CN1308345C - Gossypium barbadense lipoid transition protein and its coding gene and application - Google Patents
Gossypium barbadense lipoid transition protein and its coding gene and application Download PDFInfo
- Publication number
- CN1308345C CN1308345C CNB2005100982773A CN200510098277A CN1308345C CN 1308345 C CN1308345 C CN 1308345C CN B2005100982773 A CNB2005100982773 A CN B2005100982773A CN 200510098277 A CN200510098277 A CN 200510098277A CN 1308345 C CN1308345 C CN 1308345C
- Authority
- CN
- China
- Prior art keywords
- plant
- gene
- cotton
- protein
- ltp
- 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.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 84
- 240000000047 Gossypium barbadense Species 0.000 title claims abstract description 36
- 235000009429 Gossypium barbadense Nutrition 0.000 title claims abstract description 33
- 102000004169 proteins and genes Human genes 0.000 title abstract description 26
- 230000007704 transition Effects 0.000 title abstract 3
- 241000196324 Embryophyta Species 0.000 claims abstract description 117
- 229920000742 Cotton Polymers 0.000 claims abstract description 63
- 241000219146 Gossypium Species 0.000 claims abstract description 62
- 241000082085 Verticillium <Phyllachorales> Species 0.000 claims abstract description 50
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 19
- 244000061176 Nicotiana tabacum Species 0.000 claims abstract 2
- 108010053156 lipid transfer protein Proteins 0.000 claims description 86
- 238000000034 method Methods 0.000 claims description 36
- 239000013604 expression vector Substances 0.000 claims description 12
- 238000009396 hybridization Methods 0.000 claims description 11
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 240000007594 Oryza sativa Species 0.000 claims description 5
- 235000007164 Oryza sativa Nutrition 0.000 claims description 5
- 230000008676 import Effects 0.000 claims description 5
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims description 4
- 240000003768 Solanum lycopersicum Species 0.000 claims description 4
- 235000009566 rice Nutrition 0.000 claims description 4
- 235000021307 Triticum Nutrition 0.000 claims description 3
- 244000098338 Triticum aestivum Species 0.000 claims description 3
- 244000241235 Citrullus lanatus Species 0.000 claims description 2
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 claims description 2
- 244000068988 Glycine max Species 0.000 claims description 2
- 235000010469 Glycine max Nutrition 0.000 claims description 2
- 244000061458 Solanum melongena Species 0.000 claims description 2
- 235000002597 Solanum melongena Nutrition 0.000 claims description 2
- 241000219793 Trifolium Species 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 29
- 230000009261 transgenic effect Effects 0.000 abstract description 29
- 125000000539 amino acid group Chemical group 0.000 abstract description 14
- 230000009465 prokaryotic expression Effects 0.000 abstract description 11
- 241000219194 Arabidopsis Species 0.000 abstract description 9
- 150000003904 phospholipids Chemical class 0.000 abstract description 9
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 8
- 238000012214 genetic breeding Methods 0.000 abstract description 2
- 201000010099 disease Diseases 0.000 description 28
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 28
- 239000002299 complementary DNA Substances 0.000 description 21
- 108700019146 Transgenes Proteins 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 18
- 239000003053 toxin Substances 0.000 description 18
- 241000208125 Nicotiana Species 0.000 description 17
- 235000018102 proteins Nutrition 0.000 description 17
- 231100000765 toxin Toxicity 0.000 description 17
- 108700012359 toxins Proteins 0.000 description 17
- 208000035240 Disease Resistance Diseases 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 15
- 238000012216 screening Methods 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 13
- 239000000047 product Substances 0.000 description 13
- 230000009182 swimming Effects 0.000 description 13
- 101150054956 LTP gene Proteins 0.000 description 11
- 241000588724 Escherichia coli Species 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 239000013598 vector Substances 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 239000003550 marker Substances 0.000 description 9
- 239000012528 membrane Substances 0.000 description 9
- 241000573739 bacterium V Species 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 230000002018 overexpression Effects 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 240000008042 Zea mays Species 0.000 description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 7
- 235000005822 corn Nutrition 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 239000002689 soil Substances 0.000 description 7
- 102100035785 Acyl-CoA-binding protein Human genes 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 240000002024 Gossypium herbaceum Species 0.000 description 6
- 235000004341 Gossypium herbaceum Nutrition 0.000 description 6
- 230000002209 hydrophobic effect Effects 0.000 description 6
- 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 6
- 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 6
- 150000002632 lipids Chemical class 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 5
- 102000003867 Phospholipid Transfer Proteins Human genes 0.000 description 5
- 108090000216 Phospholipid Transfer Proteins Proteins 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 101710106492 Acyl-CoA-binding protein Proteins 0.000 description 4
- 101710169323 Acyl-CoA-binding protein homolog Proteins 0.000 description 4
- 101710083587 Antifungal protein Proteins 0.000 description 4
- 108010022172 Chitinases Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 108010015776 Glucose oxidase Proteins 0.000 description 4
- 239000004366 Glucose oxidase Substances 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 4
- 101710113369 Putative acyl-CoA-binding protein Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000002105 Southern blotting Methods 0.000 description 4
- 244000052616 bacterial pathogen Species 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 229940116332 glucose oxidase Drugs 0.000 description 4
- 235000019420 glucose oxidase Nutrition 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000006920 protein precipitation Effects 0.000 description 4
- 238000010257 thawing Methods 0.000 description 4
- 241000219195 Arabidopsis thaliana Species 0.000 description 3
- 240000005979 Hordeum vulgare Species 0.000 description 3
- 235000007340 Hordeum vulgare Nutrition 0.000 description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 description 3
- 244000300264 Spinacia oleracea Species 0.000 description 3
- 235000009337 Spinacia oleracea Nutrition 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 238000001467 acupuncture Methods 0.000 description 3
- 125000002252 acyl group Chemical group 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 230000000843 anti-fungal effect Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 244000037666 field crops Species 0.000 description 3
- 230000001408 fungistatic effect Effects 0.000 description 3
- 230000000968 intestinal effect Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 230000002103 transcriptional effect Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 235000018322 upland cotton Nutrition 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 244000291564 Allium cepa Species 0.000 description 2
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 description 2
- 102000012286 Chitinases Human genes 0.000 description 2
- 108010039287 Diazepam Binding Inhibitor Proteins 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 108091060211 Expressed sequence tag Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000305492 Gastrodia Species 0.000 description 2
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 101000763602 Manilkara zapota Thaumatin-like protein 1 Proteins 0.000 description 2
- 101000763586 Manilkara zapota Thaumatin-like protein 1a Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 101000966653 Musa acuminata Glucan endo-1,3-beta-glucosidase Proteins 0.000 description 2
- 235000019082 Osmanthus Nutrition 0.000 description 2
- 241000333181 Osmanthus Species 0.000 description 2
- 240000000528 Ricinus communis Species 0.000 description 2
- 235000004443 Ricinus communis Nutrition 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 101150054900 gus gene Proteins 0.000 description 2
- YQYJSBFKSSDGFO-FWAVGLHBSA-N hygromycin A Chemical compound O[C@H]1[C@H](O)[C@H](C(=O)C)O[C@@H]1Oc1ccc(\C=C(/C)C(=O)N[C@@H]2[C@@H]([C@H]3OCO[C@H]3[C@@H](O)[C@@H]2O)O)cc1O YQYJSBFKSSDGFO-FWAVGLHBSA-N 0.000 description 2
- 238000012613 in situ experiment Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 150000003905 phosphatidylinositols Chemical class 0.000 description 2
- 229940067626 phosphatidylinositols Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000000452 restraining effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 239000005418 vegetable material Substances 0.000 description 2
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 1
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 1
- 244000132059 Carica parviflora Species 0.000 description 1
- 235000014653 Carica parviflora Nutrition 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000701515 Commelina yellow mottle virus Species 0.000 description 1
- 241001573881 Corolla Species 0.000 description 1
- 229920000832 Cutin Polymers 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 108700003861 Dominant Genes Proteins 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 101150090421 GO gene Proteins 0.000 description 1
- 241000305491 Gastrodia elata Species 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- 241001327145 Halobaena caerulea Species 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 240000005049 Prunus salicina Species 0.000 description 1
- 241000918585 Pythium aphanidermatum Species 0.000 description 1
- 101000697856 Rattus norvegicus Bile acid-CoA:amino acid N-acyltransferase Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 1
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 1
- 244000105017 Vicia sativa Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 102220369446 c.1274G>A Human genes 0.000 description 1
- 229940027138 cambia Drugs 0.000 description 1
- 235000019994 cava Nutrition 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 239000000287 crude extract Substances 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
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 230000008641 drought stress Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000009018 li Nutrition 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 208000028284 monogenic inheritance Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 108010064343 plant lipid transfer proteins Proteins 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 230000010153 self-pollination Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention discloses a gossypium barbadense lipoid transition protein, a coding gene thereof and applications of the protein, which aims at providing a gossypium barbadense lipoid transition protein, a coding gene thereof and applications of the gene to the cultivation of plants resisting verticillium. The protein is a protein with one of the following amino acid residue sequences: 1) SEQ ID No. 1 in a sequence table and 2) a protein formed by replacing or deleting or adding one to ten amino acid residue in the amino acid residue sequence of the SEQ ID No. 1 in the sequence table, wherein the protein in 2) performs a phospholipid transportation function. A gene prokaryotic expression product of the present invention has stronger bacteriostatic activity for verticillium bacteria of cotton. Transgenic tobacco, arabidopsis and cotton all present stronger resistance capability for verticillium bacteria. The protein and the coding gene of the present invention play an important role on the genetic breeding of plants (cotton in particular) resisting verticillium.
Description
Technical field
The invention belongs to the biological high-technology field of screening, separation, transgenosis and the functional study thereof of plant disease resistance genes, particularly relate to the encoding gene and the functional study thereof of a kind of lipid transfer protein of sea island cotton, with the application of this gene in cultivating the resisting verticillium plant.
Background technology
Verticillium is one of important disease that threatens Cotton Production, because of take place year after year, prevents and treats difficulty, has now become the major obstacle that influences output of cotton and quality.According to statistics, the onset area of Cotton in China verticillium has reached the over half of whole cotton field at present.Cultivating disease-resistant variety is the most economical means of this disease of control.After deliberation, have high resisting verticillium gene in the cotton seeds such as sea island cotton, but facts have proved and utilize common cross-breeding means to be difficult to obtain the kind of stable high disease-resistant and high yield.The method of modern molecular genetics is that the separation and the transgenosis of disease-resistant gene provided convenience, and separates, obtained some disease-resistant related genes at present both at home and abroad, has also utilized some anti-fungal genes that cotton has been carried out transgenic research simultaneously.
Existing both at home and abroad anti-fungal gene is transformed into report in the cotton, the gene that transforms comprises: chitinase, β-1,3-dextranase, glucose oxidase (GO), rhizoma Gastrodiae antifungal protein genes such as (gastrodia antifungalprotein are called for short GAFP).Result of study is analyzed, and upland cotton is after transforming said gene, and the resistance to verticillium wilt of transfer-gen plant can obtain raising to a certain degree, even high anti-plant or strain occur, but does not see the high resisting verticillium variety popularization of typing as yet.These genes all belong to the broad-spectrum antimicrobial gene in addition, are not special resistant gene at verticillium wilt pathogen, also are not from disease-resistant cotton self.
Liu Huijun etc. by to change glucose oxidase (GO) gene cotton plant be B99261 (IV, VII generation) and B99267 (VIII, IV generation) with the new land of acceptor early No. 7 and anti-verticillium contrast middle cotton 12 economical character and disease resistance compare.The transgene cotton plant type becomes short as a result, and carpopodium is elongated, and single-strain bell-forming number and fibrous quality are significantly improved.B99261 to seedling stage anthrax, damping-off and bell eqpidemic disease resistance all be significantly increased but not anti-red rot; B99267 also significantly strengthens the resistance of cotton boll blight.These two strains all are significantly increased to the resistance of blight and verticillium, and wherein the 4th generation B99261 resisting verticillium performance is best, and (Liu Huijun, simple osmanthus is good, Zou Yafei to reach disease-resistant level.The GO gene imports the influence to cotton economical character and disease resistance, Molecular Plant Breeding,, the 1st volume, the 5/6th phase, 669-672 page or leaf in 2003).
Liu Guizhen etc. pass through the laser microbeam puncture method with β-1, the two valency plant expression vector pBLGC of 3-dextranase and chitinase gene import cotton immature embryos, transform generation cotton seedling, and carry out resisting verticillium screening with dipping in the root method, the seedling of survival is moved into the disease garden carried out kantlex (Kan 1%) resistant determination.The result has 9 strains to show tangible Kan resistance in the 29 strain seedling of transplanting, and the anti-Kan plant of 9 strains has been carried out the PCR detection, and wherein 7 strains show as the positive.T1 in the sick garden is experiencing verticillium onset peak after date for transfer-gen plant, and 7 strain PCR positive plants are obviously disease-resistant, and the knot bell of normally blooming, and other T1 is all dead because of the later stage morbidity for plant and adjoining tree.Preliminary proof foreign gene has been incorporated in the cotton gene group, and the render transgenic plant shows certain resistance (Liu Guizhen to verticillium, blue petrel, Yingchuan, field, Japanese plum is sincere, happy brocade is magnificent, Wang Lanlan, Chen Zhenghua utilizes the laser microbeam puncture method to obtain the research of resisting verticillium transgene cotton, Chinese laser, 27 (3): 279-283).Happy bright and beautiful China etc. with pollen tube passage method with bean chitinase gene and tobacco beta-1,3-glucanase Gene Double valency plant expression vector pBLGC importing Xinjiang cotton main breed (being) be 550,822,1304, stone is far away by 321.By kalamycin resistance detection, PCR, Southern hybridization checking and resisting verticillium, withered characteristic of disease screening, obtain the transgene cotton of the good and genetic stability of disease resistance.Through the seed selection of 6 year 10 generation, breed the disease-resistant new lines of transgenosis (Le Jinhua, the Zhu Jianbo of anti-blight, anti-(anti-) verticillium, Cui Baiming, Zhu Xinxia, Wang Aiying, Chen Zhenghua, Liu Gui, Li Guoying, simple osmanthus is good, Wu Gang, utilize the goal gene transformation technology to cultivate the cotton disease resistance new variety, Shihezi Univ's journal (natural science edition), the 6th rolls up in September, 2002 the 3rd phase).
Rhizoma Gastrodiae antifungal protein (gastrodia antifungal protein is called for short GAFP) is a kind of protein with broad-spectrum antifungal activity that is separated to from China's traditional Chinese medicine rhizoma Gastrodiae (Gastrodia elata Bl.), therefore it comprises that to many fungal diseases of plants the pathogenic bacterium of cotton wilt, verticillium etc. have very strong stripped restraining effect, and having very in plant antimycotic genetic engineering modified, important use is worth.Wang Yiqin etc. pass through pollen tube passage method, the gene gafp of coding GAFP is changed in the colored cotton variety in 3 Xinjiang, by disease-resistant screening in field and Molecular Detection, obtained the transfer-gen plant of high resisting verticillium, two strain Southern hybridization positive plant LB-5-8 and ZB-1-49 are to the whole strain immunity of verticillium performance.RT-PCR result shows that correctly transcribing of gafp all arranged among LB-5-8 and the ZB-1-49; The bacteriostatic experiment that exsomatizes shows that also their protein crude extract administration has the obvious suppression effect to the cotton verticillium wilt pathogenic bacterium, shows that gafp correctly expresses in transfer-gen plant, and translation product has activity.Numerous through further seed selection and expansion, find that the colored cotton offspring of transgenosis has stable, stronger resisting verticillium ability, this research provides a new approach (Wang Yiqin for the method by genetic engineering of plant for disease resistance for the control cotton verticillium wilt, old main forces, danger common vetch dawn, Wu Minggang, Wang Dongmei, Yao Zhengpei, Huang Quansheng, Liu Fengjiang, beautiful ancient sharp, Li Renjing, Sun Yongru, rhizoma Gastrodiae antifungus protein gene (gafp) transforms STUDY ON COLOURED COTTON, BULLETIN OF BOTANY Vol. 2003,20 (6): 703-712).
Wu Jia passes through crown gall bacillus mediated method with waiting, with the vascular bundle specificity promoter (from commelina yellow mottle virus, ComYMV) the chimeric bivalent gene of beta-1,3-glucanase that drives of chitinase of Qu Donging and CaMV 35S promoter imports upland cotton kind Ji and closes 321 and middle cotton institute 35.Utilize the kantlex streak method that the kalamycin resistance of transgenosis strain is carried out preliminary evaluation, through PCR and Southern hybridization conclusive evidence, the bivalent disease-resistant gene is incorporated in the cotton gene group with 1-2 copy respectively as a result again.Transgenic line T3 seedling is not had the end mould alms bowl bacterium liquid and water that the root method is identified and identify in sick garden, land for growing field crops, 7 transformation plants all show anti-or anti-verticillium in various degree as a result, seedling stage qualification result and land for growing field crops disease garden investigation result basically identical; Wherein 3 transgenosiss such as D9910, D9915 and D9919 land for growing field crops disease index that is that isozygotys is respectively 6.5,9.4 and 9.5, all reaches high anti-level.To isozygotying of above 3 high resisting verticilliums is to carry out genetic analysis, these 3 the disease-resistant kalamycin resistances that are that isozygoty meet a pair of dominant gene law of segregation as a result, binding molecule hybridization checking result can think that these 3 transgenosis strains all contain two disease-resistant genes of a copy.
Higher plant lipid transfer protein (lipid transfer proteins, LTP) be the basic protein of small molecule amount (about 9kDa), now from various plants such as corn seedling, the leaf of spinach, castor-oil plant, barley, onion, cotton and Arabidopis thaliana, be purified into LTP, and the coding cDNA of LTP and gene have been cloned from different plants also and have been obtained.LTP can transport phosphatide between microbial film, thereby thinks that LTP has participated in biomembranous formation in the cell.Find again that after deliberation LTP has signal peptide, can be positioned on the cell walls, thereby again it is produced query in intracellular transhipment lipid ability from the cell internal secretion to the extracellular.It is reported to be subjected under the pathogen infection situation that class LTP may rely on molecule by a fat and promote long-distance transportation (Ana M.Maldonado, Nature 419:26, September, 2002) plant.There is also evidence that LTP has participated in the adaptation to environmental change (temperature, salt, arid association compel) of the disease resistance response of the formation of cutin and cured matter, plant and plant.From cotton, separate in addition and obtain the LTP gene, and the report of studying just links together the function of this gene and the growth of fiber, and from the sea island cotton blade, separate to obtain the LTP gene, and transform this gene in upland cotton, and obtain high resisting verticillium plant and do not appear in the newspapers as yet.
The active measuring method of LTP transhipment lipid is comparatively complicated, and it needs two kinds of microbial films, i.e. donor membrane (donor) and receptor membrane (acceptor).The liposome (liposome) that donor membrane generally selects for use radio-labeled or fluorescently-labeled phosphatide to form through ultrasonication, receptor membrane then multiselect can be by centrifugal subcellular component of separating with liposome with plastosome or other.By with receptor membrane with again they are separated after donor membrane is cultivated altogether, measure in the receptor membrane radio-labeled or fluorescently-labeled phospholipids content and represent that LTP shifts the ability of lipid.When lacking LTP, the phosphatide transfer efficiency between microbial film is very low or do not have.In the test of animal and plant LTP transhipment lipid, all find the two-way exchange of phosphatide between donor membrane and receptor membrane, thereby (phospholipids exchange protein, PLEP), but the name of generally acknowledging now still is LTP again LTP to be called phospholipid exchange protein.Because LTP is the basic protein of small molecule, this proteinic isolation and purification is based on that its biochemical character carries out, and promptly separates by technology such as gel molecular sieve, ion exchange chromatography, high back voltage liquid chromatographies.The active monitoring of LTP in purge process finds to have a plurality of LTP to change the appearance at the active peak of fat, shows to have the isozyme composition in plant.
The primary structure (aminoacid sequence) of corn, wheat, castor-oil plant and oryza sativa l. TP after measured, other has the primary structure of the LTP of some plants also can derive by its cDNA sequence to get.LTP is made up of 91-95 amino-acid residue, has 1 signal peptide of being made up of 21-27 amino-acid residue, and the cysteine residues that also comprises 8 high conservatives is formed 4 disulphide bridgeses.Its primary structure differs bigger in the homology of different plant species, and the homology of plant LTP amino acid residue sequence is between 30%-70%.Although there is this polymorphism, they all have proteic constructional feature of LTP and characteristic.Utilize the three-dimensional structure of X ray crystalline diffraction and nucleus magnetic resonance (NMR) research LTP, and draw out the tomograph of LTP.These two kinds of methods are all based on corn LTP, it contains 93 amino-acid residues, constitute 4 α spirals (α-helix), form 4 disulfide linkage near C end place, and having a hydrophobic pocket that caves in (hydrophobic cavity), the hydrophobic aliphatic chain that during LTP transportation phospholipid molecule is exactly phospholipid molecule embeds in the hydrophobic pocket of LTP.The result of study of NMR shows; the hydrophobic pocket of LTP molecule is enough to hold whole phospholipid molecule; but X ray crystalline diffraction result of study shows the hydrophobic pocket that has only acyl chain to insert the LTP molecule; and the LTP that some are special; conjugated protein (acyl-CoA-binding protein, three-D space structure ACBP) are also similar to LTP as acyl-CoA.About how LTP carries out the phosphatide transhipment between film mechanism, the pattern of relatively generally acknowledging is that LTP and phosphatide form the LTP phospholipid complexes now.A little less than two acyl chains of phospholipid molecule were one strong one with the effect of LTP, when LTP carried phospholipid molecule and arrives the film surface, this specific character helped carrying out the exchange of phosphatide.Yet handle with dithiothreitol (DTT) the α spiral of LTP was reduced to 25% o'clock from 40%, the LTP of corn changes the fat activity inhibited, and the disulfide linkage that shows LTP is to keeping its active importance; Adding NaCl plasma compound also can suppress the activity of LTP, and this shows that the electrostatic balance between LTP and microbial film is also very important to keeping the LTP activity, and its active tool Ca
2+Dependency.Gene or the cDNA of coding LTP are cloned into from spinach, tomato, barley, Radix Dauci Sativae, mouseearcress etc.Nearlyly surpass 100 codings and the similar expressed sequence tag of LTP (expressed sequence tag EST) is identified.These EST are attributed to the temporary transient homologous sequence of a class, and its sequence can be obtained by the internet.But whether present still subject to confirmation they all have the function of LTP gene.All there are a plurality of copies of LTP gene in southern blotting technique analysis revealed cotton, corn, paddy rice etc., and are distributed on the different karyomit(e).
The LTP expression of gene has stronger growth and tissue specificity, and the LTP expression of gene characteristics of different plant species are not quite similar.But the expression study of the vegetative organ of most materials shows often there is not the LTP expression of gene in the root, and the LTP gene is eager to excel than the expression in the aging tissue at tender tissue, and the expression in young inflorescence is stronger.The hybridization in situ experiment result shows that the LTP gene of corn seedling, Radix Dauci Sativae embryo only expresses in epidermic cell, has stronger cell type specificity, and the LTP gene of rape all has expression in all epicotyls, and another kind of LTP gene is specifically expressing in the tapetal cell of rape then.Hybridization in situ experiment proves that also the AtLTP1 gene of mouseearcress and the origin of embryo interrelate, and mainly expresses in epidermic cell, thinks that it may be relevant with the formation of embryo top tissue.And the method by plant transgene, with the promotor of LTP gene with the gus gene combination, the specificity of research LTP genetic expression.
The secreting outside function of LTP and body embryo take place gets in touch the formation that may be interpreted as it and may participate in the rataria external protection.Nearest research shows that also LTP has participated in the disease resistance response of plant, is the disease-resistant albumen of a kind of plant.The plant LTP albumen of purifying is in external bacterium and the fungi growth of suppressing.Isolating LTP albumen all shows disease-resistant function in various degree from corn, spinach, Cauliflower, Arabidopis thaliana, and a kind of LTP albumen also shows the abduction delivering that is subjected to pathogenic fungi.Yet the disease-resistant mechanism of LTP involved in plant is indeterminate, a kind of viewpoint think it may be as a kind of polarity protein influence the cytolemma of fungi.A kind of disease-resistant albumen and the LTP of onion have homology, but it does not show the function of transhipment lipid.There are some researches show that also the expression of LTP is influenced by environmental factor, can induce the LTP expression of gene as temperature, salt and drought stress.NaCl, N.F,USP MANNITOL, dormin (ABA) are handled tomato and can be induced the expression of LTP gene at stem, and the LTP gene of barley, cotton then can be by low temperature and ABA abduction delivering.Yet whether their expression under environment stress are further studied with relevant then need of the resistance of plant.
LTP expression of gene Regulation Mechanism is also indeterminate at present.Specificity LTP is the protein of the not strong transhipment phosphatide of a class specificity; different phosphatide all had turn-over capacity; but also have the albumen that certain lipoids is had specific combination in the cell; as recent findings the protein 2 classes: a class is conjugated protein (the acyl-CoA-binding protein of acyl-CoA; ACBP); now from Arabidopis thaliana, separate, be purified to ACBP; it is a kind ofly to be made up of 92 amino-acid residues; iso-electric point is 9 basic protein; can with acyl-CoA specific combination, but different other lipid and acyl group combination.Its fundamental characteristics is similar to LTP, but does not have 8 conserved cysteine residue of signal peptide and LTP, is an intracellular protein.The cDNA of the ACBP of cotton, paddy rice, Arabidopis thaliana clones.Another kind of specificity lipid transfer protein (specific lipid transfer protein, spLTP) be the LTP of a class solubility, they find in animal, yeast, and that wherein can transport phosphatidylinositols (PI) specifically is called PI-TP (phosphatidylinositol-transfer protein); The LTP that can transport phosphatidylcholine (PC) specifically is called PC-TP (phosphatidylcholine-transfer protein).
Summary of the invention
The purpose of this invention is to provide a kind of sea island cotton lipid transfer protein and encoding gene thereof.
Sea island cotton lipid transfer protein provided by the present invention, name is called AT7, derives from Gossypium sea island cotton subspecies (Gossypium barbadense), is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 1;
2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the protein of phosphatide transhipment effect.
SEQ ID № in the sequence table: 1 is made up of 120 amino-acid residues.
The gene (At7) of coding sea island cotton lipid transfer protein At7 is one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: 1 dna sequence dna;
3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
SEQ ID № in the sequence table: 2 by 592 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 80-442 bit base.
Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.
Arbitrary segmental primer is to also within protection scope of the present invention among the amplification At7.
Another object of the present invention provides a kind of method of cultivating the resisting verticillium plant.
The method of cultivation resisting verticillium plant provided by the present invention is that described sea island cotton lipid transfer protein gene A t7 is imported plant tissue or cell, obtains the plant of resisting verticillium.
Described sea island cotton lipid transfer protein gene A t7 can import plant tissue or cell by the plant expression vector that contains described sea island cotton lipid transfer protein gene A t7; The carrier that sets out that is used to make up described plant expression vector can be any one double base agrobacterium vector or can be used for carrier of plant micropellet bombardment etc., as pCAMBIA1301, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300 or pBI121 etc.When using At7 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin (ubiquitin) gene promoter (pUbi) etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones 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 be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Be the carrier that sets out with pCAMBIA1301, the plant expression vector that contains described sea island cotton lipid transfer protein gene of structure is pCAMBIA1301-At7.
Carry At7 of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.By the plant transformed host both can be the plant of various easy infection verticillium (Verticilliumwilt) such as cotton, tobacco, Arabidopis thaliana, paddy rice, wheat, clover, soybean, tomato, eggplant and watermelon.
The invention provides a kind of lipid transfer protein At7 and encoding gene At7 thereof that derives from sea island cotton, the prokaryotic expression product of evidence At7 has stronger bacteriostatic activity to the cotton Huang bacterium that withers.Transgene tobacco, Arabidopis thaliana and cotton all show stronger tolerance to the Huang verticillium toxin that withers.Albumen of the present invention and encoding gene thereof will be played a great role in the genetic breeding of resisting verticillium plant (particularly cotton).
The present invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 induces the detected result of the proteic prokaryotic expression amount of 1-4h At7 for IPTG
Fig. 2 for At7 prokaryotic expression product A t7 to the Huang bacterium V that withers
991The inhibition effect
Fig. 3 for At7 prokaryotic expression viable bacteria (conversion has the e. coli bl21 of pET-28a-At7) to the Huang bacterium V that withers
991The inhibition effect
Fig. 3 A is the physical map of At7 overexpression vector pCAMBIA1301-At7
The young shoot of Fig. 4 for growing by At7 transgene tobacco callus
Fig. 5 is an At7 transgene tobacco positive plant
Fig. 6 is the result of RT-PCR testing goal gene A t7 transcriptional level in transfer-gen plant
Fig. 7 withers for the needle punching detection is yellow, and positive strain causes the result who withers and react to verticillium toxin to the At7 transgene tobacco
Fig. 8 is tiled in the enterprising row filter of MS substratum that contains Totomycin for the seed with At7 transgenic arabidopsis T1 generation
Fig. 9 is the plant of seed after sprouting behind the antibiotic-screening in the positive strain of At7 transgenic arabidopsis and negative strain T1 generation
Figure 10 is the PCR detected result of At7 transgenic arabidopsis
Figure 11 for the At7 transgenic arabidopsis to the wither resistance detected result of verticillium toxin of Huang
Figure 12 is the defensive ability/resistance ability of At7 transgenic positive plant to assorted bacterium
Figure 13 is the cotton seedling that just has been unearthed behind the pCAMBIA1301-At7 converting cotton
Figure 14 is the situation (the arrow indication is disease-resistant plant) that is planted in the At7 transgene cotton morbidity in seedling stage in the sick soil
Figure 15 is the survival plant of At7 transgene cotton after the disease resistance screening
Figure 16 is the field performance of the At7 transgenic positive plant of acquisition after the disease resistance screening
Figure 17 is the PCR qualification result of At7 transgenosis cotton plant
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment, and it is synthetic that the primer is given birth to the worker by Shanghai.
Embodiment 1: the clone of sea island cotton lipid transfer protein gene A t7
Vegetable material: high resisting verticillium sea island cotton AXMN.
(1) with sterile soil vegetable material is cultivated earlier, when treating that cotton seedling grows 1-2 sheet true leaf, cotton seedling is taken out and clean root system with clear water, be divided into 2 groups then, wherein one group with the strong virulence Huang bacterium V that withers
99170 times of liquid of toxin dilution soak, within 24 hours, took a sample once every 6 hours, be divided into sample time 6,12,18 and 4 time periods of 24h (take out 3 strains at every turn, sample is cut off with scissors, be divided into root and over-ground part), weigh after the sampling, use liquid nitrogen flash freezer then immediately, and place-80 ℃ of refrigerators temporary; Another group is soaked with clear water, does same treatment.(2) will handle 6-24 hour sample mix through toxin, with total RNA of CTAB method extraction cotton field top blade, the mRNA purification kit with Promega company is purified into mRNA again; Contrast is also handled with identical method.(3) with the inhibition subtractive hybridization (Suppression Subtractive Hybridization, SSH) method obtains 10 of toxin-induced sea island cotton specifically expressing fragments, concrete grammar is as follows:
Adopt the PCR-SelectTM cDNA Subtraction test kit of Clontech company and operate by the specification sheets of test kit, be template at first with above-mentioned mRNA through 6,12,18 and 24 hours cotton field top blade of toxin processing, with what handle through clear water is contrast, and reverse transcription synthesizes the first chain cDNA; Be template with the first chain cDNA again, the synthetic second chain cDNA, cut synthetic cDNA with restriction enzyme Rsal enzyme, to add two kinds of joints through the cDNA endonuclease bamhi that toxin is handled respectively then, anneal (hybridizing for the first time) with two kinds of cDNA fragments that add different joints respectively with excessive contrast cDNA fragment, two kinds of products after the hybridization for the first time carry out the hybridization second time again, be that template is carried out a regular-PCR and a nest-type PRC with the product after hybridizing through the second time again, the PCR product directly separates with PAGE for the second time, resulting electrophoresis band is sea island cotton and induces and the gene fragment of specifically expressing through the Huang verticillium toxin that withers, the result obtains 10 fragments altogether, is numbered At1-At10 respectively.Through sequence homology relatively will be wherein the 7th sequence confirm as lipid transfer protein (LTP) gene, be At7 with this unnamed gene, the design special primer, its full-length cDNA of pcr amplification, primer sequence is as follows:
Primer 1 (upstream primer): 5 '-GATATTAATTTGTAGATAAAGTTAATATTACG-3 ';
Primer 2 (downstream primer): 5 '-GACGACAATCAGCAATAGTAC-3 '
Respectively with above-mentioned through the Huang bacterium V that withers
991The reverse transcription synthetic first chain cDNA that toxin is handled 6,12,18 and 24 hours biased sample is a template, under the guiding of primer 1 and primer 2, the full-length cDNA of pcr amplification sea island cotton lipid transfer protein gene A t7, the PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 52 ℃ of 30s, 72 ℃ of 1min, totally 35 circulations; Last 72 ℃ of 10min, 4 ℃ of preservations.After reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis to be detected, reclaim the also purpose fragment of the about 592bp of purifying, it is cloned among the carrier pGEM-T (Promega company), screening positive clone, check order, the recombinant plasmid vector called after At7-pGEM-T that will contain At7 cDNA sequence, sequencing result shows that At7 has SEQ ID № in the sequence table: 2 nucleotide sequence, SEQ ID № in the sequence table: 2 by 592 based compositions, its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 80-442 bit base.
Embodiment 2: detect At7 prokaryotic expression product to the wither fungistatic effect of bacterium of Huang
One, the prokaryotic expression of At7
The cDNA sequence clone of the sea island cotton lipid transfer protein gene A t7 that embodiment 1 is obtained is gone among the carrier pET-28a (Novagen company), the recombinant vectors called after pET-28a-At7 that will contain At7 cDNA sequence, with pET-28a-At7 transformed into escherichia coli BL21, obtain positive recombinant then through screening.Picking contains single bacterium colony of pET-28a-At7, it is inoculated in the LB liquid nutrient medium, adopt ordinary method, abduction delivering 16h under 37 ℃, 1mmol/mL IPTG, the negative contrast of e. coli bl21 of pET-28a empty carrier is arranged with conversion, get sample one time every 1h, the sample that 1h, 2h, 3h, 4h are got carries out the 6%SDS-PAGE electrophoresis detection, (swimming lane Marker is the molecular weight of albumen marker to detected result as shown in Figure 1, swimming lane 1h, 2h, 3h, 4h represent respectively to induce 1h, 2h, the proteic prokaryotic expression amount of 3h, 4h At7, swimming lane CK through IPTG
1, CK
2, CK
3, CK
4Represent that respectively IPTG induces the negative control of 1h, 2h, 3h, 4h), result's expression amount of At7 when IPTG induces 4h is the highest, the differential protein At7 molecular weight of expression is 27.59kDa, pH be 9.8 o'clock water-soluble, belong to basic protein.
Two, detect At7 prokaryotic expression product A t7 to the wither fungistatic effect of bacterium of Huang
Getting step 1 induces the conversion of 4h that the e. coli bl21 bacterium liquid 1.5mL of pET-28a-At7 is arranged through IPTG, centrifugal collection thalline, add 100 μ L TE solution,, draw cleer and peaceful protein precipitation respectively and carry out the Huang bacterium V that withers with the broken thalline of alternate freezing and thawing method (under-70 ℃ and room temperature 5-6 time repeatedly)
991Inhibition test, with conversion e. coli bl21 cleer and peaceful protein precipitation on inductive of pET-28a empty carrier being arranged is contrast, method is: the bacterium that at first Huang withered is inoculated in the central authorities of PDA substratum, then Huang wither bacterium around make a call to 4 holes, two supernatant liquor 100 μ L and some insoluble albumen that add abduction delivering freeze thawing product respectively wherein.Two other hole adds respectively and transforms e. coli bl21 cleer and peaceful protein precipitation on inductive that the pET-28a empty carrier is arranged, and places down at 28 ℃ and observes fungistatic effect in 5 days, and the result (is treated to the expressed albumen of At7 to the wither inhibition effect of bacterium of Huang as shown in Figure 2; CK is contrast, and there is not restraining effect in the expressing protein that show to transform the e. coli bl21 that the pET-28a empty carrier is arranged to the Huang bacterium that withers), show that At7 prokaryotic expression product A t7 has bacteriostatic activity to the Huang bacterium that withers, especially the bacteriostatic activity with protein precipitation is higher.For further identifying the bacteriostatic activity that transforms the intestinal bacteria BL2 that pET-28a-At7 is arranged, also carried out bacteriostatic test with viable bacteria, with conversion the e. coli bl21 of pET-28a empty carrier being arranged is contrast, method is similar to inducible protein, just will go up the e. coli bl21 that cleer and peaceful albumen precipitation is changed to the intestinal bacteria BL2 that contains pET-28a-At7 and the pET-28a empty carrier is arranged, (At7-1 and At7-2 represent to transform the e. coli bl21 of pET-28a-At7 to the result as shown in Figure 3, be respectively the At7 prokaryotic expression protein (alternate freezing and thawing obtains) of inoculation at first, the survival of part bacterium is bred and is formed the BL21 bacterium colony during afterwards owing to freeze thawing; CK1 and CK2 are contrast), there is the e. coli bl21 contrast of pET-28a empty carrier to compare with conversion, transform intestinal bacteria BL2 that pET-28a-At7 is arranged the Huang bacterium that withers is also had significant bacteriostatic activity.
Embodiment 3: detect yellow cause the wither reaction of verticillium toxin to the positive strain of At7 transgene tobacco of withering
One, the acquisition of At7 transgene tobacco
1, the structure of At7 tobacco overexpression vector
After the cDNA sequence of the sea island cotton lipid transfer protein gene A t7 that embodiment 1 is obtained is cut with restriction enzyme BglII and BstE II enzyme, with be connected through the carrier pCAMBIA1301 that contains the CaMV 35S promoter of same enzyme double digestion (Australian CAMBIA company), obtain the overexpression vector of At7, called after pCAMBIA1301-At7, its physical map as shown in Figure 3A.
2, the acquisition of At7 transgene tobacco and Molecular Detection
At7 tobacco overexpression vector pCAMBIA1301-At7 agrobacterium co-cultivation transformation of tobacco coral west cigarette with step 1 structure, used Agrobacterium is GV3101, the result in the leaf section that transforms, have 75.6% grown callus, wherein major part has differentiated green sprouting (Fig. 4), transplant back final one-tenth seedling 30 strains, the transgenic positive plant as shown in Figure 5.The transfer-gen plant that is obtained is extracted genomic dna, carry out PCR with special primer primer 1 and primer 2 and detect, the result has 23 strains to detect the goal gene of about 600bp.Be the transcriptional level of testing goal gene A t7 in transfer-gen plant, method with RT-PCR detects, the result is shown in Fig. 6 (swimming lane 1-6 represents different transfer-gen plants), the object tape of swimming lane 1-3 is brighter, show that At7 has higher expression level in the 5th, 6,7 plant that detected, and the object tape of swimming lane 4-6 is darker, shows that the expression level of the 8th, 9,13 strain At7 is lower.
Two, detect yellow cause the wither reaction of verticillium toxin of withering to the positive strain of At7 transgene tobacco
The At7 transgene tobacco positive plant that step 1 is obtained is with blade acupuncture " inoculation " the Huang bacterium V that withers
991, every leaf selects four different positionss, inoculates V respectively
991Stoste (position 1), 30 times of diluents (position 2), 100 times of diluents (position 3) and clear water (position 4) that Huang withers bacterium filtrate, with the wild-type tobacco blade is contrast, observes in inoculation back 24,48 and 72h, and the result as shown in Figure 7, behind the At7 transformation of tobacco, the Huang bacterium V that withers
991Toxin obviously reduces the effect of withering of causing of blade, and contrast then shows tangible withered spot symptom, and the non-transgenic tobacco leaf is being used V
991The wither stoste of bacterium filtrate, 30 times of diluents and 100 times of diluents of Huang are handled back 24h and are shown tangible withered spot symptom, the serious more and blade flavescence to the withered spot of 72h whole blade; And contrast just after inoculation during 48h the acupuncture position just show slightly withered spot, show less withered at the acupuncture position to 72h.
Embodiment 4: detect the At7 transgenic arabidopsis to the wither resistance of verticillium toxin of Huang
One, the antibiotic-screening of At7 transgenic arabidopsis and Molecular Detection
At7 overexpression vector pCAMBIA1301-At7 agrobacterium co-cultivation arabidopsis thaliana transformation with embodiment 3 structures, used Agrobacterium is GV3101, the T1 that obtains after transforming was tiled on the MS substratum that contains 80 μ g/mL Totomycin vernalization 3 days for seed, be placed on then in the illumination box and grew 7 days, most seeds can be sprouted (Fig. 8, the arrow indication is the seed of transgenic positive plant), but the root growth of the negative plant of transgenosis is obstructed, and cotyledon can not launch, and positive transfer-gen plant leaf look darker, open and flat, plant height is also higher, root thick and long (shown in Figure 9, the arrow indication is the transgenic positive plant).Positive plant transferred to do not contain in the antibiotic MS substratum, continued growth is transplanted in the soil to 4-5 sheet true leaf, and by the results transfer-gen plant T2 of the strain system seed in generation, the result obtains 41 strain T2 altogether and for strain is.Again the Arabidopis thaliana transfer-gen plant that obtains through antibiotic-screening is carried out Molecular Detection with the method for PCR, extract the genome DNA of positive plant and as template, under the guiding of At7 cDNA special primer primer 1 and primer 2, carry out pcr amplification, after reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis to be detected, (swimming lane M is a molecular weight marker to detected result as shown in figure 10, swimming lane 5,6 positive transgenic lines, swimming lane 12,13 is the false positive transgenic line, swimming lane 35 positive contrasts, swimming lane 14 negative contrasts), can amplify the segmental positive transfer-gen plant of about 600bp purpose, and the false positive plant does not amplify the fragment of corresponding size.
Two, detect the At7 transgenic arabidopsis to the wither resistance of verticillium toxin of Huang
With concentration is the Huang of the 0.22 μ g/mL bacterium V that withers
991The T2 of positive transgenic arabidopsis the 35th strain system that bacterium liquid treatment step one obtains is for seedling, the observation growth of seedlings changes, (long arrow indication is that At7 is the transgenic positive plant to the result as shown in figure 11, short arrow indication is the non-transgenic plant), in handled culture dish, have 30 strains, wherein have 8 strain seedling to begin in the 10min to wilt after processing, the degree of after this wilting increases the weight of gradually, reaches severity to 2h; 22 strains in addition are insensitive to the wither processing of verticillium toxin of Huang, and from beginning the wilting phenomenon all not occur to 6h, promptly phenotype has anti-the withering property that causes.The anti-ratio that causes wither plant and wilting plant of statistics, the anti-ratio that causes wither plant and wilting plant of result is 22: 8, meets 3: 1 dominance monogenic inheritance rule, shows that At7 may integrate for single copy in the transgenic positive plant.In addition, when transplanting Arabidopis thaliana, a culture dish is accidental in test by living contaminants, and At7 transgenic positive plant shows stronger defensive ability/resistance ability to assorted bacterium as a result, the non-transgenic strain does not then possess resistance (shown in Figure 12, the arrow indication is that At7 is the transgenic positive plant among the figure).
Embodiment 5: detect the At7 transgene cotton to the wither resistance of verticillium toxin of Huang
One, the acquisition of At7 transgene cotton and detection
Employing period-luminosity spaces etc. are according to pollen tube passage method (pollen-tube pathway) (the Zhou G.Y.et al. of dna fragmentation hybridization theory design, Introduction of exogenous DNA into cotton embryos, MethEnzymol, 1983,101:433-481) the At7 overexpression vector pCAMBIA1301-At7 converting cotton (cotton variety is 2003-006) that embodiment 3 is made up, concrete grammar is: the 20-24h after the cotton self-pollination, remove corolla, expose gynoecium, truncation ovary top, not reveal ventricle degree of being, the pCAMBIA1301-At7 plasmid is dripped in ovary tangent plane or shallow slotting injection, every flower injects 5 μ L, to importing the flower mark of listing.Behind cotton boll blowing, gather by combination.The combined grow of pressing of future generation.In order to following method transfer-gen plant is identified then:
1, disease resistance is identified
The seed kind of the transfer-gen plant gathered in the crops behind the At7 overexpression vector pCAMBIA1301-At7 converting cotton is being contained 3.0 * 10
8Individual Huang withers in the sick soil of bacterium spore, identifies its disease resistance down at pathogenic bacteria " high pressure ".Because being used for the genetically modified cotton variety of At7 is susceptible variety, after therefore selecting through high pressure, most cotton seedling morbidities are die, and remaining minority plant has high disease resistance.When the result grows to 2 true leaves when cotton seedling, have only the cotton seedling of 15 strains not show the verticillium symptom, account for 0.4% of sum, all the other are all fallen ill withered (as Figure 13-shown in Figure 16).
2, anti-kantlex is identified
Treat that the 15 strain At7 transgenosis cotton plants of surviving grow 2 true leaves after step 1 disease resistance is identified after, with concentration is that the kantlex solution drop of 1000ppm is in seedling apical point, observe of the reaction of seedling young leaves contact kantlex solution position after several days to kantlex, it is the kalamycin resistance positive plant that blade does not have reactor, and leaf color becomes yellow person and is the negative plant to kantlex reaction sensitivity.To the kalamycin resistance positive plant of first screening, repeat the phenotypic evaluation of 2 above-mentioned kantlex solution drop seedling apical point again, finally obtain the At7 transgenosis cotton plant of 11 strain tool kalamycin resistances.
3, PCR identifies
Extract through above-mentioned two steps and be accredited as total DNA of male 11 strain At7 transgenosis cotton plant blades and as template, under the guiding of At7 cDNA special primer primer 1 and primer 2, carry out pcr amplification, after reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis to be detected, (swimming lane M is a molecular weight marker to detected result as shown in figure 17, swimming lane 1-4 is different transfer-gen plant), can amplify the segmental positive transfer-gen plant of about 600bp purpose, and the false positive plant does not amplify the fragment of corresponding size.
Two, detect the At7 transgene cotton to the wither resistance of verticillium toxin of Huang
1, indoor sick soil is identified
Fetch earth from the sick field of continuous 8 years plant cottons (this plot cotton is withered, verticillium is mixed and given birth to, and missing seedling rate is up to 67%), inoculate the verticillium V of artificial culture
991, inoculum density is 3.0 * 10
8Individual spore, inoculum size are 30 spores of every gram soil.Then with the seed of the transfer-gen plant gathered in the crops behind the At7 overexpression vector pCAMBIA1301-At7 converting cotton that obtains in the step 1 according to 100 gram seeds plantation 1m
2The amount of broadcasting plantation.Because the pathogenic bacteria amount is very big, the death of catching an illness in a large number after cotton emerges, the disease of generation comprises seedling diseases, withered and verticillium.Obtain disease-resistant At7 transfer-gen plant with this high strength screening.
2, identify naturally in the field
To identify the disease-resistant cotton transplantation of seedlings of At7 transgenosis of surviving to sick garden through the indoor sick soil of step 1, after mid-July,, finally obtain disease-resistant At7 transfer-gen plant every incidence of investigation in 10 days.
Sequence table
<160>2
<210>1
<211>120
<212>PRT
＜213〉Gossypium sea island cotton (Gossypium barbadense)
<400>1
Met?Ala?Ser?Ser?Met?Ser?Leu?Lys?Leu?Ala?Cys?Val?Ala?Val?Leu?Cys
1 5 10 15
Met?Val?Val?Gly?Ala?Pro?Leu?Ala?Gln?Gly?Ala?Val?Thr?Cys?Gly?Gln
20 25 30
Val?Thr?Ser?Ser?Leu?Ala?Pro?Cys?Ile?Gly?Tyr?Leu?Thr?Gly?Asn?Gly
35 40 45
Ala?Gly?Gly?Val?Pro?Pro?Gly?Cys?Cys?Gly?Gly?Ile?Lys?Ser?Leu?Asn
50 55 60
Ser?Ala?Ala?Gln?Thr?Thr?Pro?Asp?Arg?Gln?Ala?Ala?Cys?Lys?Cys?Ile
65 70 75 80
Lys?Ser?Ala?Ala?Ala?Gly?Ile?Ser?Gly?Ile?Asn?Tyr?Gly?Ile?Ala?Ser
85 90 95
Gly?Pro?Pro?Gly?Lys?Cys?Gly?Val?Asn?Ile?Pro?Tyr?Lys?Ile?Ser?Pro
100 105 110
Ser?Thr?Asp?Cys?Asn?Ser?Val?Lys
115 120
<210>2
<211>592
<212>DNA
＜213〉Gossypium sea island cotton (Gossypium barbadense)
<400>2
ttgggaagaa?gcagcaatag?tactactact?ccaagcaggc?attttcctta?caagtttgtt 60
ctccttgtga?ttaatcgata?tggctagctc?aatgtccctt?aagcttgcat?gtgtggcggt 120
gttgtgcatg?gtggtgggtg?cacccctggc?tcaaggggcc?gtaacctgtg?gtcaagtcac 180
aagctccctc?gcaccctgca?ttggttactt?gacagggaat?ggtgctggtg?gcgttccccc 240
aggttgctgc?ggcggcataa?aatctctcaa?ctccgccgcc?caaacaacac?cagaccggca 300
agcagcttgc?aaatgcatca?aaagtgcggc?cgccggcatt?tctggcatca?actatggtat 360
tgcaagcgga?cccccaggca?agtgcggtgt?caacatccct?tacaagatca?gccctagcac 420
tgactgcaac?agcgtcaagt?gaagttttgg?catggaaagt?tcaccagcta?gtggaagcca 480
aaataacgat?agctacagaa?taaatatgga?tgttaaaatt?ccagagttgt?gggttgtgta 540
ctatgccgct?ttatgcgact?acgtaatatt?aactttatct?acaaattaat?aa 592
Claims (6)
1, a kind of method of cultivating the resisting verticillium plant is gene transfered plant tissue or the cell with coding sea island cotton lipid transfer protein, obtains the plant of resisting verticillium; Wherein, the aminoacid sequence of described sea island cotton lipid transfer protein is shown in SEQ ID NO:1.
2, method according to claim 1 is characterized in that: the gene of described coding sea island cotton lipid transfer protein is one of following nucleotide sequence:
1) dna sequence dna of SEQ ID NO:2 in the sequence table;
2) nucleotide sequence of the dna sequence dna hybridization that under the rigorous condition of height, can limit with SEQ ID NO:2 in the sequence table.
3, method according to claim 1 and 2 is characterized in that: described sea island cotton lipid transfer protein gene imports plant tissue or cell by the plant expression vector that contains described sea island cotton lipid transfer protein gene.
4, method according to claim 3 is characterized in that: the carrier that sets out that is used to make up described plant expression vector is pCAMBIA1301, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300 or pBI121.
5, method according to claim 4 is characterized in that: described plant expression vector is pCAMBIA1301-At7.
6, method according to claim 1 and 2 is characterized in that: described plant host is cotton, tobacco, Arabidopis thaliana, paddy rice, wheat, clover, soybean, tomato, eggplant or watermelon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100982773A CN1308345C (en) | 2005-09-05 | 2005-09-05 | Gossypium barbadense lipoid transition protein and its coding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100982773A CN1308345C (en) | 2005-09-05 | 2005-09-05 | Gossypium barbadense lipoid transition protein and its coding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1740190A CN1740190A (en) | 2006-03-01 |
| CN1308345C true CN1308345C (en) | 2007-04-04 |
Family
ID=36092732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100982773A Active CN1308345C (en) | 2005-09-05 | 2005-09-05 | Gossypium barbadense lipoid transition protein and its coding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1308345C (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914147B (en) * | 2010-08-20 | 2013-01-16 | 中国农业科学院作物科学研究所 | Plant disease resistance-related protein and coding gene and application thereof |
| CN101928338B (en) * | 2010-09-03 | 2012-12-26 | 河北农业大学 | Cotton GbVe gene, encoding protein thereof and application of cotton GbVe gene in plant verticillium wilt resistance |
| WO2012083547A1 (en) * | 2010-12-23 | 2012-06-28 | Zuo Kaijing | Anti-verticillium wilt gene of gossypium barbadense and its use |
| CN102732604A (en) * | 2011-04-15 | 2012-10-17 | 吉林师范大学 | Method for fast identification of recombinant agrobacterium tumefacien plasmids |
| CN103409545B (en) * | 2013-08-16 | 2015-08-19 | 江苏省农业科学院 | Detect the allelotrope molecule marker of noise made in coughing or vomiting western eggplant resisting verticillium gene SacVe and primer and application |
| CN104086636B (en) * | 2014-07-02 | 2016-06-08 | 中国农业大学 | Derive from the resisting verticillium albumen of sea island cotton and encoding gene thereof and application |
| CN110872598B (en) * | 2019-12-13 | 2022-09-13 | 南京农业大学 | Cotton drought-resistant related gene GhDT1 and application thereof |
| CN116334094A (en) * | 2022-07-25 | 2023-06-27 | 江苏省中国科学院植物研究所 | Mint nonspecific lipid transporter gene and expression protein and application thereof |
| CN116732087B (en) * | 2023-07-18 | 2024-08-02 | 安徽荃银高科种业股份有限公司 | Method for creating broad-spectrum disease-resistant transgenic corn |
-
2005
- 2005-09-05 CN CNB2005100982773A patent/CN1308345C/en active Active
Non-Patent Citations (1)
| Title |
|---|
| AF 507141 GENE BANK 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1740190A (en) | 2006-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1308345C (en) | Gossypium barbadense lipoid transition protein and its coding gene and application | |
| CN107840872B (en) | Albumen and the application of wax plum CpWOX13 gene and its coding | |
| AU2014350744C1 (en) | Method for controlling pests | |
| WO2010042575A1 (en) | Transgenic plants with enhanced agronomic traits | |
| CN104903444B (en) | Highly yielding ability nucleic acid, the method for preparing the increased genetically modified plants of yield, the method for increasing the yield of plant are assigned to plant | |
| CN108840915A (en) | Moso bamboo PeDi19-4 albumen and its encoding gene and application | |
| CN103865939B (en) | A kind of method improving plant trait | |
| CN102965392B (en) | Cultivation of sheath blight-resisting transgenic paddy rice and special vector | |
| CN118256555A (en) | Application of potato auxin transport gene StLAX5 | |
| CN104388448B (en) | A kind of corn phospholipase A_2 gene ZmsPLA2-1 and its application | |
| CN113754748A (en) | Polypeptide immune activator for improving insect resistance and disease resistance of rice | |
| CN109468333A (en) | Citrus laccase family gene CsiLAC4 and its application | |
| CN106554964B (en) | Application of cotton GbABR1 gene in verticillium wilt resistance | |
| CN111197035B (en) | Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 and application thereof | |
| CN108559753B (en) | Application of wheat stripe rust PSTG _17694 gene in stripe rust prevention and treatment and stripe rust resistant wheat cultivation method | |
| CN115491380B (en) | Plant lipoxygenase gene LOX and application thereof in broad-spectrum disease resistance of plants | |
| CN108034662B (en) | Application of wheat stripe rust PSTG _06025 gene in stripe rust prevention and treatment and cultivation method of stripe rust resistant wheat | |
| CN108610402B (en) | Application of peanut annexin gene AhANN6 in improving high temperature resistance and oxidation stress resistance of plants and microorganisms | |
| CN102732553B (en) | Improve the gene engineering method and material of plant products | |
| CN112322651B (en) | Application of tomato autophagy gene in improving plant root-knot nematode resistance | |
| CN109439677A (en) | Purposes of the BnGLIP1 gene in enhancing disease resistance of plant | |
| CN109652425B (en) | Application of rice OsHIR3 gene and method for obtaining disease-resistant rice | |
| CN113637678A (en) | Application of gene GhSWEET42 in prevention and treatment of cotton verticillium wilt | |
| CN102191253A (en) | Plant disease-resistant regulation and control gene UEP and application thereof | |
| CN108866074B (en) | Application of herbicide-resistant gene PAR3(G311E) |
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 | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Assignee: Beijing Middle Earth Science and Technology Development Co Ltd Assignor: China Agricultural University Contract fulfillment period: 2008.1.20 to 2013.1.19 Contract record no.: 2009110000263 Denomination of invention: Gossypium barbadense lipoid transition protein and its coding gene and application Granted publication date: 20070404 License type: Exclusive license Record date: 20091103 |
|
| LIC | Patent licence contract for exploitation submitted for record |
Free format text: EXCLUSIVE LICENSE; TIME LIMIT OF IMPLEMENTING CONTACT: 2008.1.20 TO 2013.1.19; CHANGE OF CONTRACT Name of requester: BEIJING ZHONGNONG DADI DEVELOPMENT CO., LTD. Effective date: 20091103 |
|
| ASS | Succession or assignment of patent right |
Owner name: BEIJING CHINA AGRICULTURE DADI DEVELOPMENT CO., LT Free format text: FORMER OWNER: CHINA AGRICULTURAL UNIVERSITY Effective date: 20100402 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100094 NO.2, GARDEN OF PERFECT SPLENDOR WEST ROAD, HAIDIAN DISTRICT, BEIJING CITY TO: 100083 ROOM 310, JINMA HOTEL, NO. A38,XUEQING ROAD, HAIDIAN DISTRICT, BEIJING CITY |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20100402 Address after: 100083 room 310, grand code 38, a clear road, Beijing, Haidian District Patentee after: Beijing Middle Earth Science and Technology Development Co Ltd Address before: 100094 Beijing Old Summer Palace West Road, Haidian District, No. 2 Patentee before: China Agricultural University |
|
| ASS | Succession or assignment of patent right |
Owner name: TIANJIN ZTDD-GENESEED TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: BEIJING ZHONGNONG DADI TECHNOLOGY DEVELOPMENT CO., LTD. Effective date: 20110414 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100083 ROOM 310, JINMA HOTEL, NO. A-38, XUEQING ROAD, HAIDIAN DISTRICT, BEIJING TO: 300308 A2-160, NO. 166, WEST ROAD 3, TIANJIN AIRPORT LOGISTICS PROCESS AREA, TIANJIN |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20110414 Address after: 300308 A2-160, three West 166, Tianjin Airport Logistics Processing Zone, Tianjin Patentee after: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. Address before: 100083 room 310, grand code 38, a clear road, Beijing, Haidian District Patentee before: Beijing Middle Earth Science and Technology Development Co Ltd |
|
| DD01 | Delivery of document by public notice |
Addressee: Wang Hairong Document name: Notification of Passing Examination on Formalities |
|
| DD01 | Delivery of document by public notice |
Addressee: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. Document name: Review of business letter |
|
| DD01 | Delivery of document by public notice |
Addressee: Wang Hairong Document name: Notification to Pay the Fees |
|
| C56 | Change in the name or address of the patentee | ||
| CP02 | Change in the address of a patent holder |
Address after: 300308 No. 310 South River Road, Tianjin Airport Economic Zone Patentee after: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. Address before: 300308 A2-160, three West 166, Tianjin Airport Logistics Processing Zone, Tianjin Patentee before: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. |
|
| DD01 | Delivery of document by public notice |
Addressee: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. Li Yuanjing Document name: Review of business letter Addressee: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. Li Yuanjing Document name: Notification of Passing Examination on Formalities |
|
| DD01 | Delivery of document by public notice |
Addressee: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. Document name: Notification to Pay the Fees |
|
| DD01 | Delivery of document by public notice | ||
| DD01 | Delivery of document by public notice |
Addressee: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. Document name: Notification of Termination of Patent Right |
|
| DD01 | Delivery of document by public notice | ||
| DD01 | Delivery of document by public notice | ||
| DD01 | Delivery of document by public notice |
Addressee: TIANJIN ZHONGTIAN DADI SCIENCE AND TECHNOLOGY CO., LTD. Document name: Notification of Decision on Request for Restoration of Right |