CN102643804B - Method for culturing roundup ready transgene corns - Google Patents
Method for culturing roundup ready transgene corns Download PDFInfo
- Publication number
- CN102643804B CN102643804B CN201210107400.3A CN201210107400A CN102643804B CN 102643804 B CN102643804 B CN 102643804B CN 201210107400 A CN201210107400 A CN 201210107400A CN 102643804 B CN102643804 B CN 102643804B
- Authority
- CN
- China
- Prior art keywords
- sequence
- aroa
- gene
- plant
- corns
- 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
- 240000008042 Zea mays Species 0.000 title claims abstract description 79
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 79
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 76
- 235000005822 corn Nutrition 0.000 title claims abstract description 76
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 44
- 208000003643 Callosities Diseases 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 42
- 108700019146 Transgenes Proteins 0.000 title abstract description 8
- 238000012258 culturing Methods 0.000 title abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 114
- 239000005562 Glyphosate Substances 0.000 claims abstract description 48
- 229940097068 glyphosate Drugs 0.000 claims abstract description 48
- 108020004414 DNA Proteins 0.000 claims abstract description 14
- 230000009261 transgenic effect Effects 0.000 claims description 64
- 239000013604 expression vector Substances 0.000 claims description 34
- 238000003259 recombinant expression Methods 0.000 claims description 27
- 239000012634 fragment Substances 0.000 claims description 21
- 108020004511 Recombinant DNA Proteins 0.000 claims description 14
- 230000008676 import Effects 0.000 claims description 4
- 230000014509 gene expression Effects 0.000 abstract description 28
- 102000004169 proteins and genes Human genes 0.000 abstract description 20
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 3
- 102000053602 DNA Human genes 0.000 abstract 2
- 241000196324 Embryophyta Species 0.000 description 61
- 239000000523 sample Substances 0.000 description 30
- 241001057636 Dracaena deremensis Species 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 23
- 238000012360 testing method Methods 0.000 description 23
- 239000007788 liquid Substances 0.000 description 22
- 108090000790 Enzymes Proteins 0.000 description 17
- 102000004190 Enzymes Human genes 0.000 description 17
- 241000589158 Agrobacterium Species 0.000 description 16
- 108020004705 Codon Proteins 0.000 description 16
- 239000013642 negative control Substances 0.000 description 16
- 239000013612 plasmid Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 230000009182 swimming Effects 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 230000002163 immunogen Effects 0.000 description 15
- 239000000463 material Substances 0.000 description 15
- 239000002953 phosphate buffered saline Substances 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- 235000018102 proteins Nutrition 0.000 description 15
- 239000002773 nucleotide Substances 0.000 description 13
- 125000003729 nucleotide group Chemical group 0.000 description 13
- 239000000427 antigen Substances 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000001514 detection method Methods 0.000 description 11
- 108091008146 restriction endonucleases Proteins 0.000 description 11
- 230000003053 immunization Effects 0.000 description 10
- 238000002649 immunization Methods 0.000 description 10
- 241000283707 Capra Species 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- 239000006228 supernatant Substances 0.000 description 9
- 230000014616 translation Effects 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 238000013016 damping Methods 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 238000011156 evaluation Methods 0.000 description 8
- 239000012530 fluid Substances 0.000 description 8
- 206010003445 Ascites Diseases 0.000 description 7
- 230000036039 immunity Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 230000005030 transcription termination Effects 0.000 description 7
- 238000013519 translation Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 125000003275 alpha amino acid group Chemical group 0.000 description 6
- 210000003763 chloroplast Anatomy 0.000 description 6
- 238000000605 extraction Methods 0.000 description 6
- 238000002347 injection Methods 0.000 description 6
- 239000007924 injection Substances 0.000 description 6
- 231100000572 poisoning Toxicity 0.000 description 6
- 230000000607 poisoning effect Effects 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 108020004999 messenger RNA Proteins 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 4
- 108091081024 Start codon Proteins 0.000 description 4
- 239000003636 conditioned culture medium Substances 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 238000004945 emulsification Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 210000004408 hybridoma Anatomy 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 108091034057 RNA (poly(A)) Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 108020005038 Terminator Codon Proteins 0.000 description 3
- 101710120037 Toxin CcdB Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 230000024835 cytogamy Effects 0.000 description 3
- 230000030609 dephosphorylation Effects 0.000 description 3
- 238000006209 dephosphorylation reaction Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 241001233957 eudicotyledons Species 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 235000009973 maize Nutrition 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000012460 protein solution Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 235000013311 vegetables Nutrition 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108010020183 3-phosphoshikimate 1-carboxyvinyltransferase Proteins 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000209510 Liliopsida Species 0.000 description 2
- 231100000674 Phytotoxicity Toxicity 0.000 description 2
- 108700001094 Plant Genes Proteins 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000001804 emulsifying effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000002363 herbicidal effect Effects 0.000 description 2
- 239000004009 herbicide Substances 0.000 description 2
- 230000000749 insecticidal effect Effects 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000003032 molecular docking Methods 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 238000003118 sandwich ELISA Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- GEWDNTWNSAZUDX-WQMVXFAESA-N (-)-methyl jasmonate Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-WQMVXFAESA-N 0.000 description 1
- LWTDZKXXJRRKDG-KXBFYZLASA-N (-)-phaseollin Chemical compound C1OC2=CC(O)=CC=C2[C@H]2[C@@H]1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-KXBFYZLASA-N 0.000 description 1
- NWXMGUDVXFXRIG-WESIUVDSSA-N (4s,4as,5as,6s,12ar)-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O NWXMGUDVXFXRIG-WESIUVDSSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- 241001167018 Aroa Species 0.000 description 1
- 241000193388 Bacillus thuringiensis Species 0.000 description 1
- 101100061321 Bacillus thuringiensis subsp. japonensis cry8Ca gene Proteins 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- LMKYZBGVKHTLTN-NKWVEPMBSA-N D-nopaline Chemical compound NC(=N)NCCC[C@@H](C(O)=O)N[C@@H](C(O)=O)CCC(O)=O LMKYZBGVKHTLTN-NKWVEPMBSA-N 0.000 description 1
- 108010066133 D-octopine dehydrogenase Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 241001446187 Kermes Species 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 241000710118 Maize chlorotic mottle virus Species 0.000 description 1
- 101150005851 NOS gene Proteins 0.000 description 1
- 101710202365 Napin Proteins 0.000 description 1
- 101710089395 Oleosin Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108091000041 Phosphoenolpyruvate Carboxylase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 241000589540 Pseudomonas fluorescens Species 0.000 description 1
- 241000589776 Pseudomonas putida Species 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108010016634 Seed Storage Proteins Proteins 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229940097012 bacillus thuringiensis Drugs 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- CJOBVZJTOIVNNF-UHFFFAOYSA-N cadmium sulfide Chemical compound [Cd]=S CJOBVZJTOIVNNF-UHFFFAOYSA-N 0.000 description 1
- 229940027138 cambia Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- KXZOIWWTXOCYKR-UHFFFAOYSA-M diclofenac potassium Chemical compound [K+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KXZOIWWTXOCYKR-UHFFFAOYSA-M 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- ZLDFPGVILFRQON-UHFFFAOYSA-L disodium hydrogen phosphate 2-nitrophenol Chemical compound P(=O)([O-])([O-])O.[Na+].[Na+].[N+](=O)([O-])C1=C(C=CC=C1)O ZLDFPGVILFRQON-UHFFFAOYSA-L 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 230000004992 fission Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- CXORMDKZEUMQHX-UHFFFAOYSA-N kermesic acid Chemical compound O=C1C2=C(O)C(O)=CC(O)=C2C(=O)C2=C1C=C(O)C(C(O)=O)=C2C CXORMDKZEUMQHX-UHFFFAOYSA-N 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- GEWDNTWNSAZUDX-UHFFFAOYSA-N methyl 7-epi-jasmonate Natural products CCC=CCC1C(CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-UHFFFAOYSA-N 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 108010058731 nopaline synthase Proteins 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000007500 overflow downdraw method Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- LWTDZKXXJRRKDG-UHFFFAOYSA-N phaseollin Natural products C1OC2=CC(O)=CC=C2C2C1C1=CC=C3OC(C)(C)C=CC3=C1O2 LWTDZKXXJRRKDG-UHFFFAOYSA-N 0.000 description 1
- NONJJLVGHLVQQM-JHXYUMNGSA-N phenethicillin Chemical compound N([C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C(O)=O)=O)C(=O)C(C)OC1=CC=CC=C1 NONJJLVGHLVQQM-JHXYUMNGSA-N 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 108010074916 ribophorin Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- JXOHGGNKMLTUBP-HSUXUTPPSA-N shikimic acid Chemical compound O[C@@H]1CC(C(O)=O)=C[C@@H](O)[C@H]1O JXOHGGNKMLTUBP-HSUXUTPPSA-N 0.000 description 1
- JXOHGGNKMLTUBP-JKUQZMGJSA-N shikimic acid Natural products O[C@@H]1CC(C(O)=O)=C[C@H](O)[C@@H]1O JXOHGGNKMLTUBP-JKUQZMGJSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000005026 transcription initiation Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000009333 weeding Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for culturing roundup ready transgene corns. The method provided by the invention comprises the following steps that: roundup ready genes are guided into target corns, and transgene corns expressing the roundup ready genes are obtained; the transgene corns are compared with the target corns, and the glyphosate tolerance is improved; and the roundup ready genes are deoxyribose nucleic acid (DNA) molecules of a) or b): a) nucleotide sequences being sequences 2 in a sequence table; and b) nucleotide sequences with at least 98 percent identical protein with coding sequences 9 as the sequences 2 in the sequence table. Compared with the transgene corns with transformed Prokaryotic roundup ready gene G2-aroA, the transgene corns with the transformed roundup ready genes obtained through the culture by the method provided by the invention has the advantages that the G2-aroA protein expression is obviously improved, and meanwhile, the glyphosate resistance is also obviously improved.
Description
Technical field
The present invention relates to a kind of method of cultivating glyphosate tolerant transgenic corns.
Background technology
At present in plant transgene breeding applied foreign gene as Bt, EPSPS etc., mostly from prokaryotic organism, due to prokaryotic gene self, as 1) AT content is higher, surpass 60%, cause the mRNA of genetic expression to be very easily degraded in plant materials; 2) there is intron point of contact, the Transcription Termination subsequence of similar eukaryotic gene, cause and transcribe that imperfect, mRNA is abnormal to be sheared etc.; 3) there is larger difference in codon and vegetable codon, causes protein translation Efficiency Decreasing; 4) the eukaryote significant difference such as its structure and plant, adds tailer sequence if do not contained the polyA of 5 ' of eukaryotic gene-UTR sequence and 3 ' end, and often expression level is lower in plant materials to cause gene.For example, the expression amount of the wild-type insecticidal protein gene that comes from bacillus thuringiensis in plant is very low, the toxalbumin of its expression only account for total protein 0.001% or almost can't detect.The Perlak of U.S. Monsanto company (Perlak F J, Fuchs R L, Dean D A, et al.Modification of the coding SEQ ID NO.uence enhances plant expressing of insect control protein genes.Proc Natl Acad Sci USA, 1991, 88:3324-3328) and (the Iannacoe R such as Iannacone, Grieco P D, Cellini F.Specific SEQ ID NO.uence modification of a cry3B endotoxin gene result in high levels of expression and insect resistance.Plant Mol Biol, 1997, 34:485-496) do not changing under the prerequisite of toxalbumin aminoacid sequence, respectively insecticidal protein gene and cryIII gene are transformed, select the codon of plant-preference, increase GC content, and remove the polyA that exists in former sequence and be rich in the unstable element sequences such as ATTTA sequence of AT, in render transgenic plant, the expression amount of toxalbumin has increased by 30～100 times, up to 0.02～1% of soluble proteins, obtained obvious pest-resistant effect.
Weeds are large evils that farm crop produce, and since control of weeds technology in modern age appears in nineteen forty-two, chemical herbicide has a great development.Glyphosate class weedicide is a kind of broad spectrum, nonselective herbicide, it is by suppressing EPSPS (5-enol pyruvoyl oxalic acid-3-phosphate synthase, an important enzyme in die aromatischen Aminosaeuren route of synthesis in plant materials) activity, blocked the biosynthesizing of plants shikimic acid approach, strongly inhibited cell fission, all has strong restraining effect to many annual and perennial weedss.Because glyphosate is easy to be decomposed by microorganism, in soil, without residual hazard, to animal toxicological harmless, since Roundup in 1976 succeeds in developing, be widely used.
But, because the gramineous crops such as corn are responsive to glyphosate, its application is restricted.Therefore, glyphosate tolerant gene is proceeded to corn, not only can expand the use range of glyphosate, and can reduce production costs, protection corn is avoided poisoning, finally reaches the object of increasing both production and income.Although glyphosate tolerant gene G2-aroA was found in 2004, and can high efficient expression in host Pseudomonas fluorescens G2 and intestinal bacteria, show characteristic (the Yichen Sun of high glyphosate tolerant, Min Lin and Yiping Wang.Novel AroA with high tolerance to Glyposate, encoded by a gene of Pseudomonas putida 4G-1 isolated from an extremelypolluted environment in China.Aplied and environmental microbiology.2005, 71 (8): 4771-4776), but it is plant, particularly in important food crop because expression amount is compared with low and be not utilized, therefore being badly in need of application in plant to it studies, as carry out codon optimized, to accelerate it, be applied in agriculture production.
Summary of the invention
The object of this invention is to provide a kind of method of cultivating glyphosate tolerant transgenic corns.
The method of cultivation glyphosate tolerant transgenic corns provided by the present invention comprises glyphosate tolerant gene imported to object corn, obtains expressing the step of the transgenic corns of described glyphosate tolerant gene; Described transgenic corns is compared with described object corn, and the tolerance of glyphosate is improved; Described glyphosate tolerant gene is the gene of protein shown in encoding sequence 9.
Wherein, described glyphosate tolerant gene can first be modified as follows, then imports in host, to reach better expression effect:
1) modify according to actual needs and optimize, so that gene efficient expression; For example, the codon that can have a preference for according to recipient plant changes its codon to meet plant-preference when the aminoacid sequence that keeps glyphosate tolerant albumen is constant; In optimizing process, preferably can make to keep certain GC content in the encoding sequence after optimizing, to realize best the high level expression of quiding gene in plant, wherein GC content can be 35%, more than 45%, more than 50% or more than approximately 60%;
2) modify the gene order of contiguous initial methionine, so that translation is effectively initial; For example, utilize known effective sequence in plant to modify;
3) be connected with the promotor of various expression of plants, be beneficial to its expression in plant; Described promotor can comprise that composing type, induction type, sequential regulate, grow adjusting, Chemical Regulation, tissue preferably and tissue-specific promoter; The selection of promotor will be along with expression time and space requirement and is changed, and depends on target species; For example tissue or the specific expressing promoter of organ, acceptor in what period of growing is determined as required; Although proved that the many promotors that derive from dicotyledons are operational in monocotyledons, vice versa, but ideally, select dicotyledons promotor for the expression of dicotyledons, monocotyledonous promotor is for the expression of monocotyledons;
4), with applicable Transcription Termination sub-connection, also can improve the expression efficiency of gene of the present invention; For example derive from the tml of CaMV, derive from the E9 of rbcS; Any known available terminator working in plant can be connected with gene of the present invention;
5) introduce enhancer sequence, for example, for example, as intron sequences (deriving from Adhl and bronzel) and virus leader sequence (deriving from TMV, MCMV and AMV).
In the present invention, described glyphosate tolerant gene be specially following a) or b) DNA molecular:
A) nucleotides sequence is classified sequence 2 in sequence table as;
B) in nucleotide sequence and sequence table, sequence 2 at least has 98% identity, and the protein of encoding sequence 9 (called after G2-aroA albumen).
In one embodiment of the invention, described is to complete by the recombinant dna fragment of expressing described glyphosate tolerant gene is imported to described object corn by glyphosate tolerant gene importing object corn.
Described recombinant dna fragment imports described object corn by recombinant expression vector.
Recombinant dna fragment described in the present invention all refer to can be in host cell protein DNA shown in sequence 9 in expressed sequence table, this DNA not only can comprise the promotor that starts described glyphosate tolerant genetic transcription, also can comprise terminator.Further, described recombinant dna fragment also can comprise enhancer sequence.Can be used for promotor of the present invention includes but not limited to: constitutive promoter, the promotor that tissue, organ and growth are special, and inducible promoter.As the constitutive promoter 35S of cauliflower mosaic virus; Tomato proteinase inhibitor II promotor (PIN2) or LAP promotor (all available methyl jasmonate inductions); Heat-shocked promotor; Tsiklomitsin inducible promoter; Seed specific promoters, as Millet Seed specificity promoter pF128, promotor (for example, phaseollin, napin, the promotor of oleosin and soybean beta conglycin etc. that seed storage protein matter is special.Can be used for transcription terminator of the present invention includes but not limited to: Agrobacterium rouge alkali synthetase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator.
Available existing plant expression vector construction is expressed the described recombinant expression vector of described glyphosate tolerant gene.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.As pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA company) etc.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of transcribing as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean stores protein gene) 3 ' end all has similar functions.While using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.
In one embodiment of the invention, the promotor that starts described glyphosate tolerant genetic transcription in described recombinant dna fragment and described recombinant expression vector is Ubi promotor; The sequence of described Ubi promotor is sequence 5 in sequence table, or at least has 80% identity with sequence 5, and has promoter function.
In one embodiment of the invention, in described recombinant dna fragment and described recombinant expression vector, stop the transcription termination sequence of described glyphosate tolerant genetic transcription as shown in the 216-491 position of sequence in sequence table 8, or at least there is 80% identity with the 216-491 position of sequence 8, and there is Transcription Termination function.
In one embodiment of the invention, in described recombinant dna fragment and described recombinant expression vector, also comprise by Ω sequence and the sequently connected OMK sequence of Kozak sequence; Described OMK sequence, as shown in sequence in sequence table 6, or at least has 80% identity with sequence 6, and has enhanser function.
In one embodiment of the invention, in described recombinant expression vector, also comprise that chloroplast(id) leads peptide (CTP) sequence; Described chloroplast(id) is led peptide (CTP) sequence as shown in sequence in sequence table 7, or at least has 80% identity with sequence 7, and has signal peptide function.
In one embodiment of the invention, described recombinant dna fragment is led peptide sequence, described glyphosate tolerant gene and described transcription termination sequence by described Ubi promotor, described OMK sequence, described chloroplast(id) and is connected to form in turn, called after Ubi-OMK-CTP-mG2aroA-PolyA-T-NOS, its sequence is sequence 10 in sequence table.
In one embodiment of the invention, described recombinant expression vector contains described recombinant dna fragment, and by this recombinant expression vector called after pS3300-UMG2, its sequence is sequence 11 in sequence table.
Wherein, sequence 1 is comprised of 1335 Nucleotide, and its place, end is a terminator codon.Sequence 2 is comprised of 1338 Nucleotide, and its place, end is two terminator codons.The 1-1335 position of sequence 1 and sequence 2 is encoding sequence, all the G2-aroA albumen shown in sequence 9 in code sequence list.Sequence 5 forms with Nucleotide by 2010.Sequence 6 is comprised of 67 Nucleotide.Sequence 7 is comprised of 141 Nucleotide.Sequence 8 is comprised of 491 Nucleotide.Sequence 10 is comprised of 4058 Nucleotide, wherein 1-2010 position is Ubi promoter sequence, and 2022-2088 position is OMK sequence, and 2089-2229 position is that chloroplast(id) is led peptide (CTP) sequence, 2230-3567 position is mG2-aroA sequence, and 3568-4058 position is transcription termination sequence.Sequence 11 is comprised of 10488 Nucleotide, wherein 151-2165 position is Ubi promoter sequence, and 2177-2243 position is OMK sequence, and 2244-2384 position is CTP sequence, 2385-3722 position is mG2-aroA sequence, and 3723-4213 position is transcription termination sequence.
Described recombinant expression vector can be by using Ti-plasmids, plant virus carrying agent, and directly delivered DNA, microinjection, the conventional biotechnological means such as electroporation imports vegetable cell.
Utilize the method for cultivation glyphosate tolerant transgenic corns provided by the present invention to cultivate the transgenic corns obtain and also belong to protection scope of the present invention.Described transgenic corns comprises seed, callus, whole plant and cell.
In an embodiment of the present invention, described corn is specially corn variety and combines 31.
The present invention is directed to prokaryotic organism glyphosate tolerant gene G2-aroA and in plant, express lower problem, keeping under the constant prerequisite of original acid sequence, adopt corn Preference codon to be optimized it, remove and affect the structure of rna stability (as polyA, tumor-necrosis factor glycoproteins, AT and GC series connection repetition, RNA secondary structure, ribosome bind site etc.), increase GC content, make G2-aroA albumen high efficiency stable expression in corn.
Experiment confirms, utilize the method for cultivation glyphosate tolerant transgenic plant provided by the present invention, the positive milpa that proceeds to mG2-aroA gene (sequence 2) that cultivation obtains, compare with the milpa that proceeds to original G2-aroA gene, the expression amount of its G2-aroA albumen obviously improves, in the expression amount of every gram of blade G2-aroA albumen, can reach 15.057 μ g, far above 3.735 μ g of original G2-aroA gene transgenic corn., compare with the milpa that proceeds to original G2-aroA gene, the positive milpa that proceeds to mG2-aroA gene is also obviously improved the tolerance of glyphosate, proceeds to the T of mG2-aroA gene meanwhile
6for plant, after the 5 leaf phases of corn spray the agriculture of 800ml/ mu and reach, without significantly poisoning, act normally, in the vegetative period in later stage all without phytotoxicity reaction; And proceed to contain G2-aroA gene T
6for plant, in the agriculture of 800ml/ mu, reach after processing, poisoning is very serious, plant all shows yellow, deformity, in the plant strain growth phase in later stage undifferentiated, the female fringe of most of performance tassel do not weave silk or plant short and small.
Accompanying drawing explanation
Fig. 1 is the plasmid map of carrier pUC19-UG2.
Fig. 2 is the plasmid map of carrier pCAMBIA3300.
Fig. 3 is the plasmid map of carrier pS3300.
Fig. 4 is the plasmid map of recombinant expression vector pS3300-UG2.
Fig. 5 is the plasmid map of recombinant expression vector pS3300-UMG2.
Fig. 6 is the plasmid map of recombinant vectors pS3300-UG0
Fig. 7 is T
6pCR for G2-aroA transgenic corns identifies collection of illustrative plates.Wherein, the positive contrast of swimming lane 1; Swimming lane 2 is DNA Marker (D2000); Swimming lane 3 is blank group; Swimming lane 4 is not genetically modified milpa (negative control 2); Swimming lane 5-15 is the T that 11 strains proceed to G2-aroA gene
6for transgenic corn plant.
Fig. 8 is T
6pCR for mG2-aroA transgenic corns identifies collection of illustrative plates.Wherein, the positive contrast of swimming lane 1; Swimming lane 2 is DNA Marker (D2000); Swimming lane 3 is blank group; Swimming lane 4 is not genetically modified milpa (negative control 2); Swimming lane 5-24 is the T that 20 strains proceed to mG2-aroA gene
6for transgenic corn plant.
Fig. 9 is T
6for G2-aroA transgenic corn plant and T
6glyphosate-tolerant field test figure for mG2-aroA transgenic corn plant.Wherein, A is mG2-aroA transgenic corn plant, empty carrier transfer-gen plant and transfer-gen plant not; B is mG2-aroA transgenic corn plant and G2-aroA transgenic corn plant.In A and B, the corn of a line shown in 1 is mG2-aroA transgenic corn plant; The corn of a line shown in 2 is G2-aroA transgenic corn plant; The corn of a line shown in 3 is empty carrier transgenic corn plant; The corn of a line shown in 4 is not genetically modified milpa.
Figure 10 is the typical curve that double antibodies sandwich ELISA detects G2-aroA protein concentration.
Figure 11 is G2-aroA protein SDS-PAGE electrophoresis evaluation figure after purifying.Wherein, swimming lane 1 is albumen Marker, is followed successively by from top to bottom 72KD, 45KD, 32KD, 14.4KD; Swimming lane 2-4 is the G2-aroA albumen of expressing rear purifying gained in RT-PCR expression vector pET-28a-G2-aroA, and applied sample amount is respectively 5 μ l, 10 μ l, 15 μ l.
Figure 12 is the purity of monoclonal antibody after SDS-PAGE detection purifying.Wherein, swimming lane 1 is albumen Marker; Swimming lane 2 is the monoclonal antibody after purifying, and larger object band is heavy chain, and less object band is light chain.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The acquisition of embodiment 1, codon optimized type glyphosate tolerant gene
The present embodiment is according to G2-aroA gene (Chinese patent, application number 03826892.2, Granted publication CN100429311C) aminoacid sequence (nucleotide sequence is as shown in sequence in sequence table 1), guaranteeing, under the prerequisite that aminoacid sequence is constant, first to adopt corn preference codon to carry out artificial optimization's transformation to G2-aroA gene.Avoid using corn rare codon as far as possible, and adjusted the frequency of utilization (table 1) of codon, make the codon usage frequency of G2-aroA albumen and the frequency of utilization in corn approach (table 1).On this basis, remove in DNA sequence dna, exist typically cause this unsettled AT of being rich in sequence of plant gene transcription, and removed hairpin structure, the new nucleotides sequence obtaining is classified sequence 2 in sequence table as.Sequence 2 only has 84% with G2-aroA gene (sequence 1) homology, and G+C content is reduced to 62.07% by original 64.83%.Gene shown in sequence 2 is codon optimized type glyphosate tolerant gene, by its called after mG2-aroA.The frequency of utilization of codon in corn, G2-aroA gene and mG2-aroA gene is in Table 1.For convenience of clone, contriver introduces BamH I restriction enzyme site at 5 ' end of sequence 2, introduces KpnI enzyme cut restriction enzyme site at 3 ' end, and ultimate sequence is as shown in sequence in sequence table 3.The 7-1344 position of sequence 3 is sequence 2.The 1-1335 position of sequence 1, sequence 2, and the equal encoding sequence in 7-1341 position of sequence 3, albumen shown in sequence 9 in equal code sequence list, by this albumen called after G2-aroA albumen.
The preferred codon standard of table 1 corn
The acquisition of embodiment 2, mG2-aroA transgenic corns
One, the structure of recombinant expression vector pS3300-UMG2
In order to improve the expression level of mG2-aroA gene (sequence 2) in receptor biological, contriver is when building the recombinant expression vector of mG2-aroA gene, 5 ' the end at mG2-aroA gene has added Ω sequence and Kozak sequence, and Ω/Kozak sequence (being called for short OMK) is as shown in sequence in sequence table 6.Ω sequence is the translation enhancement sequences that is derived from plant virus capsid protein gene coding region, by 67bp, formed, enrichment TTAAC sequence, 5 ' end has a UAUUUUUACAACAA sequence and 4 UUAC sequences, and these sequences form rrna and rRNA binding site in the translation process of protein synthesis.Kozak sequence is the sequence that promotes foreign gene coding ribophorin of translation process in vegetable cell.Promotor is selected composition promotor Ubi promotor, and its sequence is as shown in sequence in sequence table 5.Moreover, at encoding sequence 3 ' end, design 2 continuous terminator codons, and added the PolyA+T-NOS of synthetic to stablize terminator sequence.PolyA+T-NOS sequence is as shown in sequence in sequence table 8.Wherein, PolyA has the effects such as the mRNA of maintaining is stable, and T-NOS terminator sequence has been guaranteed the accurate termination of translation.In addition, according to the special mechanism of plant glyphosate tolerant, before 5 ' initiator codon ATG, add chloroplast(id) to lead PEPC TP, the albumen (being G2-aroA albumen) of destination gene expression is transported in chloroplast(id), better to bring into play the resistance effect of mG2-aroA gene.The sequence of CTP is as shown in sequence in sequence table 7.
The recombinant expression vector pS3300-UMG2 structure specific procedure that carries mG2-aroA gene is as follows:
1, the structure of intermediate carrier pUC19-UG2
A. synthetic Ubi promotor (sequence 5), and at two ends, add Pst I restriction enzyme site; Pst I enzyme is cut pUC19 plasmid (purchased from sky, Beijing bounties Gene Tech. Company Limited, production code member 90202), after dephosphorylation, is connected with Ubi promoter fragment, obtains pUC19-Ubi.
B. synthetic G2-aroA gene (sequence 1), and at two ends, add BamHI and Kpn I restriction enzyme site; The pUC19-Ubi that is connected into the above-mentioned steps 1a gained of cutting through BamHI and Kpn I enzyme, obtains pUC19-Ubi-G2.
C. synthetic PolyA+T-NOS terminator sequence (sequence 8, wherein 261-491 position is T-NOS terminator sequence), and at two ends, add Kpn I and EcoR I restriction enzyme site; The pUC19-Ubi-G2 that is connected into the above-mentioned steps 1b gained of cutting through Kpn I and EcoR I enzyme, obtains pUC19-Ubi-G2-polyA-T-NOS.
D. synthetic OMK sequence (sequence 6)+CTP sequence (sequence 7), and at two ends, add BamH I site; BamH I enzyme is cut the pUC19-Ubi-G2-polyA-T-NOS of above-mentioned steps 1c gained, connects OMK-CTP (directly connecting between sequence 6 and sequence 7) after dephosphorylation, obtains pUC19-UG2 (plasmid map is shown in Fig. 1).
2, transformation pCAMBIA3300 carrier obtains pS3300 carrier
A. expression vector pCAMBIA3300 (plasmid map is shown in Fig. 2), purchased from Beijing DingGuo ChangSheng Biology Technology Co., Ltd (CA:MCV038), with restriction enzyme SphI, Sac II double digestion, reclaims purifying large fragment.
B. (the right border sequence from 5 ' end by T-Border of gene fragment shown in sequence 4 in artificial synthesized sequence table, the left margin sequence of catenation sequence and T-Border forms, the recognition sequence that contains Hind III and EcoRI restriction enzyme site from 5 ' end in described catenation sequence), after cutting with restriction enzyme SphI, Sac II enzyme, the large fragment reclaiming with step a is connected, and obtains pS3300 carrier (plasmid map is shown in Fig. 3).
3, carry the structure of the recombinant expression vector pS3300-UMG2 of mG2-aroA gene
A. by HindIII and EcoR I double digestion for step 2b gained pS3300 carrier, reclaim purifying.
B. by expression vector pUC19-UG2 (plasmid map is shown in Fig. 1) with reclaiming small segment (Ubi-OMK-CTP-G2-polyA-T-NOS) after Hind and EcoR I double digestion, reclaim product with step 3a gained and be connected, obtain carrying the recombinant expression vector pS3300-UG2 (plasmid map is shown in Fig. 4) of G2-aroA gene.
C. the pS3300-UG2 carrier of using BamH I and Kpn I double digestion above-mentioned steps b gained, reclaims respectively fragment pS3300-Ubi-polyA-T-NOS and OMK+CTP.
D. by newly synthetic two ends respectively with the mG2-aroA gene (sequence 3) with BamH I and Kpn I restriction enzyme site after BamH I and Kpn I enzyme are cut, the large fragment pS3300-Ubi-polyA-T-NOS obtaining with above-mentioned steps c is connected, and obtains recombinant vectors pS3300-Ubi-mG2-polyA-T-NOS.
E. with BamH I enzyme, cut the recombinant vectors pS3300-Ubi-mG2-polyA-T-NOS that above-mentioned steps d obtains, carry out afterwards dephosphorylation processing, the OMK+CTP sequence reclaiming with above-mentioned steps d is again connected, and obtains carrying the recombinant expression vector pS3300-UMG2 (plasmid map is shown in Fig. 5) of mG2-aroA gene complete reading frame.On described recombinant expression vector pS3300-UMG2, contain the recombinant dna fragment that sequence is sequence 10 in sequence table, according to entrained element by this recombinant dna fragment called after Ubi-OMK-CTP-mG2aroA-PolyA-T-NOS.。The nucleotide sequence of described recombinant expression vector pS3300-UMG2 is as shown in sequence in sequence table 11.
4, the structure of original gene G2-aroA (sequence 1) recombinant expression vector pS3300-UG2: see 3a, 3b.
5, the structure of pS3300-UG0 empty carrier (contrast)
A. the pS3300-UMG2 carrier of above-mentioned steps 3e gained of take is template, utilizes the following primer OMK+CTP that again increases:
OMK+CTP_F:5 '-
gGATCCtATTTTTACAACAATTA-3 ' (underscore is partly BamHI restriction enzyme site recognition sequence);
OMK+CTP_R:5 '-
gGTACCtTCCGCCGTTGCTGAC-3 ' (underscore is partly Kpn I restriction enzyme site recognition sequence).
B. amplified production is connected with step 3c gained pS3300-Ubi-polyA-T-NOS large fragment after Kpn I double digestion through BamH I, obtains empty carrier pS3300-UG0 (plasmid map is shown in Fig. 6).
Two, recombinant expression vector maize transformation obtains transgenic corns
1, corn transforms the acquisition of parent material
Corn variety is combined 31 (superior corn self-mating system displaying comprehensive 3 and comprehensive 31. Maize Sciences, 2009 (5)) young fringe of latter 9～13 days of pollination, peels off bract, carries out surface sterilization.From the young fringe sterilization, strip rataria, put it into and infect nutrient solution (formula reference: Methods in Molecular Biology, vol.343:Agrobacterium Protocols, 2/e, volume 1)) in clean one to twice, standby.
2, recombinant expression vector transforms Agrobacterium
Recombinant expression vector pS3300-UMG2 prepared by step 1 or pS3300-UG2 conversion Agrobacterium LBA4404 (reference: Methods in Molecular Biology, vol.343:Agrobacterium Protocols, 2/e, volume 1).The contrast that proceeds to pS3300-UG0 empty carrier is set simultaneously.
The Agrobacterium LBA4404 called after LBA4404/pS3300-UMG2 of recombinant expression vector pS3300-UMG2 will be confirmed to have proceeded to through evaluation; Proceeded to the Agrobacterium LBA4404 called after LBA4404/pS3300-UG2 of recombinant expression vector pS3300-UG2; Proceeded to the Agrobacterium LBA4404 called after LBA4404/pS3300-UG0 of pS3300-UG0 empty carrier.
3, Agrobacterium-mediated Transformation maize immature embryos
The rataria that above-mentioned steps 1 was cleaned through infection nutrient solution is put into OD
600in bacterium liquid for three kinds of Agrobacteriums of above-mentioned steps 2 preparations of about 0.3-0.5, place 5 minutes, then rataria is placed in to common culture medium (reference: Methods in Molecular Biology, vol.343:Agrobacterium Protocols, 2/e, volume 1) upper, under 20 ℃ of left and right dark conditions, cultivate altogether 3 days, to carry out the rataria of Agrobacterium-mediated Transformation, do not compare.
4, the acquisition of transgenic corns regrowth
Rataria after above-mentioned steps 3 is cultivated altogether proceeds to selects substratum (reference: Methods in Molecular Biology, vol.343:Agrobacterium Protocols, 2/e, volume 1), in selecting substratum, add final concentration be the glyphosate of 1mM as selective pressure, the material being converted is carried out to screening and culturing, every two weeks subcultures once, until grow crisp, color cadmium yellow and eugonic resistant calli.
Gained kanamycin-resistant callus tissue is proceeded to inducing culture (reference: Methods in Molecular Biology, vol.343:Agrobacterium Protocols, 2/e, volume 1) induce differentiation, after one month, can obtain ripe embryoid.Again embryoid is put on MS substratum and is taken root, obtain T
0regrowth for transgenic corns.T
0for obtaining T after transgenic corns maturation
1for the seed of transgenic corns, T
1seed continuation self propagated for transgenic corns obtains T
2seed for transgenic corns.The rest may be inferred, obtains T
6seed for transgenic corns.By T
6after planting seed for transgenic corns, obtain T
6for transgenic corn plant.
5, T
6evaluation for transgenic corn plant
To T
6for transgenic corn plant, carry out PCR evaluation, specific as follows:
First, extract respectively T
6for transgenic corn plant (proceed to mG2-aroA gene plant, proceed to the plant of G2-aroA gene, and the plant that proceeds to pS3300-UG0 empty carrier) genomic dna, concrete operations are as follows:
1) choose transgenic corns regeneration plant young leaflet tablet 0.1-0.2g, in liquid nitrogen grinding, be transferred in the Eppendorf pipe of 1.5ml;
2) add CTAB solution (Tris final concentration 100mM, NaCl final concentration 1.4M, the EDTA final concentration 20mM of 0.7ml, CTAB final concentration 2% (w/v), mercaptoethanol final concentration 0.1% (v/v)), 60 ℃, 45 minutes, every 10 minutes, put upside down and mix once.
3) add the phenol of 0.7ml: chloroform (volume ratio is 1: 1), put upside down several times, centrifugal 5 minutes of 1000rpm, shifts supernatant to new centrifuge tube.Add isopyknic chloroform: primary isoamyl alcohol (volume ratio is 24: 1), mix, centrifugal 5 minutes of 1000rpm, shifts the new centrifuge tube of supernatant to.
4) at centrifuge tube, add isopyknic Virahol, put upside down and mix, centrifugal 10 minutes of 1000rpm, abandons supernatant, with 70% ethanol, washes once, drains, and is dissolved in the sterilized water of 50 μ L, for PCR, detects.
Secondly, with the T of said extracted
6for transgenic corn plant (proceed to mG2-aroA gene plant, proceed to the plant of G2-aroA gene, and the plant that proceeds to pS3300-UG0 empty carrier) genomic dna be template, carry out PCR evaluation.Concrete operations are as follows:
1) 11 strains are proceeded to the T of G2-aroA gene
6for transgenic corn plant, carry out the detection of original gene (G2-aroA), simultaneously to proceed to the T of pS3300-UG0 empty carrier
6for transgenic corn plant, as negative control 1, with the negative control 2 of genetically modified corn not, take that not add the reaction system of template be blank.Pcr amplification primer is as follows:
Forward primer: CGGCTCCAAATCCATTACCAA (the 147-167 position of sequence 1)
Reverse primer: GCCACTTCAATCGGCGCTTC (reverse complementary sequence of the 631-650 position of sequence 1)
Reaction system (20 μ L): DNA 1 μ L (20-50ng); 10 * damping fluid, 2 μ L; MgCl
2(2.5mM) 2 μ L; DNTP (2.5mM) 2 μ L; Taq enzyme 0.2 μ L; Primer 10 μ M; Add sterilized water to 20 μ L.Reaction conditions: 94 ℃ 5 minutes; 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minute, 35 circulations; 72 ℃ are extended 5 minutes.Amplified production length 610bp.
As shown in Figure 7,11 strains shown in swimming lane 5-15 proceed to the T of G2-aroA gene to detected result
6for transgenic corn plant, all increasing, to obtain size be the object band of 610bp; And negative control group (negative control 1 and negative control 2) and blank group all do not amplify object band.The result shows that G2-aroA gene has been integrated into T
6in genome for G2-aroA transgenic corns.
2) 20 strains are proceeded to the T of mG2-aroA gene
6for transgenic corn plant, be optimized the detection of rear gene (mG2-aroA), simultaneously to proceed to the T of pS3300-UG0 empty carrier
6for transgenic corn plant, as negative control 1, using not genetically modified milpa as negative control 2, using and do not add the reaction system of template as blank.
Forward primer: CCACCTGGCTCCAAGTCTATCA (the 142-163 position of sequence 2)
Reverse primer: GCGTCAACCTGTGCTCCAAA (reverse complementary sequence of the 715-743 position of sequence 2)
Reaction system is the same.94 ℃ of reaction conditionss 5 minutes; 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 45 seconds, 35 circulations; 72 ℃ are extended 5 minutes.Amplified production length 593bp.
As shown in Figure 8,20 strains shown in swimming lane 5-24 proceed to the T of mG2-aroA gene to detected result
6for transgenic corn plant, all increasing, to obtain size be the object band of 593bp; And negative control group (negative control 1 and negative control 2) and blank group all do not amplify object band.The result shows that mG2-aroA gene has been integrated into T
6in genome for mG2-aroA transgenic corns.
The double antibodies sandwich ELISA of embodiment 3, transgenic corn plant G2-aroA protein expression detects
Sample extracting solution: Tris-Cl (pH8.0) 25mM, KCl 10mM, MgCl
26H
2o 20mM, DTT 1mM, PMSF 1mM (with front adding).
Coated damping fluid: get 1.5g Na
2cO
3, 2.93g NaHCO
3, with distilled water, be settled to 1000mL, pH9.6.
Washings (PBST): get 1mL polysorbas20, add phosphate buffered saline buffer (PBS) and be settled to 1000mL, pH7.5; Phosphate buffered saline buffer (PBS): get 8.0g NaCl, 0.2g KH
2pO
4, 2.96g Na
2hPO
412H
2o, adds 1000mL distilled water, pH7.5.
Sample buffer (PBST): get 1mL polysorbas20, add phosphate buffered saline buffer (PBS) and be settled to 1000mL, pH7.5; Phosphate buffered saline buffer (PBS): get 8.0g NaCl, 0.2g KH
2pO
4, 2.96g Na
2hPO
412H
2o, adds 1000mL distilled water, pH7.5.
Substrate buffer solution: get 0.1g MgCl
2or 0.2g MgCl
26H
2o, 97.0mL diethanolamine, is dissolved in 1000mL distilled water, pH9.8,4 ℃ of preservations.
Stop buffer: 3mol/L NaOH, pH12.0.
Confining liquid: get 3g bovine serum albumin (BSA) and be dissolved in the coated damping fluid of 100mL, coated damping fluid (carbonate is coated with damping fluid): get 1.5g Na
2cO
3, 2.93g NaHCO
3, with distilled water, be settled to 1000mL, pH9.6.
1, sample preparation
Choose the T of isometric growth stage (five leaf phases)
6for transgenic corn plant (comprising the plant that proceeds to mG2-aroA gene and the plant that proceeds to G2-aroA gene), respectively get functional leaf (upper three leaves) blade 1g (fresh weight) left and right, after liquid nitrogen grinds, proceed to and in 10ml centrifuge tube, add 3ml sample extracting solution, strenuous vibration, 4 ℃ of centrifugal 1h, get supernatant and are testing sample, standby.According to the difference of transgene, testing sample has two kinds, from the testing sample (testing sample-mG2-aroA) of mG2-aroA transfer-gen plant with from the testing sample (testing sample-G2-aroA) of G2-aroA transfer-gen plant.The T that proceeds to pS3300-UG0 empty carrier is set simultaneously
6for milpa and not genetically modified milpa in contrast.
Above-mentioned fresh weight for by blade with adding the centrifuge tube that extracting solution is housed to weigh after liquid nitrogen grinding powdered sample to deduct the centrifuge tube that extracting solution is housed to add the weight before powdered sample.
2, sample determination
Concrete operation step is as follows:
(1) coated: the primary antibodie of the anti-G2-aroA albumen that is 1.5mg/ml by concentration (rabbit is anti-) (preparation method is shown in place, article end) is diluted with coated damping fluid, after the dilution proportion that is 1: 1000 according to the volume ratio of the polyclonal antibody of anti-G2-aroA albumen and coated damping fluid, join in enzyme plate, every hole 100 μ L, 4 ℃ of wet box endoperidiums spend the night.
(2) wash plate: coating buffer is removed, be then put into wash in plate machine and wash plate 5 times with washings.
(3) sealing: every hole adds 120 μ L confining liquids, hatches 1h for 37 ℃ in wet box, then gets rid of confining liquid.
(4) reaction: on the one hand, it is 10 μ g/mL that G2-aroA protein standard substance (preparation method is shown in place, article end) is diluted to concentration with sample diluting liquid, and then 11 gradients of 2 doubling dilution (comprising 0 hole), repeat 3 times, every hole 100 μ L.0 hole (do not add standard solution and add 100 μ L sample diluting liquids) hole in contrast wherein, all the other 10 gradient pores are as test holes.Put at 37 ℃ incubation 1h.For drawing standard curve.
On the other hand, by the testing sample of step 1 preparation with sample buffer according to the dilution proportion of 1: 5 after every hole add 100 μ L, put at 37 ℃ incubation 1h.For detection of.
(5) wash plate: same step (2).
(6) add enzyme labelled antibody: by 1: 1000 use sample diluting liquid, dilute the enzyme labelled antibody (mouse monoclonal antibody) (preparation method is shown in place, article end) of anti-G2-aroA albumen, mix rear every hole and add 100 μ L.Put at 37 ℃ incubation 1h.
(7) wash plate: same step (2).
(8) add substrate colour developing: take 30mg nitrophenol Di-Sodium Phosphate (PNPP) and join in 30ml substrate buffer solution (after having prepared, 10 minutes in use), every hole 100 μ L, add 50 μ L stop buffer termination reactions after 20min.
(9) measure: under 405nm, measure OD value.
(10) drawing standard curve: using the G2-aroA protein standard substance strength of solution (ng/mL) of different concns as X-axis, the step (9) of usining measure gained G2-aroA protein standard substance OD value as Y-axis, use EXCEL drawing standard curve.
(11) the typical curve equation of the OD value substitution above-mentioned steps (10) of the testing sample of step (9) measurement gained being drawn, calculates G2-aroA protein content in testing sample.
G2-aroA albumen accounts for volume/sample fresh weight of G2-aroA protein content * testing sample in the content=testing sample of fresh weight, unit: μ g/g
3 repetitions are established in experiment, get the mean value of three experimental results, the typical curve obtaining (Figure 10), and its typical curve equation is y=9035.2x+198.75 (R
2=0.9997).
The detected result of testing sample is as shown in table 2, T
6for mG2-aroA transgenic corns and T
6for the expression amount of G2-aroA albumen in G2-aroA transgenic corns all apparently higher than the transgenic corns (mean value 0.674 μ g/g) that proceeds to empty carrier.And the G2-aroA gene (mG2-aroA) after optimization is at T
6will be far above original G2-aroA gene (G2-aroA) at T for expressing G2-aroA protein content (mean value 15.057 μ g/g) in transgenic corns
6for expressing G2-aroA protein content (mean value 3.735ug/g) in transgenic corns.The expression amount of G2-aroA albumen and the T that proceeds to pS3300-UG0 empty carrier in not genetically modified milpa
6basically identical for milpa, without significant difference.This result shows, after codon is optimized, the expression amount of G2-aroA albumen in transgenic corns is significantly improved.
The ELASA detected result of table 2 transfer-gen plant G2-aroA protein expression
Note: numbering 1,2,3,4 four different plants that represent respectively from same testing sample; The μ g/g of G2-aroA protein concentration unit represents the micrograms of every gram of blade (fresh weight) G2-aroA albumen of surveying.
1, test materials: T
6for transgenic corn plant, comprise proceed to mG2-aroA gene plant, proceed to the plant of G2-aroA gene, and the plant that proceeds to pS3300-UG0 empty carrier.Not genetically modified milpa contrast is set simultaneously.
2, capable long, the 3 row districts of test design: 5M, repeat density: 60 * 35cm for 3 times
3, test is processed: according to the consumption of 800ml/ mu, spray agriculture and reach (Roundup, containing 41% glyphosate, field recommendation dosage is 150-250ml/ mu).
4, test toeatment period: 5-6 leaf phase.After 7 days, start observation experiment result.
5, test-results
As shown in Fig. 9 (spraying agriculture reached after 15 days): (1) proceeds to the plant of G2-aroA gene, in the agriculture of 800ml/ mu, reach after processing, poisoning is very serious, and plant all shows yellow, deformity, in the plant strain growth phase in later stage undifferentiated, the female fringe of most of performance tassel do not weave silk or plant short and small; (2) proceed to the plant of mG2-aroA gene, in the agriculture of 800ml/ mu, reach after processing, without significantly poisoning, act normally, in the vegetative period in later stage all without phytotoxicity reaction; (3) proceed to the plant of pS3300-UG0 empty carrier and not genetically modified milpa poisoning is very serious, all plant are all dead.This result shows, proceeds to the corn of the mG2-aroA gene after codon optimized, and its tolerance to glyphosate is significantly improved.
(1) preparation method of the primary antibodie of the anti-G2-aroA albumen in embodiment 3 (rabbit is anti-) is as follows:
New Zealand's large ear rabbit that the G2-aroA albumen (preparation method is shown in following step (two)) of take after purifying is the about 2kg of immunogen immune body weight, the extraction of process serum and antibody purification step obtain the polyclonal antibody of anti-G2-aroA albumen, and concrete steps are as follows: utilize the method identical with embodiment 1 step 1 to prepare immunogen; By the immunogen obtaining, New Zealand's large ear rabbit of the about 2kg of body weight is carried out to immunity; Separation of serum is prepared the polyclonal antibody of anti-G2-aroA albumen afterwards.The polyclonal antibody of described anti-G2-aroA albumen can be used for when double antibodies sandwich method detects G2-aroA albumen using as coated antibody.
(2) in embodiment 3, the preparation method of G2-aroA protein standard substance is as follows:
DNA fragmentation shown in sequence 1 is connected in the multiple clone site of prokaryotic expression carrier pET-28a (+), obtains expressing the prokaryotic expression carrier pET-28a-G2-aroA of G2-aroA albumen.PET-28a-G2-aroA is transformed in e. coli bl21, with IPTG, induce it to express G2-aroA albumen, collect after thalline, thalline is passed through to ultrasonic disruption, by expressed protein delivery in extracting solution, after use His label protein purification kit (purchased from Kang Wei century bio tech ltd, Beijing, catalog number (Cat.No.) CW0009A) purifying, after G2-aroA albumen is purified, carry out SDS-PAGE electrophoresis detection.
SDS-PAGE electrophoresis qualification result as shown in figure 11.Through page glue, identify, purity can reach 95%, and through eppendorf protein nucleic acid determinator biophotometer plus, measuring concentration is 1.3mg/ml, and the concentration using the G2-aroA albumen after purifying as immunogen S780G2-aroA albumen reaches 1.3mg/ml.G2-aroA albumen after purifying is immunogen S780.
The aminoacid sequence of G2-aroA albumen is sequence 9 in sequence table.
(3) in embodiment 3, the preparation method of the enzyme labelled antibody of anti-G2-aroA albumen (mouse monoclonal antibody) is as follows:
Balb/C mouse: Beijing dimension tonneau China laboratory animal company limited
Sp2/0: Kang Wei century bio tech ltd, Beijing
1, the preparation of immunogen S780
Concrete grammar and step are with the preparation method of G2-aroA protein standard substance in (two) embodiment 3.G2-aroA albumen after purifying is immunogen S780.The aminoacid sequence of G2-aroA albumen is sequence 9 in sequence table.
2, animal immune
The female Balb/C mouse in 56 week ages of take is experimental animal, according to following immune programme for children and flow process, carries out immunity:
Before a, immunity: adopt docking blood-collecting method to gather serum, as negative control.
B, initial immunity: the G2-aroA protein solution that by concentration is 0.64 μ g/ μ l adds equal-volume Freund's complete adjuvant after sterilizing filter filters, and by the abundant stirring and emulsifying of magnetic stirring apparatus, until splash into indiffusion in water, obtains immunogen.The method immunity Balb/C mouse that adopts the subcutaneous multi-point injection in back by the good immunogen of emulsification, injected dose is the G2-aroA albumen (the good immunogen of 250 μ l emulsification) of every injected in mice 80 μ g.
C, booster immunization for the first time: initial immunity is after 21 days, the G2-aroA protein solution that by concentration is 0.32 μ g/ μ l adds equal-volume Freund's incomplete adjuvant after sterilizing filter filters, by the abundant stirring and emulsifying of magnetic stirring apparatus, until splash into indiffusion in water, obtain immunogen.The method immunity Balb/C mouse that adopts the subcutaneous multi-point injection in back by the good immunogen of emulsification, injected dose is the G2-aroA albumen (the good immunogen of 250 μ l emulsification) of every injected in mice 40 μ g.
D, booster immunization for the second time: booster immunization, after 14 days, carries out booster immunization for the second time for the first time.Concrete grammar is with the booster immunization for the first time of step c.
E, whole booster immunization: booster immunization after 14 days for the second time, by concentration, be the G2-aroA protein solution of 0.8 μ g/ μ l after sterilizing filter filters, obtain immunogen.With gained immunogen intrasplenic injection booster immunization Balb/C mouse, injected dose is the G2-aroA albumen (immunogens of 100 μ l) of every injected in mice 80 μ g.
3, cytogamy and cloning
At booster immunization for the second time, after 14 days, docking blood-collecting method gathers serum, and with indirect elisa method (take S780 as envelope antigen) mensuration serum titer.After the whole booster immunization of above-mentioned steps one the 3rd day, select above-mentioned indirect elisa method to measure mouse (tire is 1: the 720000) extracting spleen cell of serum titer the best, again by splenocyte and SP2/0 myeloma cell in the ratio of 9: 1 (quantitative proportion), by the conventional fusion method of PEG (PEG4000), carry out cytogamy.
The liquid that changes for the 3rd day and the 6th day after fusion is processed, and within the 7th day, adopts indirect elisa method (take S780 as envelope antigen) to screen fused cell supernatant, chooses positive cell hole, and hole inner cell is proceeded to respectively to 24 well culture plate amplification cultivation.When cell grows to field of microscope 1/3, collecting cell supernatant liquor, adopt indirect elisa method (usining respectively S780, K8 (Meng Shandou (Mosanto) glyphosate tolerant material mon810 extracting solution), positive (glyphosate tolerant material extraction liquid), negative (non-transgenic material extraction liquid) as envelope antigen) to carry out multiple sieve, therefrom selection and S780 and positive reaction are all stronger, simultaneously, with the equal unreacted cell (5F11 of the runic in table 3) of K8 and feminine gender, application limiting dilution assay carries out subclone.Through 3 subclones, finally obtain the monoclonal hybridoma strain of the anti-G2-aroA albumen of stably excreting, called after AntiG2-5F11.This hybridoma cell strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 7th, 2012 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.5772.
The above-mentioned K8 as envelope antigen (Meng Shandou (Mosanto) glyphosate tolerant material mon810 extracting solution), positive (my company's glyphosate tolerant material extraction liquid), negative (non-transgenic material extraction liquid) are specifically prepared as follows and obtain: get 0.3g specimen material (Meng Shandou (Mosanto) glyphosate tolerant material mon810, or the T that obtains of embodiment 2
6for mG2-aroA transgenic corns, or not genetically modified corn variety combines 31), after liquid nitrogen grinds, proceed in 2ml centrifuge tube and add 1ml sample extracting solution, strenuous vibration, 4 ℃ are extracted 1h, get supernatant after centrifugal 10 minutes for 12000 revs/min and carry out sample determination.Wherein, sample extraction liquid formula is: Tris-Cl (pH8.0) 25mm; KCl 10mm; MgCl
26H
2o 20mm; DTT 1mm; PMSF 1mm (with front adding).
The step of above-mentioned indirect elisa method is specific as follows:
1) coated: in 96 hole enzyme plates, add the antigenic solution of the 2 μ g/mL of 100 μ L, the not contrast of envelope antigen is set simultaneously, 4 ℃ of coated spending the night, with PBS damping fluid washing 3 times.
Above-mentioned antigenic solution can be S780, K8, the positive and negative solution.
2) sealing: add the confining liquid (3% bovine serum albumin) in 150 μ L/ holes, hatch 2h at 37 ℃, abandon confining liquid, wash 3 times, pat dry.Be placed in 4 ℃ of Refrigerator stores standby.
3) add testing sample:
A. for serum titer, detect, the dilution in 1: 1000 of first hole, down, with the gradient doubling dilution of 1: 3, hatches 30min for 37 ℃, washes plate 4 times, pats dry.
B. for cell conditioned medium, detect, draw cell conditioned medium 100 μ l, add in corresponding enzyme plate, hatch 30min for 37 ℃, wash plate 4 times, pat dry.
Contrast without immune mice serum/antibody/ascites/cell conditioned medium is set simultaneously; With PBS, replace the contrast (negative control hole) of detected sample.
4) add ELIAS secondary antibody: get goat anti-mouse IgG (H+L)-HRP, after 1: 5000 times of dilution, 100 μ l/ holes, hatch 20 to 30min for 37 ℃, wash 4 times, pat dry.
5) colour developing: 20 * TMB is diluted to 1 * TMB, adds by 100 μ l/ holes, 37 ℃ of colour developing 15-30min.
6) stop: add stop buffer (2M H
2sO
4) 50 μ l/ holes.
7) reading: measure each hole OD value with the mono-wavelength of 450nm, be greater than 2.1 with the ratio (P/N) with negative control hole (replacing contrasting of testing sample with PBS) OD value and be limited, as being judged as the positive or determining the stagnation point of tiring.
ELISA result decision method: during (1) screening positive cell, if P/N > 2.1 differentiates positive cell; If 1 < P/N < 2.1, detects after strengthening coated concentration again, P/N > 2.1 is still judged to the positive.(2) measure while tiring, with serum (or ascites or antibody) the maximum dilution multiple of P/N > 2.1, represent.
Table 3 adopts indirect elisa method fused cell supernatant to be carried out to the result of multiple sieve
| Envelope antigen | 1C2 | 1C11 | 1E6 | 2C1 | 2F7 | 3D12 | 3F9 | 4B11 | 5B2 | 5F11 | PBS | Positive |
| S780 | 2.398 | 2.463 | 2.655 | 2.232 | 2.067 | 2.332 | 3.431 | 3.400 | 3.474 | 3.452 | 3.489 | 3.470 |
| K8 | 0.048 | 0.057 | 0.044 | 0.049 | 0.041 | 0.053 | 0.048 | 0.045 | 0.050 | 0.046 | 0.053 | 0.104 |
| Positive | 0.297 | 0.162 | 0.398 | 0.289 | 1.424 | 0.245 | 1.762 | 0.920 | 0.393 | 2.427 | 0.068 | 1.291 |
| Negative | 0.050 | 0.046 | 0.041 | 0.045 | 0.043 | 0.045 | 0.067 | 0.043 | 0.045 | 0.044 | 0.057 | 0.052 |
Note: 1C2-5F11 represents respectively the positive cell that adopts indirect elisa method (take S780 as envelope antigen) to be screened on the 7th day after cytogamy.Light absorption value is larger, illustrates that antibody is higher to the avidity of antigen.
4, the preparation and purification of monoclonal antibody
A, increment culture method
Hybridoma cell strain AntiG2-5F11CGMCC No.5772 is placed in to RPMI 1640 substratum, cultivates 3 days collecting cell supernatant for 37 ℃.Adopt protein G affinity column (Pharmacia) to carry out purifying.Concrete operations are as follows:
(1) balance: steady to baseline with binding buffer liquid balance protein G affinity column.
(2) loading: by the cell conditioned medium upper prop of collecting, collect stream and wear liquid; Stream is worn to liquid upper prop again, continue balance steady to baseline.
(3) wash-out: add elution buffer wash-out, collect elution peak.
(4) elution peak of collecting with PBS (pH7.2) dialysis of 0.01M, is kept in the PBS (pH7.2) of 0.01M the antibody after purifying.
(5) concentration of monoclonal antibody after use protein quantification detector (Amersham Biosciences) mensuration purifying.
(6) antibody purity after SDS-PAGE detection purifying, antibody applied sample amount: 8 μ g.
B, ascites preparation
Get 2 BALB/C mice, every injection 0.5ml paraffin oil, gets hybridoma cell strain AntiG2-5F11CGMCC No.5772 and is resuspended in serum free medium, by 1 * 10 after 7 days
6individual cell/0.5ml/ only measures injection paraffin mouse, and injection cell was collected ascites after 14 days.Adopt indirect elisa method (envelope antigen is S780) to carry out titer of ascites detection, concrete operations and result decision method carry out according to a with (step 3 wherein) described in step 3), result shows that it is tired is 1: 81000.Adopt protein G affinity column (Phamacia) to carry out purifying to ascites.Concrete operations are with described in step 1.
5, the evaluation of monoclonal antibody
(1) evaluation of monoclonal antibody hypotype
Utilize the hypotype detection kit suit that Southern Biotech company produces (to comprise Goat anti Mouse IgG1-HRP (1070-50), Goat anti Mouse IgG2a-HRP (1080-05), Goat anti Mouse IgG2b-HRP (1090-05), Goat anti Mouse IgG3-HRP (1100-05), Goat anti Mouse IgA-HRP (1040-05), Goat anti Mouse IgM-HRP (1020-05), Goat anti Mouse kappa-HRP (1050-05) and Goat anti Mouse Lambda-HRP (1060-05) be totally 8 kinds of components) monoclonal antibody of above-mentioned steps 4 preparations is carried out to the detection of antibody subtype, envelope antigen is S780, concentration is 2 μ g/ml, package amount is 100 μ l, detection wavelength is 450nm.Specific operation process is carried out according to test kit specification sheets.Detected result is as shown in table 4, and the CH of monoclonal antibody is IgG1 type, and constant region of light chain is Lamda type.
The evaluation of table 4 monoclonal antibody hypotype
| IgG1 | IgG2a | IgG2b | IgG3 | IgM | IgA | Kappa | Lamda | |
| Monoclonal antibody | 2.252 | 0.051 | 0.048 | 0.058 | 0.055 | 0.056 | 0.065 | 0.461 |
(2) detection of antibody titer
Adopt indirect elisa method (envelope antigen is S780, K8, positive and negative, specifically with step 3) to step 4 gained purifying after monoclonal antibody carry out bioactivity, the alternative testing sample of the PBS solution of usining is as negative control.Concrete operations and result decision method carry out according to a with (wherein step 3) described in step 3).
Detected result is as shown in table 5, usings the positive as envelope antigen, and tiring of the monoclonal antibody of ascites purifying is 1: 81000 (0.292/0.083=3.518 > 2.1).
Table 5 antibody titer detected result
6, the SDS-PAGE glue of monoclonal antibody is identified its purity
By SDS-PAGE gel to step 4 gained purifying after monoclonal antibody carry out purity detecting, result as shown in figure 12, on gel, show two object bands, larger one is heavy chain above, below less one is light chain, and object band is clear, without assorted band, visible, after the purifying of step 4, monoclonal antibody has reached certain purity.
Claims (1)
1. cultivate a method for glyphosate tolerant transgenic corns, comprise glyphosate tolerant gene is imported to object corn, obtain expressing the step of the transgenic corns of described glyphosate tolerant gene; Described transgenic corns is compared with described object corn, and the tolerance of glyphosate is improved; Described glyphosate tolerant gene is the DNA molecular of sequence 2 in sequence table; Described is to complete by the recombinant dna fragment of expressing described glyphosate tolerant gene is imported to described object corn by glyphosate tolerant gene importing object corn; Described recombinant dna fragment imports described object corn by recombinant expression vector;
The sequence of described recombinant dna fragment is sequence 10 in sequence table;
The sequence of described recombinant expression vector is sequence 11 in sequence table.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210107400.3A CN102643804B (en) | 2012-04-12 | 2012-04-12 | Method for culturing roundup ready transgene corns |
| BR112014025529A BR112014025529A2 (en) | 2012-04-12 | 2013-04-12 | synthetic glyphosate resistance gene and its use |
| PCT/CN2013/000428 WO2013152624A1 (en) | 2012-04-12 | 2013-04-12 | Synthetic glyphosate-resistant gene and use thereof |
| US14/391,948 US20150353951A1 (en) | 2012-04-12 | 2013-04-12 | Synthetic glyphosate-resistant gene and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210107400.3A CN102643804B (en) | 2012-04-12 | 2012-04-12 | Method for culturing roundup ready transgene corns |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102643804A CN102643804A (en) | 2012-08-22 |
| CN102643804B true CN102643804B (en) | 2014-04-02 |
Family
ID=46656881
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210107400.3A Expired - Fee Related CN102643804B (en) | 2012-04-12 | 2012-04-12 | Method for culturing roundup ready transgene corns |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102643804B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BR112014025529A2 (en) * | 2012-04-12 | 2017-07-04 | Origin Agritech Ltd | synthetic glyphosate resistance gene and its use |
| CN103160529B (en) * | 2013-03-18 | 2014-10-15 | 中国农业科学院生物技术研究所 | Method for cultivating transgenosis hybrid rice |
| CN110195122A (en) * | 2019-01-02 | 2019-09-03 | 四川省农业科学院生物技术核技术研究所 | It is a kind of for detecting the nucleic acid sequence and its detection method of corn plant T anti-4 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6803501B2 (en) * | 2000-03-09 | 2004-10-12 | Monsanto Technology, Llc | Methods for making plants tolerant to glyphosate and compositions thereof using a DNA encoding an EPSPS enzyme from Eleusine indica |
| CN1720330A (en) * | 2002-12-12 | 2006-01-11 | 拜尔农科股份有限公司 | Expression cassette encoding a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and herbicide-tolerant plants containing it |
| CN100429311C (en) * | 2003-08-08 | 2008-10-29 | 四川禾本生物工程有限公司 | EPSP synzyme of high anti-cancrinia discoidea and its coding squence |
| CN101437843A (en) * | 2006-01-23 | 2009-05-20 | 密歇根州立大学评议会 | Methods for breeding glyphosate resistant plants and compositions thereof |
| CN101886088A (en) * | 2009-05-15 | 2010-11-17 | 北京大学 | Transgenic structure and transgenic plant |
-
2012
- 2012-04-12 CN CN201210107400.3A patent/CN102643804B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6803501B2 (en) * | 2000-03-09 | 2004-10-12 | Monsanto Technology, Llc | Methods for making plants tolerant to glyphosate and compositions thereof using a DNA encoding an EPSPS enzyme from Eleusine indica |
| CN1720330A (en) * | 2002-12-12 | 2006-01-11 | 拜尔农科股份有限公司 | Expression cassette encoding a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and herbicide-tolerant plants containing it |
| CN100429311C (en) * | 2003-08-08 | 2008-10-29 | 四川禾本生物工程有限公司 | EPSP synzyme of high anti-cancrinia discoidea and its coding squence |
| CN101437843A (en) * | 2006-01-23 | 2009-05-20 | 密歇根州立大学评议会 | Methods for breeding glyphosate resistant plants and compositions thereof |
| CN101886088A (en) * | 2009-05-15 | 2010-11-17 | 北京大学 | Transgenic structure and transgenic plant |
Non-Patent Citations (6)
| Title |
|---|
| Development and characterization of a CP4 EPSPS-based,Glyphosate-tolerant Corn Event;Heck等;《Corn Science》;20050130;第45卷(第1期);429-439 * |
| EU126547;登录号;《NCBI,Genbank》;20070919;全文 * |
| Heck等.DevelopmentandcharacterizationofaCP4EPSPS-based Glyphosate-tolerant Corn Event.《Corn Science》.2005 |
| 于金海.玉米同义密码子使用偏性分析.《黑龙江农业科学》.2010,(第7期),1-4. |
| 玉米同义密码子使用偏性分析;于金海;《黑龙江农业科学》;20101231(第7期);1,2 * |
| 登录号.EU126547.《NCBI,Genbank》.2007, |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102643804A (en) | 2012-08-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102643840B (en) | Recombinant DNA (deoxyribonucleic acid) fragment containing roundup ready gene and application thereof | |
| CN102676553B (en) | Artificially synthesized glyphosate-tolerant gene and application thereof | |
| CN102459315B (en) | DIG-3 insecticidal cry toxins | |
| Brar et al. | Expression of a single-chain variable-fragment antibody against a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in transgenic soybean plants | |
| WO2010025320A1 (en) | Novel hemipteran and coleopteran active toxin proteins from bacillus thuringiensis | |
| US20230183300A1 (en) | MODIFIED Cry1Ca TOXINS USEFUL FOR CONTROL OF INSECT PESTS | |
| CN102643848B (en) | Synthetic roundup ready gene expression vector and application thereof | |
| CN102643804B (en) | Method for culturing roundup ready transgene corns | |
| CN102645535B (en) | Method for detecting roundup ready protein G2-aroA and special enzyme linked immune kit | |
| CN104805106A (en) | Fusion gene containing transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) protective antigens as well as encoding protein and application thereof | |
| US6156954A (en) | Receptor-like protein kinase, RKN, and method of use for increasing growth and yield in plants | |
| US8557246B2 (en) | Fusion protein that directs vaccine antigens to antigen-presenting cells, and applications thereof | |
| CN102643786B (en) | Monoclonal antibody of glyphosate-resistant protein G2-aroA and application thereof | |
| WO2013091563A1 (en) | Methods for improving crop yield | |
| CA2754024A1 (en) | Antibody fusion-mediated plant resistance against oomycota | |
| CN102549149A (en) | Functional expression of yeast nitrate transporter (ynt1) in maize to improve nitrate uptake | |
| CN108396012B (en) | Application of monoclonal antibody 1DB4 in detection of IrrE transgenic crops | |
| CN106520721A (en) | High-glyphosate-resistance EPSP synthase and application of encoding gene thereof | |
| US20150353951A1 (en) | Synthetic glyphosate-resistant gene and use thereof | |
| US20080020464A1 (en) | Vascular plants expressing Na+ pumping ATPases | |
| Wai | Molecular Cloning and Expression of Mannose-binding Lectin from Chinese Herb, Yu Chu (Polygonatum Odoratum) in Rice | |
| Rowhani | Characterization of potato leafroll virus | |
| Nair et al. | Characterization of T | |
| Kawchuk | Molecular characterization of potato leafroll luteovirus and development of genetically engineered resistance |
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 | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140402 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |