CN116536347A - Application of calcium-dependent protein kinase gene GhCAMK in verticillium wilt resistance of plants - Google Patents
Application of calcium-dependent protein kinase gene GhCAMK in verticillium wilt resistance of plants Download PDFInfo
- Publication number
- CN116536347A CN116536347A CN202310334048.5A CN202310334048A CN116536347A CN 116536347 A CN116536347 A CN 116536347A CN 202310334048 A CN202310334048 A CN 202310334048A CN 116536347 A CN116536347 A CN 116536347A
- Authority
- CN
- China
- Prior art keywords
- gene
- cotton
- ghcamk
- verticillium wilt
- plants
- 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
- 241000082085 Verticillium <Phyllachorales> Species 0.000 title claims abstract description 72
- 108010025267 calcium-dependent protein kinase Proteins 0.000 title claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 86
- 241000196324 Embryophyta Species 0.000 claims abstract description 76
- 239000013604 expression vector Substances 0.000 claims abstract description 8
- 230000014509 gene expression Effects 0.000 claims description 38
- 239000013598 vector Substances 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 19
- 102000004169 proteins and genes Human genes 0.000 claims description 18
- 230000030279 gene silencing Effects 0.000 claims description 17
- 230000001105 regulatory effect Effects 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 239000012620 biological material Substances 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 6
- 240000002024 Gossypium herbaceum Species 0.000 claims description 5
- 235000004341 Gossypium herbaceum Nutrition 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 3
- 230000001276 controlling effect Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 2
- 244000299507 Gossypium hirsutum Species 0.000 abstract description 84
- 229920000742 Cotton Polymers 0.000 abstract description 64
- 235000009429 Gossypium barbadense Nutrition 0.000 abstract description 20
- 235000018322 upland cotton Nutrition 0.000 abstract description 20
- 201000010099 disease Diseases 0.000 abstract description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 17
- 230000028604 virus induced gene silencing Effects 0.000 abstract description 17
- 241000894006 Bacteria Species 0.000 abstract description 6
- 238000011160 research Methods 0.000 abstract description 4
- 230000009261 transgenic effect Effects 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 238000012216 screening Methods 0.000 abstract description 3
- 230000009456 molecular mechanism Effects 0.000 abstract description 2
- 238000011529 RT qPCR Methods 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 15
- 239000000725 suspension Substances 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000012226 gene silencing method Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 230000002792 vascular Effects 0.000 description 8
- 108030005456 Calcium/calmodulin-dependent protein kinases Proteins 0.000 description 7
- 241001123668 Verticillium dahliae Species 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 101150084750 1 gene Proteins 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 238000005286 illumination Methods 0.000 description 6
- ZNJFBWYDHIGLCU-HWKXXFMVSA-N jasmonic acid Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-HWKXXFMVSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 6
- 102000000584 Calmodulin Human genes 0.000 description 5
- 108010041952 Calmodulin Proteins 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 238000005520 cutting process Methods 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000010839 reverse transcription Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 239000013589 supplement Substances 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 241000589158 Agrobacterium Species 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 102000001253 Protein Kinase Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 3
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- ZNJFBWYDHIGLCU-UHFFFAOYSA-N jasmonic acid Natural products CCC=CCC1C(CC(O)=O)CCC1=O ZNJFBWYDHIGLCU-UHFFFAOYSA-N 0.000 description 3
- 235000009973 maize Nutrition 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108060006633 protein kinase Proteins 0.000 description 3
- 229960004889 salicylic acid Drugs 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical group [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 208000035240 Disease Resistance Diseases 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 238000010802 RNA extraction kit Methods 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000036579 abiotic stress Effects 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
- 208000030961 allergic reaction Diseases 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 230000008653 root damage Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 230000007226 seed germination Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000035882 stress Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 101710094045 1-deoxy-D-xylulose-5-phosphate synthase Proteins 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 101100532087 Arabidopsis thaliana RUB2 gene Proteins 0.000 description 1
- 108010032088 Calpain Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI 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
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 229930182566 Gentamicin Natural products 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
- 101000836292 Klebsiella pneumoniae Type II restriction enzyme KpnI Proteins 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108020005089 Plant RNA Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 230000004790 biotic stress Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 239000012225 czapek media Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012470 diluted sample Substances 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
- 238000004090 dissolution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000008641 drought stress Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000006353 environmental stress Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000006355 external stress Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000011331 genomic analysis Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000001965 potato dextrose agar Substances 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000001743 silencing effect Effects 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005406 washing Methods 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
- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- 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
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/11—Protein-serine/threonine kinases (2.7.11)
- C12Y207/11017—Ca2+/Calmodulin-dependent protein kinase (2.7.11.17)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses application of a calcium-dependent protein kinase gene GhCCAK in verticillium wilt resistance of plants. Belongs to the technical field of genetic engineering. The invention constructs a VIGS plant expression vector by using the GhCAMK gene to transform upland cotton TM-1, and the obtained transgenic cotton shows resistance to verticillium after being inoculated with cotton verticillium wilt bacteria V991, which shows that the GhCAMK gene is highly related to the resistance to cotton verticillium wilt. The invention lays an application foundation for molecular mechanism research of verticillium wilt resistance and cultivation of new varieties of new verticillium wilt-resistant plants, and provides new reference and reference for screening new disease-resistant genes and cultivating resistant plants.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of a calcium-dependent protein kinase gene GhCAMK in plant verticillium wilt resistance.
Background
Cotton verticillium wilt is a soil-borne disease caused by the soil filamentous fungus verticillium dahliae (Verticillium dahliae), and is also called as "cancer" of cotton because of rapid onset and difficult healing. In the 90 s, verticillium wilt of China spreads rapidly, and occurs in all cotton areas of China, so that 10% -20% of cotton yield reduction of China is caused throughout the year, and 70% of cotton yield reduction in partial cotton areas is achieved, so that the loss is huge, and the method is one of main obstacles restricting high and stable cotton yield of China. Practice proves that the cultivation of cotton disease-resistant varieties is an effective economic strategy for restricting verticillium wilt spreading. Due to the common non-disease resistance of the existing variety resources, lack of germplasm resources, poor broad spectrum and the like, breeders do not cultivate cultivars with excellent resistance to cotton verticillium wilt at present. In recent years, the development of candidate genes for resisting verticillium wilt of cotton based on genetic engineering has become another strategy for alleviating verticillium wilt invasion, and a series of genes such as GhSINAs, ghVLN2, ghSWEET42 and GhTIFY9 have been reported.
Calcium ions are important second messengers in plant cell signaling. When plants are subjected to external stress, calcium signals in cells are transmitted to downstream components through calmodulin, such as calmodulin, calmodulin-like and caldependent protein kinase (CDPK), etc., thereby regulating the expression of the related genes. In plants, the modulation of calcium ion concentration in the cytoplasm can be in response to a variety of endogenous and exogenous signals, including changes in hormone levels, abiotic stresses (e.g., drought, high, low temperature, or light, etc.), biotic stresses (e.g., pathogens and pathogenic microorganisms, etc.). Five calcium ion sensors in plants are available, including calcium pattern (CAM), calmodulin (CML), calmodulin B protein (CBL), calcium/calmodulin-dependent protein kinase (CCAMK) and calcium-dependent protein kinase (CDPK). Ca (Ca) 2+ CaM-dependent protein kinase (CCAMK) is a plant-specific protein kinase, first discovered from lily, in the smokeExpression is also found in grass, maize, rice and wheat. Studies have shown that CCAMK is expressed in roots and developing nodules, and that activation of CCAMK is the primary regulatory mode of plant root symbiosis, which can enhance tolerance of plants to environmental stresses.
CCAMK is involved in responding to plant abiotic stress processes. In arabidopsis, overexpression of TaCCAMK reduces sensitivity to ABA during seed germination and increases tolerance to salt stress during seed germination. In maize, zmCCAMK is in turn involved in ABA-induced antioxidant protection processes by phosphorylating ZmNAC 84. Recent studies have found that over-expression of ZmCCAMK and rice CCAMK enhances the antioxidant protective capacity of plants, positively regulating the tolerance of maize and rice plants to drought and oxidative stress.
In summary, how to provide a calpain gene that regulates cotton verticillium resistance is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the present invention provides the use of the calcium-dependent protein kinase gene GhCAMK in plants against verticillium wilt.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
application of calcium-dependent protein kinase gene GhCAMK in regulating and controlling verticillium wilt resistance of plants, wherein the nucleotide sequence of the gene GhCAMK is shown in SEQ ID NO: 1.
ATGGGACAAGATAAAGCGAAACTAGTAGAAGAATACGAAATCCTAGATATACTAGGACGAGGTGGATTCTCAGTCGTAAGAAAAGGTATAAAAAGAAAGAACGGATCAGATCATGAGAAAACACAAGTCGCCATTAAAACACTGAAACGATTCGGAACGACGCCGTCACCAGCTCGAGTCGAGAAAACAATTGCTTCGATGGCGGCGTTATTGCCGACGCGTAACCAGGTTTCCATCTCTGACGCGCTGTTGACGAACGAGATCCTCGTCATGAGGAAGATCGTCGAGAACGTTTCGCCGCATCCGAACGTGATCGATCTCTACGACGTTTACGAAGATCAAGCCGGGGTTCATTTGGTGCTGGAATTGTGCTCCGGCGGGGAGTTGTTCGATCGGATAGTGGCGGAAACACGGTACTCGGAAGCCGGTGCGGCAGCGGTGGTACGGCAGATCGCGGGAGGATTAGCGGCGATCCATAAGGCGAACATTGTTCATAGAGATCTGAAACCGGAGAACTGCTTGTTCTTGAATAAAAATAAAGATTCAACGTTGAAGATCATGGACTTTGGATTGAGTTCGGTGGAAGAATTTACGGATCCGGTTATCGGGTTGTTTGGATCCATAGATTATGTTTCACCTGAGGCGCTTTCTCAGGGGCAAATCACAGCAAAGAGCGATATGTGGTCTTTGGGTGTCATCTTATTCATCTTGCTTTCGGGGTATCCACCATTTATTGCTCAATCTAATCGTCAGAAACAACAGATGATAATGGCTGGAGAATACAATTTCGATGAGAGGACATGGAAAAACATTTCTTCATCAGCAAAGCATTTGATTTCTAATCTGTTGCAAGTTGATCCTGATAGAAGACCTAGTGCTGAACAACTTCTAGCTCATCCATGGGTCATCGGGGATTCAGCAAAGCAAGAACAAATAGATGCGGAGGTTGTTTCCAGATTGCAGAGTTTTAACGCGCGTCGTAAGCTACGTGCTGCCGCCATAGCCAGTGTGTTGAGCAGCAAGGTTTTACTAAGGACGAAGAGGTTAAGAAGTTTGCTTGGCTCCCATGACCTTTCAAAGGACGAAATTGATAACCTCAAGTCGAACTTTAAGAAAATATGTGCTAACGGTGACAATGCTACTTTACCCGAATTTGAGGAGGTGCTAAAAGCAATGAACATGTCTTCACTACTTCCTTTGGCTACTCGTATCTTTGACCTATTCGATAGCAATCGAGATGGTACTGTTGATATGAGAGAAATTGTGTGTGGATTTTCCAGTCTTAAGAATTCTAAAGGAGATGACGCTCTTCGCCTGTGCTTCGAGATGTATGATACAGATCGATCTGGATGCATTACTAAAGAAGAACTAGCATCAATGTTAAGAGCATTGCCCGATGACTGTCTTCCACCAGATATTACAGAACCTGGAAAGTTAGATGAAATATTCGATCGAATGGATGCAAACAGTGATGGGAAGGTTACGTTCGAAGAATTTAAGGATGCCATGCAAAGAGACAGCTCTCTCCAAGACGTAGTCCTCTCTTCTCTTCGACAACAGTAA，SEQ ID NO：1。
Application of protein or a substance for regulating and controlling expression of a protein coding gene in verticillium resistance of plants, wherein the amino acid sequence of the protein is shown in SEQ ID NO: 2.
MGQDKAKLVEEYEILDILGRGGFSVVRKGIKRKNGSDHEKTQVAIKTLKRFGTTPSPARVEKTIASMAALLPTRNQVSISDALLTNEILVMRKIVENVSPHPNVIDLYDVYEDQAGVHLVLELCSGGELFDRIVAETRYSEAGAAAVVRQIAGGLAAIHKANIVHRDLKPENCLFLNKNKDSTLKIMDFGLSSVEEFTDPVIGLFGSIDYVSPEALSQGQITAKSDMWSLGVILFILLSGYPPFIAQSNRQKQQMIMAGEYNFDERTWKNISSSAKHLISNLLQVDPDRRPSAEQLLAHPWVIGDSAKQEQIDAEVVSRLQSFNARRKLRAAAIASVLSSKVLLRTKRLRSLLGSHDLSKDEIDNLKSNFKKICANGDNATLPEFEEVLKAMNMSSLLPLATRIFDLFDSNRDGTVDMREIVCGFSSLKNSKGDDALRLCFEMYDTDRSGCITKEELASMLRALPDDCLPPDITEPGKLDEIFDRMDANSDGKVTFEEFKDAMQRDSSLQDVVLSSLRQQ*，SEQ ID NO：2。
The application of the gene GhCAMK or the protein related biological material in plant verticillium wilt resistance is that the biological material is any one of the following:
a: a nucleic acid molecule having a nucleotide sequence as described above or a nucleic acid molecule encoding the above protein;
b: an expression cassette comprising silencing of the nucleic acid molecule of a;
c: an expression vector comprising the nucleic acid molecule of A, or a recombinant vector comprising the expression cassette of B;
d: a recombinant microorganism comprising the nucleic acid molecule of A, a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.
The term "expression cassette" refers to a DNA capable of expressing a protein as described in the above applications in a host cell, which DNA may include not only a promoter for initiating transcription of a gene encoding the protein, but also a terminator for terminating transcription of the gene encoding the protein. Further, the expression cassette may also include an enhancer sequence.
Furthermore, the gene GhCAMK or the protein related biological material is applied to breeding plant varieties with enhanced or reduced verticillium wilt resistance.
Furthermore, the gene GhCAMK or the protein related biological material is applied to plant breeding.
Further, the plant is a cotton plant.
Further, the plant is cotton.
Further, the plant is upland cotton TM-1.
A method for improving verticillium wilt resistance of plants by silencing or inhibiting expression of the above gene GhCAMK or activity of the above protein in plants.
The silencing or inhibiting of the expression level of the protein-encoding gene in the plant may be achieved by any means known in the art to effect deletion, insertion or base-change mutation of the gene, thereby achieving reduced or lost gene function.
Compared with the prior art, the invention has the beneficial effects that: the invention utilizes the GhCAMK gene sequence information to amplify the gene and constructs a VIGS plant expression vector to transform upland cotton TM-1, and the obtained transgenic cotton shows resistance to verticillium after being inoculated with cotton verticillium wilt bacteria V991, which shows that the GhCAMK gene is highly relevant to the resistance of cotton verticillium wilt. The invention lays an application foundation for molecular mechanism research of verticillium wilt resistance and cultivation of new varieties of new verticillium wilt-resistant plants, and provides new reference and reference for screening new disease-resistant genes and cultivating resistant plants.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing PCR products of the GhCAMK gene in example 1 of the present invention, wherein the Marker is a 2000bp Laddermarker;
FIG. 2 is a graph showing qRT-PCR results of expression amounts of GhCAMK gene in different cotton tissues (root, stem, leaf, petal, bract and sepal) in example 2 of the present invention;
FIG. 3 is a drawing showing the signal molecules (jasmonic acid JA, salicylic acid SA, H) in example 2 of the present invention 2 O 2 Dip-dyeing) to induce qRT-PCR results of the expression amount of the GhCAMK gene;
FIG. 4 is a graph showing the results of qRT-PCR analysis of the expression pattern of the yellow wilt bacteria V991 infection-induced GhCAMK gene in example 2 of the present invention;
FIG. 5 is a graph showing phenotype of upland cotton TM-1 plants after injection of the VIGS vector in example 3 according to the present invention, wherein: a is the phenotype of the plant after 2 weeks of the VIGS vector injected with the ghca 1 gene; TRV 00 (control) and TRV GhCAMK (10) of upland cotton TM-1 were inoculated with verticillium wilt V991 spore solution by root dipping 7 condiia/mL) 21 days of morbidity;
FIG. 6 is a graph showing qRT-PCR results of expression amounts of the GhCAMK gene in three-leaf stage leaves of cotton plants of the invention in TRV 00 and TRV of upland cotton TM-1 in example 3;
FIG. 7 is a graph showing the statistical result of the disease index of transgenic upland cotton TM-1 inoculated with verticillium in example 3 of the present invention;
FIG. 8 is a graph showing cotton stalk slitters of a silent plant and a control plant of the result of analysis of resistance of a VIGS-interfered upland cotton TM-1 plant to verticillium wilt in example 3 of the present invention;
FIG. 9 is a graph showing comparison of the results of fungus recovery experiments performed on silenced plants of the VIGS-interfered upland cotton TM-1 plants of example 3 according to the results of analysis of resistance to verticillium wilt with surface sterilized stems of control plants;
FIG. 10 is a graph showing statistics of cotton verticillium recovery from a silent plant and a control plant of the analysis of resistance of a VIGS-interfered upland cotton TM-1 plant to verticillium in example 3 of the present invention;
FIG. 11 is a graph showing the relative abundance of verticillium DNA in cotton stems of a silent plant and a control plant of VIGS-interfered upland cotton TM-1 plants of example 3 according to the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The cotton material comprises:
upland cotton TM-1, supplied by cotton institute of China academy of agricultural sciences;
plant VIGS silencing expression vector:
verticillium V991: the laboratory is stored, and the public can be obtained from the institute of biotechnology of nuclear sciences of agricultural sciences of Xinjiang (the institute of biotechnology of Uygur autonomous region of Xinjiang);
primer sequence: are provided by Shanghai biochemical engineering;
other biological materials include: the escherichia coli DH5 alpha, the agrobacterium tumefaciens GV3101, the BP reaction entry vector, the VIGS interference technology vectors pTRV1 and pTRV2 and the like belong to commercial strains or vectors, and are not repeated;
experimental reagent: the plasmid extraction kit, oligo (dT) 18, RNAase inhibitor, dNTP, pMD18-T Vector, T4-DNA ligase, endonuclease EcoRI and KpnI, exTaq enzyme, PCR product recovery kit and the like are all products of TaKaRa company, and the fluorescent quantitative PCR kit is a product of TOYOBO company;
the special plate for fluorescent quantitative PCR is manufactured by Labwares company;
RNA extraction kit was purchased from TIANGEN (Beijin, china);
the culture medium and solution used: LB liquid (solid) medium, YEP liquid (solid) medium, czapek medium, PDA medium, 50 xTAE Buffer (Na) 2 EDTA·2H 2 37.2g of O, 57.1mL of glacial acetic acid, pH adjustment by NaOH=8.3, water addition and volume fixation to 1L) and the like, and are prepared according to the conventional preparation method in the field;
other undescribed antibiotics, hormones and other reagents are common in the art and will not be described again.
Example 1
Acquisition of upland cotton GhCAMK Gene
Planting upland cotton TM-1, extracting RNA of cotton seedling leaves, and performing reverse transcription to obtain cDNA;
RNA extraction procedure referring to TIANGEN plant RNA rapid extraction kit, reverse transcription system is shown in Table 1:
TABLE 1 reverse transcription system
| Reagent(s) | Dosage of |
| TotalRNA | 1μg |
| Oligo(dT)18(0.5μg/μL) | 1μL |
| 2×TSReactionMix | 10μL |
| TransScriptRT/EnzymeMix | 1μL |
| gDNARemover | 1μL |
| RNase-freeWater | Supplement to 20 mu L |
Executing the program: 42 ℃ for 30min and 85 ℃ for 5s.
cDNA with the ID number of Gh_D06G1869 gene (the nucleotide sequence is shown as SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO: 2) in a cottonFGD website (https:// CottonFGD. Org) is used as a template, primers are designed and amplified, and the primer sequences are as follows:
GhCCAMK-F:5’-ATGGGACAAGATAAAGCGAAAC-3’，SEQ ID NO：3；
GhCCAMK-R:5’-TTACTGTTGTCGAAGAGAAGAGAG-3’，SEQ ID NO：4。
the PCR amplification system is shown in Table 2:
TABLE 2PCR amplification System
| Reagent(s) | Dosage of |
| GhCCAMK-F(10μM) | 1μL |
| GhCCAMK-R(10μM) | 1μL |
| 5×TransStartFastPfuBuffer | 10μL |
| 2.5mMdNTPs | 4μL |
| TransStartFastPfuDNAPolymerase(2.5U/μL) | 1μL |
| Nuclease-freeWater | Supplement to 50 mu L |
PCR amplification procedure: 95 ℃ for 2min;95℃20s,56℃30s,72℃120s,35 cycles; 7min at 72 ℃.
The PCR electrophoresis products were subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 1. As can be seen from FIG. 1, the gene GhCAMK has a size of about 1563bp.
Example 2
1. Tissue expression pattern of GhCAMK gene under natural condition
Respectively taking leaves, roots, stems, flowers, sepals and bracts of cotton in the full-bloom stage of the cotton, extracting RNA of the cotton, obtaining cDNA by using a reverse transcription kit, and carrying out qPCR amplification by adopting the following quantitative PCR primers:
QRT-PCR-GhCCAMKF:5’-GGCGATCCATAAGGCGAACA-3’，SEQ ID NO：5；
QRT-PCR-GhCCAMKR:5’-CTCTTTGCTGTGATTTGCCCC-3’，SEQ ID NO：6。
the reference gene is Ubiquitin7 (Ubiquitin 7, UB 7), and the primer sequence is designed as follows:
UBQ7F:5’-GAAGGCATTCCACCTGACCAAC-3’，SEQ ID NO：7；
UBQ7R:5’-CTTGACCTTCTTCTTCTTGTGCTTG-3’，SEQ ID NO：8。
qRT-PCR was performed on an ABI 7500Real-time PCR sequence detection system and software (AppliedBiosystems, USA).
The 20. Mu.L reaction system was designed as follows:
the reaction system was 10. Mu. LSYBR Green Realtime PCR Master Mix, 1. Mu.L of cDNA product, 0.4. Mu.L (10. Mu.M) of each of the upstream and downstream primers, and the nucleic-free Water was supplemented to a total reaction volume of 20. Mu.L. 4 technical replicates per gene.
The corresponding procedure is:
the quantitative procedure was 94℃for 30s;95℃5s,57℃15s,72℃31s,40 cycles.
The specificity of the amplified product after 40 cycles was detected by dissolution profile analysis. Each reaction included at least three replicates. Primer amplification efficiency was measured by plotting the log of the template dilution against the Ct value of each diluted sample, diluted with a single template to different concentrations.
The detection results are shown in fig. 2, and analysis shows that the GhCCAK gene is expressed in all tissues of cotton, but the transcription level is obviously different, wherein the expression of the gene in leaves and bracts is higher than that of other tissues, and the gene is the stem; the expression level in roots, flowers and sepals is significantly lower than in other tissues, and the transcription level in flowers is slightly higher than in roots and sepals.
2. Is stressed by signal molecules (jasmonic acid JA, salicylic acid SA and H) 2 O 2 Dip dyeing) expression pattern of the gene GhCAMK
The TM-1 cotton seeds are soaked in water for 24 hours and then sowed in nutrient soil, and are subjected to illumination for 12 hours or dark for 12 hours, and are subjected to illumination culture at 26-28 ℃. The humidity is kept at 60% or above, water is poured once for 4-5 d, cotton seedlings with consistent growth vigor and size are selected when two leaves of the seedlings are centered, and 200 mu M jasmonic acid, 2mM salicylic acid and 1mM H are respectively used 2 O 2 Soaking cotton seedling root system for 10min, and planting cotton seedling in the nutrition pot. And respectively taking cotton root systems at the time points of 0.5h, 1h and 3h after the treatment, extracting RNA, and reversely transcribing cDNA for expression pattern analysis.
The qRT-PCR analysis related primers and the PCR reaction procedure were the same as in example 1, section "tissue expression pattern of the gene GhCAMK under natural conditions".
The qRT-PCR detection results are shown in FIG. 3, and the expression level of GhCAMK is shown in JA and H 2 O 2 The treatment time is highest at 0h, and then drops significantly; the expression level of ghvcmk was down-regulated after SA treatment. It was demonstrated that the expression level of GhCAMK was affected by the stress of the signal molecule.
3. Expression pattern of GhCCAK under verticillium infection condition
Root injury is carried out by selecting two-leaf and one-heart upland cotton TM-1, and the concentration of verticillium wilt V991 conidium liquid is 1 multiplied by 10 7 Spores/mL with water treatment as negative control. After the cotton seedling root system is soaked in the bacterial liquid for 10min, the cotton seedling root system is planted in a nutrition pot, and the cotton seedling root system is taken at the time points of 0h, 0.5h, 2h, 6h, 12h, 24h and 48h after inoculation, RNA is extracted, and cDNA is reversely transcribed for expression pattern analysis.
The qRT-PCR analysis related primers and the PCR reaction procedure were the same as in example 1, section "tissue expression pattern of the gene GhCAMK under natural conditions".
The detection result of qRT-PCR is shown in figure 4, after verticillium wilt V991 conidium liquid treatment, the expression level of GhCAMK is obviously reduced after inoculating verticillium wilt pathogen for 2 hours, and the expression level tends to be lowest after 48 hours. After 0.5 hour of water treatment, the significance was down-regulated. The result shows that after the root system of upland cotton TM-1 is infected by verticillium, the expression level of the gene GhCAMK is obviously changed.
Example 3
And a VIGS interference vector is constructed by using a VIGS technology, so that the gene GhCCAK is silenced. The phenotypic change of cotton after gene silencing during verticillium infection is observed, and further proves that the gene GhCAMK is highly related to verticillium resistance.
1. Construction of recombinant expression vectors
(1) Primer design and PCR amplification
Primers are designed in a non-conserved region of the gene GhCAMK, and the design principle of the primers is as follows: the restriction enzyme EcoRI cleavage site and the protecting base are added to the upstream primer, and the restriction enzyme KpnI cleavage site and the protecting base are added to the downstream primer, and the primers are designed as follows:
GhCCAMK-1F:5’-CGGAATTCTTGGTGCTGGAATTGTGCTC-3’，SEQ ID NO：9；
GhCCAMK-1R:5’-GGGGTACCCTTTGCTGTGATTTGCCCCT-3’，SEQ ID NO：10。
extracting total RNA of upland cotton TM-1, reversely transcribing the total RNA into cDNA, and carrying out PCR amplification by using the cDNA as a template and adopting a primer pair consisting of a primer GhCCAK-1F and a primer GhCCAK-1R to obtain a PCR amplification product.
The PCR amplification system is shown in Table 3:
TABLE 3PCR amplification System
| Reagent(s) | Dosage of |
| GhCCAMK-1F(10μM) | 1μL |
| GhCCAMK-1R(10μM) | 1μL |
| 5×TransStartFastPfuBuffer | 10μL |
| 2.5mMdNTPs | 4μL |
| TransStartFastPfuDNAPolymerase | 1μL |
| Nuclease-freeWater | Supplement to 50 mu L |
PCR amplification procedure: 95 ℃ for 2min;95℃for 20s,52℃for 30s,72℃for 30s,35 cycles; and at 72℃for 5min.
Amplifying a target sequence (about 318 bp) by PCR; the amplified product was subjected to 1% agarose gel electrophoresis.
(2) Sequencing to obtain recombinant expression vector
The PCR amplified product was recovered by gel ligation with pEASY-Blunt Zero Cloning Kit (cloning vector), and the positive transformants identified by PCR using the GhCCAK-1F/R primer were sequenced.
The positive transformants which were sequenced successfully were selected to extract plasmids, and the plasmids and empty vector pTRV2 were digested with the enzymes EcoRI and KpnI, respectively, in a water bath at 37℃and the digestion system was as shown in Table 4:
table 4 enzyme digestion System
| Reagent(s) | Dosage of |
| GhCCAK plasmid | 3μL |
| EcoRI | 1μL |
| KpnI | 1μL |
| 10×FlyCutBuffer | 2μL |
| Nuclease-freeWater | Supplement to 20 mu L |
And (3) enzyme cutting: 37 ℃ for 1h; 10min at 80 ℃.
The enzyme cutting product is subjected to 1% agarose gel electrophoresis, and is connected by using T4 DNA ligase, the plant expression vector recombinant plasmid TRV 2-GhCAMK is used for transforming agrobacterium GV3101, and the GhCAMK-1F/R primer is used for obtaining the VIGS interference vector TRV2: ghCAMK after PCR screening and identification.
2. Acquisition of transgenic cotton
(1) Bacterial liquid culture
Agrobacterium was transformed with pTRV1 and TRV 2-GhCAMK, respectively, and the resulting mixture was transformed into a vector containing kanamycin (50. Mu.g. Multidot.mL) -1 ) Gentamicin (50. Mu.g.mL) -1 ) And rifampicin (25. Mu.g.mL) -1 ) Resistant LB flask was incubated at 28℃to OD 600 Reaching 0.6 to 0.8.
(2) Resuspension obtaining
The cells were collected by centrifugation at 4000rpm for 5min and resuspended in the appropriate volume (formulation: 10 mmol.L) -1 MgCl 2 ，10mmol·L -1 MES and 200. Mu. Mol.L -1 Acetosyringone) was resuspended to a final concentration of OD 600 1.5. And standing the heavy suspension at room temperature in dark for more than 3 hours. Obtaining heavy suspension containing recombinant plasmid TRV 2-GhCAMK. The preparation method is similar to that of a heavy suspension containing pTRV1 vector, a heavy suspension containing TRV2-GhCLA1 vector of GhCLA 1gene fragment and a heavy suspension containing pTRV2 empty vector.
Mixing the heavy suspension containing pTRV1 vector and the heavy suspension containing target gene fragment TRV 2-GhCAMK in the volume ratio of 1:1 to obtain TRV, injecting GhCAMK solution into cotton cotyledon for obtaining gene silencing transformant; mixing the heavy suspension containing pTRV1 vector and the heavy suspension containing GhCLA 1gene fragment of TRV2-GhCLA1 vector uniformly according to the volume ratio of 1:1 to obtain TRV, wherein the TRV is GhCLA1 solution for injecting cotton cotyledons for detecting whether a gene silencing system is correct or not; the suspension containing pTRV1 carrier and suspension containing pTRV2 empty carrier are mixed uniformly according to the volume ratio of 1:1 to obtain TRV:00 solution, and the solution is injected into cotton cotyledon to be used as genetic transformation control strain.
(3) Recipient culture
The TM-1 cotton seeds are sowed in nutrient soil after being soaked in water for 24 hours, and are subjected to illumination for 12 hours or dark for 12 hours, and are subjected to illumination culture at the temperature of 26-28 ℃. The humidity is kept at 60% and above, water is poured once for 4-5 d, and the two cotyledons can be used for VIGS operation when the two cotyledons are flat and unfolded and the true cotyledons are not yet developed.
(4) Acquisition of GhCAMK Gene silencing transformants
Firstly, lightly puncturing the back of cotyledons by using a syringe needle to cause micro-wounds, and injecting the prepared heavy suspension mixed according to the volume ratio of 1:1 from the wounds by using a syringe without the needle to obtain the cotton GhCCAK gene silencing transformant. Light is prevented from 24 hours, 12 hours of illumination/12 hours of darkness, and the temperature is 26-28 ℃ of illumination culture.
A GhCLA 1gene silencing test strain and pTRV2 empty vector genetic transformation control strain were obtained in a similar manner.
Agrobacteria VIGS specific methods reference: gao, X., shan, L.functional genomic anal ysis ofcotton genes with agrobacterium-treated virus-reduced gene sizing.methods Mol Biol,2013,975:157-165.
3. Detection of VIGS genetic transformation System
(1) After 2 weeks, the phenotype of the different treated cotton was observed, and the VIGS system was examined using the upland cotton GhCLA 1gene (cloroplastos alterados gene) as a marker gene. The gene participates in chloroplast development process, codes 1-deoxyxylulose 5-phosphate synthase protein, is highly conserved in evolution, and cotton plants have obvious albino phenotype after GhCLA 1gene is silenced, thus being a marked character which is easy to identify. As shown in FIG. 5, 2 weeks after infection with VIGS, the true leaves of the plants injected with TRV1 and TRV2-GhCLA1 were almost completely whitened (FIG. 5A), while leaves injected with the empty vectors pTRV1 and pTRV2 as controls (TRV: 00) were unchanged (FIG. 5B TRV: 00). Demonstrating the successful establishment of a TRV-mediated VIGS system in upland cotton TM-1.
(2) Fluorescent quantitative Real time-PCR detection
RNA (preferably new long true leaves) is extracted from the treated cotton leaves, then qRT-PCR is performed to detect whether the target gene is expressed down or not, and the expression of the target gene is detected. For each material 30 individual plants were treated.
And extracting total RNA of the leaves of the VIGS-impregnated cotton plants by using the RNA extraction kit. The expression of the post-silencing GhCCAK gene which is disturbed and silenced is detected by fluorescent quantitative Real time-PCR by using cotton UBQ7 as an internal reference gene.
qRT-PCR analysis of the relevant primers and PCR procedure refer to example 2 part 1, "tissue expression pattern of the Gene GhCAMK in nature".
As shown in FIG. 6, compared with the empty vector (TRV: 00) control, the expression level of the GhCAMK gene in the randomly selected GhCAMK gene VIGS-infected plants is remarkably reduced, and the silencing effect is remarkable.
4. Inoculation and resistance identification for verticillium wilt resistance of cotton
(1) Inoculating verticillium wilt of cotton
The stored verticillium dahliae strain V991, a verticillium dahliae pathogen, is activated on a PDA culture medium. The selected thalli are cultured for 3-5 d in Czapek culture solution at 25 ℃ and 200 rpm. Filtering the pathogenic bacteria culture solution with 4 layers of gauze, counting pathogenic bacteria concentration with blood cell counting plate, and regulating final concentration to 1.0X10 with sterilized double distilled water 7 spores/mL, and Tween-20 was added to a final concentration of 0.001% (volume percent). The obtained transformant after 2 weeks of VIGS gene silencing was inoculated with verticillium V991 spore liquid by root injury.
(2) Verticillium wilt disease incidence statistics
And (5) investigation and statistics of verticillium wilt morbidity when the true leaves begin to turn yellow and wilt, and statistics of disease grade by adopting a 0-4 grade method. For each material 30 individual plants were treated. Let 3 biological replicates.
Disease index statistical reference: xu Li, zhu Longfu, zhang Xianlong. Research progress on the mechanism of cotton verticillium wilt resistance, crop theory, 2012,38:1553-1560; xu L, zhu L F, zhang X L.research on resistance mechanism ofcotton to Verticillium wilt. ActaAgron Sin,2012,38:1553-1560.
Disease index = [ (number of disease plants at each stage x corresponding grade)/survey total number of plants x highest grade of disease (4) ]100
And (3) test result statistics: the disease of verticillium dahliae after 2 weeks of infection with VIGS, with respect to the empty vector control (TRV: 00) plants, was inoculated with the silenced plant of TRV: ghvcak, after 21d with verticillium dahliae strain V991. As can be seen in FIG. 5B, the control (TRV: 00) plants showed more pale yellow plaques and a larger area in the leaves and more pronounced curling of the edges down than the GhCAMK gene-silenced plants. The results of the statistical analysis of 3 biological repeated observation show that the average disease index of the control plants injected with the empty vector is 72%, and the disease resistance of the plants after GhCAMK gene silencing is obviously enhanced, and the average disease index is 56%. The GhCAMK gene is shown to be involved in verticillium-induced allergic reaction. After the GhCAMK gene is silenced in upland cotton TM-1, the incidence rate and disease indication of cotton are obviously reduced when the cotton is infected by verticillium wilt bacteria, which indicates that the silencing of the GhCAMK gene improves the resistance of the cotton to verticillium wilt.
(3) Verification of the involvement of the GhCAMK Gene in the verticillium-induced allergic reaction
In order to verify the accuracy of the phenotype result, the invention continues to perform verticillium restoration culture experiments, disease plant splitting rod treatment and plant verticillium DNA relative abundance detection.
The verticillium recovery culture experimental method comprises the following steps: cutting the stem of cotton seedling with 21d after verticillium wilt treatment into segments with the length of 1cm from 6cm of cotyledons by scissors, soaking for 1min by 75% alcohol, soaking for 30min by 30% hydrogen peroxide, washing for 4-5 times by sterile water, and finally placing the segments of cotton stem after treatment on a PDA culture medium for culturing for 3d at 25 ℃, thus observing the growth condition of verticillium wilt bacteria recovery culture.
The treatment method of the disease planting dissecting rod comprises the following steps: taking a plurality of cotton stalks of 21d inoculated with verticillium wilt, longitudinally cutting the cotton stalks by using a sharp blade to expose the longitudinal section of the cotton stalks, observing the phenotype of the vascular bundle tissue of the cotton stalks, wherein if the vascular bundle tissue of the cotton stalks is brown, the vascular bundle tissue of the cotton stalks is infected with verticillium wilt and is ill, and if the vascular bundle tissue of the cotton stalks is not brown and the phenotype of the vascular bundle tissue of the cotton stalks is normal, the vascular bundle tissue of the cotton stalks is not ill.
The method for the relative abundance of the verticillium dahliae DNA of the plants comprises the following steps: RNA of the stem of cotton seedlings after verticillium treatment is extracted, inverted into cDNA and subjected to qPCR amplification.
The primers used were:
ITS1-F:5’-AAAGTTTTAATGGTTCGCTAAGA-3’，SEQ ID NO：11；
ST-VE1-R:5’-CTTGGTCATTTAGAGGAAGTAA-3’，SEQ ID NO：12。
reverse transcription and qPCR reaction procedure conditions and procedures refer to example 2, part 1, "tissue expression pattern of the naturally occurring gene gccamk".
As shown in fig. 8-11, after the stalks are longitudinally cut, the stalk vascular bundles of the control plants injected with the empty vector become light brown, while the stalk vascular bundles of the plants with the silence GhCCAK genes are normal, and the browning degree of the stalk cutting material of the GhCCAK plant materials is lower than that of the control plant materials injected with the empty vector (fig. 8); the bacterial colony of verticillium bacteria cultured by the GhCCAK plant material in the PDA culture medium is less than that of the TRV 00 plant material (figure 9); the recovery rate of verticillium of the TRV 00 control plants reaches 83%, and the recovery rate of verticillium of the plants after the TRV GhCAMK gene silencing reaches 27% (figure 10); the relative abundance of the verticillium DNA of the TRV 00 plant is about 3 times that of the plant with the TRV GhCAMK gene silenced (figure 11).
These results also indicate that cotton has increased resistance to verticillium after silencing the gccamk gene, indicating that the gccamk gene is involved in regulating verticillium resistance.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. The application of the calcium-dependent protein kinase gene GhCAMK in regulating and controlling verticillium wilt resistance of plants is characterized in that the nucleotide sequence of the gene GhCAMK is shown in SEQ ID NO: 1.
2. The application of protein or a substance for regulating the expression of a protein coding gene in plant verticillium wilt resistance is characterized in that the amino acid sequence of the protein is shown as SEQ ID NO: 2.
3. Use of the gene ghvcak according to claim 1 or the protein-related biomaterial according to claim 2 for plant verticillium wilt resistance, characterized in that the biomaterial is any of the following:
a: a nucleic acid molecule according to claim 1 or a nucleic acid molecule encoding a protein according to claim 2;
b: an expression cassette comprising silencing of the nucleic acid molecule of a;
c: an expression vector comprising the nucleic acid molecule of A, or a recombinant vector comprising the expression cassette of B;
d: a recombinant microorganism comprising the nucleic acid molecule of A, a recombinant microorganism comprising the expression cassette of B, or a recombinant microorganism comprising the recombinant vector of C.
4. Use according to any one of claims 1 to 3, wherein the plant is a cotton plant.
5. A method for increasing verticillium wilt resistance in a plant, comprising increasing verticillium wilt resistance in a plant by silencing or inhibiting expression of the gene ghvcak according to claim 1 or activity of the protein according to claim 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310334048.5A CN116536347B (en) | 2023-03-31 | 2023-03-31 | Application of calpain gene GhCCAMK in verticillium resistance of plants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310334048.5A CN116536347B (en) | 2023-03-31 | 2023-03-31 | Application of calpain gene GhCCAMK in verticillium resistance of plants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116536347A true CN116536347A (en) | 2023-08-04 |
| CN116536347B CN116536347B (en) | 2024-10-11 |
Family
ID=87456755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310334048.5A Active CN116536347B (en) | 2023-03-31 | 2023-03-31 | Application of calpain gene GhCCAMK in verticillium resistance of plants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116536347B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080034453A1 (en) * | 1999-05-06 | 2008-02-07 | Nordine Cheikh | Annotated Plant Genes |
| CN110343157A (en) * | 2019-07-31 | 2019-10-18 | 新疆农业科学院核技术生物技术研究所（新疆维吾尔自治区生物技术研究中心） | Cotton verticillium wilt related gene GhBONI and its coding albumen and application |
| CN114790449A (en) * | 2022-05-11 | 2022-07-26 | 新疆农业科学院核技术生物技术研究所（新疆维吾尔自治区生物技术研究中心） | Application of calcium-dependent protein kinase gene GhCPK4 in resisting verticillium wilt of plants |
-
2023
- 2023-03-31 CN CN202310334048.5A patent/CN116536347B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080034453A1 (en) * | 1999-05-06 | 2008-02-07 | Nordine Cheikh | Annotated Plant Genes |
| CN110343157A (en) * | 2019-07-31 | 2019-10-18 | 新疆农业科学院核技术生物技术研究所（新疆维吾尔自治区生物技术研究中心） | Cotton verticillium wilt related gene GhBONI and its coding albumen and application |
| CN114790449A (en) * | 2022-05-11 | 2022-07-26 | 新疆农业科学院核技术生物技术研究所（新疆维吾尔自治区生物技术研究中心） | Application of calcium-dependent protein kinase gene GhCPK4 in resisting verticillium wilt of plants |
Non-Patent Citations (3)
| Title |
|---|
| HU Q 等: ""GhCPK33 Negatively Regulates Defense against Verticillium dahliae by Phosphorylating GhOPR3"", PLANT PHYSIOLOGY, vol. 178, no. 2, 27 August 2018 (2018-08-27), pages 876 * |
| ZENG H 等: ""Calmodulin and calmodulin‐like protein‐mediated plant responses to biotic stresses"", PLANT, CELL & ENVIRONMENT, vol. 46, no. 12, 14 August 2023 (2023-08-14), pages 3680 - 3703 * |
| 邓亚辉: ""陆地棉黄萎病抗病基因的挖掘及功能鉴定"", 中国博士学位论文全文数据库 农业科技辑, no. 3, 15 March 2023 (2023-03-15), pages 046 - 8 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116536347B (en) | 2024-10-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110331145B (en) | Application of miR156 and related biological materials thereof in regulation and control of plant disease resistance | |
| CN116334023A (en) | Application of calpain gene GhCDPK29 in verticillium resistance of plants | |
| CN110004156A (en) | GhCML20 gene relevant to resistance to verticillium wilt and its application | |
| CN115992153A (en) | Wheat stem rot resistance gene TaP CS, encoding protein and application thereof | |
| CN114790449B (en) | Application of calpain gene GhCPK4 in verticillium resistance of plants | |
| CN116425847B (en) | Rice OsGLP8-10 for inhibiting sclerotinia and application thereof | |
| CN116355067B (en) | Rice OsGLP8-12 for inhibiting sclerotinia and application thereof | |
| CN103451225B (en) | Application of rice WRKY transcription factor gene OsWRKY21 in cultivation of resistant varieties of plants | |
| CN103352038B (en) | Corn disease-resistance related gene MR4, and application of the same in corn disease-resistance improvement | |
| CN116004660A (en) | Melon powdery mildew resistance related gene CmVDAC and application thereof | |
| CN116536347B (en) | Application of calpain gene GhCCAMK in verticillium resistance of plants | |
| CN115976052A (en) | Wheat stem basal rot resistance gene TaHSP18.6, expression product, recombinant vector and application thereof | |
| CN115976051A (en) | Potato StRTP7 gene and application thereof in disease-resistant breeding | |
| CN108715854A (en) | Applications of the cotton AINTEGUMENTA genes GhANT in cotton breeding | |
| CN113832166A (en) | Verticillium dahliae PEX30 gene target gene fragment for resisting pathogenic bacteria, interference vector and application thereof | |
| CN117165599B (en) | Application of transcription factor GhTIFY b in verticillium resistance of plants | |
| CN116496372B (en) | Rice OsGLP8-11 for inhibiting sclerotinia and application thereof | |
| CN118147168B (en) | GHAP2-DREB gene and application thereof in improving verticillium wilt resistance of upland cotton | |
| CN116926088B (en) | Verticillium dahliae VdNRPS6 gene antipathogenic target gene fragment, interference vector and application thereof | |
| CN116496371B (en) | Rice OsGLP3-5 for inhibiting sclerotinia and application thereof | |
| CN117247949B (en) | Notoginseng disease course related protein 1 gene PnPR1-8 and its application | |
| CN114990156B (en) | Recombinant vector based on RNAi interference sclerotinia sclerotiorum OAH gene, construction method and application thereof | |
| CN116768992B (en) | Gene for regulating and controlling poplar leaf surface coat development and application thereof | |
| CN117821499B (en) | Biological material for regulating expression of TaWRKY24 protein coding gene and application thereof | |
| CN111454987B (en) | Application of GhNAC091 gene in improving plant photosynthesis efficiency and strong light tolerance |
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 |