CN106754992A - A kind of application of grape disease-resistant related gene VvPUB17, expression vector and its application - Google Patents
A kind of application of grape disease-resistant related gene VvPUB17, expression vector and its application Download PDFInfo
- Publication number
- CN106754992A CN106754992A CN201710051147.7A CN201710051147A CN106754992A CN 106754992 A CN106754992 A CN 106754992A CN 201710051147 A CN201710051147 A CN 201710051147A CN 106754992 A CN106754992 A CN 106754992A
- Authority
- CN
- China
- Prior art keywords
- vvpub17
- plant
- grape
- disease
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 73
- 235000014787 Vitis vinifera Nutrition 0.000 title claims abstract description 58
- 235000009754 Vitis X bourquina Nutrition 0.000 title claims abstract description 57
- 235000012333 Vitis X labruscana Nutrition 0.000 title claims abstract description 57
- 201000010099 disease Diseases 0.000 title claims abstract description 43
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 43
- 239000013604 expression vector Substances 0.000 title claims abstract description 17
- 240000006365 Vitis vinifera Species 0.000 title description 2
- 241000196324 Embryophyta Species 0.000 claims abstract description 61
- 241000219095 Vitis Species 0.000 claims abstract description 58
- 230000002018 overexpression Effects 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 208000035240 Disease Resistance Diseases 0.000 claims description 18
- 239000012634 fragment Substances 0.000 claims description 12
- 230000029087 digestion Effects 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 239000002299 complementary DNA Substances 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 4
- 239000013598 vector Substances 0.000 claims description 4
- 239000012467 final product Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- 239000002253 acid Substances 0.000 claims 1
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 17
- 241000219194 Arabidopsis Species 0.000 abstract description 14
- 230000009466 transformation Effects 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000000034 method Methods 0.000 description 17
- 239000000243 solution Substances 0.000 description 14
- 241000221785 Erysiphales Species 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 10
- 239000012530 fluid Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000011081 inoculation Methods 0.000 description 8
- 244000052769 pathogen Species 0.000 description 8
- 230000001717 pathogenic effect Effects 0.000 description 8
- 229930027917 kanamycin Natural products 0.000 description 7
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class 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 7
- 239000007788 liquid Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 229930182566 Gentamicin Natural products 0.000 description 6
- 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 6
- 229960002518 gentamicin Drugs 0.000 description 6
- 235000015097 nutrients Nutrition 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 241000589158 Agrobacterium Species 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 238000005498 polishing Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 102100032814 ATP-dependent zinc metalloprotease YME1L1 Human genes 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 229920003266 Leaf® Polymers 0.000 description 4
- 108091008193 PUBs Proteins 0.000 description 4
- 101800000795 Proadrenomedullin N-20 terminal peptide Proteins 0.000 description 4
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 4
- 230000000149 penetrating effect Effects 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 101100246069 Arabidopsis thaliana PUB22 gene Proteins 0.000 description 3
- 101100246070 Arabidopsis thaliana PUB23 gene Proteins 0.000 description 3
- 101150022658 PUB24 gene Proteins 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 108010083111 Ubiquitin-Protein Ligases Proteins 0.000 description 3
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 3
- 230000007123 defense Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- PIRWNASAJNPKHT-SHZATDIYSA-N pamp Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)N)C(C)C)C1=CC=CC=C1 PIRWNASAJNPKHT-SHZATDIYSA-N 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000005720 sucrose Substances 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
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 102000006382 Ribonucleases Human genes 0.000 description 2
- 108010083644 Ribonucleases Proteins 0.000 description 2
- 102000006275 Ubiquitin-Protein Ligases Human genes 0.000 description 2
- 235000009392 Vitis Nutrition 0.000 description 2
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 230000002363 herbicidal effect Effects 0.000 description 2
- 239000004009 herbicide Substances 0.000 description 2
- 230000009610 hypersensitivity Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000008823 permeabilization Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 101100532034 Drosophila melanogaster RTase gene 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
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000896246 Golovinomyces cichoracearum Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 102000036382 U-box-type E3 ubiquitin tranferases Human genes 0.000 description 1
- 108091007057 U-box-type E3 ubiquitin tranferases Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 230000004665 defense response Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000002635 electroconvulsive therapy Methods 0.000 description 1
- ZRPZZJCWIPRUMJ-BEVCUQGISA-N elf18 Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O)[C@@H](C)O)C(C)C)C(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(C)=O)[C@@H](C)O)C1=CNC=N1 ZRPZZJCWIPRUMJ-BEVCUQGISA-N 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000019254 respiratory burst Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical class OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
- C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
Abstract
The invention discloses a kind of application of grape disease-resistant related gene VvPUB17, expression vector and its application, belong to plant genetic engineering field.The present invention clones and isolates VvPUB17 genes from grape leave, and the plant overexpression carrier containing VvPUB17 genes is transformed into arabidopsis and grape leave, VvPUB17 gene expression amounts can significantly increase the expression high of the resistance against diseases and grape disease-resistant related gene of the arabidopsis of genetic transformation, prove that VvPUB17 genes play a significant role in plant disease-resistant is strengthened, the research of VvPUB17 genes has great importance to cultivating disease-resistant plants new varieties.
Description
Technical field
The present invention relates to a kind of applications of grape disease-resistant related gene VvPUB17 in disease resistance of plant, also relate to
Expression vector and its application comprising grape disease-resistant related gene VvPUB17, belong to plant genetic engineering field.
Background technology
Research in recent years finds that protein ubiquitination wide participation plant defense is adjusted, proteins ubiquitin modification
Key enzyme be ubiquitin ligase E3, it determines the specific recognition of substrate protein, is related to Plant pathogen interaction, including
The defense reaction of early stage, the interaction of gene-for-gene and inductivity are disease-resistant.U-box albumen is a kind of new E3 albumen, is had
E3 activity.Research finds that U-box albumen is widely present in fungi, plant, animal, in the eucaryote U- for having identified at present
In box albumen, the number of plant U-box (plant U-box protein, PUB) albumen is far more than other biological.Exist at present
Have been found that more than 60 PUB albumen in arabidopsis, and the PUB albumen for confirming and plant defense pathogen infect reaction
Relevant, 3 expression of U-box type E3 ligases of PUB22, PUB23 and PUB24 can be by flg22 types PAMP such as in arabidopsis
(Pathogen-associated molecular pattern) induces (Trujillo M etc., 2008, Current
Biology 18(18):1396-1401).Under the invasion and attack of pathogen, the producing ratio of the Trimutant active oxygens of PUB 22/23/24
Wild type is significantly improved, and also can substantially suppress the growth of pathogen.Consistent with this, active oxygen produces related gene three
Also substantially induced in mutant material.In Trimutant material, other PAMP, such as elf18 and chitin can also draw
Play oxidative burst.These results indicate that the E3 ligases such as PUB22, PUB23 and PUB24 are PAMPs activation PTI in plant
(PAMP-triggered immunity) downstream signal factor institute is required, it was demonstrated that PUB22, PUB23 and PUB24 are that plant is supported
The disease-resistant related gene that anti-microbial pathogen is invaded.
However, at present ubiquitin ligase gene especially most of new U-box GFPs in Plant defense responses
Middle effect is still not very clear.VvPUB17 genes of the present invention are grape list subunit U-box families ubiquitinbond enzyme families
One of member.Screened and identified by grape U-box gene families, demonstrated VvPUB17 genes by the various diseases of grape
The induced expression of opportunistic pathogen.Therefore, to the exploitation of grape VvPUB17 genes it is the new way of studying plant disease-resistant, is turned by heredity
Change related gene improvement Disease Resistance of Grapevine weak kind and other plant varieties, will further enhance plant to the disease-resistant of pathogen
Property, the anti-spectrum of plant is widened, it is significant for cultivating plant disease-resistant new varieties.
The content of the invention
The answering in disease resistance of plant is strengthened it is an object of the invention to provide a kind of grape disease-resistant related gene VvPUB17
With.
Second object of the present invention is to provide a kind of plant overexpression carrier.
Third object of the present invention is to provide a kind of application of plant overexpression carrier in disease resistance of plant is strengthened.
In order to realize the above object the technical solution adopted in the present invention is：
A kind of applications of grape disease-resistant related gene VvPUB17 in disease resistance of plant is strengthened, the nucleotides sequence of the gene
Row such as SEQ ID NO:Shown in 5, length 2695bp, ORFs (ORF) is from the 274th~2418 bit base (SEQ ID NO:
1), the amino acid sequence of the protein coded by it is as shown in SEQ ID NO.2 (terminator codon is not translated), the protein
Molecular weight is 78.2kDa, and isoelectric point is 6.82, comprising U-box and two ARMrepeat conserved domain.
Specifically, applications of the grape disease-resistant related gene VvPUB17 in disease resistance of plant is strengthened, comprises the following steps：
1) grape disease-resistant related gene VvPUB17 is connected in expression vector, forms plant overexpression carrier；
2) plant overexpression carrier transfection Agrobacterium is taken, then is transferred in plant cell, you can.
A kind of plant overexpression carrier, the expression vector contains such as SEQ ID NO:Grape shown in 5 is disease-resistant related
Because of the ORFs of VvPUB17, such as SEQ ID NO:Shown in 1.
Specifically, the expression vector is pCAMBIA3301-VvPUB17, its preparation process is as follows：
1) with grape cDNA as template, P1, P2 are primer, carry out pcr amplification reaction, and amplified production is returned after electrophoretic analysis
Receive purpose fragment；
Forward primer P1：5’-GGGAGATCTATGGCCTCCGCTGCGATAGTAT-3 ',
Reverse primer P2：5’-GGGGGTGACCCTACAAAACAGGCACTGAAATGGGC-3’；
It is for restriction enzyme site BstE II at P1 underscores at restriction enzyme site Bgl II, P2 underscores；
2) distinguish double digestion step 1 with Bgl II, BstE II) in purpose fragment and plant expression vector
PCAMBIA3301, reclaims target fragment and linearized vector, and screening, checking, obtain final product after connection, conversion.
Above-mentioned plant overexpression carrier (contains such as SEQ ID NO:The VvPUB17's of grape disease-resistant related gene shown in 5
The expression vector of ORFs) strengthen disease resistance of plant in application.
Beneficial effects of the present invention：
The present invention provides a kind of new gene VvPUB17 to the resistance of pathogen for enhancing plant.By separating clone Portugal
The DNA fragmentation of VvPUB17 genes in grape blade, is connected with the carrier for being capable of the overexpression target gene, is transformed into plant thin
Born of the same parents, resistance of the plant to pathogen can be improved by overexpression VvPUB17 genes, control the generation of disease, reduced or kept away
Exempt from the loss that plant disease is brought.Meanwhile, the VvPUB17 genes of present invention exploitation are also for plant disease-resistant has widened the anti-of plant
Spectrum, overcomes traditional breeding method and is unable to the problem that plant inter-species shifts disease-resistant related gene.
Brief description of the drawings
Fig. 1 is agarose gel electrophoretogram after plant overexpression carrier pCAMBIA3301-VvPUB17 digestions；
Fig. 2 is the expression quantity change of different time VvPUB17 genes after grape leave inoculation powdery mildew；
Fig. 3 is the identification of VvPUB17 genetic transformation Arabidopsis plant disease resistances；
Fig. 4 is the identification of disease resistance after VvPUB17 gene instantaneous conversion grape leaves.
Specific embodiment
Following embodiments are only described in further detail to the present invention, but do not constitute any limitation of the invention.
Embodiment 1
The separation clone of grape disease-resistant related gene VvPUB17, specific method is as follows：
1) grape leave total serum IgE is extracted according to SDS/ phenol method
Prepare extract solution：To added in 2.0mL centrifuge tubes 850 μ L Extraction buffers (140mM LiCl, 10mM EDTA,
10mM Tris, 5% (w/v) SDS, 2% (w/v) PVP) and 30 μ L beta -mercaptoethanols, fully mix, it is stand-by.
After Liquid nitrogen precooler mortar, takes 0.2g grape young leaflet tablets in mortar, plus appropriate liquid nitrogen is fully ground, packing is to containing
Have in the 2.0mL centrifuge tubes for preparing extract solution in advance, be fully vortexed and mix, 12000rpm centrifugations 5min under the conditions of 4 DEG C；In absorption
In clear liquid to another 2.0mL centrifuge tubes, isometric chloroform-isoamyl alcohol (24 is added:1), fully it is vortexed and mixes, under the conditions of 4 DEG C
12000rpm is centrifuged 15min；In Aspirate supernatant to another 2.0mL centrifuge tubes, the KAc (pH4.8) of 1/3 volume 5M is added, filled
Divide to be vortexed and mix, 12000rpm centrifugations 10min under the conditions of 4 DEG C；In Aspirate supernatant to another 2.0mL centrifuge tubes, the body such as addition
Long-pending chloroform-isoamyl alcohol (24:1), fully it is vortexed and mixes, 12000rpm centrifugations 10min under the conditions of 4 DEG C；Aspirate supernatant is to another
In one 1.5mL centrifuge tubes, 1/3 volume 8M LiCl are added, being fully vortexed mixes, -20 DEG C of placement more than 2h, then under the conditions of 4 DEG C
12000rpm is centrifuged 15min；Supernatant is lightly removed, precipitation is washed with the ethanol of 800 μ L 75% twice (under the conditions of 4 DEG C
12000rpm is centrifuged 8min)；Air drying 10min treats that ethanol fully volatilizees, and adds 30 μ L DEPC-H2O dissolution precipitations, i.e.,
Obtain grape leave total serum IgE.
2) total serum IgE is purified
Added in 1.5mL centrifuge tubes：The μ L of 100 μ g, DNase I 10U, 10 × DNase buffer of total serum IgE 10,
RNasin100U, DEPC-H2O polishings are fully mixed to 100 μ L, 37 DEG C of incubation 30min；The isometric phenol-chloroform of addition-different
Amylalcohol (25:24:1), fully it is vortexed and mixes, 12000rpm centrifugations 10min under the conditions of 4 DEG C；Aspirate supernatant to another 1.5mL from
In heart pipe, isometric chloroform-isoamyl alcohol (24 is added:1), fully it is vortexed and mixes, 12000rpm centrifugations 10min under the conditions of 4 DEG C；
In Aspirate supernatant to another 2mL centrifuge tubes, 1/10 volume 3M NaAC (pH 5.2), 2.5 times of volume absolute ethyl alcohols, -20 are added
DEG C place more than 2h；Then 12000rpm centrifugations 15min under the conditions of 4 DEG C；Supernatant is lightly removed, with the ethanol of 800 μ L 75%
Washing precipitation is twice (12000rpm centrifugations 8min under the conditions of 4 DEG C)；Air drying 10min treats that ethanol fully volatilizees, and adds 20 μ
L DEPC-H2O dissolution precipitations；RNA purifying is completed.
3) chains of cDNA first are synthesized by template reverse transcription of RNA
Added in PCR pipe：Random 6mers (50 μM) 1 μ L, dNTP Mixture (10mM each) 1 μ L, Total
μ g, the RNase free dH of RNA 22O polishings are fully mixed to 10 μ L, and brief centrifugation makes solution to PCR pipe bottom.In PCR instrument
Upper 65 DEG C are reacted 5min, on ice chilling；Added in PCR pipe：Buffer 4 μ L, RNase
Inhibitor (40U/ μ L) 0.5 μ L,μ L, the RNase Free dH of RTase (200U/ μ L) 12O polishings are to 20 μ
L.Following reactions are carried out in PCR instrument：30℃10min；42℃60min；95℃5min；4 DEG C of preservations；Obtained final product after the completion of reverse transcription
The chains of cDNA first；- 40 DEG C save backup.
4) performing PCR amplification is entered by template of cDNA
PCR clones VvPUB17 gene PCR reaction systems：CDNA templates 1 μ L, forward and reverse primer P3, P4 each 2 μ L, PCR
μ L, PCR-Grade the Water polishings of 5 μ L, dNTP Mix of Buffer, 2.5 μ L, DNA Polymerase 1.0 are to 50 μ L, PCR
Response procedures are：94 DEG C of 30sec, 94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 3min, 30cycles, 72 DEG C of 10min, 4 DEG C
Forever。
Forward primer P3：5’-AGAGTCTGAGAAACGAGATTCAGAC-3’(SEQ ID NO:3)；
Reverse primer P4：5’-AAGCAGCTTTTCCACATTATACTATTACC-3’(SEQ ID NO:4).
5) detection and recovery
The product that PCR is expanded is carried out 1% agarose gel electrophoresis, target fragment is reclaimed, pMD19-T loads are connected to
Body, coupled reaction system is：0.5 μ L, Solution I of pMD19-T carriers 2.5 μ L, the μ L of target fragment 2.0, fully mix,
2h conversions are reacted under the conditions of 16 DEG C, by connection product transformed competence colibacillus TOP10 cells, on additional 100mg/L Amp culture mediums
Blue hickie screening is carried out, picking positive colony is seeded to LB fluid nutrient mediums and shakes bacterium culture, extracts plasmid and obtains positive plasmid
PMD19-T-VvPUB17, send company that detection, VvPUB17 gene orders such as SEQ ID NO is sequenced:Shown in 5.
Embodiment 2
The structure of grape disease-resistant related gene VvPUB17 overexpression carriers, specific method is as follows：
PMD19-T-VvPUB17 plasmids with the preparation of embodiment 1, with P1, P2 as primer, enter performing PCR amplification anti-as template
Should, PCR primer reclaims purpose fragment after agarose gel electrophoresis；It is restriction enzyme site Bgl II, P2 underscores at P1 underscores
It is restriction enzyme site BstE II to locate.
Forward primer P1：5’-GGGAGATCTATGGCCTCCGCTGCGATAGTAT-3’(SEQ ID NO:10)；
Reverse primer P2：5’-GGGGGTGACCCTACAAAACAGGCACTGAAATGGGC-3’(SEQ ID NO:11)。
Distinguish double digestion above-mentioned purpose fragment and plant expression vector pCAMBIA3301 with Bgl II, BstE II, reclaim
Target fragment is attached with linearized vector；Coupled reaction system is：PCAMBIA3301 linearized vectors 5 μ L, Solution
The μ L of I 10, the μ L of target fragment 5, are fully mixed, and 2h is reacted under the conditions of 16 DEG C；Connection product converts TOP10 competent cells, attached
Plus screened on the LB culture mediums of kanamycins, picking positive colony be seeded to LB fluid nutrient mediums (containing 100mg/L card that
Mycin) in shake bacterium culture, extract plasmid, send company that detection is sequenced, the correct plasmid of sequence is named as plant overexpression carrier
pCAMBIA3301-VvPUB17.Correct sequence is carried out into double digestion, endonuclease reaction system is：μ L, Bgl the II restriction endonucleases of plasmid 4
0.5 μ L, BstE II restriction endonucleases 0.5 μ L, ddH2The μ L of O 15, are fully mixed, and 2h is reacted under the conditions of 37 DEG C；Digestion products are through fine jade
Sepharose electrophoresis, as a result as shown in the band 2 of Fig. 1, discharges 2100bp's or so after pCAMBIA3301-VvPUB17 digestions
Electrophoretic band.M is DNA Marker in Fig. 1, and 1 for before plasmid enzyme restriction, 2 for after plasmid enzyme restriction.
Embodiment 3
Plant overexpression carrier pCAMBIA3301-VvPUB17 converts Agrobacterium, and specific method is as follows：
Electric shock cup processes 20min under being placed in uviol lamp；Agrobacterium GV3101 competent cells are taken out, thawed on ice is placed in,
It is transferred to after thawing in electric shock cup, and adds 10 μ L plasmids, fully mixed, is placed in 5min on ice；Electric shock cup is transferred to electric shock instrument
In carry out shock treatment；The conversion fluid in the cup that shocks by electricity is taken out in the centrifuge tube of 1.5mL after treatment, adds 1mL LB cultures
Base, 1h is cultivated under the conditions of 28 DEG C；Brief centrifugation 15s in centrifuge, removes part supernatant, is coated on after suspension and contains antibiotic
On the flat board of (gentamicin of 60mg/L, the kanamycins of 100mg/L), 28 DEG C of cultures are placed in.Picking monoclonal has been seeded to
In the LB liquid medium of antibiotic (gentamicin of 60mg/L, the kanamycins of 100mg/L), 24h is cultivated under the conditions of 28 DEG C,
Detected through bacterium solution PCR, positive colony is tested for next step.
Embodiment 4
Plant overexpression carrier pCAMBIA3301-VvPUB17 is transformed into plant
1) plant overexpression carrier pCAMBIA3301-VvPUB17 is transformed into arabidopsis
By the Agrobacterium inoculation containing plant expression vector pCAMBIA3301-VvPUB17 to 10mL LB fluid nutrient mediums
In (gentamicin containing 60mg/L, the kanamycins of 100mg/L), 28 DEG C of culture 24h；Taking 5mL bacterium solutions, to be transferred to 50mL fresh
LB fluid nutrient mediums (containing 60mg/L gentamicin and 100mg/L kanamycins), 28 DEG C continue cultivate, to bacterium solution OD600
Reach 0.6 or so；During bacterium solution is transferred into centrifugal bottle or centrifuge tube, under room temperature condition, rotating speed is that 4000rpm is centrifuged 10min, is gone
Except supernatant collection thalline；Permeabilization buffer (0.5 × MS, 5% sucrose, 0.03%Silwet L-77) is resuspended in, OD is adjusted600Extremely
0.8；Existing Fruit pod in arabidopsis floral is removed, inflorescence is completely immersed in the (10~30s, or with moving of 20s in penetrating fluid
Liquid device is directly dropped on inflorescence penetrating fluid), the penetrating fluid in arabidopsis leaf or stalk is removed immediately, plant is lain in into pallet
In, covered rearing with plastic film pallet is used, film is removed after 24h, continue to cultivate in greenhouse；To improve transformation efficiency, use afterwards within 7 days
Same method infects again；Normal management is carried out by the Arabidopsis plant for converting, seed is harvested when Fruit pod existing white；To receive
The transgenic seed for obtaining soaks 10min with 0.2% TritonX-100；Again with 10% sodium hypochlorite surface sterilization, 12min；
Sterilizing water washing five times, each 2min；With yellow liquid transfer gun head by seed be layered on containing 20mg/L herbicide bastas MS (0.5 ×
MS, 1% sucrose, 1% agar, pH 5.7) on flat board, 4 DEG C of dark culturings two days；Incubator is then transferred to be cultivated, if
Put condition of culture：22 DEG C of temperature, illumination 16h.Still grown fine after being screened through herbicide (20mg/L Basta), true leaf blade
And growing point is dark green and primarily determines that to be transfer-gen plant by the seedling for penetrating culture medium is stretched in root, transplants to nutritive cube
It is middle to continue to cultivate.
2) plant overexpression carrier pCAMBIA3301-VvPUB17 is transformed into grape
The Agrobacterium inoculation of plant expression vector pCAMBIA3301-VvPUB17 to 10mL LB fluid nutrient mediums will be converted
In (gentamicin containing 60mg/L, the kanamycins of 100mg/L), 28 DEG C of culture 24h；Taking 5mL bacterium solutions, to be transferred to 50mL fresh
LB fluid nutrient mediums (containing 60mg/L gentamicin and 100mg/L kanamycins), 28 DEG C continue cultivate, to bacterium solution OD600
Reach 0.6 or so；During bacterium solution is transferred into centrifugal bottle or centrifuge tube, under room temperature condition, rotating speed is that 4000rpm is centrifuged 10min, is gone
Except supernatant collection thalline；Add permeabilization buffer (10mM MES pH5.6, the 10mM MgCl of same volume2, 2% (w/v)
Sucrose and 150mM acetosyringone) it is resuspended；Grape leave is placed in appropriate re-suspension liquid, ability of swimming is placed in true
Vacuumize process is carried out in idle loop pump, timing is started when vacuum is pumped when strong pointer is 0.085MPa, slowly discharged after 30min
Gas；The grape leave that will be handled well is taken out, and the bacterium solution of blade surface is removed with sterilized filter paper, and blade distal shaft is faced up
It is placed in pallet, petiole absorbent cotton moisturizing, pallet is covered with preservative film, is placed in incubator and cultivates.
Test example
1st, expression pattern analysis of the grape disease-resistant related gene VvPUB17 in grape
In order to confirm that VvPUB17 genes participate in the regulation and control of disease resistance response, using quantitative fluorescent PCR (Real-time PCR)
Expression pattern of the technical Analysis VvPUB17 genes after grape leave is inoculated with powdery mildew (powdery mildew, PM).Inoculation side
Method according to Wang method (Wang Y etc., Vitis 34:159-164), SDS/ phenol method extracts different time after inoculation powdery mildew
The total serum IgE of (0,12,24,36,48,60,72h) grape leave, by PrimeScriptTMRT-PCR Kit specifications (are purchased from
TAKARA companies) reverse transcription is carried out, 7 reverse transcription products of different time detect grape as Real-time pcr templates
Response after VvPUB17 gene pairs grape leave inoculation powdery mildew.Real-time PCR use SYBR Premix Ex TMTaq
II kits (are purchased from TAKARA companies), and according to kit specification, in the iCycler iQ5Real-time PCR instruments (U.S.
Bio-Rad companies) on carry out real-time quantitative PCR reaction.Reaction system is：1 μ L, SYBR Mix of template 12.5 μ L, it is forward and reverse
Each 1 μ L of primer, sterile purified water polishing to 25 μ L；Response procedures are：95 DEG C of 5min, 95 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 30s, 30
Individual circulation, 72 DEG C of 5min, 4 DEG C of 10min.P5, P6 are the forward and reverse primer of VvPUB17 gene magnifications, and VvActin is internal reference base
Cause, P7, P8 are the forward and reverse primer of VvActin gene magnifications.
Forward primer P5：5’-CTGTAACGGCAATGCTTAACCTAT-3’(SEQ ID NO:6)；
Reverse primer P6：5’-CCTCGTCCGCTATTCTCTTCTTA-3’(SEQ ID NO:7).
Forward primer P7：5’-TCCTGTGGACAATGGATGGA-3’(SEQ ID NO:8)；
Reverse primer P8：5’-CTTGCATCCCTCAGCACCTT-3’(SEQ ID NO:9).
Thresh values are defaulted as 30 by PCR instrument, each reaction fluorescence signal is recorded respectively and enters exponential increase rank by background
Period (threshold cycle, Ct) corresponding to the flex point of section, then with 2- △ △CtMethod is not being inoculated with the leaf of powdery mildew
Piece is control, and the relative expression quantity to different time points VvPUB17 genes carries out normalization.Result is as shown in Fig. 2 grape leave
Middle VvPUB17 genes 12h expression quantity after powdery mildew is inoculated with is sharply increased, and expression quantity peaks during 24h, is expressed in 36h
Amount has declined, and subsequent expression quantity is sharply increased again, reaches a peak, subsequent and on a declining curve, explanation again in 60h
VvPUB17 genes are by powdery mildew induced expression.
2nd, the identification of grape disease-resistant related gene VvPUB17 disease resistances
1) grape disease-resistant related gene VvPUB17 Disease Resistance Identifications in arabidopsis
The arabidopsis T3 of conversion carries out Disease Resistance Identification after being grown 60 days for transfer-gen plant.Arabidopsis powdery mildew
(Golovinomyces cichoracearum UCSC1) is bred and is preserved on pad4 Arabidopsis Mutants.Selection life
The consistent transfer-gen plant of length, according to method (Wang W etc., 2007, Molecular Plant-Microbe of Wang
Interactions 20:966-976) it is inoculated with powdery mildew.Inoculation detects transfer-gen plant with WT lines to white powder after 6 days
The disease resistance of disease, apoptosis are detected using Trypan blue decoration methods, and as a result such as Fig. 3, (WT is wild in Fig. 3
Type, #1, #2, #4 are followed successively by transgenosis system 1, transgenosis system 2 and transgenosis system 3) shown in, 3 transgenosis systems are relative to wild type
It is more disease-resistant, show lighter susceptible symptom；3 transgenosis system plant leafs produce hypersensitivity, WT lines without or
Produce slight hypersensitivity；These results suggest that overexpression VvPUB17 genes cause the enhancing of Arabidopsis plant disease resistance.
2) grape disease-resistant related gene VvPUB17 Disease Resistance Identifications in grape
Checking grape disease-resistant related gene VvSTS, VvPR1, VvPR10, VvNPR1 enters grape in VvPUB17 genetic transformation
Expression after blade.After VvPUB17 gene instantaneous conversion grape leaves 24h, according to Wang method (Wang Y etc., 1995,
Vitis 34:Powdery mildew 159-164) is inoculated with, continues to cultivate 24h after the completion of inoculation.Using to grape leave water spray treatment as right
According to, conversion VvPUB17 genomes and control group grape leave total serum IgE are extracted, detect that grape is disease-resistant with Real-time round pcrs
The expression of related gene VvSTS, VvPR1, VvPR10 and VvNPR1.Draw by the forward and reverse of VvSTS gene magnifications of P9, P10
Thing, P11, P12 are the forward and reverse primer of VvPR1 gene magnifications, and P13, P14 are the forward and reverse primer of VvPR10 gene magnifications,
P15, P16 are the forward and reverse primer of VvNPR1 gene magnifications, primer and the phase in test example 1 of internal reference VvActin gene magnifications
Together.
Forward primer P9：5’-GAAACGCTCAACGTGCCAAGG-3’(SEQ ID NO:12)；
Reverse primer P10：5’-GTAACCATAGGAATGCTATGTAGC-3'(SEQ ID NO:13).
Forward primer P11：5’-GGAGTCCATTAGCACTCCTTTG-3’(SEQ ID NO:14)；
Reverse primer P12：5’-CATAATTCTGGGCGTAGGCAG-3’(SEQ ID NO:15).
Forward primer P13：5’-CCAACCAATCCTCCTCCTCTTC-3’(SEQ ID NO:16)；
Reverse primer P14：5’-CATCTCCGTCAACCACAGTGTA-3’(SEQ ID NO:17).
Forward primer P15：5’-TCTCCGATTCCAACGACTTCAG-3’(SEQ ID NO:18)；
Reverse primer P16：5’-CATCATCAACGCACGCACAA-3’(SEQ ID NO:19).
PCR reactions are carried out in iCycler iQ5Real-time PCR instruments (Bio-Rad companies of the U.S.).With 2- △ △Ct
Method calculates the relative expression quantity of gene.Result is as shown in figure 4, after powdery mildew is inoculated with, convert the grape leave of VvPUB17 genes
Its expression quantity significantly increases relative to unconverted grape leave for middle disease-resistant related gene VvSTS, VvPR1, VvPR10, VvNPR1
Plus, show that each disease-resistant related gene expression quantity rises in grape leave after conversion VvPUB17 genes, enhances the anti-of transformed plant
Characteristic of disease.
Sequence table
SEQUENCE LISTING
<110>University Of Science and Technology Of He'nan
<120>A kind of application of grape disease-resistant related gene VvPUB17, expression vector and its application
<170> PatentIn version 3.5
<211> 2145
<212> DNA
<213>Sequence
<221>The ORFs of grape disease-resistant related gene VvPUB17
<222> (1)..(2145)
<400> 1
ATGGCCTCCG CTGCGATAGT ATCGTCTCTG CGGCGGCGGA GATCGCCGTC TTTGGAGGCT 60
TTCTTGGCGC CGGTGGATCT AAACGAGGTG GCTCTTGTAC GGACACTGGC CACCATCTCC 120
ATGGAGCTTA TATTTGCGTT TTCCGATAGG CCTTGGCCGT TTCAGCGCAA GAATTCGAGG 180
TCCTTGATTC GGAAAATTGA GGTCTTTCTG GTGCTGTTAG AGTTCCTGAG GGATTGCAAT 240
TTGAGTCTGC CTTCGGCGGC GGTGCTTTGC TTCAAGGAGC TTTACCTGCT TCTGTATCGG 300
TCGAAGATAC TTCTCGATTA TTGCTTGCAA TCAAGTAAAT TGTGGCTTCT GTTGCAGAAC 360
CAGTCGATTT CGGGGCATTT TCATGATCTT AATCAGGAAA TTTCGACGCT GTTGGATGTA 420
TTTCCGATGG AGGAACTTGA ATTGACTGAA GATATAAGGG AGCAGCTTGA GCTTTTGCAG 480
AAACAGGTGA GGAGGGCGAA GTTGTTTCTT GATAAGAATG ATGAGGGGTT GAGGCTGAGG 540
TTGTATTCTT TTCTTGATGA CTTTGGGAGT GGGCGGATTC CTGATCCAGT GGAGCTGCGG 600
TTGTTTTTTG TGGATAGATT AGGGATTCGG GATGCGAAGA GTTGTAGGGC TGAAATTGAG 660
TTTTTGGAGG AGCAGATTTA TAGTCACGAG GGAGATGTTG AGCCGAATGT TGCTGTGCTT 720
AATGGGTTCG TTGCGCTTAC TCGATATTGC AGATTCTTGC TATTTGGATT TGAGGAGAGC 780
GAGGTAGAAA TGAGTTTTGG GATTAAGAAG CCGAGGAAAG GGTTGATTAC TCAAGAAATT 840
GGGGATACCT TCATAACAGT CCCCAAGGAC TTTTGTTGCC CCATATCTTT GGATGTGATG 900
CGAGACCCAG TAATAATTTC AACAGGACAG ACATATGATC GAACTTCAAT ATCAAGGTGG 960
ATGGAAGAAG GGCATTGTAG TTGCCCAAAG ACAGGGCAGA TGCTTGCTCA CCCTCGCCTT 1020
GTTCCCAATC GAGCTCTCAG GAATTTGATC ACACAATGGT GCACTGCTTA TGGAATCACC 1080
TTGGATCCAC CAGACAGCCC AGATAGTGTT GTAGAAACTT TTGCAGCAGC TTTGCCAACC 1140
AAAGCTGCTA TTGAAGCCAA CAAAGCCACA GCAGCGCTTC TCGTCCAACA GCTTGCAAGT 1200
GGTTCACAGG GTGCAAAGAC TGTAGCTGCC CGTGAGATAC GTTTATTAGC AAAAACAGGG 1260
AAGGAAAACC GTGCGTATAT AGCAGAAGCT GGGGCAATCC CCCATCTTCT CAAGCTACTC 1320
TCATCTCCAA ATTCTGTCGC ACAAGAGAAT TCTGTAACGG CAATGCTTAA CCTATCAATA 1380
TATGACAAGA ACAAAAGCCG AATTATGGAT GAGGATGGGT GTTTAGGTTT GATTGTTGAA 1440
GTTCTGATAT TTGGGCACAC AACGGAAGCA AGGGAAAATG CAGCTGCGAC ATTGTTCAGC 1500
CTTTCTGCTG TTCATGATTA TAAGAAGAGA ATAGCGGACG AGGGTGGGGC AGTTGAAGCC 1560
CTGGCAGGGC TGTTGAGAGA GGGAACCCCA AGAGGAAGGA AAGATGCTGT AACGGCTCTA 1620
TTTAATCTAT CAACCCACAC AGATAATTGT GCCAGAATGG TAGCGTCAGG GGCTGTAACT 1680
GCATTAGTAG CGGCCTTGGG AACTGAGGGG GTTGCAGAGG AAGCAGCGGG AGCATTGGCC 1740
TTGATTGTTA GGCGGCCAAT TGGGGCTGAA GCAGTAGGGA GGGAGGAAAT GGCAGTGGCT 1800
GGGTTATTAG GAATGATGCG GTGTGGGACT CCAAGGGGGA AAGAGAATGC AGTTGCAGCA 1860
TTGCTTGAAC TGTGCAGAAG TGGCGGGACA GCTGCAACAG AAAGGGTCCT TAAGGCTCCA 1920
GCGCTGGCTG GTTTGCTTCA GACTCTGTTG TTTACAGGTA CAAAGCGCGC TAGGAGAAAG 1980
GCTGCATCCC TTGCCAGAGT TTTCCAGAGG TGTGAGAATG CAGCCTTGCA TTTTGGTGGA 2040
CTTGGTGTAG GGTATGCATT TGCCAGAAAC TCATCTGCAA ATACCGATGC AAGCTTTTCT 2100
AGTGAGGTTT CCATGCAAAT GCCCATTTCA GTGCCTGTTT TGTAG 2145
<211> 714
<212> PRT
<213>Sequence
<221>The protein of grape disease-resistant related gene VvPUB17 codings
<222> (1)..(714)
<400> 2
MASAAIVSSL RRRRSPSLEA FLAPVDLNEV ALVRTLATIS MELIFAFSDR PWPFQRKNSR 60
SLIRKIEVFL VLLEFLRDCN LSLPSAAVLC FKELYLLLYR SKILLDYCLQ SSKLWLLLQN 120
QSISGHFHDL NQEISTLLDV FPMEELELTE DIREQLELLQ KQVRRAKLFL DKNDEGLRLR 180
LYSFLDDFGS GRIPDPVELR LFFVDRLGIR DAKSCRAEIE FLEEQIYSHE GDVEPNVAVL 240
NGFVALTRYC RFLLFGFEES EVEMSFGIKK PRKGLITQEI GDTFITVPKD FCCPISLDVM 300
RDPVIISTGQ TYDRTSISRW MEEGHCSCPK TGQMLAHPRL VPNRALRNLI TQWCTAYGIT 360
LDPPDSPDSV VETFAAALPT KAAIEANKAT AALLVQQLAS GSQGAKTVAA REIRLLAKTG 420
KENRAXIAEA GAIPHLLKLL SSPNSVAQEN SVTAMLNLSI YDKNKSRIMD EDGCLGLIVE 480
VLIFGHTTEA RENAAATLFS LSAVHDYKKR IADEGGAVEA LAGLLREGTP RGRKDAVTAL 540
FNLSTHTDNC ARMVASGAVT ALVAALGTEG VAEEAAGALA LIVRRPIGAE AVGREEMAVA 600
GLLGMMRCGT PRGKENAVAA LLELCRSGGT AATERVLKAP ALAGLLQTLL FTGTKRARRK 660
AASLARVFQR CENAALHFGG LGVGYAFARN SSANTDASFS SEVSMQMPIS VPVL 714
<211> 25
<212> DNA
<213>Sequence
<221>Forward primer P3
<222> (1)..(25)
<400> 3
AGAGTCTGAG AAACGAGATT CAGAC 25
<211> 29
<212> DNA
<213>Sequence
<221>Reverse primer P4
<222> (1)..(29)
<400> 4
AAGCAGCTTT TCCACATTAT ACTATTACC 29
<211> 2695
<212> DNA
<213>Sequence
<221>Grape disease-resistant related gene VvPUB17
<222> (1)..(2695)
<400> 5
AGAGTCTGAG AAACGAGATT CAGACAAGTA GTAGAATCAC ACACAAAAAT AAACAATAAG 60
TGTAAGAGAG TTGCACTGTT TTCCTTGTTT TTGTTTTTAT TGTTTTGTTT TTTGTTTCTG 120
GGTTTGTTTA TTAGAGAGAG AGAGAGATTT TGGTGCTGCA GAGGTCTTGA TTTTCCAAGA 180
GATTTGTGCA TCTTCAAAAT TTGTTTGCAG GTAAATTGAG GGGAAATATT TCGCAGTGTT 240
TTTTTTAGTA ACCCTAGATA ACGTTCGAAT TCCATGGCCT CCGCTGCGAT AGTATCGTCT 300
CTGCGGCGGC GGAGATCGCC GTCTTTGGAG GCTTTCTTGG CGCCGGTGGA TCTAAACGAG 360
GTGGCTCTTG TACGGACACT GGCCACCATC TCCATGGAGC TTATATTTGC GTTTTCCGAT 420
AGGCCTTGGC CGTTTCAGCG CAAGAATTCG AGGTCCTTGA TTCGGAAAAT TGAGGTCTTT 480
CTGGTGCTGT TAGAGTTCCT GAGGGATTGC AATTTGAGTC TGCCTTCGGC GGCGGTGCTT 540
TGCTTCAAGG AGCTTTACCT GCTTCTGTAT CGGTCGAAGA TACTTCTCGA TTATTGCTTG 600
CAATCAAGTA AATTGTGGCT TCTGTTGCAG AACCAGTCGA TTTCGGGGCA TTTTCATGAT 660
CTTAATCAGG AAATTTCGAC GCTGTTGGAT GTATTTCCGA TGGAGGAACT TGAATTGACT 720
GAAGATATAA GGGAGCAGCT TGAGCTTTTG CAGAAACAGG TGAGGAGGGC GAAGTTGTTT 780
CTTGATAAGA ATGATGAGGG GTTGAGGCTG AGGTTGTATT CTTTTCTTGA TGACTTTGGG 840
AGTGGGCGGA TTCCTGATCC AGTGGAGCTG CGGTTGTTTT TTGTGGATAG ATTAGGGATT 900
CGGGATGCGA AGAGTTGTAG GGCTGAAATT GAGTTTTTGG AGGAGCAGAT TTATAGTCAC 960
GAGGGAGATG TTGAGCCGAA TGTTGCTGTG CTTAATGGGT TCGTTGCGCT TACTCGATAT 1020
TGCAGATTCT TGCTATTTGG ATTTGAGGAG AGCGAGGTAG AAATGAGTTT TGGGATTAAG 1080
AAGCCGAGGA AAGGGTTGAT TACTCAAGAA ATTGGGGATA CCTTCATAAC AGTCCCCAAG 1140
GACTTTTGTT GCCCCATATC TTTGGATGTG ATGCGAGACC CAGTAATAAT TTCAACAGGA 1200
CAGACATATG ATCGAACTTC AATATCAAGG TGGATGGAAG AAGGGCATTG TAGTTGCCCA 1260
AAGACAGGGC AGATGCTTGC TCACCCTCGC CTTGTTCCCA ATCGAGCTCT CAGGAATTTG 1320
ATCACACAAT GGTGCACTGC TTATGGAATC ACCTTGGATC CACCAGACAG CCCAGATAGT 1380
GTTGTAGAAA CTTTTGCAGC AGCTTTGCCA ACCAAAGCTG CTATTGAAGC CAACAAAGCC 1440
ACAGCAGCGC TTCTCGTCCA ACAGCTTGCA AGTGGTTCAC AGGGTGCAAA GACTGTAGCT 1500
GCCCGTGAGA TACGTTTATT AGCAAAAACA GGGAAGGAAA ACCGTGCGTA TATAGCAGAA 1560
GCTGGGGCAA TCCCCCATCT TCTCAAGCTA CTCTCATCTC CAAATTCTGT CGCACAAGAG 1620
AATTCTGTAA CGGCAATGCT TAACCTATCA ATATATGACA AGAACAAAAG CCGAATTATG 1680
GATGAGGATG GGTGTTTAGG TTTGATTGTT GAAGTTCTGA TATTTGGGCA CACAACGGAA 1740
GCAAGGGAAA ATGCAGCTGC GACATTGTTC AGCCTTTCTG CTGTTCATGA TTATAAGAAG 1800
AGAATAGCGG ACGAGGGTGG GGCAGTTGAA GCCCTGGCAG GGCTGTTGAG AGAGGGAACC 1860
CCAAGAGGAA GGAAAGATGC TGTAACGGCT CTATTTAATC TATCAACCCA CACAGATAAT 1920
TGTGCCAGAA TGGTAGCGTC AGGGGCTGTA ACTGCATTAG TAGCGGCCTT GGGAACTGAG 1980
GGGGTTGCAG AGGAAGCAGC GGGAGCATTG GCCTTGATTG TTAGGCGGCC AATTGGGGCT 2040
GAAGCAGTAG GGAGGGAGGA AATGGCAGTG GCTGGGTTAT TAGGAATGAT GCGGTGTGGG 2100
ACTCCAAGGG GGAAAGAGAA TGCAGTTGCA GCATTGCTTG AACTGTGCAG AAGTGGCGGG 2160
ACAGCTGCAA CAGAAAGGGT CCTTAAGGCT CCAGCGCTGG CTGGTTTGCT TCAGACTCTG 2220
TTGTTTACAG GTACAAAGCG CGCTAGGAGA AAGGCTGCAT CCCTTGCCAG AGTTTTCCAG 2280
AGGTGTGAGA ATGCAGCCTT GCATTTTGGT GGACTTGGTG TAGGGTATGC ATTTGCCAGA 2340
AACTCATCTG CAAATACCGA TGCAAGCTTT TCTAGTGAGG TTTCCATGCA AATGCCCATT 2400
TCAGTGCCTG TTTTGTAGTG TTAACATCTG CCTTGTGTTA CTGTGTTCCC TGGTTATTTA 2460
TGCACCATTT TTGTTGGTAA TGTTCACAAA TTCTTTCATT GAAATGTTAA GAGGAGGTGG 2520
GAAAAGAAAG TTATAGGAAT ACCATATTTG ATGTTTTAGA AAGATTGATG AATGTGTTTA 2580
TCATTTAGTT TCATTTTCTG CTATATTTTG TAATGCCCAA TTACTTGACA AATCAGAATG 2640
AGGAATTCTC TAAAAATGGT CAAGTTGGTA ATAGTATAAT GTGGAAAAGC TGCTT 2695
<211> 24
<212> DNA
<213>Sequence
<221>Forward primer P5
<222> (1)..(24)
<400> 6
CTGTAACGGC AATGCTTAAC CTAT 24
<211> 23
<212> DNA
<213>Sequence
<221>Reverse primer P6
<222> (1)..(23)
<400> 7
CCTCGTCCGC TATTCTCTTC TTA 23
<211> 20
<212> DNA
<213>Sequence
<221>Forward primer P7
<222> (1)..(20)
<400> 8
TCCTGTGGAC AATGGATGGA 20
<211> 20
<212> DNA
<213>Sequence
<221>Reverse primer P8
<222> (1)..(20)
<400> 9
CTTGCATCCC TCAGCACCTT 20
<211> 31
<212> DNA
<213>Sequence
<221>Forward primer P1
<222> (1)..(31)
<400> 10
GGGAGATCTA TGGCCTCCGC TGCGATAGTA T 31
<211> 35
<212> DNA
<213>Sequence
<221>Reverse primer P2
<222> (1)..(35)
<400> 11
GGGGGTGACC CTACAAAACA GGCACTGAAA TGGGC 35
<211> 21
<212> DNA
<213>Sequence
<221>Forward primer P9
<222> (1)..(21)
<400> 12
GAAACGCTCA ACGTGCCAAG G 21
<211> 24
<212> DNA
<213>Sequence
<221>Reverse primer P10
<222> (1)..(24)
<400> 13
GTAACCATAG GAATGCTATG TAGC 24
<211> 22
<212> DNA
<213>Sequence
<221>Forward primer P11
<222> (1)..(22)
<400> 14
GGAGTCCATT AGCACTCCTT TG 22
<211> 21
<212> DNA
<213>Sequence
<221>Reverse primer P12
<222> (1)..(21)
<400> 15
CATAATTCTG GGCGTAGGCA G 21
<211> 22
<212> DNA
<213>Sequence
<221>Forward primer P13
<222> (1)..(22)
<400> 16
CCAACCAATC CTCCTCCTCT TC 22
<211> 22
<212> DNA
<213>Sequence
<221>Reverse primer P14
<222> (1)..(22)
<400> 17
CATCTCCGTC AACCACAGTG TA 22
<211> 22
<212> DNA
<213>Sequence
<221>Forward primer P15
<222> (1)..(22)
<400> 18
TCTCCGATTC CAACGACTTC AG 22
<211> 20
<212> DNA
<213>Sequence
<221>Reverse primer P16
<222> (1)..(20)
<400> 19
CATCATCAAC GCACGCACAA 20
Claims (4)
1. applications of a kind of grape disease-resistant related gene VvPUB17 in disease resistance of plant is strengthened, it is characterised in that：The gene
Nucleotide sequence such as SEQ ID NO:Shown in 5, ORFs such as SEQ ID NO:Shown in 1, the ammonia of the protein coded by it
Base acid sequence is as shown in SEQ ID NO.2.
2. a kind of plant overexpression carrier, it is characterised in that：The expression vector contains such as SEQ ID NO:Grape shown in 5 resists
The ORFs of sick related gene VvPUB17, ORFs such as SEQ ID NO:Shown in 1.
3. expression vector according to claim 2, it is characterised in that：The expression vector is pCAMBIA3301-
VvPUB17, preparation process is as follows：
1) with grape cDNA as template, P1, P2 are primer, carry out pcr amplification reaction, and amplified production reclaims mesh after electrophoretic analysis
Fragment；
Forward primer P1：5’-GGGAGATCTATGGCCTCCGCTGCGATAGTAT-3 ',
Reverse primer P2：5’-GGGGGTGACCCTACAAAACAGGCACTGAAATGGGC-3’；
2) distinguish double digestion step 1 with Bgl II, BstE II) in purpose fragment and plant expression vector pCAMBIA3301, return
Target fragment and linearized vector are received, screening, checking, obtain final product after connection, conversion.
4. application of a kind of plant overexpression carrier as claimed in claim 2 or claim 3 in disease resistance of plant is strengthened.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710051147.7A CN106754992B (en) | 2017-01-23 | 2017-01-23 | A kind of application, expression vector and its application of grape disease-resistant related gene VvPUB17 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710051147.7A CN106754992B (en) | 2017-01-23 | 2017-01-23 | A kind of application, expression vector and its application of grape disease-resistant related gene VvPUB17 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106754992A true CN106754992A (en) | 2017-05-31 |
| CN106754992B CN106754992B (en) | 2019-06-14 |
Family
ID=58942426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710051147.7A Expired - Fee Related CN106754992B (en) | 2017-01-23 | 2017-01-23 | A kind of application, expression vector and its application of grape disease-resistant related gene VvPUB17 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754992B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102121005A (en) * | 2010-12-03 | 2011-07-13 | 西北农林科技大学 | Grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof |
-
2017
- 2017-01-23 CN CN201710051147.7A patent/CN106754992B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102121005A (en) * | 2010-12-03 | 2011-07-13 | 西北农林科技大学 | Grapevine powdery mildew resistance transcription factor gene VpRFP1 promoter sequence and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| LI JIAO等: "Overexpression of a stress-responsive U-box protein gene VaPUB affects the accumulation of resistance related proteins in Vitis vinifera ‘Thompson Seedless’", 《PLANT PHYSIOLOGY AND BIOCHEMISTRY》 * |
| NCBI: "GenBank登录号：XM_019222704.1", 《NCBI GENBANK》 * |
| 鲁玉清等: "拟南芥U-box蛋白的研究进展", 《分子植物育种》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106754992B (en) | 2019-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109825457B (en) | Salt-tolerant bacillus E40207a2 and application thereof | |
| CN105255915B (en) | Application of the arabidopsis AtGDSL genes in the anti-sclerotiniose of rape and in promoting seed to sprout | |
| CN103756975B (en) | A kind of preparation method and application thereof infecting the soybean mosaic virus of tobacco | |
| CN106244607B (en) | Application of the cotton cells cytochrome p 450 CYP94C1 gene in resisting verticillium | |
| CN104450740B (en) | A kind of alfalfa MsWRKY33 transcription factor and encoding proteins, preparation method and application | |
| CN102392080B (en) | Method for identifying tomato yellow leaf curl virus resistance | |
| CN103214564B (en) | Clone of MYB transcription factor MaMYB in Musa spp. and expression vector construction | |
| CN110004156A (en) | GhCML20 gene relevant to resistance to verticillium wilt and its application | |
| CN108660140A (en) | Application of the SlSL4 genes in regulating and controlling Fruit Ripening of Tomato | |
| CN105274122B (en) | The RNAi expression vector and its construction method of diamondback moth TOR gene and application | |
| CN106520791B (en) | Grape disease-resistant related gene VvPUB21 and its plant expression vector and application | |
| CN111635904B (en) | Gene CsWRKY10 for enhancing cucumber target spot disease resistance and application thereof | |
| CN104046639A (en) | Wheat methionine sulfoxide reductase gene TaMsrB3.1 and application thereof | |
| CN115820685B (en) | Citrus CsGSTF1 gene and application thereof | |
| CN106754992B (en) | A kind of application, expression vector and its application of grape disease-resistant related gene VvPUB17 | |
| CN102659933A (en) | Bacillus thuringiensis gene cry8like and cry8G combination and application thereof | |
| CN116162645A (en) | Application of induction transcription factor CoWRKY3 in plant anthracnose resistance | |
| CN109825456B (en) | Marina marine bacillus E40208a1 and application thereof | |
| CN109666655B (en) | Fusarium graminearum single-stranded circular DNA virus FgGMTV1/HB58 and application thereof | |
| CN104131014B (en) | Lilium regale wilson class sprouts the application of fibroin gene LrGLP1 | |
| CN106834337A (en) | The DON tolerances and FHB resistances of arabidopsis are improved using wheat cdna | |
| CN106520723A (en) | Protein VvMas and encoding gene, and application thereof in improvement of salt tolerance of plants | |
| CN102140446A (en) | Application of rape iMyAP gene over-expression in sclerotinia sclerotiorum resistance of rape | |
| CN105779476A (en) | Tea tree cold-proof gene CsSPMS and plant expression vector construction and application thereof | |
| CN101892242A (en) | Ammopiptanthus mongolicus cold-resistant gene AmEBP1 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20170531 Assignee: LUOYANG JIENTE BIOTECHNOLOGY CO.,LTD. Assignor: Henan University of Science and Technology Contract record no.: X2019980000341 Denomination of invention: Application of anti-disease related gene VvPUB17 of grape, expression vector and application thereof Granted publication date: 20190614 License type: Common License Record date: 20191111 |
|
| EE01 | Entry into force of recordation of patent licensing contract | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190614 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |