的豇豆CPRD65基因的氨基酸序列具有高度同源性(图9)。为了检验AtNCED3基因是否编码新黄素裂解酶，分析了由大肠杆菌表达的重组AtNCED3蛋白的生化特性。
以克隆的AtNCED3cDNA为模板，以5’-ATTGAATTCATGGCTTCTTTCACGGCAACGGC-3’(SEQ ID NO：31)和5’-GTTTTCCCAGTCACGAC-3’(SEQ ID NO：32)为引物，通过PCR方法扩增编码AtNCED3蛋白的DNA。将该PCR片段克隆到pBluescript II SK+(Stratagene)中EcoRV位点上。证实插入的PCR片段以确定通过PCR合成的核苷酸序列是否产生了突变。将其中没有检测到任何突变的DNA片段以框架形式克隆到含有谷胱甘肽S-转移酶(GST)基因的pGEX4T-1中的EcoRI位点上(Amersham Pharmacia Biotech)来构建嵌合质粒pGST-AtNCED3。用pGST-AtNCED3或pGEX4T-1转化大肠杆菌菌株JM109的细胞并在37℃下于L肉汤培养基中进行培养。当OD600达到大约0.5时，加入异丙基β-D-硫代半乳糖吡喃糖苷(IPTG)，在17℃下继续培养12小时。收集大肠杆菌细胞，洗涤并悬浮在提取缓冲液(10mMTris-HCl(PH8.0)、5mM MgCl2，5％d的甘油、0.1mM苯甲基磺酰氟(PMSF)和0.1mM二硫苏糖醇(DTT))中。使用谷胱甘肽-Sepharose 4B[GST基因融合系统(Amersham Pharmacia Biotech)]按其说明书纯化融合蛋白并用凝血酶消化。用蛋白分析试剂盒(Bio-Rad，CA，USA)测定蛋白浓度。
新黄素裂解酶活性分析的结果表明，在含有GST-AtCED3蛋白和顺-新黄素的反应混合物中检测到了预期的C25产物和黄氧素，证明AtNCED3蛋白具有新黄素裂解活性。类似的实验在AtNCED1和AtNCED5中检测到了新黄素裂解活性。
实施例13：转基因植物的制备
以拟南芥属(Arabidopsis thaliana(L.)Heynh.ecotype Columbia)作为样品。采用培养土壤在9cm-直径的塑料盆中播种野生型拟南芥属植物，以16小时的光周期在22℃下生长6星期，然后用于转化。
由含有卡那霉素抗性标记和花椰菜花叶病毒的35S启动子的pBE2113载体构建不含GUS报道基因的载体(pBE2113NOT)(Mitsuhara，I.等，植物细胞生理学，37：49-59，1996)，将从拟南芥属中分离到的AtNCED3的cDNA以右向(有义方向)或反向(反义方向)连接到载体的BamHI位点。通过混合载体和细菌，将获得的载体引进土壤细菌(根癌农杆菌菌株GV3101(pMP90))中。通过卡那霉素(Km)抗性筛选含有靶基因的根癌农杆菌，采用真空渗入方法感染野生型拟南芥属植物(Bechtold，N.等，C.R.Acad.Sci.Paris，LifeSci.，316：1194-1199，1993)。从感染的植物中获得干种籽，播种在加了Km的琼脂平板中进行生长以便选择转化体第一代(T1)的个体。将从转化体第一代得到的转化体第二代(T2)的种籽播种在加了Km的琼脂平板上，种植后从显示Km抗性的植物中收集第三代(T3)的种籽。此外，将第三代的种籽以类似的方式播种在加了Km的平板中，表现出抗药性的所有种籽作为T3同源株系被用于下面实验。最后，AtNCED3基因的每一有义和反义转化体分离出两个株系。
实施例14：转化体中AtNCED3基因表达的评价
通过Northern杂交方法评价AtNCED3基因在野生型拟南芥属和AtCED3-基因转化的植物中的表达。
培育了一个月的植物用于分析转化体中AtNCED3基因的表达。拔出植物，并在滤纸上风干(相对湿度为50％)作为干旱逆境处理。在液氮中裂解经上述环境逆境处理的植物以便提取全长RNA(Nagy F，Kay SA和Chua N.-H.(1998)转基因植物中基因表达的分析。在Gelvin和Schilperoort编，植物分子生物学手册B4.Kluwer Academic Publishers，Dordrechrt，1-29页)。RNA在1％琼脂糖凝胶中进行电泳(每一泳道20μg)，从电泳凝胶中转移到尼龙膜上，使用[32P]-标记的RNA探针进行Northern杂交(Sambrook，J.，Fritsch，E.F.和Maniatis T.(1989)“分子克隆实验室手册”第二版，冷泉港实验室出版社，冷泉港，纽约)。
结果，在受到干旱逆境作用前，AtNCED3基因在野生型植物中不表达，但却在有义植物中表达了。另一方面，干旱处理后，AtNCED3基因在野生型植物中受干旱诱导进行表达，但是在反义植物中即使受到干旱逆境处理也不表达(图13)。
实施例15：转化体中内源ABA含量的评价
测定野生型拟南芥属和AtNCED3-基因转化的植物中的内源ABA含量。
培育了一个月的植物被用于评价野生型拟南芥属和转化体中的内源ABA含量。将样品在液氮中进行均质并用液体甲醇(20-80％)提取两次。加入[2H3]ABA，浓缩提取物，并进行标准溶剂分馏过程来得到酸性-乙酸乙酯可溶级分。按照Wijayanti，L.，等，Biosci.Biotech.Biochem.，59：1533-1535，1995中描述的方法，采用结合洗脱柱体(C18和DEA，Varian)纯化这些级分。然后采用Senshu PakODS-2101-N柱(100mm长，6mm的内径)(SenshuScientific Co.)对从没有干燥的植物中纯化的样品进行HPLC分析。分析条件与Wijayanti，L.等，Biosci.Biotech.Biochem.，59：1533-1535，1995中描述的相同。采用醚性重氮甲烷将纯化样品甲基化并进行GC-SIM分析。
结果，与其野生型相比，在有义植物中ABA含量增加了，在反义植物中ABA含量降低了(图14)。
实施例16：干旱耐受性的评价结果
将获得的转化体植物的种籽播种在加了营养盐的琼脂平板上(Valvekens，D.等，美国国家科学院院报，85：5536-5540，1988)，在上述种植条件下生长两周以便进行下面的实验。
将4株上述植物转种到填满土的9cm-直径的塑料盆(蛭石∶珍珠岩＝1∶1)中，在22℃和光周期为16小时的条件下生长。播种三周后(转种后两周)，通过中止浇水，使盆脱水以便自然产生干旱逆境。开始停止浇灌后14天和17天，对植物进行拍照。其中以反义方向引进AtNCED3基因的植物在开始停止浇灌后14天枯萎(图15)。相反，以有义方向引进基因的转化体植物没有枯萎。野生株系也在开始停止浇灌后17天枯萎，然而以有义方向引进基因的转化体植物对干旱表现出很高的耐受性(图16)。
序列表
<110>RIKEN
<120>携带新黄素裂解酶基因的转基因植物
<130>R3-102DP1
<140>
<141>
<150>JP 2000-010056
<151>2000-01-13
<160>32
<170>PatentIn Ver.2.0
<210>1
<211>1752
<212>DNA
<213>拟南芥
<220>
<221>CDS
<222>(1)..(1752)
<400>1
atg gtt tct ctt ctt aca atg ccg atg agt ggt ggt att aaa aca tgg 48
Met Val Ser Leu Leu Thr Met Pro Met Ser Gly Gly Ile Lys Thr Trp
1 5 10 15
cct caa gcc caa att gat ttg ggt ttt agg ccc att aaa aga caa ccg 96
Pro Gln Ala Gln Ile Asp Leu Gly Phe Arg Pro Ile Lys Arg Gln Pro
20 25 30
aag gtt att aaa tgc acg gtg cag atc gac gta acg gaa tta acc aaa 144
Lys Val Ile Lys Cys Thr Val Gln Ile Asp Val Thr Glu Leu Thr Lys
35 40 45
aaa cgc caa tta ttt aca ccc aga acc acc gct act ccg ccg cag cat 192
Lys Arg Gln Leu Phe Thr Pro Arg Thr Thr Ala Thr Pro Pro Gln His
50 55 60
aat cct ctc cgg cta aac atc ttc cag aaa gcg gcg gcg att gcg atc 240
Asn Pro Leu Arg Leu Asn Ile Phe Gln Lys Ala Ala Ala Ile Ala Ile
65 70 75 80
gac gcg gct gag cgt gca tta atc tca cac gag caa gat tct cca ctt 288
Asp Ala Ala Glu Arg Ala Leu Ile Ser His Glu Gln Asp Ser Pro Leu
85 90 95
ccc aaa acc gct gat cca cgt gtt cag att gcc ggg aat tat tcc ccg 336
Pro Lys Thr Ala Asp Pro Arg Val Gln Ile Ala Gly Asn Tyr Ser Pro
100 105 110
gta ccg gaa tct tcc gtc cgg cga aac ctc acc gtc gaa gga aca atc 384
Val Pro Glu Ser Ser Val Arg Arg Asn Leu Thr Val Glu Gly Thr Ile
115 120 125
cct gac tgc att gac ggt gtt tat atc cgt aac ggc gcg aat ccg atg 432
Pro Asp Cys Ile Asp Gly Val Tyr Ile Arg Asn Gly Ala Asn Pro Met
130 135 140
ttt gag cca aca gct ggg cac cat tta ttc gac gga gac gga atg gtt 480
Phe Glu Pro Thr Ala Gly His His Leu Phe Asp Gly Asp Gly Met Val
145 150 155 160
cac gca gtt aaa ata acc aac ggt tca gct agc tac gca tgc cgg ttt 528
His Ala Val Lys Ile Thr Asn Gly Ser Ala Ser Tyr Ala Cys Arg Phe
165 170 175
aca aaa acc gag aga ttg gtt cag gaa aaa cga ttg ggt cga cca gtt 576
Thr Lys Thr Glu Arg Leu Val Gln Glu Lys Arg Leu Gly Arg Pro Val
180 185 190
ttc ccg aaa gca atc ggc gag ctt cac ggt cac tcg gga atc gca cgt 624
Phe Pro Lys Ala Ile Gly Glu Leu His Gly His Ser Gly Ile Ala Arg
195 200 205
ttg atg ctg ttt tac gca cgt ggg ctt tgt ggt ctg atc aac aac caa 672
Leu Met Leu Phe Tyr Ala Arg Gly Leu Cys Gly Leu Ile Asn Asn Gln
210 215 220
aac ggc gtc gga gta gca aac gcc ggt ttg gtt tac ttt aat aac cgg 720
Asn Gly Val Gly Val Ala Asn Ala Gly Leu Val Tyr Phe Asn Asn Arg
225 230 235 240
ctt tta gct atg tca gaa gac gat tta ccg tac caa tta aaa att act 768
Leu Leu Ala Met Ser Glu Asp Asp Leu Pro Tyr Gln Leu Lys Ile Thr
245 250 255
caa acc ggc gat ctc caa acc gtt gga cgt tac gat ttc gac ggt cag 816
Gln Thr Gly Asp Leu Gln Thr Val Gly Arg Tyr Asp Phe Asp Gly Gln
260 265 270
tta aaa tcc gca atg ata gct cac ccg aaa ctg gac ccg gtt acg aag 864
Leu Lys Ser Ala Met Ile Ala His Pro Lys Leu Asp Pro Val Thr Lys
275 280 285
gag ctt cac gcg tta agc tac gac gtc gtt aag aaa cct tac ctg aaa 912
Glu Leu His Ala Leu Ser Tyr Asp Val Val Lys Lys Pro Tyr Leu Lys
290 295 300
tac ttc aga ttc tcg cca gac ggc gtt aaa tcg ccg gaa ttg gag atc 960
Tyr Phe Arg Phe Ser Pro Asp Gly Val Lys Ser Pro Glu Leu Glu Ile
305 310 315 320
ccg ctc gaa act ccg acg atg att cac gat ttc gct ata acg gag aat 1008
Pro Leu Glu Thr Pro Thr Met Ile His Asp Phe Ala Ile Thr Glu Asn
325 330 335
ttt gtg gtg att cct gat caa caa gtc gtg ttc aag ctc ggc gag atg 1056
Phe Val Val Ile Pro Asp Gln Gln Val Val Phe Lys Leu Gly Glu Met
340 345 350
att tcc ggt aaa tct ccg gtt gtt ttc gac gga gaa aag gtt tcc cga 1104
Ile Ser Gly Lys Ser Pro Val Val Phe Asp Gly Glu Lys Val Ser Arg
355 360 365
ttg ggg ata atg ccc aag gac gcg aca gaa gct tct cag ata atc tgg 1152
Leu Gly Ile Met Pro Lys Asp Ala Thr Glu Ala Ser Gln Ile Ile Trp
370 375 380
gtg aac tct ccg gag acg ttc tgt ttt cat ctc tgg aat gca tgg gaa 1200
Val Asn Ser Pro Glu Thr Phe Cys Phe His Leu Trp Asn Ala Trp Glu
385 390 395 400
tcg ccg gag acg gag gag att gtg gtg atc gga tcg tgt atg tcg ccg 1248
Ser Pro Glu Thr Glu Glu Ile Val Val Ile Gly Ser Cys Met Ser Pro
405 410 415
gcg gat tca atc ttc aac gag aga gac gag agc ttg aga agc gtt ttg 1296
Ala Asp Ser Ile Phe Asn Glu Arg Asp Glu Ser Leu Arg Ser Val Leu
420 425 430
tcg gag atc agg ata aac ctc aga aca cgt aaa acc acg cgt cgt tcg 1344
Ser Glu Ile Arg Ile Asn Leu Arg Thr Arg Lys Thr Thr Arg Arg Ser
435 440 445
ttg ttg gtt aac gag gat gta aat tta gag att ggt atg gtt aac cgg 1392
Leu Leu Val Asn Glu Asp Val Asn Leu Glu Ile Gly Met Val Asn Arg
450 455 460
aac cgg tta gga aga aaa acc cgg ttc gcg ttt ttg gct att gct tat 1440
Asn Arg Leu Gly Arg Lys Thr Arg Phe Ala Phe Leu Ala Ile Ala Tyr
465 470 475 480
cct tgg cca aaa gtt tcc ggt ttc gct aag gtc gat ctt tgc acc ggt 1488
Pro Trp Pro Lys Val Ser Gly Phe Ala Lys Val Asp Leu Cys Thr Gly
485 490 495
gag atg aaa aaa tat att tac ggc ggt gag aaa tat ggc ggc gaa ccg 1536
Glu Met Lys Lys Tyr Ile Tyr Gly Gly Glu Lys Tyr Gly Gly Glu Pro
500 505 510
ttt ttc ttg ccc ggc aac tcc ggt aac ggc gaa gaa aat gaa gat gac 1584
Phe Phe Leu Pro Gly Asn Ser Gly Asn Gly Glu Glu Asn Glu Asp Asp
515 520 525
ggt tat ata ttt tgt cac gtt cat gac gaa gaa aca aag aca tca gag 1632
Gly Tyr Ile Phe Cys His Val His Asp Glu Glu Thr Lys Thr Ser Glu
530 535 540
ctt cag att att aac gct gtt aat tta aag ctt gaa gct acg att aaa 1680
Leu Gln Ile Ile Asn Ala Val Asn Leu Lys Leu Glu Ala Thr Ile Lys
545 550 555 560
cta ccg tct aga gta ccg tat ggg ttt cat ggc aca ttt gtg gat tcg 1728
Leu Pro Ser Arg Val Pro Tyr Gly Phe His Gly Thr Phe Val Asp Ser
565 570 575
aat gaa ctc gtt gat caa tta taa 1752
Asn Glu Leu Val Asp Gln Leu
580
<210>2
<211>583
<212>PRT
<213>拟南芥
<400>2
Met Val Ser Leu Leu Thr Met Pro Met Ser Gly Gly Ile Lys Thr Trp
1 5 10 15
Pro Gln Ala Gln Ile Asp Leu Gly Phe Arg Pro Ile Lys Arg Gln Pro
20 25 30
Lys Val Ile Lys Cys Thr Val Gln Ile Asp Val Thr Glu Leu Thr Lys
35 40 45
Lys Arg Gln Leu Phe Thr Pro Arg Thr Thr Ala Thr Pro Pro Gln His
50 55 60
Asn Pro Leu Arg Leu Asn Ile Phe Gln Lys Ala Ala Ala Ile Ala Ile
65 70 75 80
Asp Ala Ala Glu Arg Ala Leu Ile Ser His Glu Gln Asp Ser Pro Leu
85 90 95
Pro Lys Thr Ala Asp Pro Arg Val Gln Ile Ala Gly Asn Tyr Ser Pro
100 105 110
Val Pro Glu Ser Ser Val Arg Arg Asn Leu Thr Val Glu Gly Thr Ile
115 120 125
Pro Asp Cys Ile Asp Gly Val Tyr Ile Arg Asn Gly Ala Asn Pro Met
130 135 140
Phe Glu Pro Thr Ala Gly His His Leu Phe Asp Gly Asp Gly Met Val
145 150 155 160
His Ala Val Lys Ile Thr Asn Gly Ser Ala Ser Tyr Ala Cys Arg Phe
165 170 175
Thr Lys Thr Glu Arg Leu Val Gln Glu Lys Arg Leu Gly Arg Pro Val
180 185 190
Phe Pro Lys Ala Ile Gly Glu Leu His Gly His Ser Gly Ile Ala Arg
195 200 205
Leu Met Leu Phe Tyr Ala Arg Gly Leu Cys Gly Leu Ile Asn Asn Gln
210 215 220
Asn Gly Val Gly Val Ala Asn Ala Gly Leu Val Tyr Phe Asn Asn Arg
225 230 235 240
Leu Leu Ala Met Ser Glu Asp Asp Leu Pro Tyr Gln Leu Lys Ile Thr
245 250 255
Gln Thr Gly Asp Leu Gln Thr Val Gly Arg Tyr Asp Phe Asp Gly Gln
260 265 270
Leu Lys Ser Ala Met Ile Ala His Pro Lys Leu Asp Pro Val Thr Lys
275 280 285
Glu Leu His Ala Leu Ser Tyr Asp Val Val Lys Lys Pro Tyr Leu Lys
290 295 300
Tyr Phe Arg Phe Ser Pro Asp Gly Val Lys Ser Pro Glu Leu Glu Ile
305 310 315 320
Pro Leu Glu Thr Pro Thr Met Ile His Asp Phe Ala Ile Thr Glu Asn
325 330 335
Phe Val Val Ile Pro Asp Gln Gln Val Val Phe Lys Leu Gly Glu Met
340 345 350
Ile Ser Gly Lys Ser Pro Val Val Phe Asp Gly Glu Lys Val Ser Arg
355 360 365
Leu Gly Ile Met Pro Lys Asp Ala Thr Glu Ala Ser Gln Ile Ile Trp
370 375 380
Val Asn Ser Pro Glu Thr Phe Cys Phe His Leu Trp Asn Ala Trp Glu
385 390 395 400
Ser Pro Glu Thr Glu Glu Ile Val Val Ile Gly Ser Cys Met Ser Pro
405 410 415
Ala Asp Ser Ile Phe Asn Glu Arg Asp Glu Ser Leu Arg Ser Val Leu
420 425 430
Ser Glu Ile Arg Ile Asn Leu Arg Thr Arg Lys Thr Thr Arg Arg Ser
435 440 445
Leu Leu Val Asn Glu Asp Val Asn Leu Glu Ile Gly Met Val Asn Arg
450 455 460
Asn Arg Leu Gly Arg Lys Thr Arg Phe Ala Phe Leu Ala Ile Ala Tyr
465 470 475 480
Pro Trp Pro Lys Val Ser Gly Phe Ala Lys Val Asp Leu Cys Thr Gly
485 490 495
Glu Met Lys Lys Tyr Ile Tyr Gly Gly Glu Lys Tyr Gly Gly Glu Pro
500 505 510
Phe Phe Leu Pro Gly Asn Ser Gly Asn Gly Glu Glu Asn Glu Asp Asp
515 520 525
Gly Tyr Ile Phe Cys His Val His Asp Glu Glu Thr Lys Thr Ser Glu
530 535 540
Leu Gln Ile Ile Asn Ala Val Asn Leu Lys Leu Glu Ala Thr Ile Lys
545 550 555 560
Leu Pro Ser Arg Val Pro Tyr Gly Phe His Gly Thr Phe Val Asp Ser
565 570 575
Asn Glu Leu Val Asp Gln Leu
580
<210>3
<211>1788
<212>DNA
<213>拟南芥
<220>
<221>CDS
<222>(1)..(1788)
<400>3
atg gac tct gtt tct tct tct tcc ttc ctc tcc tcc aca ttc tct ctt 48
Met Asp Ser Val Ser Ser Ser Ser Phe Leu Ser Ser Thr Phe Ser Leu
1 5 10 15
cat cac tct ctt ctt cgc cgc cga tct tcc tct cct act ctc ctc cgt 96
His His Ser Leu Leu Arg Arg Arg Ser Ser Ser Pro Thr Leu Leu Arg
20 25 30
atc aac tcc gcc gtc gtc gaa gaa cgt tct cca atc aca aac cca agc 144
Ile Asn Ser Ala Val Val Glu Glu Arg Ser Pro Ile Thr Asn Pro Ser
35 40 45
gac aac aat gat cgt cgt aac aaa ccc aaa aca ctc cac aac cga acc 192
Asp Asn Asn Asp Arg Arg Asn Lys Pro Lys Thr Leu His Asn Arg Thr
50 55 60
aat cac acc tta gtc tca tca cca ccg aaa ctc cga cca gaa atg act 240
Asn His Thr Leu Val Ser Ser Pro Pro Lys Leu Arg Pro Glu Met Thr
65 70 75 80
ctc gca aca gct ctc ttc acc acc gtc gaa gat gta atc aac acg ttc 288
Leu Ala Thr Ala Leu Phe Thr Thr Val Glu Asp Val Ile Asn Thr Phe
85 90 95
atc gat cca cct tca cgt cct tcc gtt gat cca aaa cat gtc ctc tct 336
Ile Asp Pro Pro Ser Arg Pro Ser Val Asp Pro Lys His Val Leu Ser
100 105 110
gat aac ttc gct cct gtc ctc gac gag ctt cct cca aca gac tgt gaa 384
Asp Asn Phe Ala Pro Val Leu Asp Glu Leu Pro Pro Thr Asp Cys Glu
115 120 125
atc atc cac ggc act ctt cca ctg tca ctt aac ggc gct tac atc cgt 432
Ile Ile His Gly Thr Leu Pro Leu Ser Leu Asn Gly Ala Tyr Ile Arg
130 135 140
aac ggt cca aat cca cag ttt ctc cct cgt ggt cct tac cat ctc ttc 480
Asn Gly Pro Asn Pro Gln Phe Leu Pro Arg Gly Pro Tyr His Leu Phe
145 150 155 160
gac ggc gac ggt atg ctt cac gcc ata aaa atc cac aac ggt aaa gcc 528
Asp Gly Asp Gly Met Leu His Ala Ile Lys Ile His Asn Gly Lys Ala
165 170 175
act ctc tgt agc aga tac gtc aag act tat aaa tac aac gtc gag aaa 576
Thr Leu Cys Ser Arg Tyr Val Lys Thr Tyr Lys Tyr Asn Val Glu Lys
180 185 190
caa acc gga gct ccg gtt atg cct aac gtg ttt tcc gga ttc aac ggt 624
Gln Thr Gly Ala Pro Val Met Pro Asn Val Phe Ser Gly Phe Asn Gly
195 200 205
gta acg gcg tca gta gct cgt gga gct tta acg gca gct agg gtt tta 672
Val Thr Ala Ser Val Ala Arg Gly Ala Leu Thr Ala Ala Arg Val Leu
210 215 220
acc gga cag tat aat ccg gtt aac ggc att ggt tta gct aat aca agt 720
Thr Gly Gln Tyr Asn Pro Val Asn Gly Ile Gly Leu Ala Asn Thr Ser
225 230 235 240
cta gct ttc ttc agt aac cgt ctc ttt gct tta ggt gaa tct gat tta 768
Leu Ala Phe Phe Ser Asn Arg Leu Phe Ala Leu Gly Glu Ser Asp Leu
245 250 255
ccc tac gcc gtc cga tta acc gaa tca gga gat att gaa acg atc gga 816
Pro Tyr Ala Val Arg Leu Thr Glu Ser Gly Asp Ile Glu Thr Ile Gly
260 265 270
cgg tac gat ttc gac ggg aaa tta gcg atg agt atg aca gct cat cct 864
Arg Tyr Asp Phe Asp Gly Lys Leu Ala Met Ser Met Thr Ala His Pro
275 280 285
aaa acc gat cca ata acc gga gaa act ttc gct ttc cgg tac ggt ccg 912
Lys Thr Asp Pro Ile Thr Gly Glu Thr Phe Ala Phe Arg Tyr Gly Pro
290 295 300
gtt cca ccg ttt tta aca tat ttc cgg ttt gat tcc gcc ggg aaa aaa 960
Val Pro Pro Phe Leu Thr Tyr Phe Arg Phe Asp Ser Ala Gly Lys Lys
305 310 315 320
caa aga gac gtt ccg ata ttc tcg atg acg tct ccg tcg ttt ctc cat 1008
Gln Arg Asp Val Pro Ile Phe Ser Met Thr Ser Pro Ser Phe Leu His
325 330 335
gac ttc gcg atc acg aaa cgt cac gcg att ttc gca gag att cag ctt 1056
Asp Phe Ala Ile Thr Lys Arg His Ala Ile Phe Ala Glu Ile Gln Leu
340 345 350
ggc atg agg atg aac atg ttg gat ttg gtt ctc gaa ggt ggt tct ccg 1104
Gly Met Arg Met Asn Met Leu Asp Leu Val Leu Glu Gly Gly Ser Pro
355 360 365
gtt ggt act gat aac gga aaa act cca agg ctt gga gtg att cct aag 1152
Val Gly Thr Asp Asn Gly Lys Thr Pro Arg Leu Gly Val Ile Pro Lys
370 375 380
tac gcc gga gat gag tcg gag atg aaa tgg ttc gaa gtt cct gga ttc 1200
Tyr Ala Gly Asp Glu Ser Glu Met Lys Trp Phe Glu Val Pro Gly Phe
385 390 395 400
aat atc att cac gct att aat gct tgg gat gaa gat gat gga aac agc 1248
Asn Ile Ile His Ala Ile Asn Ala Trp Asp Glu Asp Asp Gly Asn Ser
405 410 415
gtc gtt ttg att gca ccg aat att atg tcg att gaa cat act tta gag 1296
Val Val Leu Ile Ala Pro Asn Ile Met Ser Ile Glu His Thr Leu Glu
420 425 430
agg atg gat ctg gtt cat gct ttg gtg gag aag gtg aag atc gat ctc 1344
Arg Met Asp Leu Val His Ala Leu Val Glu Lys Val Lys Ile Asp Leu
435 440 445
gtc acc ggg att gtg aga cgt cat ccg atc tca gcg agg aat ctc gat 1392
Val Thr Gly Ile Val Arg Arg His Pro Ile Ser Ala Arg Asn Leu Asp
450 455 460
ttc gct gtg att aat ccg gcg ttt ctc ggg aga tgt agc agg tac gtt 1440
Phe Ala Val Ile Asn Pro Ala Phe Leu Gly Arg Cys Ser Arg Tyr Val
465 470 475 480
tac gcg gcg att gga gat ccg atg ccg aag atc tcc ggt gtg gtg aag 1488
Tyr Ala Ala Ile Gly Asp Pro Met Pro Lys Ile Ser Gly Val Val Lys
485 490 495
ctt gat gtg tct aaa gga gat cgg gat gat tgt acg gtg gcc cgt aga 1536
Leu Asp Val Ser Lys Gly Asp Arg Asp Asp Cys Thr Val Ala Arg Arg
500 505 510
atg tac ggt tca ggt tgt tac ggc gga gaa ccg ttt ttc gta gct agg 1584
Met Tyr Gly Ser Gly Cys Tyr Gly Gly Glu Pro Phe Phe Val Ala Arg
515 520 525
gat cct ggt aat ccg gag gcg gag gag gat gat ggt tat gtg gtg acg 1632
Asp Pro Gly Asn Pro Glu Ala Glu Glu Asp Asp Gly Tyr Val Val Thr
530 535 540
tat gtt cac gat gaa gtg act gga gaa tcg aag ttt ctg gtg atg gac 1680
Tyr Val His Asp Glu Val Thr Gly Glu Ser Lys Phe Leu Val Met Asp
545 550 555 560
gct aaa tcg ccg gag ctt gaa atc gtc gcc gcc gtg agg ttg ccg cga 1728
Ala Lys Ser Pro Glu Leu Glu Ile Val Ala Ala Val Arg Leu Pro Arg
565 570 575
agg gtt ccg tac gga ttc cat ggg tta ttt gtc aag gaa agt gac ctt 1776
Arg Val Pro Tyr Gly Phe His Gly Leu Phe Val Lys Glu Ser Asp Leu
580 585 590
aat aag ctt taa 1788
Asn Lys Leu
595
<210>4
<211>595
<212>PRT
<213>拟南芥
<400>4
Met Asp Ser Val Ser Ser Ser Ser Phe Leu Ser Ser Thr Phe Ser Leu
1 5 10 15
His His Ser Leu Leu Arg Arg Arg Ser Ser Ser Pro Thr Leu Leu Arg
20 25 30
Ile Asn Ser Ala Val Val Glu Glu Arg Ser Pro Ile Thr Asn Pro Ser
35 40 45
Asp Asn Asn Asp Arg Arg Asn Lys Pro Lys Thr Leu His Asn Arg Thr
50 55 60
Asn His Thr Leu Val Ser Ser Pro Pro Lys Leu Arg Pro Glu Met Thr
65 70 75 80
Leu Ala Thr Ala Leu Phe Thr Thr Val Glu Asp Val Ile Asn Thr Phe
85 90 95
Ile Asp Pro Pro Ser Arg Pro Ser Val Asp Pro Lys His Val Leu Ser
100 105 110
Asp Asn Phe Ala Pro Val Leu Asp Glu Leu Pro Pro Thr Asp Cys Glu
115 120 125
Ile Ile His Gly Thr Leu Pro Leu Ser Leu Asn Gly Ala Tyr Ile Arg
130 135 140
Asn Gly Pro Asn Pro Gln Phe Leu Pro Arg Gly Pro Tyr His Leu Phe
145 150 155 160
Asp Gly Asp Gly Met Leu His Ala Ile Lys Ile His Asn Gly Lys Ala
165 170 175
Thr Leu Cys Ser Arg Tyr Val Lys Thr Tyr Lys Tyr Asn Val Glu Lys
180 185 190
Gln Thr Gly Ala Pro Val Met Pro Asn Val Phe Ser Gly Phe Asn Gly
195 200 205
Val Thr Ala Ser Val Ala Arg Gly Ala Leu Thr Ala Ala Arg Val Leu
210 215 220
Thr Gly Gln Tyr Asn Pro Val Asn Gly Ile Gly Leu Ala Asn Thr Ser
225 230 235 240
Leu Ala Phe Phe Ser Asn Arg Leu Phe Ala Leu Gly Glu Ser Asp Leu
245 250 255
Pro Tyr Ala Val Arg Leu Thr Glu Ser Gly Asp Ile Glu Thr Ile Gly
260 265 270
Arg Tyr Asp Phe Asp Gly Lys Leu Ala Met Ser Met Thr Ala His Pro
275 280 285
Lys Thr Asp Pro Ile Thr Gly Glu Thr Phe Ala Phe Arg Tyr Gly Pro
290 295 300
Val Pro Pro Phe Leu Thr Tyr Phe Arg Phe Asp Ser Ala Gly Lys Lys
305 310 315 320
Gln Arg Asp Val Pro Ile Phe Ser Met Thr Ser Pro Ser Phe Leu His
325 330 335
Asp Phe Ala Ile Thr Lys Arg His Ala Ile Phe Ala Glu Ile Gln Leu
340 345 350
Gly Met Arg Met Asn Met Leu Asp Leu Val Leu Glu Gly Gly Ser Pro
355 360 365
Val Gly Thr Asp Asn Gly Lys Thr Pro Arg Leu Gly Val Ile Pro Lys
370 375 380
Tyr Ala Gly Asp Glu Ser Glu Met Lys Trp Phe Glu Val Pro Gly Phe
385 390 395 400
Asn Ile Ile His Ala Ile Asn Ala Trp Asp Glu Asp Asp Gly Asn Ser
405 410 415
Val Val Leu Ile Ala Pro Asn Ile Met Ser Ile Glu His Thr Leu Glu
420 425 430
Arg Met Asp Leu Val His Ala Leu Val Glu Lys Val Lys Ile Asp Leu
435 440 445
Val Thr Gly Ile Val Arg Arg His Pro Ile Ser Ala Arg Asn Leu Asp
450 455 460
Phe Ala Val Ile Asn Pro Ala Phe Leu Gly Arg Cys Ser Arg Tyr Val
465 470 475 480
Tyr Ala Ala Ile Gly Asp Pro Met Pro Lys Ile Ser Gly Val Val Lys
485 490 495
Leu Asp Val Ser Lys Gly Asp Arg Asp Asp Cys Thr Val Ala Arg Arg
500 505 510
Met Tyr Gly Ser Gly Cys Tyr Gly Gly Glu Pro Phe Phe Val Ala Arg
515 520 525
Asp Pro Gly Asn Pro Glu Ala Glu Glu Asp Asp Gly Tyr Val Val Thr
530 535 540
Tyr Val His Asp Glu Val Thr Gly Glu Ser Lys Phe Leu Val Met Asp
545 550 555 560
Ala Lys Ser Pro Glu Leu Glu Ile Val Ala Ala Val Arg Leu Pro Arg
565 570 575
Arg Val Pro Tyr Gly Phe His Gly Leu Phe Val Lys Glu Ser Asp Leu
580 585 590
Asn Lys Leu
595
<210>5
<211>1800
<212>DNA
<213>拟南芥
<220>
<221>CDS
<222>(1)..(1800)
<400>5
atg gct tct ttc acg gca acg gct gcg gtt tct ggg aga tgg ctt ggt 48
Met Ala Ser Phe Thr Ala Thr Ala Ala Val Ser Gly Arg Trp Leu Gly
1 5 10 15
ggc aat cat act cag ccg cca tta tcg tct tct caa agc tcc gac ttg 96
Gly Asn His Thr Gln Pro Pro Leu Ser Ser Ser Gln Ser Ser Asp Leu
20 25 30
agt tat tgt agc tcc tta cct atg gcc agt cgt gtc aca cgt aag ctc 144
Ser Tyr Cys Ser Ser Leu Pro Met Ala Ser Arg Val Thr Arg Lys Leu
35 40 45
aat gtt tca tct gcg ctt cac act cct cca gct ctt cat ttc cct aag 192
Asn Val Ser Ser Ala Leu His Thr Pro Pro Ala Leu His Phe Pro Lys
50 55 60
caa tca tca aac tct ccc gcc att gtt gtt aag ccc aaa gcc aaa gaa 240
Gln Ser Ser Asn Ser Pro Ala Ile Val Val Lys Pro Lys Ala Lys Glu
65 70 75 80
tcc aac act aaa cag atg aat ttg ttc cag aga gcg gcg gcg gca gcg 288
Ser Asn Thr Lys Gln Met Asn Leu Phe Gln Arg Ala Ala Ala Ala Ala
85 90 95
ttg gac gcg gcg gag ggt ttc ctt gtc agc cac gag aag cta cac ccg 336
Leu Asp Ala Ala Glu Gly Phe Leu Val Ser His Glu Lys Leu His Pro
100 105 110
ctt cct aaa acg gct gat cct agt gtt cag atc gcc gga aat ttt gct 384
Leu Pro Lys Thr Ala Asp Pro Ser Val Gln Ile Ala Gly Asn Phe Ala
115 120 125
ccg gtg aat gaa cag ccc gtc cgg cgt aat ctt ccg gtg gtc gga aaa 432
Pro Val Asn Glu Gln Pro Val Arg Arg Asn Leu Pro Val Val Gly Lys
130 135 140
ctt ccc gat tcc atc aaa gga gtg tat gtg cgc aac gga gct aac cca 480
Leu Pro Asp Ser Ile Lys Gly Val Tyr Val Arg Asn Gly Ala Asn Pro
145 150 155 160
ctt cac gag ccg gtg aca ggt cac cac ttc ttc gac gga gac ggt atg 528
Leu His Glu Pro Val Thr Gly His His Phe Phe Asp Gly Asp Gly Met
165 170 175
gtt cac gcc gtc aaa ttc gaa cac ggt tca gct agc tac gct tgc cgg 576
Val His Ala Val Lys Phe Glu His Gly Ser Ala Ser Tyr Ala Cys Arg
180 185 190
ttt act cag act aac cgg ttt gtt cag gaa cgt caa ttg ggt cga ccg 624
Phe Thr Gln Thr Asn Arg Phe Val Gln Glu Arg Gln Leu Gly Arg Pro
195 200 205
gtt ttc ccc aaa gcc atc ggt gag ctt cac ggc cac acc ggt att gcc 672
Val Phe Pro Lys Ala Ile Gly Glu Leu His Gly His Thr Gly Ile Ala
210 215 220
cga ctc atg cta ttc tac gcc aga gct gca gcc ggt ata gtc gac ccg 720
Arg Leu Met Leu Phe Tyr Ala Arg Ala Ala Ala Gly Ile Val Asp Pro
225 230 235 240
gca cac gga acc ggt gta gct aac gcc ggt ttg gtc tat ttc aat ggc 768
Ala His Gly Thr Gly Val Ala Asn Ala Gly Leu Val Tyr Phe Asn Gly
245 250 255
cgg tta ttg gct atg tcg gag gat gat tta cct tac caa gtt cag atc 816
Arg Leu Leu Ala Met Ser Glu Asp Asp Leu Pro Tyr Gln Val Gln Ile
260 265 270
act ccc aat gga gat tta aaa acc gtt ggt cgg ttc gat ttt gat gga 864
Thr Pro Asn Gly Asp Leu Lys Thr Val Gly Arg Phe Asp Phe Asp Gly
275 280 285
caa tta gaa tcc aca atg att gcc cac ccg aaa gtc gac ccg gaa tcc 912
Gln Leu Glu Ser Thr Met Ile Ala His Pro Lys Val Asp Pro Glu Ser
290 295 300
ggt gaa ctc ttc gct tta agc tac gac gtc gtt tca aag cct tac cta 960
Gly Glu Leu Phe Ala Leu Ser Tyr Asp Val Val Ser Lys Pro Tyr Leu
305 310 315 320
aaa tac ttc cga ttc tca ccg gac gga act aaa tca ccg gac gtc gag 1008
Lys Tyr Phe Arg Phe Ser Pro Asp Gly Thr Lys Ser Pro Asp Val Glu
325 330 335
att cag ctt gat cag cca acg atg atg cac gat ttc gcg att aca gag 1056
Ile Gln Leu Asp Gln Pro Thr Met Met His Asp Phe Ala Ile Thr Glu
340 345 350
aac ttc gtc gtc gta cct gac cag caa gtc gtt ttc aag ctg ccg gag 1104
Asn Phe Val Val Val Pro Asp Gln Gln Val Val Phe Lys Leu Pro Glu
355 360 365
atg atc cgc ggt ggg tct ccg gtg gtt tac gac aag aac aag gtc gca 1152
Met Ile Arg Gly Gly Ser Pro Val Val Tyr Asp Lys Asn Lys Val Ala
370 375 380
aga ttc ggg att tta gac aaa tac gcc gaa gat tca tcg aac att aag 1200
Arg Phe Gly Ile Leu Asp Lys Tyr Ala Glu Asp Ser Ser Asn Ile Lys
385 390 395 400
tgg att gat gct cca gat tgc ttc tgc ttc cat ctc tgg aac gct tgg 1248
Trp Ile Asp Ala Pro Asp Cys Phe Cys Phe His Leu Trp Asn Ala Trp
405 410 415
gaa gag cca gaa aca gat gaa gtc gtc gtg ata ggg tcc tgt atg act 1296
Glu Glu Pro Glu Thr Asp Glu Val Val Val Ile Gly Ser Cys Met Thr
420 425 430
cca cca gac tca att ttc aac gag tct gac gag aat ctc aag agt gtc 1344
Pro Pro Asp Ser Ile Phe Asn Glu Ser Asp Glu Asn Leu Lys Ser Val
435 440 445
ctg tct gaa atc cgc ctg aat ctc aaa acc ggt gaa tca act cgc cgt 1392
Leu Ser Glu Ile Arg Leu Asn Leu Lys Thr Gly Glu Ser Thr Arg Arg
450 455 460
ccg atc atc tcc aac gaa gat caa caa gtc aac ctc gaa gca ggg atg 1440
Pro Ile Ile Ser Asn Glu Asp Gln Gln Val Asn Leu Glu Ala Gly Met
465 470 475 480
gtc aac aga aac atg ctc ggc cgt aaa acc aaa ttc gct tac ttg gct 1488
Vat Asn Arg Asn Met Leu Gly Arg Lys Thr Lys Phe Ala Tyr Leu Ala
485 490 495
tta gcc gag ccg tgg cct aaa gtc tca gga ttc gct aaa gtt gat ctc 1536
Leu Ala Glu Pro Trp Pro Lys Val Ser Gly Phe Ala Lys Val Asp Leu
500 505 510
act act gga gaa gtt aag aaa cat ctt tac ggc gat aac cgt tac gga 1584
Thr Thr Gly Glu Val Lys Lys His Leu Tyr Gly Asp Asn Arg Tyr Gly
515 520 525
gga gag cct ctg ttt ctc ccc gga gaa gga gga gag gaa gac gaa gga 1632
Gly Glu Pro Leu Phe Leu Pro Gly Glu Gly Gly Glu Glu Asp Glu Gly
530 535 540
tac atc ctc tgt ttc gtt cac gac gag aag aca tgg aaa tcg gag tta 1680
Tyr Ile Leu Cys Phe Val His Asp Glu Lys Thr Trp Lys Ser Glu Leu
545 550 555 560
cag ata gtt aac gcc gtt agc tta gag gtt gaa gca acg gtt aaa ctt 1728
Gln Ile Val Asn Ala Val Ser Leu Glu Val Glu Ala Thr Val Lys Leu
565 570 575
ccg tca agg gtt ccg tac gga ttt cac ggt aca ttc atc gga gcc gat 1776
Pro Ser Arg Val Pro Tyr Gly Phe His Gly Thr Phe Ile Gly Ala Asp
580 585 590
gat ttg gcg aag cag gtc gtg tga 1800
Asp Leu Ala Lys Gln Val Val
595 600
<210>6
<211>599
<212>PRT
<213>拟南芥
<400>6
Met Ala Ser Phe Thr Ala Thr Ala Ala Val Ser Gly Arg Trp Leu Gly
1 5 10 15
Gly Asn His Thr Gln Pro Pro Leu Ser Ser Ser Gln Ser Ser Asp Leu
20 25 30
Ser Tyr Cys Ser Ser Leu Pro Met Ala Ser Arg Val Thr Arg Lys Leu
35 40 45
Asn Val Ser Ser Ala Leu His Thr Pro Pro Ala Leu His Phe Pro Lys
50 55 60
Gln Ser Ser Asn Ser Pro Ala Ile Val Val Lys Pro Lys Ala Lys Glu
65 70 75 80
Ser Asn Thr Lys Gln Met Asn Leu Phe Gln Arg Ala Ala Ala Ala Ala
85 90 95
Leu Asp Ala Ala Glu Gly Phe Leu Val Ser His Glu Lys Leu His Pro
100 105 110
Leu Pro Lys Thr Ala Asp Pro Ser Val Gln Ile Ala Gly Asn Phe Ala
115 120 125
Pro Val Asn Glu Gln Pro Val Arg Arg Asn Leu Pro Val Val Gly Lys
130 135 140
Leu Pro Asp Ser Ile Lys Gly Val Tyr Val Arg Asn Gly Ala Asn Pro
145 150 155 160
Leu His Glu Pro Val Thr Gly His His Phe Phe Asp Gly Asp Gly Met
165 170 175
Val His Ala Val Lys Phe Glu His Gly Ser Ala Ser Tyr Ala Cys Arg
180 185 190
Phe Thr Gln Thr Asn Arg Phe Val Gln Glu Arg Gln Leu Gly Arg Pro
195 200 205
Val Phe Pro Lys Ala Ile Gly Glu Leu His Gly His Thr Gly Ile Ala
210 215 220
Arg Leu Met Leu Phe Tyr Ala Arg Ala Ala Ala Gly Ile Val Asp Pro
225 230 235 240
Ala His Gly Thr Gly Val Ala Asn Ala Gly Leu Val Tyr Phe Asn Gly
245 250 255
Arg Leu Leu Ala Met Ser Glu Asp Asp Leu Pro Tyr Gln Val Gln Ile
260 265 270
Thr Pro Asn Gly Asp Leu Lys Thr Val Gly Arg Phe Asp Phe Asp Gly
275 280 285
Gln Leu Glu Ser Thr Met Ile Ala His Pro Lys Val Asp Pro Glu Ser
290 295 300
Gly Glu Leu Phe Ala Leu Ser Tyr Asp Val Val Ser Lys Pro Tyr Leu
305 310 315 320
Lys Tyr Phe Arg Phe Ser Pro Asp Gly Thr Lys Ser Pro Asp Val Glu
325 330 335
Ile Gln Leu Asp Gln Pro Thr Met Met His Asp Phe Ala Ile Thr Glu
340 345 350
Asn Phe Val Val Val Pro Asp Gln Gln Val Val Phe Lys Leu Pro Glu
355 360 365
Met Ile Arg Gly Gly Ser Pro Val Val Tyr Asp Lys Asn Lys Val Ala
370 375 380
Arg Phe Gly Ile Leu Asp Lys Tyr Ala Glu Asp Ser Ser Asn Ile Lys
385 390 395 400
Trp Ile Asp Ala Pro Asp Cys Phe Cys Phe His Leu Trp Asn Ala Trp
405 410 415
Glu Glu Pro Glu Thr Asp Glu Val Val Val Ile Gly Ser Cys Met Thr
420 425 430
Pro Pro Asp Ser Ile Phe Asn Glu Ser Asp Glu Asn Leu Lys Ser Val
435 440 445
Leu Ser Glu Ile Arg Leu Asn Leu Lys Thr Gly Glu Ser Thr Arg Arg
450 455 460
Pro Ile Ile Ser Asn Glu Asp Gln Gln Val Asn Leu Glu Ala Gly Met
465 470 475 480
Val Asn Arg Asn Met Leu Gly Arg Lys Thr Lys Phe Ala Tyr Leu Ala
485 490 495
Leu Ala Glu Pro Trp Pro Lys Val Ser Gly Phe Ala Lys Val Asp Leu
500 505 510
Thr Thr Gly Glu Val Lys Lys His Leu Tyr Gly Asp Asn Arg Tyr Gly
515 520 525
Gly Glu Pro Leu Phe Leu Pro Gly Glu Gly Gly Glu Glu Asp Glu Gly
530 535 540
Tyr Ile Leu Cys Phe Val His Asp Glu Lys Thr Trp Lys Ser Glu Leu
545 550 555 560
Gln Ile Val Asn Ala Val Ser Leu Glu Val Glu Ala Thr Val Lys Leu
565 570 575
Pro Ser Arg Val Pro Tyr Gly Phe His Gly Thr Phe Ile Gly Ala Asp
580 585 590
Asp Leu Ala Lys Gln Val Val
595
<210>7
<211>1617
<212>DNA
<213>拟南芥
<220>
<221>CDS
<222>(1)..(1617)
<400>7
atg gcg gag aaa ctc agt gat ggc agc agc atc atc tca gtc cat cct 48
Met Ala Glu Lys Leu Ser Asp Gly Ser Ser Ile Ile Ser Val His Pro
1 5 10 15
aga ccc tcc aag ggt ttc tcc tcg aag ctt ctc gat ctt ctc gag aga 96
Arg Pro Ser Lys Gly Phe Ser Ser Lys Leu Leu Asp Leu Leu Glu Arg
20 25 30
ctt gtt gtc aag ctc atg cac gat gct tct ctc cct ctc cac tac ctc 144
Leu Val Val Lys Leu Met His Asp Ala Ser Leu Pro Leu His Tyr Leu
35 40 45
tca ggc aac ttc gct ccc atc cgt gat gaa act cct ccc gtc aag gat 192
Ser Gly Asn Phe Ala Pro Ile Arg Asp Glu Thr Pro Pro Val Lys Asp
50 55 60
ctc ccc gtc cat gga ttt ctt ccc gaa tgc ttg aat ggt gaa ttt gtg 240
Leu Pro Val His Gly Phe Leu Pro Glu Cys Leu Asn Gly Glu Phe Val
65 70 75 80
agg gtt ggt cca aac ccc aag ttt gat gct gtc gct gga tat cac tgg 288
Arg Val Gly Pro Asn Pro Lys Phe Asp Ala Val Ala Gly Tyr His Trp
85 90 95
ttt gat gga gat ggg atg att cat ggg gta cgc atc aaa gat ggg aaa 336
Phe Asp Gly Asp Gly Met Ile His Gly Val Arg Ile Lys Asp Gly Lys
100 105 110
gct act tat gtt tct cga tat gtt aag aca tca cgt ctt aag cag gaa 384
Ala Thr Tyr Val Ser Arg Tyr Val Lys Thr Ser Arg Leu Lys Gln Glu
115 120 125
gag ttc ttc gga gct gcc aaa ttc atg aag att ggt gac ctt aag ggg 432
Glu Phe Phe Gly Ala Ala Lys Phe Met Lys Ile Gly Asp Leu Lys Gly
130 135 140
ttt ttc gga ttg cta atg gtc aat gtc caa cag ctg aga acg aag ctc 480
Phe Phe Gly Leu Leu Met Val Asn Val Gln Gln Leu Arg Thr Lys Leu
145 150 155 160
aaa ata ttg gac aac act tat gga aat gga act gcc aat aca gca ctc 528
Lys Ile Leu Asp Asn Thr Tyr Gly Asn Gly Thr Ala Asn Thr Ala Leu
165 170 175
gta tat cac cat gga aaa ctt cta gca tta cag gag gca gat aag ccg 576
Val Tyr His His Gly Lys Leu Leu Ala Leu Gln Glu Ala Asp Lys Pro
180 185 190
tac gtc atc aaa gtt ttg gaa gat gga gac ctg caa act ctt ggt ata 624
Tyr Val Ile Lys Val Leu Glu Asp Gly Asp Leu Gln Thr Leu Gly Ile
195 200 205
ata gat tat gac aag aga ttg acc cac tcc ttc act gct cac cca aaa 672
Ile Asp Tyr Asp Lys Arg Leu Thr His Ser Phe Thr Ala His Pro Lys
210 215 220
gtt gac ccg gtt acg ggt gaa atg ttt aca ttc ggc tat tcg cat acg 720
Val Asp Pro Val Thr Gly Glu Met Phe Thr Phe Gly Tyr Ser His Thr
225 230 235 240
cca cct tat ctc aca tac aga gtt atc tcg aaa gat ggc att atg cat 768
Pro Pro Tyr Leu Thr Tyr Arg Val Ile Ser Lys Asp Gly Ile Met His
245 250 255
gac cca gtc cca att act ata tca gag cct atc atg atg cat gat ttt 816
Asp Pro Val Pro Ile Thr Ile Ser Glu Pro Ile Met Met His Asp Phe
260 265 270
gct att act gag act tat gca atc ttc atg gat ctt cct atg cac ttc 864
Ala Ile Thr Glu Thr Tyr Ala Ile Phe Met Asp Leu Pro Met His Phe
275 280 285
agg cca aag gaa atg gtg aaa gag aag aaa atg ata tac tca ttt gat 912
Arg Pro Lys Glu Met Val Lys Glu Lys Lys Met Ile Tyr Ser Phe Asp
290 295 300
ccc aca aaa aag gct cgt ttt ggt gtt ctt cca cgc tat gcc aag gat 960
Pro Thr Lys Lys Ala Arg Phe Gly Val Leu Pro Arg Tyr Ala Lys Asp
305 310 315 320
gaa ctt atg att aga tgg ttt gag ctt ccc aac tgc ttt att ttc cac 1008
Glu Leu Met Ile Arg Trp Phe Glu Leu Pro Asn Cys Phe Ile Phe His
325 330 335
aac gcc aat gct tgg gaa gaa gag gat gaa gtc gtc ctc atc act tgt 1056
Asn Ala Asn Ala Trp Glu Glu Glu Asp Glu Val Val Leu Ile Thr Cys
340 345 350
cgt ctt gag aat cca gat ctt gac atg gtc agt ggg aaa gtg aaa gaa 1104
Arg Leu Glu Asn Pro Asp Leu Asp Met Val Ser Gly Lys Val Lys Glu
355 360 365
aaa ctc gaa aat ttt ggc aac gaa ctg tac gaa atg aga ttc aac atg 1152
Lys Leu Glu Asn Phe Gly Asn Glu Leu Tyr Glu Met Arg Phe Asn Met
370 375 380
aaa acg ggc tca gct tct caa aaa aaa cta tcc gca tct gcg gtt gat 1200
Lys Thr Gly Ser Ala Ser Gln Lys Lys Leu Ser Ala Ser Ala Val Asp
385 390 395 400
ttc ccc aga atc aat gag tgc tac acc gga aag aaa cag aga tac gta 1248
Phe Pro Arg Ile Asn Glu Cys Tyr Thr Gly Lys Lys Gln Arg Tyr Val
405 410 415
tat gga aca att ctg gac agt atc gca aag gtt acc gga atc atc aag 1296
Tyr Gly Thr Ile Leu Asp Ser Ile Ala Lys Val Thr Gly Ile Ile Lys
420 425 430
ttt gat ctg cat gca gaa gct gag aca ggg aaa aga atg ctg gaa gta 1344
Phe Asp Leu His Ala Glu Ala Glu Thr Gly Lys Arg Met Leu Glu Val
435 440 445
gga ggt aat atc aaa gga ata tat gac ctg gga gaa ggc aga tat ggt 1392
Gly Gly Asn Ile Lys Gly Ile Tyr Asp Leu Gly Glu Gly Arg Tyr Gly
450 455 460
tca gag gct atc tat gtt ccg cgt gag aca gca gaa gaa gac gac ggt 1440
Ser Glu Ala Ile Tyr Val Pro Arg Glu Thr Ala Glu Glu Asp Asp Gly
465 470 475 480
tac ttg ata ttc ttt gtt cat gat gaa aac aca ggg aaa tca tgc gtg 1488
Tyr Leu Ile Phe Phe Val His Asp Glu Asn Thr Gly Lys Ser Cys Val
485 490 495
act gtg ata gac gca aaa aca atg tcg gct gaa ccg gtg gca gtg gtg 1536
Thr Val Ile Asp Ala Lys Thr Met Ser Ala Glu Pro Val Ala Val Val
500 505 510
gag ctg ccg cac agg gtc cca tat ggc ttc cat gcc ttg ttt gtt aca 1584
Glu Leu Pro His Arg Val Pro Tyr Gly Phe His Ala Leu Phe Val Thr
515 520 525
gag gaa caa ctc cag gaa caa act ctt ata taa 1617
Glu Glu Gln Leu Gln Glu Gln Thr Leu Ile
530 535
<210>8
<211>538
<212>PRT
<213>拟南芥
<400>8
Met Ala Glu Lys Leu Ser Asp Gly Ser Ser Ile Ile Ser Val His Pro
1 5 10 15
Arg Pro Ser Lys Gly Phe Ser Ser Lys Leu Leu Asp Leu Leu Glu Arg
20 25 30
Leu Val Val Lys Leu Met His Asp Ala Ser Leu Pro Leu His Tyr Leu
35 40 45
Ser Gly Asn Phe Ala Pro Ile Arg Asp Glu Thr Pro Pro Val Lys Asp
50 55 60
Leu Pro Val His Gly Phe Leu Pro Glu Cys Leu Asn Gly Glu Phe Val
65 70 75 80
Arg Val Gly Pro Asn Pro Lys Phe Asp Ala Val Ala Gly Tyr His Trp
85 90 95
Phe Asp Gly Asp Gly Met Ile His Gly Val Arg Ile Lys Asp Gly Lys
100 105 110
Ala Thr Tyr Val Ser Arg Tyr Val Lys Thr Ser Arg Leu Lys Gln Glu
115 120 125
Glu Phe Phe Gly Ala Ala Lys Phe Met Lys Ile Gly Asp Leu Lys Gly
130 135 140
Phe Phe Gly Leu Leu Met Val Asn Val Gln Gln Leu Arg Thr Lys Leu
145 150 155 160
Lys Ile Leu Asp Asn Thr Tyr Gly Asn Gly Thr Ala Asn Thr Ala Leu
165 170 175
Val Tyr His His Gly Lys Leu Leu Ala Leu Gln Glu Ala Asp Lys Pro
180 185 190
Tyr Val Ile Lys Val Leu Glu Asp Gly Asp Leu Gln Thr Leu Gly Ile
195 200 205
Ile Asp Tyr Asp Lys Arg Leu Thr His Ser Phe Thr Ala His Pro Lys
210 215 220
Val Asp Pro Val Thr Gly Glu Met Phe Thr Phe Gly Tyr Ser His Thr
225 230 235 240
Pro Pro Tyr Leu Thr Tyr Arg Val Ile Ser Lys Asp Gly Ile Met His
245 250 255
Asp Pro Val Pro Ile Thr Ile Ser Glu Pro Ile Met Met His Asp Phe
260 265 270
Ala Ile Thr Glu Thr Tyr Ala Ile Phe Met Asp Leu Pro Met His Phe
275 280 285
Arg Pro Lys Glu Met Val Lys Glu Lys Lys Met Ile Tyr Ser Phe Asp
290 295 300
Pro Thr Lys Lys Ala Arg Phe Gly Val Leu Pro Arg Tyr Ala Lys Asp
305 310 315 320
Glu Leu Met Ile Arg Trp Phe Glu Leu Pro Asn Cys Phe Ile Phe His
325 330 335
Asn Ala Asn Ala Trp Glu Glu Glu Asp Glu Val Val Leu Ile Thr Cys
340 345 350
Arg Leu Glu Asn Pro Asp Leu Asp Met Val Ser Gly Lys Val Lys Glu
355 360 365
Lys Leu Glu Asn Phe Gly Asn Glu Leu Tyr Glu Met Arg Phe Asn Met
370 375 380
Lys Thr Gly Ser Ala Ser Gln Lys Lys Leu Ser Ala Ser Ala Val Asp
385 390 395 400
Phe Pro Arg Ile Asn Glu Cys Tyr Thr Gly Lys Lys Gln Arg Tyr Val
405 410 415
Tyr Gly Thr Ile Leu Asp Ser Ile Ala Lys Val Thr Gly Ile Ile Lys
420 425 430
Phe Asp Leu His Ala Glu Ala Glu Thr Gly Lys Arg Met Leu Glu Val
435 440 445
Gly Gly Asn Ile Lys Gly Ile Tyr Asp Leu Gly Glu Gly Arg Tyr Gly
450 455 460
Ser Glu Ala Ile Tyr Val Pro Arg Glu Thr Ala Glu Glu Asp Asp Gly
465 470 475 480
Tyr Leu Ile Phe Phe Val His Asp Glu Asn Thr Gly Lys Ser Cys Val
485 490 495
Thr Val Ile Asp Ala Lys Thr Met Ser Ala Glu Pro Val Ala Val Val
500 505 510
Glu Leu Pro His Arg Val Pro Tyr Gly Phe His Ala Leu Phe Val Thr
515 520 525
Glu Glu Gln Leu Gln Glu Gln Thr Leu Ile
530 535
<210>9
<211>1734
<212>DNA
<213>拟南芥
<220>
<221>CDS
<222>(1)..(1734)
<400>9
atg caa cac tct ctt cgt tct gat ctt ctt ccg acg aag act tct cct 48
Met Gln His Ser Leu Arg Ser Asp Leu Leu Pro Thr Lys Thr Ser Pro
1 5 10 15
cgt tct cat tta ctt cca caa ccc aaa aat gca aat att tct cga cga 96
Arg Ser His Leu Leu Pro Gln Pro Lys Asn Ala Asn Ile Ser Arg Arg
20 25 30
att ctc att aac cct ttc aag ata ccg aca ctt cct gat ctc act tct 144
Ile Leu Ile Asn Pro Phe Lys Ile Pro Thr Leu Pro Asp Leu Thr Ser
35 40 45
ccg gtt ccg tca ccg gtt aag ctc aaa cca acg tat cca aac tta aac 192
Pro Val Pro Ser Pro Val Lys Leu Lys Pro Thr Tyr Pro Asn Leu Asn
50 55 60
ctt ctt cag aag cta gcg gct acg atg ctc gac aag att gag tcc tct 240
Leu Leu Gln Lys Leu Ala Ala Thr Met Leu Asp Lys Ile Glu Ser Ser
65 70 75 80
atc gtt att cct atg gag cag aat cgc ccg ctt cct aaa ccg acc gac 288
Ile Val Ile Pro Met Glu Gln Asn Arg Pro Leu Pro Lys Pro Thr Asp
85 90 95
ccg gcg gtt caa tta tca ggt aac ttc gct ccg gtt aat gaa tgt ccg 336
Pro Ala Val Gln Leu Ser Gly Asn Phe Ala Pro Val Asn Glu Cys Pro
100 105 110
gtt cag aac ggt tta gaa gtg gtt ggt cag att cct tct tgt cta aaa 384
Val Gln Asn Gly Leu Glu Val Val Gly Gln Ile Pro Ser Cys Leu Lys
115 120 125
gga gtt tac atc cgt aac ggt gca aac cct atg ttt ccg ccg tta gcc 432
Gly Val Tyr Ile Arg Asn Gly Ala Asn Pro Met Phe Pro Pro Leu Ala
130 135 140
gga cat cat tta ttt gac ggt gac gga atg att cac gcc gtt agt atc 480
Gly His His Leu Phe Asp Gly Asp Gly Met Ile His Ala Val Ser Ile
145 150 155 160
ggt ttt gat aac cag gtt agt tac agc tgc cgg tac act aaa aca aac 528
Gly Phe Asp Asn Gln Val Ser Tyr Ser Cys Arg Tyr Thr Lys Thr Asn
165 170 175
cgg ctt gtt caa gaa acc gcg ctt gga cga tcg gtt ttc cct aaa cca 576
Arg Leu Val Gln Glu Thr Ala Leu Gly Arg Ser Val Phe Pro Lys Pro
180 185 190
atc ggc gag ctt cac ggc cat tcc ggt cta gct cga ctc gct ctc ttc 624
Ile Gly Glu Leu His Gly His Ser Gly Leu Ala Arg Leu Ala Leu Phe
195 200 205
acg gct cga gct ggg atc ggt cta gtg gac ggg aca cgt ggc atg ggc 672
Thr Ala Arg Ala Gly Ile Gly Leu Val Asp Gly Thr Arg Gly Met Gly
210 215 220
gta gct aac gcc ggt gtg gtt ttc ttt aac ggc agg tta tta gcc atg 720
Val Ala Asn Ala Gly Val Val Phe Phe Asn Gly Arg Leu Leu Ala Met
225 230 235 240
tca gaa gat gat ctt cct tac caa gtg aag atc gac ggt caa gga gat 768
Ser Glu Asp Asp Leu Pro Tyr Gln Val Lys Ile Asp Gly Gln Gly Asp
245 250 255
ctt gag acg atc gga cgg ttc gga ttc gat gac cag att gac tct tca 816
Leu Glu Thr Ile Gly Arg Phe Gly Phe Asp Asp Gln Ile Asp Ser Ser
260 265 270
gtg ata gcg cat cct aag gtg gac gcg acc aca gga gat ctc cat aca 864
Val Ile Ala His Pro Lys Val Asp Ala Thr Thr Gly Asp Leu His Thr
275 280 285
ctg agc tac aac gtt ttg aag aaa cct cat ctc agg tat ctt aaa ttc 912
Leu Ser Tyr Asn Val Leu Lys Lys Pro His Leu Arg Tyr Leu Lys Phe
290 295 300
aac acg tgc ggg aaa aag aca cgt gac gtg gag atc acg ctc cct gaa 960
Asn Thr Cys Gly Lys Lys Thr Arg Asp Val Glu Ile Thr Leu Pro Glu
305 310 315 320
cca acg atg att cat gat ttc gcg ata acc gag aat ttt gtc gtt ata 1008
Pro Thr Met Ile His Asp Phe Ala Ile Thr Glu Asn Phe Val Val Ile
325 330 335
ccg gat cag caa atg gta ttc aaa tta tcc gaa atg att cgg ggc ggg 1056
Pro Asp Gln Gln Met Val Phe Lys Leu Ser Glu Met Ile Arg Gly Gly
340 345 350
tca ccc gtt atc tac gtt aaa gaa aaa atg gcg aga ttt gga gtt ttg 1104
Ser Pro Val Ile Tyr Val Lys Glu Lys Met Ala Arg Phe Gly Val Leu
355 360 365
tca aag cag gat ctg acc ggg tcg gat ata aat tgg gtt gat gta ccg 1152
Ser Lys Gln Asp Leu Thr Gly Ser Asp Ile Asn Trp Val Asp Val Pro
370 375 380
gat tgt ttc tgt ttc cat cta tgg aat gcg tgg gaa gag aga acc gaa 1200
Asp Cys Phe Cys Phe His Leu Trp Asn Ala Trp Glu Glu Arg Thr Glu
385 390 395 400
gag gga gac cca gtt atc gtc gta atc ggg tca tgt atg agc cca ccc 1248
Glu Gly Asp Pro Val Ile Val Val Ile Gly Ser Cys Met Ser Pro Pro
405 410 415
gac acg atc ttt agt gaa tca gga gaa cca acc cgg gtt gaa tta agt 1296
Asp Thr Ile Phe Ser Glu Ser Gly Glu Pro Thr Arg Val Glu Leu Ser
420 425 430
gag atc cgg tta aac atg cgt aca aaa gaa tcg aac cgt aag gtt atc 1344
Glu Ile Arg Leu Asn Met Arg Thr Lys Glu Ser Asn Arg Lys Val Ile
435 440 445
gta acc gga gtg aat tta gaa gcg ggt cac ata aac cgt agt tac gtg 1392
Val Thr Gly Val Asn Leu Glu Ala Gly His Ile Asn Arg Ser Tyr Val
450 455 460
ggc cgg aaa agc cag ttc gtt tac ata gca ata gcc gat cct tgg ccc 1440
Gly Arg Lys Ser Gln Phe Val Tyr Ile Ala Ile Ala Asp Pro Trp Pro
465 470 475 480
aaa tgc agt ggc att gcg aag gta gat ata caa aac ggc acc gtt tca 1488
Lys Cys Ser Gly Ile Ala Lys Val Asp Ile Gln Asn Gly Thr Val Ser
485 490 495
gag ttt aat tac gga ccg agc cgg ttc ggt gga gaa ccg tgc ttt gta 1536
Glu Phe Asn Tyr Gly Pro Ser Arg Phe Gly Gly Glu Pro Cys Phe Val
500 505 510
ccg gag gga gaa gga gaa gaa gac aaa ggt tat gta atg ggg ttt gtg 1584
Pro Glu Gly Glu Gly Glu Glu Asp Lys Gly Tyr Val Met Gly Phe Val
515 520 525
aga gac gaa gag aaa gac gag tcg gag ttt gtg gtg gtc gac gcg acg 1632
Arg Asp Glu Glu Lys Asp Glu Ser Glu Phe Val Val Val Asp Ala Thr
530 535 540
gat atg aag caa gtc gcg gcg gtg cgc ttg ccg gag agg gta cct tat 1680
Asp Met Lys Gln Val Ala Ala Val Arg Leu Pro Glu Arg Val Pro Tyr
545 550 555 560
ggt ttc cat gga acg ttc gtg agc gag aat cag ttg aag gaa caa gtt 1728
Gly Phe His Gly Thr Phe Val Ser Glu Asn Gln Leu Lys Glu Gln Val
565 570 575
ttc tga 1734
Phe
<210>10
<211>577
<212>PRT
<213>拟南芥
<400>10
Met Gln His Ser Leu Arg Ser Asp Leu Leu Pro Thr Lys Thr Ser Pro
1 5 10 15
Arg Ser His Leu Leu Pro Gln Pro Lys Asn Ala Asn Ile Ser Arg Arg
20 25 30
Ile Leu Ile Asn Pro Phe Lys Ile Pro Thr Leu Pro Asp Leu Thr Ser
35 40 45
Pro Val Pro Ser Pro Val Lys Leu Lys Pro Thr Tyr Pro Asn Leu Asn
50 55 60
Leu Leu Gln Lys Leu Ala Ala Thr Met Leu Asp Lys Ile Glu Ser Ser
65 70 75 80
Ile Val Ile Pro Met Glu Gln Asn Arg Pro Leu Pro Lys Pro Thr Asp
85 90 95
Pro Ala Val Gln Leu Ser Gly Asn Phe Ala Pro Val Asn Glu Cys Pro
100 105 110
Val Gln Asn Gly Leu Glu Val Val Gly Gln Ile Pro Ser Cys Leu Lys
115 120 125
Gly Val Tyr Ile Arg Asn Gly Ala Asn Pro Met Phe Pro Pro Leu Ala
130 135 140
Gly His His Leu Phe Asp Gly Asp Gly Met Ile His Ala Val Ser Ile
145 150 155 160
Gly Phe Asp Asn Gln Val Ser Tyr Ser Cys Arg Tyr Thr Lys Thr Asn
165 170 175
Arg Leu Val Gln Glu Thr Ala Leu Gly Arg Ser Val Phe Pro Lys Pro
180 185 190
Ile Gly Glu Leu His Gly His Ser Gly Leu Ala Arg Leu Ala Leu Phe
195 200 205
Thr Ala Arg Ala Gly Ile Gly Leu Val Asp Gly Thr Arg Gly Met Gly
210 215 220
Val Ala Asn Ala Gly Val Val Phe Phe Asn Gly Arg Leu Leu Ala Met
225 230 235 240
Ser Glu Asp Asp Leu Pro Tyr Gln Val Lys Ile Asp Gly Gln Gly Asp
245 250 255
Leu Glu Thr Ile Gly Arg Phe Gly Phe Asp Asp Gln Ile Asp Ser Ser
260 265 270
Val Ile Ala His Pro Lys Val Asp Ala Thr Thr Gly Asp Leu His Thr
275 280 285
Leu Ser Tyr Asn Val Leu Lys Lys Pro His Leu Arg Tyr Leu Lys Phe
290 295 300
Asn Thr Cys Gly Lys Lys Thr Arg Asp Val Glu Ile Thr Leu Pro Glu
305 310 315 320
Pro Thr Met Ile His Asp Phe Ala Ile Thr Glu Asn Phe Val Val Ile
325 330 335
Pro Asp Gln Gln Met Val Phe Lys Leu Ser Glu Met Ile Arg Gly Gly
340 345 350
Ser Pro Val Ile Tyr Val Lys Glu Lys Met Ala Arg Phe Gly Val Leu
355 360 365
Ser Lys Gln Asp Leu Thr Gly Ser Asp Ile Asn Trp Val Asp Val Pro
370 375 380
Asp Cys Phe Cys Phe His Leu Trp Asn Ala Trp Glu Glu Arg Thr Glu
385 390 395 400
Glu Gly Asp Pro Val Ile Val Val Ile Gly Ser Cys Met Ser Pro Pro
405 410 415
Asp Thr Ile Phe Ser Glu Ser Gly Glu Pro Thr Arg Val Glu Leu Ser
420 425 430
Glu Ile Arg Leu Asn Met Arg Thr Lys Glu Ser Asn Arg Lys Val Ile
435 440 445
Val Thr Gly Val Asn Leu Glu Ala Gly His Ile Asn Arg Ser Tyr Val
450 455 460
Gly Arg Lys Ser Gln Phe Val Tyr Ile Ala Ile Ala Asp Pro Trp Pro
465 470 475 480
Lys Cys Ser Gly Ile Ala Lys Val Asp Ile Gln Asn Gly Thr Val Ser
485 490 495
Glu Phe Asn Tyr Gly Pro Ser Arg Phe Gly Gly Glu Pro Cys Phe Val
500 505 510
Pro Glu Gly Glu Gly Glu Glu Asp Lys Gly Tyr Val Met Gly Phe Val
515 520 525
Arg Asp Glu Glu Lys Asp Glu Ser Glu Phe Val Val Val Asp Ala Thr
530 535 540
Asp Met Lys Gln Val Ala Ala Val Arg Leu Pro Glu Arg Val Pro Tyr
545 550 555 560
Gly Phe His Gly Thr Phe Val Ser Glu Asn Gln Leu Lys Glu Gln Val
565 570 575
Phe
<210>11
<211>1839
<212>DNA
<213>Vigna unguiculata
<220>
<221>CDS
<222>(1)..(1839)
<400>11
atg cct tca tca gct tca aac act tgg ttt aac gcc aca ctc cca tct 48
Met Pro Ser Ser Ala Ser Asn Thr Trp Phe Asn Ala Thr Leu Pro Ser
1 5 10 15
ccc ccc ttc aaa gac cta cct tcc aca tct tct ccc aca aac tta ctt 96
Pro Pro Phe Lys Asp Leu Pro Ser Thr Ser Ser Pro Thr Asn Leu Leu
20 25 30
cct tta agg aaa aca tcc tct tcc aac acc atc aca tgt tcc ctt caa 144
Pro Leu Arg Lys Thr Ser Ser Ser Asn Thr Ile Thr Cys Ser Leu Gln
35 40 45
aca ctc cac ttc ccc aaa cag tac caa cca aca tcc aca tcc aca tcc 192
Thr Leu His Phe Pro Lys Gln Tyr Gln Pro Thr Ser Thr Ser Thr Ser
50 55 60
aca gcc acc acc aca aca ccc acc cca atc aaa act acc acc atc acc 240
Thr Ala Thr Thr Thr Thr Pro Thr Pro Ile Lys Thr Thr Thr Ile Thr
65 70 75 80
acc acc aca ccg ccc agg gaa acc aac cct ctc tct gac acc aac caa 288
Thr Thr Thr Pro Pro Arg Glu Thr Asn Pro Leu Ser Asp Thr Asn Gln
85 90 95
cca tta cct caa aaa tgg aac ttt ctc cag aaa gcc gct gcc acg gcc 336
Pro Leu Pro Gln Lys Trp Asn Phe Leu Gln Lys Ala Ala Ala Thr Ala
100 105 110
ttg gac ctg gtc gaa acg gcg ctc gtc tcg cac gag cgc aaa cac ccg 384
Leu Asp Leu Val Glu Thr Ala Leu Val Ser His Glu Arg Lys His Pro
115 120 125
ctc ccc aaa acg gcg gac ccg agg gtc caa atc gcc ggg aac ttc gcg 432
Leu Pro Lys Thr Ala Asp Pro Arg Val Gln Ile Ala Gly Asn Phe Ala
130 135 140
ccg gtg ccg gag cat gcc gcg gat caa gga ctc ccg gtg gtc gga aaa 480
Pro Val Pro Glu His Ala Ala Asp Gln Gly Leu Pro Val Val Gly Lys
145 150 155 160
atc ccc aaa tgc att gac ggc gtg tac gtg cgc aac ggt gcc aat ccg 528
Ile Pro Lys Cys Ile Asp Gly Val Tyr Val Arg Asn Gly Ala Asn Pro
165 170 175
ctc tac gag cct gtg gcc ggg cac cac ttc ttc gac ggc gac ggc atg 576
Leu Tyr Glu Pro Val Ala Gly His His Phe Phe Asp Gly Asp Gly Met
180 185 190
gtc cac gcc gtg aag ttc acg aac ggc gcc gcc agc tac gcc tgc cgc 624
Val His Ala Val Lys Phe Thr Asn Gly Ala Ala Ser Tyr Ala Cys Arg
195 200 205
ttc acc gag acg cag cgt ctc tcg cag gag aaa tct cta ggc cgc ccg 672
Phe Thr Glu Thr Gln Arg Leu Ser Gln Glu Lys Ser Leu Gly Arg Pro
210 215 220
gtg ttc ccg aag gcc atc ggg gag ctc cac ggc cac tcc ggc atc gcg 720
Val Phe Pro Lys Ala Ile Gly Glu Leu His Gly His Ser Gly Ile Ala
225 230 235 240
cgg ctc ctc ctc ttc tac gcg cgc ggt ctc ttc ggg ctc gtt gat ggg 768
Arg Leu Leu Leu Phe Tyr Ala Arg Gly Leu Phe Gly Leu Val Asp Gly
245 250 255
tcc cag ggc atg ggc gtg gcg aac gcc ggt ctc gtc tac ttc aac aac 816
Ser Gln Gly Met Gly Val Ala Asn Ala Gly Leu Val Tyr Phe Asn Asn
260 265 270
cac ctc ttg gcc atg tcc gaa gac gat tta ccc tac cac gtg aga atc 864
His Leu Leu Ala Met Ser Glu Asp Asp Leu Pro Tyr His Val Arg Ile
275 280 285
acc cct aac ggc gac tta acc acc gtt ggc cgt tac gac ttc aac ggg 912
Thr Pro Asn Gly Asp Leu Thr Thr Val Gly Arg Tyr Asp Phe Asn Gly
290 295 300
cag ctc aac tca aca atg atc gcc cac ccg aaa ctg gac ccc gtc gac 960
Gln Leu Asn Ser Thr Met Ile Ala His Pro Lys Leu Asp Pro Val Asp
305 310 315 320
ggc gac ctc cac gcg ctc agc tac gac gtc att cag aag cct tac ctc 1008
Gly Asp Leu His Ala Leu Ser Tyr Asp Val Ile Gln Lys Pro Tyr Leu
325 330 335
aag tac ttc cgt ttc tcc ccc gac ggc gtc aag tcc ccc gac gtg gaa 1056
Lys Tyr Phe Arg Phe Ser Pro Asp Gly Val Lys Ser Pro Asp Val Glu
340 345 350
atc ccc ctg aag gag ccc acc atg atg cac gat ttc gcc ata acg gag 1104
Ile Pro Leu Lys Glu Pro Thr Met Met His Asp Phe Ala Ile Thr Glu
355 360 365
aat ttc gtc gtc gtc ccc gac cag cag gtg gtc ttc aaa cta acg gag 1152
Asn Phe Val Val Val Pro Asp Gln Gln Val Val Phe Lys Leu Thr Glu
370 375 380
atg atc acc ggc ggg tcc ccc gtg gtc tac gac aag aac aaa acc tca 1200
Met Ile Thr Gly Gly Ser Pro Val Val Tyr Asp Lys Asn Lys Thr Ser
385 390 395 400
cgg ttt ggg att ctg cac aag aat gcg aag gac gcg aat gcg atg cgg 1248
Arg Phe Gly Ile Leu His Lys Asn Ala Lys Asp Ala Asn Ala Met Arg
405 410 415
tgg atc gac gcg ccg gat tgt ttc tgc ttc cac ctc tgg aac gcg tgg 1296
Trp Ile Asp Ala Pro Asp Cys Phe Cys Phe His Leu Trp Asn Ala Trp
420 425 430
gag gag ccc gaa acc gag gag gtt gtg gtg att ggg tcc tgc atg acc 1344
Glu Glu Pro Glu Thr Glu Glu Val Val Val Ile Gly Ser Cys Met Thr
435 440 445
cct gcg gac tcc att ttc aac gaa tgc gag gag agt ttg aag agc gtg 1392
Pro Ala Asp Ser Ile Phe Asn Glu Cys Glu Glu Ser Leu Lys Ser Val
450 455 460
ctg tca gag ata agg ctg aac ttg agg acc ggc aag tcc act cgg cgc 1440
Leu Ser Glu Ile Arg Leu Asn Leu Arg Thr Gly Lys Ser Thr Arg Arg
465 470 475 480
ccc att atc tcc gac gcc gaa caa gtg aac ctg gaa gcc ggc atg gtg 1488
Pro Ile Ile Ser Asp Ala Glu Gln Val Asn Leu Glu Ala Gly Met Val
485 490 495
aac aga aac aag ctc gga agg aag acc cag ttc gcg tat ctg gct ctg 1536
Asn Arg Asn Lys Leu Gly Arg Lys Thr Gln Phe Ala Tyr Leu Ala Leu
500 505 510
gcg gag ccc tgg ccc aaa gtc tcg ggc ttt gcg aaa gtt gat ttg ctg 1584
Ala Glu Pro Trp Pro Lys Val Ser Gly Phe Ala Lys Val Asp Leu Leu
515 520 525
agt ggg gaa gtg aag aag tac atg tat gga gaa gag aag ttc ggt ggg 1632
Ser Gly Glu Val Lys Lys Tyr Met Tyr Gly Glu Glu Lys Phe Gly Gly
530 535 540
gag cct ctg ttt ctt ccc aac ggc caa aaa gaa gac gat ggg tat att 1680
Glu Pro Leu Phe Leu Pro Asn Gly Gln Lys Glu Asp Asp Gly Tyr Ile
545 550 555 560
ctg gca ttc gtg cac gac gag aaa gaa tgg aaa tcc gag ctg cag att 1728
Leu Ala Phe Val His Asp Glu Lys Glu Trp Lys Ser Glu Leu Gln Ile
565 570 575
gtg aat gcc caa aat tta aag ctc gaa gct tcc atc aaa ctc ccc tct 1776
Val Asn Ala Gln Asn Leu Lys Leu Glu Ala Ser Ile Lys Leu Pro Ser
580 585 590
cgt gtt ccc tac ggt ttt cat gga act ttc att cat tcc aag gat ttg 1824
Arg Val Pro Tyr Gly Phe His Gly Thr Phe Ile His Ser Lys Asp Leu
595 600 605
agg aaa caa gct tga 1839
Arg Lys Gln Ala
610
<210>12
<211>612
<212>PRT
<213>Vigna unguiculata
<400>12
Met Pro Ser Ser Ala Ser Asn Thr Trp Phe Asn Ala Thr Leu Pro Ser
1 5 10 15
Pro Pro Phe Lys Asp Leu Pro Ser Thr Ser Ser Pro Thr Asn Leu Leu
20 25 30
Pro Leu Arg Lys Thr Ser Ser Ser Asn Thr Ile Thr Cys Ser Leu Gln
35 40 45
Thr Leu His Phe Pro Lys Gln Tyr Gln Pro Thr Ser Thr Ser Thr Ser
50 55 60
Thr Ala Thr Thr Thr Thr Pro Thr Pro Ile Lys Thr Thr Thr Ile Thr
65 70 75 80
Thr Thr Thr Pro Pro Arg Glu Thr Asn Pro Leu Ser Asp Thr Asn Gln
85 90 95
Pro Leu Pro Gln Lys Trp Asn Phe Leu Gln Lys Ala Ala Ala Thr Ala
100 105 110
Leu Asp Leu Val Glu Thr Ala Leu Val Ser His Glu Arg Lys His Pro
115 120 125
Leu Pro Lys Thr Ala Asp Pro Arg Val Gln Ile Ala Gly Asn Phe Ala
130 135 140
Pro Val Pro Glu His Ala Ala Asp Gln Gly Leu Pro Val Val Gly Lys
145 150 155 160
Ile Pro Lys Cys Ile Asp Gly Val Tyr Val Arg Asn Gly Ala Asn Pro
165 170 175
Leu Tyr Glu Pro Val Ala Gly His His Phe Phe Asp Gly Asp Gly Met
180 185 190
Val His Ala Val Lys Phe Thr Asn Gly Ala Ala Ser Tyr Ala Cys Arg
195 200 205
Phe Thr Glu Thr Gln Arg Leu Ser Gln Glu Lys Ser Leu Gly Arg Pro
210 215 220
Val Phe Pro Lys Ala Ile Gly Glu Leu His Gly His Ser Gly Ile Ala
225 230 235 240
Arg Leu Leu Leu Phe Tyr Ala Arg Gly Leu Phe Gly Leu Val Asp Gly
245 250 255
Ser Gln Gly Met Gly Val Ala Asn Ala Gly Leu Val Tyr Phe Asn Asn
260 265 270
His Leu Leu Ala Met Ser Glu Asp Asp Leu Pro Tyr His Val Arg Ile
275 280 285
Thr Pro Asn Gly Asp Leu Thr Thr Val Gly Arg Tyr Asp Phe Asn Gly
290 295 300
Gln Leu Asn Ser Thr Met Ile Ala His Pro Lys Leu Asp Pro Val Asp
305 310 315 320
Gly Asp Leu His Ala Leu Ser Tyr Asp Val Ile Gln Lys Pro Tyr Leu
325 330 335
Lys Tyr Phe Arg Phe Ser Pro Asp Gly Val Lys Ser Pro Asp Val Glu
340 345 350
Ile Pro Leu Lys Glu Pro Thr Met Met His Asp Phe Ala Ile Thr Glu
355 360 365
Asn Phe Val Val Val Pro Asp Gln Gln Val Val Phe Lys Leu Thr Glu
370 375 380
Met Ile Thr Gly Gly Ser Pro Val Val Tyr Asp Lys Asn Lys Thr Ser
385 390 395 400
Arg Phe Gly Ile Leu His Lys Asn Ala Lys Asp Ala Asn Ala Met Arg
405 410 415
Trp Ile Asp Ala Pro Asp Cys Phe Cys Phe His Leu Trp Asn Ala Trp
420 425 430
Glu Glu Pro Glu Thr Glu Glu Val Val Val Ile Gly Ser Cys Met Thr
435 440 445
Pro Ala Asp Ser Ile Phe Asn Glu Cys Glu Glu Ser Leu Lys Ser Val
450 455 460
Leu Ser Glu Ile Arg Leu Asn Leu Arg Thr Gly Lys Ser Thr Arg Arg
465 470 475 480
Pro Ile Ile Ser Asp Ala Glu Gln Val Asn Leu Glu Ala Gly Met Val
485 490 495
Asn Arg Asn Lys Leu Gly Arg Lys Thr Gln Phe Ala Tyr Leu Ala Leu
500 505 510
Ala Glu Pro Trp Pro Lys Val Ser Gly Phe Ala Lys Val Asp Leu Leu
515 520 525
Ser Gly Glu Val Lys Lys Tyr Met Tyr Gly Glu Glu Lys Phe Gly Gly
530 535 540
Glu Pro Leu Phe Leu Pro Asn Gly Gln Lys Glu Asp Asp Gly Tyr Ile
545 550 555 560
Leu Ala Phe Val His Asp Glu Lys Glu Trp Lys Ser Glu Leu Gln Ile
565 570 575
Val Asn Ala Gln Asn Leu Lys Leu Glu Ala Ser Ile Lys Leu Pro Ser
580 585 590
Arg Val Pro Tyr Gly Phe His Gly Thr Phe Ile His Ser Lys Asp Leu
595 600 605
Arg Lys Gln Ala
610
<210>13
<211>1815
<212>DNA
<213>玉米
<220>
<221>CDS
<222>(1)..(1815)
<400>13
atg cag ggt ctc gcc ccg ccc acc tct gtt tcc ata cac cgg cac ctg 48
Met Gln Gly Leu Ala Pro Pro Thr Ser Val Ser Ile His Arg His Leu
1 5 10 15
ccg gcc cgg tcc agg gcc cgg gcc tcc aat tcc gtc agg ttc tcg ccg 96
Pro Ala Arg Ser Arg Ala Arg Ala Ser Asn Ser Val Arg Phe Ser Pro
20 25 30
cgc gcc gtc agc tcc gtg ccg ccc gcc gag tgc ctc cag gcg ccg ttc 144
Arg Ala Val Ser Ser Val Pro Pro Ala Glu Cys Leu Gln Ala Pro Phe
35 40 45
cac aag ccc gtc gcc gac ctg cct gcg ccg tcc agg aag ccc gcc gcc 192
His Lys Pro Val Ala Asp Leu Pro Ala Pro Ser Arg Lys Pro Ala Ala
50 55 60
att gcc gtc cca ggg cac gcc gcg gcg ccg agg aaa gcg gag ggc ggc 240
Ile Ala Val Pro Gly His Ala Ala Ala Pro Arg Lys Ala Glu Gly Gly
65 70 75 80
aag aag cag ctc aac ttg ttc cag cgc gcc gcg gcg gcc gcg ctc gac 288
Lys Lys Gln Leu Asn Leu Phe Gln Arg Ala Ala Ala Ala Ala Leu Asp
85 90 95
gcg ttc gag gaa ggg ttc gtg gcc aac gtc ctc gag cgg ccc cac ggg 336
Ala Phe Glu Glu Gly Phe Val Ala Asn Val Leu Glu Arg Pro His Gly
100 105 110
ctg ccc agc acg gcc gac ccg gcc gtg cag atc gcc ggc aac ttc gcg 384
Leu Pro Ser Thr Ala Asp Pro Ala Val Gln Ile Ala Gly Asn Phe Ala
115 120 125
ccc gtc ggg gag agg ccg ccc gtg cac gag ctc ccc gtc tcc ggc cgc 432
Pro Val Gly Glu Arg Pro Pro Val His Glu Leu Pro Val Ser Gly Arg
130 135 140
atc ccg ccc ttc atc gac ggg gtc tac gcg cgc aac ggc gcc aac ccc 480
Ile Pro Pro Phe Ile Asp Gly Val Tyr Ala Arg Asn Gly Ala Asn Pro
145 150 155 160
tgc ttc gac ccc gtc gcg ggg cac cac ctc ttc gac ggc gac ggc atg 528
Cys Phe Asp Pro Val Ala Gly His His Leu Phe Asp Gly Asp Gly Met
165 170 175
gtg cac gcg ctg cgg ata cgc aac ggc gcc gcc gag tcc tac gcc tgc 576
Val His Ala Leu Arg Ile Arg Asn Gly Ala Ala Glu Ser Tyr Ala Cys
180 185 190
cgc ttc acg gag acc gcg cgc ctg cgc cag gag cgc gcg atc ggc cgc 624
Arg Phe Thr Glu Thr Ala Arg Leu Arg Gln Glu Arg Ala Ile Gly Arg
195 200 205
ccc gtc ttc ccc aag gcc att ggc gag ctg cac ggg cac tcc ggg atc 672
Pro Val Phe Pro Lys Ala Ile Gly Glu Leu His Gly His Ser Gly Ile
210 215 220
gcg cgc ctc gcc ctg ttc tac gcg cgc gcc gcg tgc ggc ctc gtg gac 720
Ala Arg Leu Ala Leu Phe Tyr Ala Arg Ala Ala Cys Gly Leu Val Asp
225 230 235 240
ccc tcg gcc ggc acc ggc gtg gcc aac gcc ggc ctc gtc tac ttc aac 768
Pro Ser Ala Gly Thr Gly Val Ala Asn Ala Gly Leu Val Tyr Phe Asn
245 250 255
ggc cgc ctg ctc gcc atg tcc gag gac gac ctc ccc tac cac gtc cgc 816
Gly Arg Leu Leu Ala Met Ser Glu Asp Asp Leu Pro Tyr His Val Arg
260 265 270
gtg gcg gac gac ggc gac ctc gag acc gtc ggc cgc tac gac ttc gac 864
Val Ala Asp Asp Gly Asp Leu Glu Thr Val Gly Arg Tyr Asp Phe Asp
275 280 285
ggg cag ctc ggc tgc gcc atg atc gcg cac ccc aag ctg gac ccg gcc 912
Gly Gln Leu Gly Cys Ala Met Ile Ala His Pro Lys Leu Asp Pro Ala
290 295 300
acc ggg gag ctc cac gcg ctc agc tac gac gtc atc aag agg ccg tac 960
Thr Gly Glu Leu His Ala Leu Ser Tyr Asp Val Ile Lys Arg Pro Tyr
305 310 315 320
ctc aag tac ttc tac ttc agg ccc gac ggc acc aag tcc gac gac gtg 1008
Leu Lys Tyr Phe Tyr Phe Arg Pro Asp Gly Thr Lys Ser Asp Asp Val
325 330 335
gag atc ccg ctg gag cag ccc acg atg atc cac gac ttc gcc atc acc 1056
Glu Ile Pro Leu Glu Gln Pro Thr Met Ile His Asp Phe Ala Ile Thr
340 345 350
gag aac ttc gtg gtt gtg ccc gac cac cag gtg gtg ttc aag ctc cag 1104
Glu Asn Phe Val Val Val Pro Asp His Gln Val Val Phe Lys Leu Gln
355 360 365
gag atg ctg cgc ggc ggg tcg ccc gtg gtg ctg gac aag gag aag acg 1152
Glu Met Leu Arg Gly Gly Ser Pro Val Val Leu Asp Lys Glu Lys Thr
370 375 380
tcg cgg ttc ggc gtg ctc ccc aag cac gcc gcg gac gcg tcg gag atg 1200
Ser Arg Phe Gly Val Leu Pro Lys His Ala Ala Asp Ala Ser Glu Met
385 390 395 400
gcg tgg gtg gac gtg ccg gac tgc ttc tgc ttc cac ctg tgg aac gcg 1248
Ala Trp Val Asp Val Pro Asp Cys Phe Cys Phe His Leu Trp Asn Ala
405 410 415
tgg gag gac gag gcg acg ggc gag gtg gtg gtg atc ggc tcc tgc atg 1296
Trp Glu Asp Glu Ala Thr Gly Glu Val Val Val Ile Gly Ser Cys Met
420 425 430
acc ccc gcc gac tcc atc ttc aac gag tcc gac gag cgc ctg gag agc 1344
Thr Pro Ala Asp Ser Ile Phe Asn Glu Ser Asp Glu Arg Leu Glu Ser
435 440 445
gtg ctg acc gag atc cgc ctg gac gcg cgc acg ggc cgg tcc acg cgc 1392
Val Leu Thr Glu Ile Arg Leu Asp Ala Arg Thr Gly Arg Ser Thr Arg
450 455 460
cgc gcc gtc ctg ccg ccg tcg cag cag gag aac ctg gag gtg ggc atg 1440
Arg Ala Val Leu Pro Pro Ser Gln Gln Glu Asn Leu Glu Val Gly Met
465 470 475 480
gtg aac cgc aac ctg ctg ggc cgc gag agc cgg tac gcg tac ctc gcg 1488
Val Asn Arg Asn Leu Leu Gly Arg Glu Ser Arg Tyr Ala Tyr Leu Ala
485 490 495
gtg gcg gag ccg tgg ccc aag gag tcg ggc ttc gcc aag gag gac ctg 1536
Val Ala Glu Pro Trp Pro Lys Glu Ser Gly Phe Ala Lys Glu Asp Leu
500 505 510
tcc acg ggc gag ctc acc aag ttc gag tac ggc gag ggc cgg ttc ggc 1584
Ser Thr Gly Glu Leu Thr Lys Phe Glu Tyr Gly Glu Gly Arg Phe Gly
515 520 525
ggc gag ccc tgc ttc gtt ccc atg gac ccg gcc gcg gcc cac ccg cgc 1632
Gly Glu Pro Cys Phe Val Pro Met Asp Pro Ala Ala Ala His Pro Arg
530 535 540
ggc gag gac gac ggg tac gtg ctc acc ttc gtc cac gac gag cgc gcc 1680
Gly Glu Asp Asp Gly Tyr Val Leu Thr Phe Val His Asp Glu Arg Ala
545 550 555 560
ggc acg tcg gag cta ctt gtg gtc aat gcc gcc gac atc cgg ctg gag 1728
Gly Thr Ser Glu Leu Leu Val Val Asn Ala Ala Asp Ile Arg Leu Glu
565 570 575
gcc acg gtt cag ctg ccg tcc cgc gtg ccc ttc ggc ttc cac ggc acc 1776
Ala Thr Val Gln Leu Pro Ser Arg Val Pro Phe Gly Phe His Gly Thr
580 585 590
ttc atc acg ggc cag gag ctc gag gcc cag gcg gcc tga 1815
Phe Ile Thr Gly Gln Glu Leu Glu Ala Gln Ala Ala
595 600 605
<210>14
<211>604
<212>PRT
<213>玉米
<400>14
Met Gln Gly Leu Ala Pro Pro Thr Ser Val Ser Ile His Arg His Leu
1 5 10 15
Pro Ala Arg Ser Arg Ala Arg Ala Ser Asn Ser Val Arg Phe Ser Pro
20 25 30
Arg Ala Val Ser Ser Val Pro Pro Ala Glu Cys Leu Gln Ala Pro Phe
35 40 45
His Lys Pro Val Ala Asp Leu Pro Ala Pro Ser Arg Lys Pro Ala Ala
50 55 60
Ile Ala Val Pro Gly His Ala Ala Ala Pro Arg Lys Ala Glu Gly Gly
65 70 75 80
Lys Lys Gln Leu Asn Leu Phe Gln Arg Ala Ala Ala Ala Ala Leu Asp
85 90 95
Ala Phe Glu Glu Gly Phe Val Ala Asn Val Leu Glu Arg Pro His Gly
100 105 110
Leu Pro Ser Thr Ala Asp Pro Ala Val Gln Ile Ala Gly Asn Phe Ala
115 120 125
Pro Val Gly Glu Arg Pro Pro Val His Glu Leu Pro Val Ser Gly Arg
130 135 140
Ile Pro Pro Phe Ile Asp Gly Val Tyr Ala Arg Asn Gly Ala Asn Pro
145 150 155 160
Cys Phe Asp Pro Val Ala Gly His His Leu Phe Asp Gly Asp Gly Met
165 170 175
Val His Ala Leu Arg Ile Arg Asn Gly Ala Ala Glu Ser Tyr Ala Cys
180 185 190
Arg Phe Thr Glu Thr Ala Arg Leu Arg Gln Glu Arg Ala Ile Gly Arg
195 200 205
Pro Val Phe Pro Lys Ala Ile Gly Glu Leu His Gly His Ser Gly Ile
210 215 220
Ala Arg Leu Ala Leu Phe Tyr Ala Arg Ala Ala Cys Gly Leu Val Asp
225 230 235 240
Pro Ser Ala Gly Thr Gly Val Ala Asn Ala Gly Leu Val Tyr Phe Asn
245 250 255
Gly Arg Leu Leu Ala Met Ser Glu Asp Asp Leu Pro Tyr His Val Arg
260 265 270
Val Ala Asp Asp Gly Asp Leu Glu Thr Val Gly Arg Tyr Asp Phe Asp
275 280 285
Gly Gln Leu Gly Cys Ala Met Ile Ala His Pro Lys Leu Asp Pro Ala
290 295 300
Thr Gly Glu Leu His Ala Leu Ser Tyr Asp Val Ile Lys Arg Pro Tyr
305 310 315 320
Leu Lys Tyr Phe Tyr Phe Arg Pro Asp Gly Thr Lys Ser Asp Asp Val
325 330 335
Glu Ile Pro Leu Glu Gln Pro Thr Met Ile His Asp Phe Ala Ile Thr
340 345 350
Glu Asn Phe Val Val Val Pro Asp His Gln Val Val Phe Lys Leu Gln
355 360 365
Glu Met Leu Arg Gly Gly Ser Pro Val Val Leu Asp Lys Glu Lys Thr
370 375 380
Ser Arg Phe Gly Val Leu Pro Lys His Ala Ala Asp Ala Ser Glu Met
385 390 395 400
Ala Trp Val Asp Val Pro Asp Cys Phe Cys Phe His Leu Trp Asn Ala
405 410 415
Trp Glu Asp Glu Ala Thr Gly Glu Val Val Val Ile Gly Ser Cys Met
420 425 430
Thr Pro Ala Asp Ser Ile Phe Asn Glu Ser Asp Glu Arg Leu Glu Ser
435 440 445
Val Leu Thr Glu Ile Arg Leu Asp Ala Arg Thr Gly Arg Ser Thr Arg
450 455 460
Arg Ala Val Leu Pro Pro Ser Gln Gln Glu Asn Leu Glu Val Gly Met
465 470 475 480
Val Asn Arg Asn Leu Leu Gly Arg Glu Ser Arg Tyr Ala Tyr Leu Ala
485 490 495
Val Ala Glu Pro Trp Pro Lys Glu Ser Gly Phe Ala Lys Glu Asp Leu
500 505 510
Ser Thr Gly Glu Leu Thr Lys Phe Glu Tyr Gly Glu Gly Arg Phe Gly
515 520 525
Gly Glu Pro Cys Phe Val Pro Met Asp Pro Ala Ala Ala His Pro Arg
530 535 540
Gly Glu Asp Asp Gly Tyr Val Leu Thr Phe Val His Asp Glu Arg Ala
545 550 555 560
Gly Thr Ser Glu Leu Leu Val Val Asn Ala Ala Asp Ile Arg Leu Glu
565 570 575
Ala Thr Val Gln Leu Pro Ser Arg Val Pro Phe Gly Phe His Gly Thr
580 585 590
Phe Ile Thr Gly Gln Glu Leu Glu Ala Gln Ala Ala
595 600
<210>15
<211>1818
<212>DNA
<213>番茄
<220>
<221>CDS
<222>(1)..(1818)
<400>15
atg gca act act act tca cat gcc aca aat aca tgg att aag act aag 48
Met Ala Thr Thr Thr Ser His Ala Thr Asn Thr Trp Ile Lys Thr Lys
1 5 10 15
ttg tca atg cca tca tca aag gag ttt ggt ttt gca tca aac tct att 96
Leu Ser Met Pro Ser Ser Lys Glu Phe Gly Phe Ala Ser Asn Ser Ile
20 25 30
tct cta ctc aaa aat caa cat aat agg caa agt ctc aac att aat tcc 144
Ser Leu Leu Lys Asn Gln His Asn Arg Gln Ser Leu Asn Ile Asn Ser
35 40 45
tct ctt caa gct cca cct ata ctt cat ttt cct aaa caa tct tca aat 192
Ser Leu Gln Ala Pro Pro Ile Leu His Phe Pro Lys Gln Ser Ser Asn
50 55 60
tat caa aca cca aag aat aat aca att tca cac cca aaa caa gaa aac 240
Tyr Gln Thr Pro Lys Asn Asn Thr Ile Ser His Pro Lys Gln Glu Asn
65 70 75 80
aac aac tcc tct tct tct tca act tcc aag tgg aat tta gtg cag aaa 288
Asn Asn Ser Ser Ser Ser Ser Thr Ser Lys Trp Asn Leu Val Gln Lys
85 90 95
gca gca gca atg gct tta gat gct gta gaa agt gct tta act aaa cat 336
Ala Ala Ala Met Ala Leu Asp Ala Val Glu Ser Ala Leu Thr Lys His
100 105 110
gaa ctt gaa cac cct ttg ccg aaa aca gcc gac cca cga gtc cag att 384
Glu Leu Glu His Pro Leu Pro Lys Thr Ala Asp Pro Arg Val Gln Ile
115 120 125
tct ggg aat ttt gct ccg gta ccg gaa aat cca gtc tgt caa tct ctt 432
Ser Gly Asn Phe Ala Pro Val Pro Glu Asn Pro Val Cys Gln Ser Leu
130 135 140
ccg gtc acc gga aaa ata ccc aaa tgt gtt caa ggc gtt tac gtt cga 480
Pro Val Thr Gly Lys Ile Pro Lys Cys Val Gln Gly Val Tyr Val Arg
145 150 155 160
aac gga gct aac cct ctt ttt gaa cca acc gcc gga cac cat ttc ttc 528
Asn Gly Ala Asn Pro Leu Phe Glu Pro Thr Ala Gly His His Phe Phe
165 170 175
gac ggc gac ggt atg gtt cac gcc gtt caa ttc aaa aat ggg tcg gct 576
Asp Gly Asp Gly Met Val His Ala Val Gln Phe Lys Asn Gly Ser Ala
180 185 190
agt tac gct tgc cgt ttc act gaa aca gag agg ctt gtt caa gaa aaa 624
Ser Tyr Ala Cys Arg Phe Thr Glu Thr Glu Arg Leu Val Gln Glu Lys
195 200 205
gct ttg ggt cgc cct gtt ttc cct aaa gcc att ggt gaa tta cat ggt 672
Ala Leu Gly Arg Pro Val Phe Pro Lys Ala Ile Gly Glu Leu His Gly
210 215 220
cac tct gga att gca agg ctt atg ctg ttt tac gct cgt ggg ctc ttc 720
His Ser Gly Ile Ala Arg Leu Met Leu Phe Tyr Ala Arg Gly Leu Phe
225 230 235 240
gga ctt gtt gat cac agt aaa gga act ggt gtt gca aac gcc ggt tta 768
Gly Leu Val Asp His Ser Lys Gly Thr Gly Val Ala Asn Ala Gly Leu
245 250 255
gtc tat ttc aat aac cga tta ctt gct atg tct gaa gat gat ttg cct 816
Val Tyr Phe Asn Asn Arg Leu Leu Ala Met Ser Glu Asp Asp Leu Pro
260 265 270
tac cat gta aag gta aca ccc acc ggc gat ctt aaa aca gag ggt cga 864
Tyr His Val Lys Val Thr Pro Thr Gly Asp Leu Lys Thr Glu Gly Arg
275 280 285
ttc gat ttc gac ggc cag cta aaa tcc acc atg ata gct cac cca aag 912
Phe Asp Phe Asp Gly Gln Leu Lys Ser Thr Met Ile Ala His Pro Lys
290 295 300
ctc gac cca gtt tcc ggt gag cta ttt gct ctt agc tac gat gtg att 960
Leu Asp Pro Val Ser Gly Glu Leu Phe Ala Leu Ser Tyr Asp Val Ile
305 310 315 320
cag aag cca tac ctc aag tac ttc aga ttt tca aaa aat ggg gaa aaa 1008
Gln Lys Pro Tyr Leu Lys Tyr Phe Arg Phe Ser Lys Asn Gly Glu Lys
325 330 335
tca aat gat gtt gaa att cca gtt gaa gac cca aca atg atg cat gat 1056
Ser Asn Asp Val Glu Ile Pro Val Glu Asp Pro Thr Met Met His Asp
340 345 350
ttc gca att act gag aac ttc gtc gtc att cct gat caa caa gtc gtt 1104
Phe Ala Ile Thr Glu Asn Phe Val Val Ile Pro Asp Gln Gln Val Val
355 360 365
ttc aag atg tct gaa atg atc cgt gga ggt tca ccg gtg gtt tac gac 1152
Phe Lys Met Ser Glu Met Ile Arg Gly Gly Ser Pro Val Val Tyr Asp
370 375 380
aag aac aaa gtt tcc cga ttt ggt att ctg gat aag tac gcg aaa gat 1200
Lys Asn Lys Val Ser Arg Phe Gly Ile Leu Asp Lys Tyr Ala Lys Asp
385 390 395 400
ggg tct gat ttg aaa tgg gtt gaa gta cct gat tgt ttc tgt ttc cac 1248
Gly Ser Asp Leu Lys Trp Val Glu Val Pro Asp Cys Phe Cys Phe His
405 410 415
ctc tgg aat gct tgg gaa gaa gca gaa aca gat gaa atc gtt gta att 1296
Leu Trp Asn Ala Trp Glu Glu Ala Glu Thr Asp Glu Ile Val Val Ile
420 425 430
ggt tca tgt atg aca cca cca gac tcc att ttc aat gaa tgt gat gaa 1344
Gly Ser Cys Met Thr Pro Pro Asp Ser Ile Phe Asn Glu Cys Asp Glu
435 440 445
ggg cta aag agt gtt tta tcc gaa atc cgt ctc aat ttg aaa aca ggg 1392
Gly Leu Lys Ser Val Leu Ser Glu Ile Arg Leu Asn Leu Lys Thr Gly
450 455 460
aaa tca aca aga aaa tcc ata atc gaa aac ccg gat gaa caa gtg aat 1440
Lys Ser Thr Arg Lys Ser Ile Ile Glu Asn Pro Asp Glu Gln Val Asn
465 470 475 480
tta gaa gct gga atg gtg aac cga aac aaa ctc gga agg aaa aca gag 1488
Leu Glu Ala Gly Met Val Asn Arg Asn Lys Leu Gly Arg Lys Thr Glu
485 490 495
tat gct tat ttg gct atc gct gaa cca tgg cca aaa gtt tct ggt ttt 1536
Tyr Ala Tyr Leu Ala Ile Ala Glu Pro Trp Pro Lys Val Ser Gly Phe
500 505 510
gca aaa gta aac ctg ttc acc ggt gaa gtt gag aaa ttc att tat ggt 1584
Ala Lys Val Asn Leu Phe Thr Gly Glu Val Glu Lys Phe Ile Tyr Gly
515 520 525
gac aac aaa tat ggt ggg gaa cct ctt ttt tta cca aga gac ccc aac 1632
Asp Asn Lys Tyr Gly Gly Glu Pro Leu Phe Leu Pro Arg Asp Pro Asn
530 535 540
agc aag gaa gaa gac gat ggt tat att tta gct ttc gtt cac gat gag 1680
Ser Lys Glu Glu Asp Asp Gly Tyr Ile Leu Ala Phe Val His Asp Glu
545 550 555 560
aaa gaa tgg aaa tca gaa ctg caa att gtt aac gca atg agt ttg aag 1728
Lys Glu Trp Lys Ser Glu Leu Gln Ile Val Asn Ala Met Ser Leu Lys
565 570 575
ttg gag gca act gtg aag ctt cca tea aga gtt cct tat gga ttt cat 1776
Leu Glu Ala Thr Val Lys Leu Pro Ser Arg Val Pro Tyr Gly Phe His
580 585 590
gga aca ttc ata aac gcc aat gat ttg gca aat cag gca tga 1818
Gly Thr Phe Ile Asn Ala Asn Asp Leu Ala Asn Gln Ala
595 600 605
<210>16
<211>605
<212>PRT
<213>番茄
<400>16
Met Ala Thr Thr Thr Ser His Ala Thr Asn Thr Trp Ile Lys Thr Lys
1 5 10 15
Leu Ser Met Pro Ser Ser Lys Glu Phe Gly Phe Ala Ser Asn Ser Ile
20 25 30
Ser Leu Leu Lys Asn Gln His Asn Arg Gln Ser Leu Asn Ile Asn Ser
35 40 45
Ser Leu Gln Ala Pro Pro Ile Leu His Phe Pro Lys Gln Ser Ser Asn
50 55 60
Tyr Gln Thr Pro Lys Asn Asn Thr Ile Ser His Pro Lys Gln Glu Asn
65 70 75 80
Asn Asn Ser Ser Ser Ser Ser Thr Ser Lys Trp Asn Leu Val Gln Lys
85 90 95
Ala Ala Ala Met Ala Leu Asp Ala Val Glu Ser Ala Leu Thr Lys His
100 105 110
Glu Leu Glu His Pro Leu Pro Lys Thr Ala Asp Pro Arg Val Gln Ile
115 120 125
Ser Gly Asn Phe Ala Pro Val Pro Glu Asn Pro Val Cys Gln Ser Leu
130 135 140
Pro Val Thr Gly Lys Ile Pro Lys Cys Val Gln Gly Val Tyr Val Arg
145 150 155 160
Asn Gly Ala Asn Pro Leu Phe Glu Pro Thr Ala Gly His His Phe Phe
165 170 175
Asp Gly Asp Gly Met Val His Ala Val Gln Phe Lys Asn Gly Ser Ala
180 185 190
Ser Tyr Ala Cys Arg Phe Thr Glu Thr Glu Arg Leu Val Gln Glu Lys
195 200 205
Ala Leu Gly Arg Pro Val Phe Pro Lys Ala Ile Gly Glu Leu His Gly
210 215 220
His Ser Gly Ile Ala Arg Leu Met Leu Phe Tyr Ala Arg Gly Leu Phe
225 230 235 240
Gly Leu Val Asp His Ser Lys Gly Thr Gly Val Ala Asn Ala Gly Leu
245 250 255
Val Tyr Phe Asn Asn Arg Leu Leu Ala Met Ser Glu Asp Asp Leu Pro
260 265 270
Tyr His Val Lys Val Thr Pro Thr Gly Asp Leu Lys Thr Glu Gly Arg
275 280 285
Phe Asp Phe Asp Gly Gln Leu Lys Ser Thr Met Ile Ala His Pro Lys
290 295 300
Leu Asp Pro Val Ser Gly Glu Leu Phe Ala Leu Ser Tyr Asp Val Ile
305 310 315 320
Gln Lys Pro Tyr Leu Lys Tyr Phe Arg Phe Ser Lys Asn Gly Glu Lys
325 330 335
Ser Asn Asp Val Glu Ile Pro Val Glu Asp Pro Thr Met Met His Asp
340 345 350
Phe Ala Ile Thr Glu Asn Phe Val Val Ile Pro Asp Gln Gln Val Val
355 360 365
Phe Lys Met Ser Glu Met Ile Arg Gly Gly Ser Pro Val Val Tyr Asp
370 375 380
Lys Asn Lys Val Ser Arg Phe Gly Ile Leu Asp Lys Tyr Ala Lys Asp
385 390 395 400
Gly Ser Asp Leu Lys Trp Val Glu Val Pro Asp Cys Phe Cys Phe His
405 410 415
Leu Trp Asn Ala Trp Glu Glu Ala Glu Thr Asp Glu Ile Val Val Ile
420 425 430
Gly Ser Cys Met Thr Pro Pro Asp Ser Ile Phe Asn Glu Cys Asp Glu
435 440 445
Gly Leu Lys Ser Val Leu Ser Glu Ile Arg Leu Asn Leu Lys Thr Gly
450 455 460
Lys Ser Thr Arg Lys Ser Ile Ile Glu Asn Pro Asp Glu Gln Val Asn
465 470 475 480
Leu Glu Ala Gly Met Val Asn Arg Asn Lys Leu Gly Arg Lys Thr Glu
485 490 495
Tyr Ala Tyr Leu Ala Ile Ala Glu Pro Trp Pro Lys Val Ser Gly Phe
500 505 510
Ala Lys Val Asn Leu Phe Thr Gly Glu Val Glu Lys Phe Ile Tyr Gly
515 520 525
Asp Asn Lys Tyr Gly Gly Glu Pro Leu Phe Leu Pro Arg Asp Pro Asn
530 535 540
Ser Lys Glu Glu Asp Asp Gly Tyr Ile Leu Ala Phe Val His Asp Glu
545 550 555 560
Lys Glu Trp Lys Ser Glu Leu Gln Ile Val Asn Ala Met Ser Leu Lys
565 570 575
Leu Glu Ala Thr Val Lys Leu Pro Ser Arg Val Pro Tyr Gly Phe His
580 585 590
Gly Thr Phe Ile Asn Ala Asn Asp Leu Ala Asn Gln Ala
595 600 605
<210>17
<211>1617
<212>DNA
<213>拟南芥
<220>
<221>CDS
<222>(1)..(1617)
<400>17
atg gcg gag aaa ctc agt gat ggc agc atc atc atc tca gtc cat cct 48
Met Ala Glu Lys Leu Ser Asp Gly Ser Ile Ile Ile Ser Val His Pro
1 5 10 15
aga ccc tcc aag ggt ttc tcc tcg aag ctt ctc gat ctt ctc gag aga 96
Arg Pro Ser Lys Gly Phe Ser Ser Lys Leu Leu Asp Leu Leu Glu Arg
20 25 30
ctt gtc gtc aag ctc atg cac gat gct tct ctc cct ctc cac tac ctc 144
Leu Val Val Lys Leu Met His Asp Ala Ser Leu Pro Leu His Tyr Leu
35 40 45
tca ggc aac ttc gct ccc atc cgt gat gaa act cct ccc gtc aag gat 192
Ser Gly Asn Phe Ala Pro Ile Arg Asp Glu Thr Pro Pro Val Lys Asp
50 55 60
ctc ccc gtc cat gga ttt ctt ccc gaa tgc ttg aat ggt gaa ttt gtg 240
Leu Pro Val His Gly Phe Leu Pro Glu Cys Leu Asn Gly Glu Phe Val
65 70 75 80
agg gtt ggt cca aac ccc aag ttt gat gct gtc gct gga tat cac tgg 288
Arg Val Gly Pro Asn Pro Lys Phe Asp Ala Val Ala Gly Tyr His Trp
85 90 95
ttt gat gga gat ggg atg att cat ggg gta cgc atc aaa gat ggg aaa 336
Phe Asp Gly Asp Gly Met Ile His Gly Val Arg Ile Lys Asp Gly Lys
100 105 110
gct act tat gtt tct cga tat gtt aag aca tca cgt ctt aag cag gaa 384
Ala Thr Tyr Val Ser Arg Tyr Val Lys Thr Ser Arg Leu Lys Gln Glu
115 120 125
gag ttc ttc gga gct gcc aaa ttc atg aag att ggt gac ctt aag ggg 432
Glu Phe Phe Gly Ala Ala Lys Phe Met Lys Ile Gly Asp Leu Lys Gly
130 135 140
ttt ttc gga ttg cta atg gtc aat atc caa cag ctg aga acg aag ctc 480
Phe Phe Gly Leu Leu Met Val Asn Ile Gln Gln Leu Arg Thr Lys Leu
145 150 155 160
aaa ata ttg gac aac act tat gga aat gga act gcc aat aca gca ctc 528
Lys Ile Leu Asp Asn Thr Tyr Gly Asn Gly Thr Ala Asn Thr Ala Leu
165 170 175
gta tat cac cat gga aaa ctt cta gca tta cag gag gca gat aag ccg 576
Val Tyr His His Gly Lys Leu Leu Ala Leu Gln Glu Ala Asp Lys Pro
180 185 190
tac gtc atc aaa gtt ttg gaa gat gga gac ctg caa act ctt ggt ata 624
Tyr Val Ile Lys Val Leu Glu Asp Gly Asp Leu Gln Thr Leu Gly Ile
195 200 205
ata gat tat gac aag aga ttg acc cac tcc ttc act gct cac cca aaa 672
Ile Asp Tyr Asp Lys Arg Leu Thr His Ser Phe Thr Ala His Pro Lys
210 215 220
gtt gac ccg gtt acg ggt gaa atg ttt aca ttc ggc tat tcg cat acg 720
Val Asp Pro Val Thr Gly Glu Met Phe Thr Phe Gly Tyr Ser His Thr
225 230 235 240
cca cct tat ctc aca tac aga gtt atc tcg aaa gat ggc att atg cat 768
Pro Pro Tyr Leu Thr Tyr Arg Val Ile Ser Lys Asp Gly Ile Met His
245 250 255
gac cca gtc cca att act ata tca gag cct atc atg atg cat gat ttt 816
Asp Pro Val Pro Ile Thr Ile Ser Glu Pro Ile Met Met His Asp Phe
260 265 270
gct att act gag act tat gca atc ttc atg gat ctt cct atg cac ttc 864
Ala Ile Thr Glu Thr Tyr Ala Ile Phe Met Asp Leu Pro Met His Phe
275 280 285
agg cca aag gaa atg gtg aaa gag aag aaa atg ata tac tca ttt gat 912
Arg Pro Lys Glu Met Val Lys Glu Lys Lys Met Ile Tyr Ser Phe Asp
290 295 300
ccc aca aaa aag gct cgt ttt ggt gtt ctt ccg cgc tat gcc aag gat 960
Pro Thr Lys Lys Ala Arg Phe Gly Val Leu Pro Arg Tyr Ala Lys Asp
305 310 315 320
gaa ctt atg att aga tgg ttt gag ctt ccc aac tgc ttt att ttc cac 1008
Glu Leu Met Ile Arg Trp Phe Glu Leu Pro Asn Cys Phe Ile Phe His
325 330 335
aac gcc aat gct tgg gaa gaa gag gat gaa gtc gtc ctc atc act tgt 1056
Asn Ala Asn Ala Trp Glu Glu Glu Asp Glu Val Val Leu Ile Thr Cys
340 345 350
cgt ctt gag aat cca gat ctt gac atg gtc agt ggg aaa gtg aaa gaa 1104
Arg Leu Glu Asn Pro Asp Leu Asp Met Val Ser Gly Lys Val Lys Glu
355 360 365
aaa ctc gaa aat ttt ggc aac gaa ctg tac gaa atg aga ttc aac atg 1152
Lys Leu Glu Asn Phe Gly Asn Glu Leu Tyr Glu Met Arg Phe Asn Met
370 375 380
aaa acg ggc tca gct tct caa aaa aaa cta tcc gca tct gcg gtt gat 1200
Lys Thr Gly Ser Ala Ser Gln Lys Lys Leu Ser Ala Ser Ala Val Asp
385 390 395 400
ttc ccc aga atc aat gag tgc tac acc gga aag aaa cag aga tac gta 1248
Phe Pro Arg Ile Asn Glu Cys Tyr Thr Gly Lys Lys Gln Arg Tyr Val
405 410 415
tat gga aca att ctg gac agt atc gca aag gtt acc gga atc atc aag 1296
Tyr Gly Thr Ile Leu Asp Ser Ile Ala Lys Val Thr Gly Ile Ile Lys
420 425 430
ttt gat ctg cat gca gaa gct gag aca ggg aaa aga atg ctg gaa gta 1344
Phe Asp Leu His Ala Glu Ala Glu Thr Gly Lys Arg Met Leu Glu Val
435 440 445
gga ggt aat atc aaa gga ata tat gac ctg gga gaa ggc aga tat ggt 1392
Gly Gly Asn Ile Lys Gly Ile Tyr Asp Leu Gly Glu Gly Arg Tyr Gly
450 455 460
tca gag gct atc tat gtt ccg cgt gag aca gca gaa gaa gac gac ggt 1440
Ser Glu Ala Ile Tyr Val Pro Arg Glu Thr Ala Glu Glu Asp Asp Gly
465 470 475 480
tac ttg ata ttc ttt gtt cat gat gaa aac aca ggg aaa tca tgc gtg 1488
Tyr Leu Ile Phe Phe Val His Asp Glu Asn Thr Gly Lys Ser Cys Val
485 490 495
act gtg ata gac gca aaa aca atg tcg gct gaa ccg gtg gca gtg gtg 1536
Thr Val Ile Asp Ala Lys Thr Met Ser Ala Glu Pro Val Ala Val Val
500 505 510
gag ctg ccg cac agg gtc cca tat ggc ttc cat gcc ttg ttt gtt aca 1584
Glu Leu Pro His Arg Val Pro Tyr Gly Phe His Ala Leu Phe Val Thr
515 520 525
gag gaa caa ctc cag gaa caa act ctt ata taa 1617
Glu Glu Gln Leu Gln Glu Gln Thr Leu Ile
530 535
<210>18
<211>538
<212>PRT
<213>拟南芥
<400>18
Met Ala Glu Lys Leu Ser Asp Gly Ser Ile Ile Ile Ser Val His Pro
1 5 10 15
Arg Pro Ser Lys Gly Phe Ser Ser Lys Leu Leu Asp Leu Leu Glu Arg
20 25 30
Leu Val Val Lys Leu Met His Asp Ala Ser Leu Pro Leu His Tyr Leu
35 40 45
Ser Gly Asn Phe Ala Pro Ile Arg Asp Glu Thr Pro Pro Val Lys Asp
50 55 60
Leu Pro Val His Gly Phe Leu Pro Glu Cys Leu Asn Gly Glu Phe Val
65 70 75 80
Arg Val Gly Pro Asn Pro Lys Phe Asp Ala Val Ala Gly Tyr His Trp
85 90 95
Phe Asp Gly Asp Gly Met Ile His Gly Val Arg Ile Lys Asp Gly Lys
100 105 110
Ala Thr Tyr Val Ser Arg Tyr Val Lys Thr Ser Arg Leu Lys Gln Glu
115 120 125
Glu Phe Phe Gly Ala Ala Lys Phe Met Lys Ile Gly Asp Leu Lys Gly
130 135 140
Phe Phe Gly Leu Leu Met Val Asn Ile Gln Gln Leu Arg Thr Lys Leu
145 150 155 160
Lys Ile Leu Asp Asn Thr Tyr Gly Asn Gly Thr Ala Asn Thr Ala Leu
165 170 175
Val Tyr His His Gly Lys Leu Leu Ala Leu Gln Glu Ala Asp Lys Pro
180 185 190
Tyr Val Ile Lys Val Leu Glu Asp Gly Asp Leu Gln Thr Leu Gly Ile
195 200 205
Ile Asp Tyr Asp Lys Arg Leu Thr His Ser Phe Thr Ala His Pro Lys
210 215 220
Val Asp Pro Val Thr Gly Glu Met Phe Thr Phe Gly Tyr Ser His Thr
225 230 235 240
Pro Pro Tyr Leu Thr Tyr Arg Val Ile Ser Lys Asp Gly Ile Met His
245 250 255
Asp Pro Val Pro Ile Thr Ile Ser Glu Pro Ile Met Met His Asp Phe
260 265 270
Ala Ile Thr Glu Thr Tyr Ala Ile Phe Met Asp Leu Pro Met His Phe
275 280 285
Arg Pro Lys Glu Met Val Lys Glu Lys Lys Met Ile Tyr Ser Phe Asp
290 295 300
Pro Thr Lys Lys Ala Arg Phe Gly Val Leu Pro Arg Tyr Ala Lys Asp
305 310 315 320
Glu Leu Met Ile Arg Trp Phe Glu Leu Pro Asn Cys Phe Ile Phe His
325 330 335
Asn Ala Asn Ala Trp Glu Glu Glu Asp Glu Val Val Leu Ile Thr Cys
340 345 350
Arg Leu Glu Asn Pro Asp Leu Asp Met Val Ser Gly Lys Val Lys Glu
355 360 365
Lys Leu Glu Asn Phe Gly Asn Glu Leu Tyr Glu Met Arg Phe Asn Met
370 375 380
Lys Thr Gly Ser Ala Ser Gln Lys Lys Leu Ser Ala Ser Ala Val Asp
385 390 395 400
Phe Pro Arg Ile Asn Glu Cys Tyr Thr Gly Lys Lys Gln Arg Tyr Val
405 410 415
Tyr Gly Thr Ile Leu Asp Ser Ile Ala Lys Val Thr Gly Ile Ile Lys
420 425 430
Phe Asp Leu His Ala Glu Ala Glu Thr Gly Lys Arg Met Leu Glu Val
435 440 445
Gly Gly Asn Ile Lys Gly Ile Tyr Asp Leu Gly Glu Gly Arg Tyr Gly
450 455 460
Ser Glu Ala Ile Tyr Val Pro Arg Glu Thr Ala Glu Glu Asp Asp Gly
465 470 475 480
Tyr Leu Ile Phe Phe Val His Asp Glu Asn Thr Gly Lys Ser Cys Val
485 490 495
Thr Val Ile Asp Ala Lys Thr Met Ser Ala Glu Pro Val Ala Val Val
500 505 510
Glu Leu Pro His Arg Val Pro Tyr Gly Phe His Ala Leu Phe Val Thr
515 520 525
Glu Glu Gln Leu Gln Glu Gln Thr Leu Ile
530 535
<210>19
<211>27
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>19
attgaattca tgccttcagc ttcaaac 27
<210>20
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>20
attggatccc aaaagctaca cgctggtccc c 31
<210>21
<211>38
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>21
atatatctag aatgccttca tcagcttcaa acacttgg 38
<210>22
<211>36
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>22
atataggatc cctccggcac cggcgcgaag ttcccg 36
<210>23
<211>29
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>23
cccgggatcc ctcaagcctc tctataccg 29
<210>24
<211>31
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>24
cccgggatcc tttatacgga ttctgaggga g 31
<210>25
<211>33
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>25
attgaattca tggactctgt ttcttcttct tcc 33
<210>26
<211>34
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>26
attgaattct taaagcttat taaggtcact ttcc 34
<210>27
<211>35
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>27
aagaattcat ggcggagaaa ctcagtgatg gcagc 35
<210>28
<211>35
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>28
aaaagaattc ggcttatata agagtttgtt cctgg 35
<210>29
<211>39
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>29
cgggatccat gcaacactct cttcgttctg atcttcttc 39
<210>30
<211>40
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>30
cgggatcctc agaaaacttg ttccttcaac tgattctcgc 40
<210>31
<211>32
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>31
attgaattca tggcttcttt cacggcaacg gc 32
<210>32
<211>17
<212>DNA
<213>人工序列
<220>
<223>人工序列的描述：人工合成序列
<400>32
gttttcccag tcacgac 17
Claims (7)
1.一种编码选自下述蛋白质的DNA：
(a)具有SEQ ID NO：6所示氨基酸序列并且还具有新黄素裂解活性的蛋白质；
(b)具有新黄素裂解活性并且其氨基酸序列具有SEQ ID NO：6所示氨基酸序列中取代，缺失，添加和/或插入了一个或几个氨基酸的氨基酸序列的蛋白质；和
(c)蛋白质，其(i)由在高度严谨条件下与具有SEQ ID NO：5的核苷酸序列的DNA杂交之DNA编码，其中所述高度严谨条件是：在0.1X SSC，0.1％SDS，60℃洗涤两次，15分钟；(ii)具有新黄素裂解活性。
2.如权利要求1中所述的DNA，其中具有新黄素裂解活性的蛋白源于拟南芥属植物。
3.一种携带有如权利要求1或2中所述的DNA的转化体植物细胞。
4.一种含有权利要求1或2中所述的DNA的载体。
5.一种生产转基因植物的方法，包括将权利要求1至2中的任意一项权利要求所述的DNA引进植物细胞并由该植物细胞再生植物的步骤。
6.一种用于增强或降低植物逆境耐性的方法，包括在植物细胞中表达权利要求1至2中的任意一项权利要求所述的DNA。
7.权利要求1所述的DNA用于改进植物逆境耐性的用途。
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10056/00 | 2000-01-13 | ||
| JP2000010056 | 2000-01-13 | ||
| JP10056/2000 | 2000-01-13 | ||
| JP003476/2001 | 2001-01-11 | ||
| JP2001003476A JP4621361B2 (ja) | 2000-01-13 | 2001-01-11 | ネオザンチン開裂酵素遺伝子を用いるトランスジェニック植物 |
| JP003476/01 | 2001-01-11 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1307137A CN1307137A (zh) | 2001-08-08 |
| CN1294267C true CN1294267C (zh) | 2007-01-10 |
Family
ID=26583754
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011116285A Expired - Fee Related CN1294267C (zh) | 2000-01-13 | 2001-01-13 | 携带新黄素裂解酶基因的转基因植物 |
Country Status (8)
| Country | Link |
|---|---|
| US (2) | US7049487B2 (zh) |
| EP (1) | EP1116794B1 (zh) |
| JP (1) | JP4621361B2 (zh) |
| CN (1) | CN1294267C (zh) |
| AT (1) | ATE434048T1 (zh) |
| AU (1) | AU785477B2 (zh) |
| DE (1) | DE60138972D1 (zh) |
| ES (1) | ES2326789T3 (zh) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2326789T3 (es) * | 2000-01-13 | 2009-10-20 | Riken | Plantas transgenicas con el gen de la enzima para la segmentacion de neoxantina. |
| GB0007291D0 (en) * | 2000-03-24 | 2000-05-17 | Univ York | Control of aerial branching |
| BR0316076A (pt) * | 2002-11-06 | 2005-09-27 | Pioneer Hi Bred Int | Promotor auxin-reprimido, dormência-associado e uso do mesmo |
| BRPI0409326A (pt) * | 2003-04-11 | 2006-04-25 | Cropdesign Nv | método para identificação e obtenção de ácidos nucléicos capazes de modificar a toleráncia ao estresse em plantas |
| CN1973044B (zh) * | 2004-04-23 | 2010-07-07 | 塞雷斯公司 | 用于提高植物耐旱性的方法和材料 |
| US20070259397A1 (en) * | 2004-07-28 | 2007-11-08 | Danisco A/S | Enzymes |
| WO2006098626A2 (en) * | 2005-03-18 | 2006-09-21 | Plant Research International B.V. | Resistance against parasitic weeds |
| CA2647900C (en) * | 2008-12-23 | 2017-05-16 | National Research Council Of Canada | Inhibitors of a 9-cis-epoxycarotenoid dioxygenase |
| CN113215180B (zh) * | 2021-06-08 | 2023-03-28 | 吉林大学 | 玉米9-顺-环氧类胡萝卜素双加氧酶蛋白基因ZmVP14及应用 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996038566A1 (fr) * | 1995-05-31 | 1996-12-05 | Institut National De La Recherche Agronomique | Fragment d'acide nucleique codant pour un enzyme implique dans la voie de biosynthese de l'acide abscissique (aba) chez les plantes |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990008828A2 (en) * | 1989-02-02 | 1990-08-09 | Paladin Hybrids Inc. | Molecular methods of hybrid seed production |
| UA39169C2 (uk) * | 1991-04-16 | 2001-06-15 | Моген Інтернешнл Н.В. | Рекомбінантна молекула днк, яка може бути використана для одержання рослини з чоловічою стерильністю, спосіб одержання рослин із чоловічою стерильністю, спосіб одержання самозаплідненого насіння рослин з чоловічою стерильністю, олігонуклеотид |
| DE19503359C1 (de) * | 1995-02-02 | 1996-02-22 | Kws Kleinwanzlebener Saatzucht | Streßtolerante Pflanzen und Verfahren zu deren Herstellung |
| NZ321080A (en) * | 1995-10-12 | 1999-05-28 | Cornell Res Foundation Inc | Production of water stress or salt stress tolerant transgenic cereal plants |
| AU1775299A (en) * | 1997-12-23 | 1999-07-19 | Ascorbex Limited | Plant galactose dehydrogenase |
| AU6795000A (en) * | 1999-08-18 | 2001-03-13 | Curagen Corporation | Defense-related signaling genes and methods of use |
| ES2326789T3 (es) | 2000-01-13 | 2009-10-20 | Riken | Plantas transgenicas con el gen de la enzima para la segmentacion de neoxantina. |
-
2001
- 2001-01-11 ES ES01300218T patent/ES2326789T3/es not_active Expired - Lifetime
- 2001-01-11 AT AT01300218T patent/ATE434048T1/de not_active IP Right Cessation
- 2001-01-11 JP JP2001003476A patent/JP4621361B2/ja not_active Expired - Fee Related
- 2001-01-11 EP EP01300218A patent/EP1116794B1/en not_active Expired - Lifetime
- 2001-01-11 DE DE60138972T patent/DE60138972D1/de not_active Expired - Lifetime
- 2001-01-12 AU AU13688/01A patent/AU785477B2/en not_active Ceased
- 2001-01-12 US US09/758,269 patent/US7049487B2/en not_active Expired - Lifetime
- 2001-01-13 CN CNB011116285A patent/CN1294267C/zh not_active Expired - Fee Related
-
2006
- 2006-03-22 US US11/385,832 patent/US7482509B2/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996038566A1 (fr) * | 1995-05-31 | 1996-12-05 | Institut National De La Recherche Agronomique | Fragment d'acide nucleique codant pour un enzyme implique dans la voie de biosynthese de l'acide abscissique (aba) chez les plantes |
Also Published As
| Publication number | Publication date |
|---|---|
| US20020104120A1 (en) | 2002-08-01 |
| US7049487B2 (en) | 2006-05-23 |
| EP1116794A3 (en) | 2001-12-19 |
| AU785477B2 (en) | 2007-08-23 |
| EP1116794B1 (en) | 2009-06-17 |
| JP4621361B2 (ja) | 2011-01-26 |
| US7482509B2 (en) | 2009-01-27 |
| JP2001258579A (ja) | 2001-09-25 |
| CN1307137A (zh) | 2001-08-08 |
| EP1116794A2 (en) | 2001-07-18 |
| US20060212969A1 (en) | 2006-09-21 |
| AU1368801A (en) | 2001-07-19 |
| ES2326789T3 (es) | 2009-10-20 |
| ATE434048T1 (de) | 2009-07-15 |
| DE60138972D1 (de) | 2009-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1252273C (zh) | 编码植物脱氧海普赖氨酸合成酶的dna,植物真核起始因子5a，转基因植物和控制植物衰老和程序性细胞死亡的方法 | |
| CN1170940C (zh) | 生长得到改造的植物以及获得此种植物的方法 | |
| CN1151183A (zh) | Rps2基因及其应用 | |
| CN1823168A (zh) | 提高植物非生物胁迫耐受性和/或生物量的方法及用该方法所产生的植物 | |
| CN1496403A (zh) | 植物来源分子、其编码遗传序列及其用途 | |
| CN1285875A (zh) | 突变的羟基苯丙酮酸双氧化酶、基dna序列和含该基因且耐除草剂的植物的分离 | |
| CN1836039A (zh) | 改变了花色的蔷薇的制造方法 | |
| CN1878463A (zh) | 不育性植物体的生产方法以及使用此方法得到的植物体及其利用 | |
| CN101048508A (zh) | 植物发育和形态学的修饰 | |
| CN1694962A (zh) | 植物的胁迫耐受和延缓衰老 | |
| CN101048507A (zh) | 一种增大种子大小的方法 | |
| CN1487997A (zh) | 诱导植物开花的hd3a基因及其应用 | |
| CN1810977A (zh) | 增强植物和真菌中的2－乙酰基－1－吡咯的合成的核酸 | |
| CN1155714C (zh) | 抗真菌蛋白及其编码dna和掺入此dna的宿主 | |
| CN1594571A (zh) | 百草枯抗性基因及维管束和毛状体特异性启动子 | |
| CN1294267C (zh) | 携带新黄素裂解酶基因的转基因植物 | |
| CN1842601A (zh) | 通过靶向抑制内源贮藏蛋白增强植物种子中异源多肽的累积 | |
| CN101037693A (zh) | 新的CkNHX基因及其剪切修饰基因CkNHXn,以及培育耐逆植物的方法 | |
| CN1639337A (zh) | 具有催泪成分合成酶活性的蛋白质或多肽、编码该蛋白质或多肽的 DNA、利用该 DNA制造具有催泪成分合成酶活性的蛋白质或多肽的制造方法以及具有抑制该蛋白质或多肽的mRNA翻译的功能的核酸分子 | |
| CN1646005A (zh) | 具有改进形态发生的植物及其制备方法 | |
| CN1202254C (zh) | 水稻抗白叶枯病基因Xa26（t） | |
| CN101037696A (zh) | 一种水稻冷害基因及应用 | |
| CN1246464C (zh) | 可增强植物对渗透压的抵抗力的新转录因子 | |
| CN1195063C (zh) | 蛋白酶抑制剂融合蛋白 | |
| CN101033472A (zh) | 产生聚谷氨酸的转基因植物 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070110 Termination date: 20150113 |
|
| EXPY | Termination of patent right or utility model |