CN101161675B - Rice big grain gene and uses thereof - Google Patents
Rice big grain gene and uses thereof Download PDFInfo
- Publication number
- CN101161675B CN101161675B CN2006101171135A CN200610117113A CN101161675B CN 101161675 B CN101161675 B CN 101161675B CN 2006101171135 A CN2006101171135 A CN 2006101171135A CN 200610117113 A CN200610117113 A CN 200610117113A CN 101161675 B CN101161675 B CN 101161675B
- Authority
- CN
- China
- Prior art keywords
- polynucleotide
- gene
- albumen
- grain
- seq
- 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
- 235000013339 cereals Nutrition 0.000 title claims abstract description 95
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 68
- 235000009566 rice Nutrition 0.000 title claims abstract description 66
- 108090000623 proteins and genes Proteins 0.000 title claims description 79
- 240000007594 Oryza sativa Species 0.000 title claims description 66
- 238000000034 method Methods 0.000 claims abstract description 70
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 64
- 229920001184 polypeptide Polymers 0.000 claims description 61
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 61
- 210000004027 cell Anatomy 0.000 claims description 55
- 108091033319 polynucleotide Proteins 0.000 claims description 52
- 102000040430 polynucleotide Human genes 0.000 claims description 52
- 239000002157 polynucleotide Substances 0.000 claims description 52
- 241000196324 Embryophyta Species 0.000 claims description 45
- 239000008187 granular material Substances 0.000 claims description 35
- 235000018102 proteins Nutrition 0.000 claims description 18
- 102000004169 proteins and genes Human genes 0.000 claims description 18
- 235000013311 vegetables Nutrition 0.000 claims description 17
- 239000013604 expression vector Substances 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 15
- 125000003729 nucleotide group Chemical group 0.000 claims description 15
- 230000009261 transgenic effect Effects 0.000 claims description 14
- 241000589158 Agrobacterium Species 0.000 claims description 13
- 239000003550 marker Substances 0.000 claims description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 11
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 claims description 9
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 claims description 9
- 230000014509 gene expression Effects 0.000 claims description 9
- 125000000539 amino acid group Chemical group 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 7
- 230000008676 import Effects 0.000 claims description 7
- 210000000056 organ Anatomy 0.000 claims description 5
- 238000013459 approach Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 230000017854 proteolysis Effects 0.000 claims description 4
- 230000009467 reduction Effects 0.000 claims description 4
- 230000008929 regeneration Effects 0.000 claims description 4
- 238000011069 regeneration method Methods 0.000 claims description 4
- 210000004899 c-terminal region Anatomy 0.000 claims description 3
- 210000001519 tissue Anatomy 0.000 claims description 3
- 108010010256 Dietary Proteins Proteins 0.000 claims description 2
- 102000015781 Dietary Proteins Human genes 0.000 claims description 2
- 108090000848 Ubiquitin Proteins 0.000 claims description 2
- 102000044159 Ubiquitin Human genes 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 claims description 2
- 235000021245 dietary protein Nutrition 0.000 claims description 2
- 101150102138 GW2 gene Proteins 0.000 abstract description 116
- 238000009395 breeding Methods 0.000 abstract description 7
- 230000001488 breeding effect Effects 0.000 abstract description 7
- 241000209094 Oryza Species 0.000 abstract description 4
- 239000003147 molecular marker Substances 0.000 abstract description 4
- 230000032823 cell division Effects 0.000 abstract 1
- 239000012634 fragment Substances 0.000 description 29
- 235000001014 amino acid Nutrition 0.000 description 24
- 150000001413 amino acids Chemical class 0.000 description 24
- 108020004414 DNA Proteins 0.000 description 23
- 238000005516 engineering process Methods 0.000 description 20
- 230000006870 function Effects 0.000 description 20
- 108700026244 Open Reading Frames Proteins 0.000 description 13
- 230000008034 disappearance Effects 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- 230000000692 anti-sense effect Effects 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 108700019146 Transgenes Proteins 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 108091008146 restriction endonucleases Proteins 0.000 description 7
- 230000009466 transformation Effects 0.000 description 7
- 239000000370 acceptor Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 230000009465 prokaryotic expression Effects 0.000 description 6
- 230000008521 reorganization Effects 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 108091026890 Coding region Proteins 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 108020005038 Terminator Codon Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 150000007523 nucleic acids Chemical group 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108020004485 Nonsense Codon Proteins 0.000 description 3
- 241000276498 Pollachius virens Species 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 239000000556 agonist Substances 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 230000004992 fission Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003259 recombinant expression Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- -1 Polyoxyethylene Polymers 0.000 description 2
- 101100066315 Trypanosoma brucei brucei (strain 927/4 GUTat10.1) FAZ1 gene Proteins 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 101150052418 gw gene Proteins 0.000 description 2
- 210000004408 hybridoma Anatomy 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000002493 microarray Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 230000004853 protein function Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 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
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- RDIKFPRVLJLMER-BQBZGAKWSA-N Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)N RDIKFPRVLJLMER-BQBZGAKWSA-N 0.000 description 1
- PTVGLOCPAVYPFG-CIUDSAMLSA-N Arg-Gln-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O PTVGLOCPAVYPFG-CIUDSAMLSA-N 0.000 description 1
- PTNFNTOBUDWHNZ-GUBZILKMSA-N Asn-Arg-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O PTNFNTOBUDWHNZ-GUBZILKMSA-N 0.000 description 1
- LJUOLNXOWSWGKF-ACZMJKKPSA-N Asn-Asn-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N LJUOLNXOWSWGKF-ACZMJKKPSA-N 0.000 description 1
- KHCNTVRVAYCPQE-CIUDSAMLSA-N Asn-Lys-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O KHCNTVRVAYCPQE-CIUDSAMLSA-N 0.000 description 1
- FANQWNCPNFEPGZ-WHFBIAKZSA-N Asp-Asp-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O FANQWNCPNFEPGZ-WHFBIAKZSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical class 0.000 description 1
- 238000000018 DNA microarray 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
- 102100037840 Dehydrogenase/reductase SDR family member 2, mitochondrial Human genes 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- NUSWUSKZRCGFEX-FXQIFTODSA-N Glu-Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O NUSWUSKZRCGFEX-FXQIFTODSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- IOVUXUSIGXCREV-DKIMLUQUSA-N Ile-Leu-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 IOVUXUSIGXCREV-DKIMLUQUSA-N 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 101150005851 NOS gene Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- WEMYTDDMDBLPMI-DKIMLUQUSA-N Phe-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N WEMYTDDMDBLPMI-DKIMLUQUSA-N 0.000 description 1
- YTILBRIUASDGBL-BZSNNMDCSA-N Phe-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 YTILBRIUASDGBL-BZSNNMDCSA-N 0.000 description 1
- KIQUCMUULDXTAZ-HJOGWXRNSA-N Phe-Tyr-Tyr Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O KIQUCMUULDXTAZ-HJOGWXRNSA-N 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710188053 Protein D Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 101710132893 Resolvase Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- FZXOPYUEQGDGMS-ACZMJKKPSA-N Ser-Ser-Gln Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O FZXOPYUEQGDGMS-ACZMJKKPSA-N 0.000 description 1
- DKGRNFUXVTYRAS-UBHSHLNASA-N Ser-Ser-Trp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O DKGRNFUXVTYRAS-UBHSHLNASA-N 0.000 description 1
- 244000046109 Sorghum vulgare var. nervosum Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 108700007696 Tetrahydrofolate Dehydrogenase Proteins 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- ARJASMXQBRNAGI-YESZJQIVSA-N Tyr-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N ARJASMXQBRNAGI-YESZJQIVSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000005314 correlation function Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000003208 gene overexpression Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 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
- 239000008101 lactose Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000010297 mechanical methods and process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- DCWXELXMIBXGTH-QMMMGPOBSA-N phosphonotyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-QMMMGPOBSA-N 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- BZQFBWGGLXLEPQ-REOHCLBHSA-N phosphoserine Chemical compound OC(=O)[C@@H](N)COP(O)(O)=O BZQFBWGGLXLEPQ-REOHCLBHSA-N 0.000 description 1
- USRGIUJOYOXOQJ-GBXIJSLDSA-N phosphothreonine Chemical compound OP(=O)(O)O[C@H](C)[C@H](N)C(O)=O USRGIUJOYOXOQJ-GBXIJSLDSA-N 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000004960 subcellular localization Effects 0.000 description 1
- 239000002512 suppressor factor Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012256 transgenic experiment Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 101150085703 vir gene Proteins 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明公开了一种水稻大粒基因GW2及其应用，GW2基因可用于控制作物籽粒的大小、提高作物的产量或品质、调节细胞分裂的速度、以及作为鉴定作物大粒品种和小粒品种的分子标记。本发明还公开了一种改良作物的方法。GW2基因在水稻等作物高产育种中具有广泛的应用前景。The invention discloses a rice large-grain gene GW2 and its application. The GW2 gene can be used to control the size of crop grains, improve the yield or quality of crops, regulate the speed of cell division, and be used as a molecular marker for identifying large-grain varieties and small-grain varieties. The invention also discloses a method for improving crops. The GW2 gene has broad application prospects in high-yield breeding of rice and other crops.
Description
Technical field
The invention belongs to gene engineering and phytology field.More particularly, the present invention relates to a kind of new rice big grain gene and application thereof.
Background technology
Rice yield is heavy by grain, spike number, grain several three big proterties determine.These proterties are the quantitative characters by a plurality of quantitative character genes (QTL) control.The applied molecular labeling technique has been located the QTL of many output correlated character over nearly 10 years, but by successful cloned genes also seldom.
Japanese scientists in 2005 have successfully cloned the gene (Gnla) of 1 control paddy rice grain number, and have illustrated the molecule mechanism of this gene, and relevant achievement is published on the Science, causes international extensive concern.In addition, be positioned a trisomic report that control grain length gene GS3 is cloned, it does not carried out the correlation function analysis though have recently.
Yet the clone and the functional study of the genes involved that the control grain is heavy also do not obtain real success so far.Grain heavily is one of most important proterties of decision output, carries out the clone and the functional study of a heavy gene, so that be applied to the high crop yield molecular breeding, has great theory significance and practical value.
Summary of the invention
The object of the present invention is to provide a kind of new can be used for to control the rice big grain gene and the application thereof of crop kernel size.
In first aspect of the present invention, a kind of isolating rice big grain albumen is provided, this albumen is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence; Or
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the wide or heavy function of grain of control crop grain by (a) polypeptides derived.
In another preference of the present invention, described dietary protein origin is in paddy rice.
In another preference of the present invention, it is active that described albumen has the E3 ligase enzyme.
In second aspect of the present invention, a kind of isolating polynucleotide are provided, these polynucleotide are selected from down group:
(i) the proteic polynucleotide of the described rice big grain of coding; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
In another preference of the present invention, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
In another preference of the present invention, these polynucleotide are selected from down group:
(1) nucleotide sequence shown in the SEQ ID NO:1; Or
(2) nucleotide sequence shown in the 255-1532 position among the SEQ ID NO:1.
In the third aspect of the invention, a kind of carrier is provided, it contains described polynucleotide.
In fourth aspect of the present invention, a kind of genetically engineered host cell is provided, it contains described carrier; Or be integrated with described polynucleotide in its genome.
Aspect the of the present invention the 5th, the purposes of described rice big grain albumen or its encoding sox is provided, be used for: the grain of control crop kernel is wide or grain is heavy (preferred, wide or grain is heavy for the grain that increases crop kernel, thereby increase crop yield); Regulate cell processes; Or as the molecule marker of identifying crop big grain kind and granule kind.
In another preference of the present invention, described cell processes includes but not limited to: cell fission, signal conduction, cell elongation.
Aspect the of the present invention the 6th, the method for a kind of improvement crop (preferred, wide or grain is heavy for the grain that increases crop kernel) is provided, this method comprises: (A) reduce the expression of rice big grain gene in the said crop; Or (B) function is reduced or the rice big grain gene or the albumen of afunction import in the crop.
In another preference of the present invention, function is reduced or the rice big grain gene or the albumen of afunction import in the crop of granule kind, thereby can promote crop kernel to become big.Preferred, in the rice big grain gene or albumen importing crop with afunction.
In another preference of the present invention, the GW2 gene fragment of selecting technology from the crop of big grain kind, to obtain with molecule marker imports in the crop of granule kind.This method is a non-transgenic method, does not have potential safety hazard.
Aspect the of the present invention the 7th, the agonist or the antagonist of a kind of described rice big grain albumen or its encoding sox is provided.
In eight aspect of the present invention, provide a kind of can with described rice big grain gene or its coded protein specific bonded antibody.
Aspect the of the present invention the 9th, a kind of method for preparing transgenic plant is provided, described method comprises step: described polynucleotide are imported in the vegetable cell, cultivate described vegetable cell, the regeneration plant.
In another preference of the present invention, said method comprises step:
(s1) Agrobacterium of carrying expression vector is provided, described expression vector contains described proteic encoding sox;
(s2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (s1), thereby make said proteic encoding sox change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell or tissue or the organ that changes described protein coding gene over to; And
(s4) vegetable cell in the step (S3) or tissue or neomorph are become plant.
Aspect the of the present invention the tenth, provide a kind of preparation described proteic method, said method comprises: cultivate described host cell, collect and obtain described albumen.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown paddy rice GW2 antisense transgene plant (AS3, AS24, the comparison of the grain of grain AS25) and acceptor kind " in spend 11 (ZH11) ".
Fig. 2 has shown paddy rice GW2 justice transfer-gen plant (S21, S22, the comparison of the grain of grain S24) and acceptor kind " in spend 11 (ZH11) ".
Fig. 3 has shown will the influence heavy to granule kind grain of big grain GW2 gene fragment importing granule kind with method for selecting molecular marker (non-transgenic method).
Embodiment
The inventor has at first found a kind of new gene that the crop kernel grain is wide and/or grain is heavy of controlling through extensive and deep research, and the disappearance of the function of this gene can produce the phenotype of big grain, inventor general's called after rice big grain gene (GW2).Test confirms; The grain type of the transfer-gen plant of GW2 gene overexpression diminishes, grain heavily reduces; The grain type that the GW2 gene reduces the transfer-gen plant of expressing becomes greatly, grain is brought up again height, and visible GW2 gene will play an important role in the higher yield of crops breeding, be with a wide range of applications.Accomplished the present invention on this basis.
As used herein, described " crop " includes but not limited to: grass.Preferred, described grass includes but not limited to: wheat, barley, corn, Chinese sorghum etc.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating GW2 albumen or polypeptide " is meant that described GW2 albumen does not contain natural relative other albumen, lipid, carbohydrate or other material basically.Those skilled in the art can use the purified technology of protein purifying GW2 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises the proteic fragment of GW2, verivate and analogue.As used herein, term " fragment ", " verivate " are meant biological function or the active polypeptide that keeps natural GW2 albumen of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, verivate or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue); And so substituted amino-acid residue can be also can not encoded by genetic code; Or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical; Or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period; Polyoxyethylene glycol for example) merges formed polypeptide; Or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (like leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying or fusion rotein).These fragments of definition, verivate and analogue according to this paper belong to the known scope of those skilled in the art.
In the present invention, term " GW2 albumen " refers to have the SEQ ID NO:2 polypeptide of sequence of GW2 protein-active.This term also comprises having and variant form GW2 albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50; Preferably 1-30, more preferably 1-20,1-10 best; Also better for 1-8,1-5) amino acid whose disappearance, insertion and/or replacement; And at C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of GW2 and reactive derivative.
The variant form of polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with coded albumen of the DNA of GW2 protein D NA hybridization and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of anti-GW2 to obtain.The present invention also provides other polypeptide, as comprises GW2 albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of GW2.Usually, this fragment have the GW2 protein sequence at least about 20 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of GW2 albumen or polypeptide.These analogues and the proteic difference of natural GW2 can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps have both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain through various technology, as through radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to above-mentioned representational polypeptide of giving an example.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modify and also comprise glycosylation.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the polypeptide that has improved its anti-proteolyze performance or optimized solubility property.
In the present invention; " GW2 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8; More preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.For example, these conservative propertys variation polypeptide can carry out the amino acid replacement according to table 1 and produce.
Table 1
| Amino-acid residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The present invention also provides the polynucleotide sequence of code book invention GW2 albumen or its conservative property variation polypeptide.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the verivate of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it possibly be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide according to the invention.In the present invention, " stringent condition " is meant: (1) than hybridization under LIS and the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, like 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (like PCR) of nucleic acid to confirm and/or the proteic polynucleotide of separation coding GW2.
Should understand; Though GW2 gene of the present invention preferably derives from paddy rice; But that derive from other plant and other gene paddy rice GW2 gene height homology (as have more than 80%, like 85%, 90%, 95% even 98% sequence homogeny) are also within the scope that the present invention considers.The Method and kit for of aligned sequences homogeny also is that this area is known, for example BLAST.
GW2 pyrenoids thuja acid full length sequence of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.For the pcr amplification method; Can be disclosed according to the present invention about nucleotide sequence; Especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need carries out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, through first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) through chemosynthesis.Can this dna sequence dna be introduced in various existing dna moleculars as known in the art (or like carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention through chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or GW2 albumen coded sequence, and produce the method for polypeptide according to the invention through recombinant technology.
Recombinant DNA technology (Science, 1984 through routine; 224:1431), polymerized nucleoside acid sequence of the present invention capable of using can be used to express or produce the GW2 albumen of reorganization.In general following steps are arranged:
(1). with the proteic polynucleotide of coding GW2 of the present invention (or varient), or with recombinant expression vector conversion that contains these polynucleotide or transduction proper host cell;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, the GW2 egg can be inserted in the recombinant expression vector from polynucleotide sequence.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carriers.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains GW2 encoding histone dna sequence dna and suitable transcribing/the translate expression vector of wave.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness like eukaryotic cell, or be used for colibacillary kantlex or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell such as yeast; Vegetable cell etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Transform plant and also can use methods such as Agrobacterium-mediated Transformation or particle gun conversion, for example leaf dish method, paddy rice rataria conversion method etc.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain genetically modified plant.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (like temperature transition or chemically induced), cell is cultivated for some time again.
The extracellular can expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
The GW2 albumen or the polypeptide of reorganization are of use in many ways.For example be used to screen antibody, polypeptide or other part that promotes or resist the GW2 protein function.Can be used for seeking the valuable peptide molecule that can suppress or stimulate the GW2 protein function with the reorganization GW2 protein screening peptide library of expressing, proteic antagonist of the GW2 that is obtained or agonist are also included within the scope of the present invention.
On the other hand, the present invention also comprises GW2DNA or the polypeptide of its segment encoding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into GW2 gene product or fragment.Preferably, refer to that those can combine with GW2 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Preferable, antibody is those molecules that can combine and suppress the GW2 function among the present invention.
Antibody of the present invention can prepare through the known various technology of those skilled in that art.For example, the GW2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing GW2 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology system each (see people such as Kohler,
Nature256; 495,1975; People such as Kohler,
Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Each antibody-like of the present invention can utilize the fragment or the functional zone of GW2 gene product, obtains through the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can use the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) to come immune animal and produce with the unmodified form bonded antibody of GW2 gene product; With posttranslational modification form bonded antibody (like the albumen or the polypeptide of glycosylation or phosphorylation), can use the gene product that produces in the eukaryotic cell (for example yeast or insect cell) to come immune animal and obtain.
Antibody among the present invention can be used for suppressing the function of GW2 in the crop.
Utilize albumen of the present invention,, can filter out with GW2 albumen or gene interactional material takes place, like acceptor, suppressor factor, agonist or antagonist etc. through various conventional screening methods.
Polynucleotide of the present invention a part or all can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), be used for analyzing the differential expression analysis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of GW2 albumen and also can detect the proteic transcription product of GW2.
The invention still further relates to a kind of crop kernel that makes and become big method, this method comprises GW2 gene or its homogenic expression in the said plant of reduction.The method that reduces the expression of GW2 gene or its homologous gene is that this area is known, such as waiting the expression that neutralizes or stop GW2 through adopting the proteic antagonist of GW2, antibody.Perhaps, can realize that the seed of crop becomes big through in plant, importing the GW2 gene fragment of forfeiture GW2 gene function.
GW2 gene of the present invention also can be used as the cue mark of true hybrid in gene transformation plant offspring's tracking mark and the hybrid seeding process.
In addition, the invention still further relates to the application of molecule marker selection technology in the big grain of farm crop SOYBEAN IN HIGH-YIELD BREEDING of GW2 gene.
As an instance of the present invention; The inventor is with WY3 (especially big grain kind) and rich short No. 1 (granule kind) hybridization structure genetic group that accounts for; The applied molecular labeling technique has been located one and has been positioned at the wide new gene GW2 heavy with grain of the second chromosomal control paddy rice grain, and has cloned this gene through the map based cloning technology.The genome length of GW2 gene is 5.877kb, includes 8 exons, 7 introns; Its cDNA length is 1.634Kb; Total length ORF (Open Reading-Frame) length is 1.278Kb, 1 albumen that contains 425 bases of encoding, and the protein product molecular weight is estimated as 47KDa.
In an instance of the present invention, find to take place on a kind of the 4th exon of GW2 gene the disappearance of 1 base, produce the premature termination codon, cause to lack 310 amino acid, make GW2 gene function disappearance, thereby produce the phenotype of big grain.
As another instance of the present invention; The inventor obtains the mutant gene of a GW2 from rice varieties " spend 11 "; (be ORF the 787th) C → T variation causes occurring in advance terminator codon because the 1041st of the base of this gene, causes its encoded protein disappearance partial amino-acid series.Yet this plant still shows the granule phenotype, although prove deletion mutation has taken place, yet has still kept the proteic function of GW2.
As another instance of the present invention; The inventor obtains the mutant gene of a GW2 from rice varieties " Huang Huazhan "; C → T variation has also taken place in the 1041st of the base of this gene (be ORF the 787th); Occur terminator codon in advance, cause its encoded protein disappearance partial amino-acid series.Yet this plant still shows the granule phenotype, has also promptly kept the proteic function of GW2.
GW2 albumen contains a RING domain, and in an instance of the present invention, the inventor shows that through protease activity functional analysis result the E3 ligase enzyme that GW2 has in the proteolytic degradation approach is active.The proteolytic degradation approach is participated in cell processes such as cell fission, so the size of seed maybe to have E3 ligase enzyme activity relevant with GW2.
In an instance of the present invention, the inventor places the full-length cDNA of the GW2 gene of granule rice varieties under the control of CaMV35S promotor by justice and antisense orientation respectively, is building up to respectively on the paddy rice expression vector.Through agrobacterium-mediated transformation 2 kinds of expression vectors are imported in the rice varieties and to spend in 11.Positive transformed plant is carried out the phenotype observation to be shown; Antisense GW2 obviously increases owing to this expression of gene of reduction causes the grain type of transfer-gen plant, grain is heavy obviously increases weight (Fig. 1), and just overexpression GW2 then causes that owing to increasing this expression of gene the grain type of transfer-gen plant diminishes, grain heavily reduces (Fig. 2).Show that the GW2 gene plays an important role, and can significantly improve rice yield through this gene of molecular designing in control paddy rice grain is heavy.
In an instance of the present invention, the inventor selects GW2 gene fragment that technology will big grain kind to import to improve grain in the granule kind significantly heavy through molecule marker, and thousand seed weight is increased to 32 grams by 22 original grams, and amplification is up to 46% (Fig. 3).This technology can be exempted the transgenic safety evaluation owing to be non-transgenic method, does not have potential safety hazard.Show that the GW2 gene is with a wide range of applications in high-yield breeding of crops such as paddy rice.
Major advantage of the present invention is:
(1) separation obtains a kind of new rice big grain gene first, reduces this expression of gene and can make the seed of crop (like paddy rice) become big, thereby can increase the output of crop.
(2) rice big grain gene GW2 of the present invention can be used as control crop kernel size, and a gene that improves the yield and quality is applied to the improvement of crop varieties.And, can the molecule marker selection technology of GW2 gene be used for the big grain of farm crop SOYBEAN IN HIGH-YIELD BREEDING.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or according to the method for announcing in the following document: Carl W.Di effenbach and Gabriela S.Devksler eds.PCRPrimer:A Laboratory Manual.Cold Spring Harbor Laboratory Press, 1995.Or the condition of advising according to manufacturer.
The acquisition of embodiment 1 rice big grain gene
WY3 is huge grain rice varieties (thousand seed weight reaches 48 grams), and richly shortly accounts for No. 1 (FAZ1) and promote fine quality (thousand seed weight only 22 restrains) for the granule paddy rice.The inventor utilizes molecule marker to locate wide new gene (or QTL) GW2 heavy with grain of control paddy rice grain with WY3 and the rich short hybridization structure genetic group that accounts for No. 1, and this gene is positioned on second karyomit(e).Further utilize the map based cloning technology to clone the GW2 gene, its gene order is shown in SEQ ID NO:1, and the albumen that contains 425 bases of encoding is shown in SEQ ID NO:2.
Sequence comparing analysis shows that there are 3 nucleotide variations in the GW2 gene of big grain kind, and 2 are the base replacement, but do not cause amino acid change; And in addition 1 variation is the disappearance that 1 base (A316, ORF the 316th) takes place on the 4th exon, causes the reading frame displacement; 30 bases thereafter produce the premature termination codon at the back; Cause to lack 310 amino acid, make GW2 gene function disappearance, thereby produce the phenotype of big grain.
The cDNA sequence of paddy rice GW2 gene (from rich short the accounting for No. 1 of granule kind, being normal gene) (SEQ ID NO:1) as follows, wherein, underscore is depicted as ORFs.
ccaccccgagaaagccaaaaaaaagaaaaattgtttttcaaaaaaaaagctcgcctagccctcgcgtcgtcgtccccatcaccccccctcct
ccgctccgagtacgcgtgcgtataccaccacctccatctccaccaccgtatgtatctacggtgaggcggcggcggcggaggaggaggaggag
ggggagtggtgagggtttcatctgcggaggaggagggaggaggagggaggagggtagatctgggaggggg
ATGGGGAACAGGATAGGGGGGA
GGAGGAAGGCGGGGGTGGAGGAGAGGTACACGAGGCCGCAGGGGCTGTACGAGCACAGGGACATCGACCAGAAGAAGCTCCGGAAGCTGATT
CTCGAGGCCAAGCTCGCGCCGTGCTACATGGGCGCCGACGACGCCGCCGCCGCCGCCGACCTCGAGGAGTGCCCCATCTGCTTCCTGTACTA
CCCAAGTCTTAACCGATCAAAGTGTTGCTCAAAAGGGATATGCACCGAGTGCTTTCTCCAAATGAAACCAACTCACACTGCTCAGCCTACAC
AATGTCCATTCTGCAAAACTCCCAGTTATGCTGTGGAGTATCGTGGTGTAAAGACAAAGGAGGAAAGGAGCATAGAACAATTTGAAGAGCAG
AAAGTCATAGAAGCACAAATGAGGATGCGCCAGCAAGCACTTCAAGATGAAGAAGATAAGATGAAAAGAAAACAGAACAGGTGCTCTTCTAG
CAGAACAATCACACCGACCAAAGAAGTGGAGTATAGAGATATTTGCAGCACATCCTTTTCAGTGCCGTCATACCGATGTGCTGAGCAAGAAA
CTGAATGCTGTTCATCGGAACCTTCATGCTCTGCCCAGACTAGCATGCGCCCTTTCCATTCTAGGCATAACCGTGATGATAACATTGACATG
AATATAGAGGATATGATGGTTATGGAAGCGATTTGGCGTTCCATTCAGGAGCAGGGAAGTATAGGGAATCCTGTCTGTGGCAACTTTATGCC
TGTAACTGAGCCATCTCCGCGTGAACGCCAGCCATTCGTTCCAGCTGCTTCTCTAGAAATACCTCATGGTGGTGGATTTTCCTGTGCGGTTG
CGGCAATGGCTGAGCACCAGCCACCCAGTATGGACTTCTCTTACATGGCTGGCAGCAGCGCATTCCCAGTTTTCGACATGTTCCGGCGACCA
TGCAACATTGCTGGTGGAAGCATGTGTAATCTGGAGAGCTCACCGGAGAGCTGGAGCGGGATAGCACCAAGCTGCAGCAGGGAAGTGGTAAG
AGAAGAAGGAGAGTGCTCGGCTGACCACTGGTCGGAGGGTGCAGAGGCCGGAACAAGCTACGCGGGCTCAGACATCGTGGCGGATGCCGGGA
CCATGCCGCAGCTGCCTTTCGCCGAGAACTTCGCCATGGCGCCAAGCCACTTCCGCCCGGAGAGCATCGAAGAACAGATGATGTTTTCCATG
GCTCTTTCTTTAGCAGATGGTCATGGAAGAACACACTCGCAAGGGTTGGCATGGTTGTAGgtagagcactctaattttgacgcctTgctgcc
ctctcccttgcgctgctgttgctgcccttctctcccctgcctcctgcttctgcctcctttttgccaccag
The aminoacid sequence of paddy rice GW2 coded by said gene is (SEQ ID NO:2) as follows, wherein, after the base deletion variation takes place, produces the premature termination codon, cause to lack 310 amino acid (shown in the underscore),
MetGlyAsnArgIleGlyGlyArgArgLysAlaGlyValGluGluArgTyrThrArgProGlnGlyLeuTyrGluHis
ArgAspIleAspGlnLysLysLeuArgLysLeuIleLeuGluAlaLysLeuAlaProCysTyrMetGlyAlaAspAsp
AlaAlaAlaAlaAlaAspLeuGluGluCysProIleCysPheLeuTyrTyrProSerLeuAsnArgSerLysCysCys
SerLysGlyIleCysThrGluCysPheLeuGlnMetLysProThrHisThrAlaGlnProThrGlnCysProPheCys
LysThrProSerTyrAlaValGluTyrArgGly
ValLysThrLysGluGluArgSerIleGluGlnPheGluGluGln
LysValIleGluAlaGlnMetArgMetArgGlnGlnAlaLeuGlnAspGluGluAspLysMetLysArgLysGlnAsn
ArgCysSerSerSerArgThrIleThrProThrLysGluValGluTyrArgAspIleCysSerThrSerPheSerVal
ProSerTyrArgCysAlaGluGlnGluThrGluCysCysSerSerGluProSerCysSerAlaGlnThrSerMetArg
ProPheHisSerArgHisAsnArgAspAspAsnIleAspMetAsnIleGluAspMetMetValMetGluAlaIleTrp
ArgSerIleGlnGluGlnGlySerIleGlyAsnProValCysGlyAsnPheMetProValThrGluProSerProArg
GluArgGlnProPheValProAlaAlaSerLeuGluIleProHisGlyGlyGlyPheSerCysAlaValAlaAlaMet
AlaGluHisGlnProProSerMetAspPheSerTyrMetAlaGlySerSerAlaPheProValPheAspMetPheArg
ArgProCysAsnIleAlaGlyGlySerMetCysAsnLeuGluSerSerProGluSerTrpSerGlyIleAlaProSer
CysSerArgGluValValArgGluGluGlyGluCysSerAlaAspHisTrpSerGluGlyAlaGluAlaGlyThrSer
TyrAlaGlySerAspIleValAlaAspAlaGlyThrMetProGlnLeuProPheAlaGluAsnPheAlaMetAlaPro
SerHisPheArgProGluSerIleGluGluGlnMetMetPheSerMetAlaLeuSerLeuAlaAspGlyHisGlyArg
ThrHisSerGlnGlyLeuAlaTrpLeu
The variant form of embodiment 2 rice big grain genes
The inventor is a research object with rice varieties " in spend 11 "; From " spend 11 " of granule phenotype, obtain the mutant gene of a GW2; (ORF the 787th) C → T variation causes occurring in advance terminator codon because the 1041st of the base of this gene, causes its encoded protein disappearance partial amino-acid series.This plant shows the granule phenotype, although prove deletion mutation has taken place, yet has still kept the proteic function of GW2.
The inventor is a research object with rice varieties " Huang Huazhan "; Obtain the mutant gene of a GW2; C → T variation has also taken place in the 1041st of the base of this gene (ORF the 787th), occurs terminator codon in advance, causes its encoded protein disappearance partial amino-acid series.This plant still shows the granule phenotype, has also promptly kept the proteic function of GW2.
In addition, the inventor has also obtained the 368th of GW2 gene (ORF the 114th) T → C variation from the plant of paddy rice granule phenotype, and this variation does not cause the variation of amino acid (Ile).This plant still shows the granule phenotype, also promptly should make a variation for the not influence of function of GW2 gene.
In addition, the inventor has also obtained the 1370th of GW2 gene (ORF the 1116th) G → A variation from the plant of paddy rice granule phenotype, and this variation does not cause the variation of amino acid (Ala).This plant still shows the granule phenotype, also promptly should make a variation for the not influence of function of GW2 gene.
The molecular marker assisted selection breeding experiment of embodiment 3GW2
In the GW2 gene, design PCR Oligonucleolide primers (SEQ ID NO:3 and SEQ IDNO:4) in the present embodiment; Carry out the DNA of pcr amplification big grain kind and granule kind with the Taq enzyme; Cut amplified production with restriction enzyme EcoR I enzyme; Detect between big grain kind and the granule kind through 1% agarose gel electrophoresis and to have dna polymorphism (difference); The molecular weight of big grain kind is 1.159kb, and the molecular weight of granule kind is 0.88kb, so this primer develops into the molecule marker that specificity is differentiated big grain GW2 gene and granule GW2 gene.In the filial generation colony of big grain kind and granule kind, can apace the individuality that carries big grain gene be picked out, reach the purpose of the big grain of further cultivation high-yield variety with this molecule marker.
5 ' end Oligonucleolide primers sequence is:
5’-TGTTGTTGCGAGAGTAGGG-3’(SEQ?ID?NO:3)；
3 ' end primer sequence is:
5’-TCGGTTAAGACTTGGGTAG-3’(SEQ?ID?NO:4)。
Embodiment 4GW2 paddy rice transgenic experiments
Present embodiment adopt the binary vector pHB derive from plant expression vector pCAMBIA3301 (Mao etc., 2005, PNAS102:12270-12275) as the paddy rice transgene carrier.A bacterium replication orgin of this vector encoded (ori), kalamycin resistance gene (Kan
r), hygromycin gene (Hyg
r), the termination signal sequence of herbicide resistance gene (Bar), CaMV35S promotor, NOS gene and after between the two restriction enzyme cloning site (MCS).The cDNA that can insert GW2 at the restriction enzyme cloning site forward or backwards is built into the transgenic plasmid.
1.GW2 the transgenic plasmid construction of just overexpression
In the present embodiment; With the RNA that derives from the granule kind is template; Synthetic article one chain cDNA; The PCR oligonucleotide of holding with 5 ' and 3 ' of this dna sequence dna is primer (SEQ ID NO:5 and SEQ ID NO:6), increases with high-fidelity Taq enzyme pfu Taq, obtains the full-length cDNA amplified production of 1.634kb.This amplified production through Hind III and the Bam HI restriction enzyme digestion corresponding restriction enzyme cloning site of PMD18-T (available from TAKARA) of recombinating into, and is checked order to a plurality of recons, with the exactness of checking sequence.The transition plasmid vector of this reorganization is called GW2-PMD.
5 ' end Oligonucleolide primers sequence is:
5’-CCACCCCGAGAAAGCCAAAA-3’(SEQ?ID?NO：5)；
3 ' end primer sequence is:
5’-CTGGTGGCAAAAAGGAGG-3’(SEQ?ID?NO：6)。
With Hind III and Bam HI digestion GW2-PMD and carrier pHB, the postdigestive 1.634kb purpose of GW2-PMD fragment is connected among the Hind III and Bam HI restriction enzyme site of carrier pHB.Connector transformed into escherichia coli bacterial strain DH5 α; On the LB substratum that contains Kan (50 μ g/ml), screen transformant; Select single bacterium colony and extract plasmid; Pick out the clone that the 1.6Kb fragment of having an appointment is inserted with Hind III and Bam HI enzymolysis, and whether correct with M13 universal primer order-checking check nucleotide sequence.So successfully make up the pHB-35S-GW2 plasmid.
2.GW2 the transgenic plasmid construction of antisense overexpression
With the RNA that derives from the granule kind is template; Synthetic article one chain cDNA; The PCR oligonucleotide of holding with 5 ' and 3 ' of this dna sequence dna is primer (SEQ ID NO:7 and SEQ ID NO:8); Increase with high-fidelity Taq enzyme pfu Taq, obtain the total length ORF amplified production of 1.278kb, be connected to then among the Hind III and Bam HI restriction enzyme site of carrier pHB.
The evaluation of recon is identical with preceding method, so successfully makes up pHB-35S-GW2 (antisense) plasmid.
5 ' end Oligonucleolide primers sequence is:
5’-ATGGGGAACAGGATAGGGGGGAG-3’(SEQ?ID?NO：7)；
3 ' end primer sequence is:
5’-CTACAACCATGCCAACCCTTGCGAG-3’(SEQ?ID?NO：8)。
3.GW2 rice transformation
Above-mentioned two kinds of recombinant plasmids import agrobacterium strains EHA105 through freeze-thaw method.Per 200 μ l EHA105 competent cells and 0.5-1 μ g (about 10 μ l) DNA mixing, successively on ice, in the liquid nitrogen and respectively placed 5 minutes in 37 ℃ of water-baths; YEB liquid nutrient medium with fresh is diluted to 1ml, cultivates 2-4 hour in 28 ℃ of joltings; Get 200 μ l and coat on the YEB flat board that contains microbiotic Kan (50 μ g/ml), cultivated 2-3 days for 28 ℃.The bacterium colony that grows is drawn 3 times containing stroke single bacterium on the antibiotic YEB flat board continuously.The single colony inoculation of picking Agrobacterium contains the antibiotic YEB liquid nutrient medium in 28 ℃ of jolting overnight cultures to 3ml from the YEB flat board, contains in the antibiotic AB liquid nutrient medium by the 1% inoculum size 50ml that transfers in the 2nd day, and 200rpm continues jolting and is cultured to OD
600When being 0.6 to 0.8 left and right sides, fresh Agrobacterium bacterium liquid in centrifugal 5 minutes of 5000rpm, 4 ℃, is collected and is resuspended in the AAM liquid nutrient medium of 1/3 volume, promptly can be used for the various acceptor materials of rice transformation this moment.
Present embodiment adopts in the conventional conversion method for agrobacterium rice transformation and spends 11 rataria callus.Get pollination back 12-15 days in spend 11 immature seeds through 70% alcohol immersion after 1 minute; (1:3 mixes with water in NaClO solution; Adding 2-3 and drip polysorbas20) sterilization is more than 90 minutes, with aseptic water washing 4-5 time, then with scalper with take the photograph son and choose rataria and be inoculated in N6D
2Evoked callus on the substratum is cultivated under 26 ± 1 ℃, lucifuge condition, can be used for after 4 days transforming.The rataria callus is soaked in the fresh AAM Agrobacterium bacterium liquid and shakes frequently, after 20 minutes rice material is shifted out, on aseptic filter paper, inhale and remove too much bacterium liquid, transfer to N6D immediately
2On the C substratum, cultivated altogether 3 days in 26 ℃.When cultivating altogether, adding Syringylethanone as Agrobacterium Vir gene activation thing in the culture medium altogether, working concentration is 100 μ mol/L.After 3 days, take out callus, cut plumule and change over to and select substratum N6D from being total to culture medium
2S1 (Hyg25mg/l) selects to cultivate.Forward resistant calli to N6D after 7-12 days
2S2 (Hyg50mg/l) selects to continue on the substratum screening.Eugonic resistant calli is transferred on the presorting substratum and is cultivated about a week after 10-12 days, moves to differentiation (12 hours illumination/skies) on the division culture medium again.The regenerated seedling is at 1/2MS
0Strong plantlets and rootage on the H substratum moves into the cultivation of phytotron basin soil subsequently.
Screen transformed plant once more with weedicide behind the regeneration plant transplant survival that obtains; Positive plant extracts the total DNA of blade, further identifies transformed plant through PCR.For observing paddy rice grain type phenotype, verify the GW2 gene function with transgenic T1.
Embodiment 5
The grain size of paddy rice GW antisense transgene plant and paddy rice GW justice transfer-gen plant and the big grain of molecule marker (non-transgenic) importing GW2 fragment plant relatively
1.GW the comparison of antisense transgene plant and wild-type
Obtain paddy rice GW antisense transgene plant like embodiment 4 described methods, for observing paddy rice grain type phenotype, observe of the influence of GW gene for the grain size with transgenic T1.
The result sees Fig. 1.Visible by Fig. 1, the grain of paddy rice GW antisense transgene plant (AS3,24,25) is obviously big than spending 11 (ZH11) in the acceptor kind.
2.GW the comparison of just transfer-gen plant and wild-type
Obtain paddy rice GW justice transfer-gen plant like embodiment 2 described methods, for observing paddy rice grain type phenotype, observe of the influence of GW gene for the grain size with transgenic T1.
The result sees Fig. 2.Visible by Fig. 2, the grain of paddy rice GW justice transfer-gen plant (S21,22,24) is obviously little than spending 11 (ZH11) in the acceptor kind.
3. big grain GW2 gene fragment imports the influence heavy to granule kind grain of granule kind
Big grain kind WY3 and rich short the accounting for of granule kind are hybridized for No. 1 acquisition F1 generation; Shortly account for No. 1 and repeatedly backcross so that the granule kind is rich as recurrent parent; Use molecule marker described in the embodiment 3 from backcross progeny, to select then to carry big grain GW2 gene fragment and genetic background is the plant of granule kind, like this will big grain GW2 gene fragment importing granule kind (FAZ1) in.Method for selecting molecular marker is a non-transgenic method, has the advantage that does not have potential safety hazard.
The result is as shown in Figure 3.The result shows, big grain GW2 gene fragment is imported in the granule kind, and the paddy rice grain that can obviously improve the granule kind is heavy.The thousand seed weight of paddy rice is increased to 32 grams by 22 original grams, and amplification is up to 46%.
The analysis of protein of embodiment 6GW2
1. Antibody Preparation
With the 1.278kb total length ORF that the foregoing description was obtained, with HindIII and the BamHI pET32a (+) (available from Noyagen company) that recombinates.Have the prokaryotic expression carrier pET32a (+) of GW2 to be transformed into e. coli bl21 (DE3) reorganization, IPDG induces prokaryotic expression, uses His-tag pearl purifying protein then.With purified protein immunization rabbit, obtain the antibody of anti-GW2 through ordinary method.
2. detection antibody titer
The expression amount of Western Blotting detection GW2 overexpression, and do negative contrast with unconverted parent, empty plasmid and antisense transgene plant simultaneously, the conduct of GW2 prokaryotic expression protein is over against photograph.
3. immunohistochemical analysis
Detect the variation of GW2 protein content, and carry out tissue and the Subcellular Localization of GW2.
4.E3 the active research of ligase enzyme
There is the prokaryotic expression carrier pET32a (+) of GW2 to be transformed into e. coli bl21 (DE3) reorganization; 0.5% lactose, low temperature (12 ℃) are induced prokaryotic expression; Whether from the prokaryotic expression bacterial strain, extract and the active GW2 albumen of purifying with the not damaged method, being used to study GW2 has the E3 ligase enzyme active.
Similar methods (seeing Xiuren Zhang etc., Genes & Dev.200519:1532-1543) such as Xiuren Zhang are adopted in ubiquitin ligase active reaction in the present embodiment.Be summarized as follows: the 30ul system includes 5mM MgCl
2, 0.33mM DTT, 50mM Tris-HCl (PH7.4), 2mM ATP, 0.125ug E1,0.2ug E2 (UbcH5b), 1ug E3 adds 2ul intestinal bacteria crude extract in addition in reaction system.Be reflected at warm the bath 3 hours in 30 degrees centigrade.Add 100 degrees centigrade of temperature of 1 * SDS-PAGE sample loading buffer afterwards and bathe 5 minutes termination reactions.Reaction effect is identified through the 12%SDS-PAGE gel electrophoresis.Ubiquitin protein utilizes Western blotting to detect through anti-Ubiquitin.
Above-mentioned experimental result shows that the E3 ligase enzyme that GW2 has in the proteolytic degradation approach is active, thereby points out it relevant with cell incident such as cell fission.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
< 110>Shanghai Inst. of Life Science, CAS
< 120>rice big grain gene and application thereof
<130>065312
<160>9
<170>PatentIn?version3.3
<210>1
<211>1634
<212>DNA
< 213>Oryza (Oryza sativa L.)
<220>
<221>CDS
<222>(255)..(1532)
<400>1
<210>2
<211>425
<212>PRT
< 213>Oryza (Oryza sativa L)
<400>2
<210>3
<211>19
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>primer
<400>3
<210>4
<211>19
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>primer
<400>4
<210>5
<211>20
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>primer
<400>5
<210>6
<211>18
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>primer
<400>6
<210>7
<211>23
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>primer
<400>7
<210>8
<211>25
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>primer
<400>8
Claims (17)
1. an isolating albumen is characterized in that, this albumen is selected from down group:
(a) polypeptide shown in SEQ ID NO:2 aminoacid sequence; Or
(b) interpolation through 1-10 amino-acid residue of the C-terminal of SEQ ID NO:2 aminoacid sequence and/or N-terminal is formed, and have the wide or grain weight function of control crop grain by (a) polypeptides derived.
2. albumen as claimed in claim 1 is characterized in that described dietary protein origin is in paddy rice.
3. albumen as claimed in claim 1 is characterized in that, it is active that described albumen has the E3 ligase enzyme.
4. isolating polynucleotide is characterized in that, these polynucleotide are selected from down group:
(i) the described proteic polynucleotide of coding claim 1; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
5. polynucleotide as claimed in claim 4 is characterized in that, the polypeptide of this polynucleotide encoding aminoacid sequence shown in SEQ ID NO:2.
6. polynucleotide as claimed in claim 4 is characterized in that, these polynucleotide are selected from down group:
(1) nucleotide sequence shown in the SEQ ID NO:1; Or
(2) nucleotide sequence shown in the 255-1532 position among the SEQ ID NO:1.
7. isolating polynucleotide is characterized in that, it is the mutant gene of the polynucleotide of the nucleotide sequence shown in the SEQ ID NO:1, and C → T variation has taken place the 1041st of its base.
8. the albumen of the described polynucleotide encoding of claim 7.
9. a carrier is characterized in that, it contains claim 4 or 7 described polynucleotide.
10. a genetically engineered host cell is characterized in that,
It contains the described carrier of claim 9; Or
Be integrated with claim 4 or 7 described polynucleotide in its genome.
11. the purposes of the described albumen of claim 1 or its encoding sox or the described polynucleotide of claim 7 or its encoded protein is characterized in that, is used for:
The grain of control crop kernel is wide or grain is heavy;
Performance E3 ligase enzyme is active; Or
As the molecule marker of identifying crop big grain kind and granule kind.
12. purposes as claimed in claim 11 is characterized in that, described performance E3 ligase enzyme activity comprises: ubiquitin protein or participation proteolytic degradation approach.
13. a method that improves crop is characterized in that, this method comprises:
(A) the proteic expression of described albumen of claim 1 or the described polynucleotide encoding of claim 7 in the said crop of reduction; Or
(B) the described albumen of claim 1 or its encoding sox with function reduction or afunction imports in the crop.
14. an ability and the described albumen of claim 1 or its encoding sox specificity bonded antibody.
15. a method for preparing transgenic plant is characterized in that, described method comprises step: claim 4 or 7 described polynucleotide are imported in the vegetable cell, cultivate described vegetable cell, the regeneration plant.
16. method as claimed in claim 15 is characterized in that, said method comprises step:
(s1) Agrobacterium of carrying expression vector is provided, described expression vector contains described proteic encoding sox of claim 1 or the described polynucleotide of claim 7;
(s2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (s1), thereby make described proteic encoding sox of claim 1 or the described polynucleotide of claim 7 change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell or tissue or the organ that changes described protein coding gene of claim 1 or the described polynucleotide of claim 7 over to; And
(s4) vegetable cell in the step (s3) or tissue or neomorph are become plant.
17. one kind prepares the described proteic method of claim 1, it is characterized in that, cultivates the described host cell of claim 10, collects to obtain the described albumen of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101171135A CN101161675B (en) | 2006-10-13 | 2006-10-13 | Rice big grain gene and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101171135A CN101161675B (en) | 2006-10-13 | 2006-10-13 | Rice big grain gene and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101161675A CN101161675A (en) | 2008-04-16 |
| CN101161675B true CN101161675B (en) | 2012-07-04 |
Family
ID=39296664
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2006101171135A Expired - Fee Related CN101161675B (en) | 2006-10-13 | 2006-10-13 | Rice big grain gene and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101161675B (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993482B (en) | 2009-08-24 | 2013-04-03 | 夏新界 | Protein associated with long grain foliaceous of paddy rice and coding gene and application thereof |
| CN102094012B (en) * | 2010-11-30 | 2013-09-18 | 深圳华大基因科技有限公司 | BGIos1027 gene and application thereof |
| CN102094013B (en) * | 2010-11-30 | 2013-02-27 | 深圳华大基因科技有限公司 | BGIos1005 gene and application thereof |
| CN102094011B (en) * | 2010-11-30 | 2014-01-08 | 深圳华大基因科技有限公司 | BGIos1036 gene and application thereof |
| CN102532290B (en) * | 2010-12-27 | 2013-12-04 | 中国农业大学 | Protein for controlling rice grain weight as well as coding gene and application thereof |
| CN102532289B (en) * | 2010-12-31 | 2014-06-18 | 中国农业大学 | Protein relevant to rice grain weight and encoding gene and application thereof |
| CN102329806B (en) * | 2011-09-30 | 2014-10-15 | 中国科学院遗传与发育生物学研究所 | Gene for controlling rice grain width, grain weight and yield and application thereof |
| CN102533782B (en) * | 2012-01-05 | 2014-07-02 | 华南师范大学 | Clone and application of OsAGSw1 gene for controlling width and weight of rice grains |
| CN103408648B (en) * | 2013-08-08 | 2015-02-25 | 中国科学院遗传与发育生物学研究所 | Application of paddy rice BG1 proteins and encoding genes of paddy rice BG1 proteins to adjusting growth and development of plants |
| CN106939312B (en) * | 2015-12-30 | 2020-08-11 | 中国科学院分子植物科学卓越创新中心 | A kind of grain weight related gene and its application |
| CN108395472A (en) * | 2018-01-16 | 2018-08-14 | 中国水稻研究所 | A kind of gene and its application of control rice class grain length and grain weight |
| CN110079532A (en) * | 2018-01-24 | 2019-08-02 | 深圳市作物分子设计育种研究院 | Regulate and control gene and its application of size of plant seed |
| CN110747288A (en) * | 2019-11-14 | 2020-02-04 | 扬州大学 | Rice large grain gene function marker and application |
| CN110923245B (en) * | 2019-12-24 | 2020-11-24 | 江西省农业科学院水稻研究所 | Rice granule heterosis regulation gene and breeding application thereof |
| CN119391906A (en) * | 2024-12-31 | 2025-02-07 | 云南省农业科学院粮食作物研究所 | Method for creating new germplasm of large-grain rice by using rice grain-related gene GW2-SNP molecular marker |
-
2006
- 2006-10-13 CN CN2006101171135A patent/CN101161675B/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| Chuchuan Fan et al.GS3,a major QTL for grain length and weight and minor QTLfor grain width and thickness in rice,encodes a putativetransmembrane protein.Theor Appl Genet 112.2006,(112),1164-1171. |
| Chuchuan Fan et al.GS3,a major QTL for grain length and weight and minor QTLfor grain width and thickness in rice,encodes a putativetransmembrane protein.Theor Appl Genet 112.2006,(112),1164-1171. * |
| Ken Ishimaru.Identification of a locus increasing rice yield and physiologicalanalysis of its function.Plant Physiology133.2003,1331083-1090. * |
| Zhikang Li et al.Epistasis for three grain yield components in rice(Oryza sativaL.).Genetics145.1997,145453-465. * |
| Zhou LiangQiang et al.Genetic analysis and physical mapping of Lk-4(t), a majorgene controlling grain length in rice,with a BC2F2 population.遗传学报33 1.2006,33(1),72-79. |
| Zhou LiangQiang et al.Genetic analysis and physical mapping of Lk-4(t), a majorgene controlling grain length in rice,with a BC2F2 population.遗传学报33 1.2006,33(1),72-79. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101161675A (en) | 2008-04-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101161675B (en) | Rice big grain gene and uses thereof | |
| CN101932596B (en) | The gene of regulating plant plant height and application thereof | |
| CN105646684B (en) | A rice grain shape-related protein GLW2 and its encoding gene and application | |
| CN103289972A (en) | Rice long-grain related gene and application for same | |
| CN105524152A (en) | Rice high-temperature-resistance gene and application thereof in crop high-temperature-resistance breeding | |
| CN108395472A (en) | A kind of gene and its application of control rice class grain length and grain weight | |
| CN114958867B (en) | Corn ear grain weight and yield regulation gene KWE2, coded protein, functional marker, expression vector and application thereof | |
| CN101535472B (en) | Grain incomplete filling gene (GIF1) and uses thereof | |
| CN113846117B (en) | A method for enhancing the fragrance of plants | |
| CN102776161A (en) | Preparation and use of high-glyphosate-resistance EPSP (5-enolpyruvylshikimate-3-phosphate) synthase separated from soil and coding sequence thereof | |
| CN103172716A (en) | Heat-resistant plant gene and application thereof | |
| CN100393744C (en) | Rice stem elongation gene and its encoded protein and application | |
| CN105693835B (en) | A kind of rice grain shape GAP-associated protein GAP GIF1 and its encoding gene and application | |
| WO2021155753A1 (en) | Herbicide-resistant gene, polypeptide, and application thereof in plant breeding | |
| CN114807172A (en) | Salt-responsive corn functional gene | |
| CN100467599C (en) | Flower type and inflorescence structure regulation genes, encoded proteins and their applications in japonicus japonicus | |
| CN101906154B (en) | Protein regulating the greening process of plant leaves, its coding gene and application | |
| CN101575366B (en) | Rice plant type gene and application thereof | |
| CN100355883C (en) | Paddg rice anti reverse related gene trehalose-6-phosphoric acid phosphorylase gene and its application | |
| CN1710076A (en) | Use of boea crassifolia BcBCP1 gene for breeding drought-salt-tolerant plants | |
| CN101173001B (en) | Gene for regulation of auxin sensibility and blade area, and uses thereof | |
| CN105399806B (en) | A kind of relevant albumen of rice male and female fertility, its encoding gene and its application | |
| CN117844829B (en) | Soybean heat-resistant protein GmBSK1 and application of encoding gene thereof in regulation and control of plant stress resistance | |
| CN1480464A (en) | Ethane cyclic amp receptor protein of wheat and its coding sequence | |
| CN102675437A (en) | Method for adjusting size of plant organs and inside asymmetry of floral organs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200609 Address after: 200032 building 4, No. 300 Fenglin Road, Xuhui District, Shanghai Patentee after: Center for excellence and innovation in molecular plant science, Chinese Academy of Sciences Address before: 200031 No. 320, Yueyang Road, Shanghai Patentee before: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120704 |