CN116396972B - 一种大豆甲基转移酶基因GmCCOMT及其应用 - Google Patents
一种大豆甲基转移酶基因GmCCOMT及其应用 Download PDFInfo
- Publication number
- CN116396972B CN116396972B CN202310292645.6A CN202310292645A CN116396972B CN 116396972 B CN116396972 B CN 116396972B CN 202310292645 A CN202310292645 A CN 202310292645A CN 116396972 B CN116396972 B CN 116396972B
- Authority
- CN
- China
- Prior art keywords
- soybean
- gmccomt
- gene
- smv
- silencing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 244000068988 Glycine max Species 0.000 title claims abstract description 48
- 235000010469 Glycine max Nutrition 0.000 title claims abstract description 47
- 108060004795 Methyltransferase Proteins 0.000 title claims abstract description 13
- 241000723811 Soybean mosaic virus Species 0.000 claims abstract description 38
- 230000030279 gene silencing Effects 0.000 claims abstract description 16
- 230000014509 gene expression Effects 0.000 claims abstract description 13
- 238000009825 accumulation Methods 0.000 claims abstract description 6
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 230000001737 promoting effect Effects 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 29
- 108090000623 proteins and genes Proteins 0.000 abstract description 25
- 230000008859 change Effects 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 229920005610 lignin Polymers 0.000 description 16
- 238000000034 method Methods 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 10
- 239000000758 substrate Substances 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 7
- 208000024891 symptom Diseases 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 101150060228 CCOMT gene Proteins 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 239000006180 TBST buffer Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000030833 cell death Effects 0.000 description 5
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 5
- 239000011536 extraction buffer Substances 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 208000035240 Disease Resistance Diseases 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 3
- 229960001570 ademetionine Drugs 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 239000012192 staining solution Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000723596 Bean pod mottle virus Species 0.000 description 2
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102100023321 Ceruloplasmin Human genes 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 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 2
- 238000002835 absorbance Methods 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000008260 defense mechanism Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000009610 hypersensitivity Effects 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- ODLMAHJVESYWTB-UHFFFAOYSA-N propylbenzene Chemical compound CCCC1=CC=CC=C1 ODLMAHJVESYWTB-UHFFFAOYSA-N 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 239000005723 virus inoculator Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 101150084750 1 gene Proteins 0.000 description 1
- XMIIGOLPHOKFCH-UHFFFAOYSA-N 3-phenylpropionic acid Chemical compound OC(=O)CCC1=CC=CC=C1 XMIIGOLPHOKFCH-UHFFFAOYSA-N 0.000 description 1
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 241000219195 Arabidopsis thaliana Species 0.000 description 1
- 101100438260 Arabidopsis thaliana At1g67980 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101710095827 Cyclopropane mycolic acid synthase 1 Proteins 0.000 description 1
- 101710095826 Cyclopropane mycolic acid synthase 2 Proteins 0.000 description 1
- 101710095828 Cyclopropane mycolic acid synthase 3 Proteins 0.000 description 1
- 101710110342 Cyclopropane mycolic acid synthase MmaA2 Proteins 0.000 description 1
- 101710177638 Hydroxymycolate synthase MmaA4 Proteins 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710174850 Methoxy mycolic acid synthase MmaA3 Proteins 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 101710204417 Mycolic acid methyltransferase MmaA1 Proteins 0.000 description 1
- 102000012064 NLR Proteins Human genes 0.000 description 1
- 108010036473 NLR Proteins Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 240000009164 Petroselinum crispum Species 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 102000055027 Protein Methyltransferases Human genes 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 101900173213 Soybean mosaic virus Capsid protein Proteins 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 235000001465 calcium Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000002796 luminescence method Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 235000011197 perejil Nutrition 0.000 description 1
- 229930015704 phenylpropanoid Natural products 0.000 description 1
- 150000002995 phenylpropanoid derivatives Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910010271 silicon carbide Inorganic materials 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 230000028604 virus induced gene silencing Effects 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000001262 western blot 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
-
- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8203—Virus mediated transformation
-
- 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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
本发明属于植物基因工程领域，涉及大豆甲基转移酶基因GmCCOMT及其应用。一种大豆甲基转移酶基因GmCCOMT，该基因的核苷酸序列如SEQ ID No：1所示。本发明还提供了该基因在培育大豆抗SMV品种中的应用。本发明实验表明沉默该基因并不改变大豆的表型，沉默GmCCOMT的表达能促进SMV的积累，因此可以通过上调该基因从而实现抗SMV的目的。本发明为改善大豆对大豆花叶病毒抗性的分子育种提供重要基因资源。
Description
技术领域
本发明属于植物基因工程领域，涉及大豆甲基转移酶基因GmCCOMT及其应用。
背景技术
CCOMT基因首先在欧芹中被克隆到(Schmit等人，1991)，是苯丙烷途径中调控木质素合成的关键酶之一(Liu等人，2022；Li等人，2021)，木质素沉积在植物-病原体相互作用期间快速诱导，作为ETI反应，在空间上将病原体限制在感染部位，从而赋予植物抗病能力(Lee等人，2019；Kim等人，2020)。大豆花叶病是由SMV(大豆花叶病毒)引起的世界性大豆病毒病害，在我国各大豆产区也普遍发生，对大豆的实际产量与质量有着重要的影响。为了防止病原体感染，植物进化出了多种复杂的防御机制，包括细胞壁木质化，植物主要防御机制之一是由抗性(R)基因介导的。CCOMT蛋白被证明富含亮氨酸重复结构域(NLR蛋白)，在介导的超敏反应(HR)和植物抗病性中起关键作用(Wang和Balint-Kurti，2016；Yang等人，2017)。SMV侵染后上调GmCCOMT基因表达，暗示大豆可能遵循类似的途径抵抗SMV侵染。大豆含有丰富的优质蛋白、不饱和脂肪酸、钙及B族维生素是我国居民膳食中优质蛋白质的重要来源，然而在大豆生长发育的过程中，会受到诸多因素的影响而感染SMV，进而对大豆的生长发育产生严重的影响。因此进行大豆抗SMV的研究有助于阐明大豆对SMV的调控网络，有效提升大豆的实际产量与质量，进而为大豆产业的可持续发展奠定良好的基础。
发明内容
本发明的目的是通过研究SMV侵染大豆的分子机制，进而寻找与SMV发病机制相关的基因，通过对该基因的表达进行调控，来达到抑制SMV的目的，为培育抗病新品种提供基础。
为了实现上述目的，本发明采用的技术手段是提供一种大豆甲基转移酶基因GmCCOMT，该基因的核苷酸序列如SEQ ID No：1所示。
本发明还提供了所述大豆甲基转移酶基因GmCCOMT的应用，该基因可用于培育大豆抗SMV品种。
本发明还进一步提供了一种抗SMV植物品种的培育方法，该方法通过上调所述的大豆甲基转移酶基因GmCCOMT的表达，实现抗SMV的功能。
本发明的有益效果是：本发明从大豆中克隆了1个在大豆花叶病毒(SMV)侵染大豆中发挥重要调控作用的基因GmCCOMT，该基因mRNA表达分析表明其在茎和花中表达最多，且受SMV诱导极显著上调，可能参与大豆调控大豆花叶病毒繁殖的过程。实验表明沉默该基因并不改变大豆的表型，沉默GmCCOMT的表达能促进SMV的积累，因此可以通过上调该基因从而实现抗SMV的目的。本发明为改善大豆对大豆花叶病毒抗性的分子育种提供重要基因资源。
附图说明
图1为GmCCOMT在大豆不同组织中的表达情况；
图2为GmCCOMT对SMV的响应；
图3为GmCCOMT的氨基酸序列分析；其中，At1g67980为拟南芥CCOMT的氨基酸，Glyma05g27960为本发明GmCCOMT的氨基酸；a表示甲基转移酶；b表示S-腺苷甲硫氨酸结合位点；
图4为大豆中沉默GmCCOMT基因的检测结果；
图5为沉默GmCCOMT的大豆植株形态学特征；
图6为沉默GmCCOMT的植物中SMV G7的累积；
图7为SilCCOMT植物中SMV病毒粒子ELISA分析结果；
图8为Rsv1 SilCCOMT植物SMV-G7感染与系统性细胞死亡症状的相关性；
图9为SilCCOMT植物中木质素水平测定结果。
具体实施方式
下面将结合本发明的实施例和附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，绝不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一、GmCCOMT的组织表达分析
参照Trizol说明书从大豆不同组织中提取RNA，利用超微量分光光度计测定各组织RNA浓度和纯度，并利用1％琼脂糖凝胶电泳检测总RNA的完整性，并将将28S和18S条带的亮度比例为2:1的总RNA利用反转录酶(TIANScript RT Kit)反转录成cDNA。设计GmCCOMT特异性荧光定量引物，以大豆组成型表达的Tubulin基因为内部参照，使用TAKARA的Premix Ex TaqTM PCR试剂盒，反应体系：SYBRMIX 10μl；Forward primer 0.5μl；Reverseprimer0.5μl；ddH2O 8.2μl；cDNA0.8μl。反应条件：50℃2min,95℃10min，95℃20s，60℃1min，40个循环。每个样品做3次生物学重复。相对定量(2-ΔΔCT)方法对基因表达进行定量。如图1所示，荧光定量PCR显示，GmCCOMT在根、茎、子叶、叶、花中均有表达，且在茎和花中表达量最高。
二、GmCCOMT对SMV的响应
采用汁液摩擦接种的方法，将SMV G5接种于大豆感病品种南农1138-2上进行扩繁。当供试豆苗第一对真叶完全展开时，取南农1138-2上症状典型的叶片加入0.1M、pH 7.3～7.4的磷酸缓冲液(约每1g新鲜叶片加5ml缓冲液)，再加少许金刚砂(600目)在研钵中研成匀浆，用毛刷将匀浆涂到第一对展开的真叶上，然后用自来水冲洗此为实验组；对照组是利用摩擦方法接种磷酸缓冲液PBS。采集0dpi(取接种病毒前的叶片为0dpi，作为对照)和4dpi叶片提取RNA并反转录为cDNA，进行荧光定量分析。结果如图2所示，与对照相比，GmCCOMT的表达量明显上调，表明SMV病毒侵染诱导了GmCCOMT基因的上调表达。
三、大豆中沉默GmCCOMT基因
对GmCCOMT的氨基酸序列与模式作物拟南芥中的CCOMT At1g67980进行比对，如图3所示，两条序列含有62％的相似性，且含有I类蛋白质S-腺苷甲硫氨酸依赖性甲基转移酶的两个保守位点，甲基转移酶(如图3中a所示)以及S-腺苷甲硫氨酸结合位点(如图3中b所示)。为了研究GmCCOMT的功能，基于菜豆豆荚斑驳病毒(BPMV)介导的VIGS体系,选取含有保守S-腺苷甲硫氨酸结合位点的区域来构建沉默载体(S33-E98)。以南农1138-2cDNA为模板，进行PCR扩增并进行线性化。构建好的沉默载体接种大豆叶片，侵染完成后将植株培养在条件为16h光照(25℃)/8h黑暗(20℃)、湿度60％的光照培养箱中，同时在南农1138-2的叶片上用同样方法接种空载V对照。荧光定量PCR显示Glyma05g27960的转录物比对照显著减少，如图4所示，接种两周后开始观察表型表明沉默GmCCOMT不影响大豆形态学特征，如图5所示，说明沉默材料SilCCOMT构建成功。
四、沉默GmCCOMT的植株对SMV的抗病性变化
为检测GmCCOMT是否参与大豆调控SMV侵染，沉默材料SilCCOMT与对照空载V分别接种在Essex和Rsv1大豆上，待第一对三出复叶展开并有明显的花叶症状时分别接种SMVG5和G7株系，并随时间监测病毒增殖情况。取接种病毒前的叶片为0dpi，作为对照，分别采集4dpi和7dpi的接种叶片，10dpi和14dpi的系统叶片，于1.5ml离心管中，保存于-80℃冰箱。对收集的样品采用GTEN法提取总蛋白，GTEN提取缓冲液配置:
溶于水中，4℃保存。
具体方法如下：叶片先在液氮中充分研磨，加入500μl GTEN提取液，充分研磨，4℃、12000rpm离心10min，将上清转移到新的离心管中，此为总蛋白。首先制备SDS-PAGE蛋白胶，其中分离胶为10％，浓缩胶为3.5％。以100V、120min的电泳条件，分离上述变性蛋白。电泳结束后，将尼龙膜、胶、滤纸在转膜缓冲液中充分浸泡，依续叠放滤纸、膜、胶、滤纸，胶和膜之间不能有气泡，否则影响转膜效果。用转膜仪转印，400mA恒电流，120min。转好的膜，用立春红染色，检测总蛋白浓度。用TBST配制5％脱脂牛奶，将膜室温封闭1h。再将膜浸在含有一抗(Anti-SMV CP)的TBST脱脂牛奶中2h，TBST漂洗3次，每次10min；将漂洗后的膜浸在含有二抗的TBST脱脂牛奶中2h，TBST漂洗3次后，用HRP-ECL发光法显色，标定Marker，并进行分析与扫描。或者用碱性磷酸酶显色法，加入NBT和BCIP显色底物，直至目的条带清晰。大豆花叶病毒衣壳蛋白(Capsid Protein，CP)的数量代表病毒的含量，Western blot分析表明，与V对比在Rsv1背景下，沉默CCOMT基因的植物在更早时间点检测到SMV G7的积累，如图6所示。同时进行双酶联免疫夹心反应(ELISA)进一步检测SilCCOMT植物病毒含量变化，ELISA的试验程序如下：
(1)样品制备：选取新鲜的、具有典型症状的叶片0.3g为一个样，置于灭过菌的研钵中，加抽提缓冲液研磨至匀浆状。按大豆叶片重量(mg)：抽提缓冲液体积(μl)＝1：10的比例稀释样品。
(2)包被抗体：按照1：200稀释比例用包被抗体缓冲液稀释抗体，每孔加200ml，37℃孵育3-6h。
(3)洗板：快速甩掉板内的液体，每孔加满PBST缓冲液，然后迅速倒掉。重复4～6次。最后反转ELISA板，在吸水纸上反复拍打几次以去除残存在板内的PBST缓冲液。
(4)加样、孵育：每孔加样200μl，每个样品重复2次，放在保湿盒子内存4℃冰箱中过夜。同时加阳性对照、阴性对照。
(5)酶标抗体孵育：洗板同前，重复6-8次。在酶标板中每孔加100μl酶标抗体，37℃4-6h。
(6)准备底物溶液：底物pNPP溶液的浓度为1mg/ml。
(7)放在保湿盒内室温(21～24℃)孵育2.5h。洗板，同前，重复6～8次。
(8)底物孵育：加入底物溶液(200μl/孔)，在避光、室温(21～24℃)下显色，约20～30min，待颜色变黄时读数。
(10)结果测定：在酶标仪405nm波长下，测定光密度值(OD)。
根据所提取的病毒粒子做标准样，双酶联免疫夹心反应测定吸光值，制作标准曲线。将样品吸光值带入标准曲线，得到所含病毒粒子的浓度。
其中包被缓冲液、洗涤缓冲液、样品抽提缓冲液、酶标稀释缓冲液、底物(4-硝基苯酚磷酸酯(4-nitrophenyl phosphate，ρNPP))的配制表如下：
(1)包被缓冲液
无水碳酸钠 1.59g
碳酸氢钠 2.93g
叠氮化钠 0.2g
溶解于蒸馏水中使容积为1000ml，调整pH至9.6，4℃贮存。
(2)磷酸盐-吐温(PBST)缓冲液
溶解于蒸馏水至1000ml，pH 7.3。
(3)抽提缓冲液
溶解于100l 1 PBST中，pH 7.3，4℃贮存。
(4)酶标抗体缓冲液
小牛血清白蛋白 2.0g
PVP(分子量24，000～40，000) 10.0g
叠氮化钠 0.2g
溶解于1000ml 1 PBST中，pH 7.3，4℃贮存。
(5)底物缓冲液
二乙醇胺 97.0ml
氯化镁 0.1g
叠氮化钠 0.2g
溶解于800ml蒸馏水中，用HCl调pH至9.8，再加蒸馏水到1000ml，4℃贮存。
(6)底物溶液
底物4-硝基苯酚磷酸酯(4-nitrophenyl phosphate，NPP)用底物缓冲液按1mg.ml-1配制。
ELISA分析显示在SilCCOMT植物的接种叶片(0、4、7dpi)和上位叶片(10、14dpi)中SMV(G5和G7)的病毒粒子含量明显更多，如图7所示。有趣的是，与Rsv1 V植物相比，Rsv1SilCCOMT植物表现出与SMV-G7感染相关的更严重的系统性细胞死亡症状，如图8的上部所示。
对采集的7dpi和10dpi新鲜叶片，用剪刀剪取半片新鲜的叶片置于在65℃预热过的台盼蓝染色液中，以染色液刚浸没过叶片为准。用真空泵抽真空5-10min，反复3次，直到没有气泡冒出，表明染料进入叶片。接着放到沸水表面，煮3min。室温下固定过夜，固定好后去除多余的染色液，加入适量脱色液脱色，每半小时换一次脱色液，重复2次。脱色好的叶片，用镊子放平在载玻片上，50％甘油覆盖，压好盖玻片，制片完成后，在显微镜下观察是否有细胞被染成蓝色。
台盼蓝染色液配置：
脱色液：称量25g氢氯酸溶解于10ml水中。
显微细胞死亡分析证实了SMV G7感染的Rsv1 SilCCOMT植物的细胞死亡表型增加，如图8的下部所示。这些结果表明，降低GmCCOMT的表达促进了大豆中SMV的积累。
GmCCOMT是在SilVma12植物中诱导的，根据CCOMT蛋白在木质素生物合成中的已知功能，GmCCOMT可能在类苯丙烷途径中发挥作用。为了测试增强的SMV应答性细胞死亡症状是否与木质素合成缺陷相关。已知木质素普遍存在于植物体主干中，植物体主要成份为纤维素和木质素，纤维素溶于酸性液体、木质素溶于碱性液体，植物体中含有木质素10％～30％，使用如下方法提取木质素：将40mg SilCCOMT植物及对照V植物中叶组织提取物在80％甲醇中进行两次连续提取，然后用甲醇和丙酮进行第三次提取，以去除可溶性酚类成分。使用碱法(1M NaOH，80℃)水解去除细胞结合的酚类成分。在乙酸乙酯中溶解后，使用2MHCl和巯基乙酸进一步提取木质素，并将其溶解在0.5M NaOH中。通过测量340nm处的吸光度并使用纯化的木质素作为标准曲线(1至0.0001mg/ml)，用分光光度法定量木质素水平。然而，如图9所示，结果表明在SilCCOMT植物中观察到的SMV相关症状不太可能与木质素合成缺陷有关，因为SilCCOMT植物中的木质素水平与对照植物相当，而且SilCCOMT植物的SMV表型不可能与木质素合成苯丙酸途径的缺陷有关。
Claims (1)
1. 一种大豆甲基转移酶基因GmCCOMT 在促进大豆花叶病毒SMV 的积累中的应用，其特征在于：所述应用通过沉默大豆甲基转移酶基因GmCCOMT在大豆中的表达来实现；沉默大豆甲基转移酶基因GmCCOMT的表达并不改变大豆的表型；所述大豆花叶病毒SMV 为 G5或G7株系；所述大豆甲基转移酶基因GmCCOMT的核苷酸序列如SEQ ID No 1 所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310292645.6A CN116396972B (zh) | 2023-03-23 | 2023-03-23 | 一种大豆甲基转移酶基因GmCCOMT及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310292645.6A CN116396972B (zh) | 2023-03-23 | 2023-03-23 | 一种大豆甲基转移酶基因GmCCOMT及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116396972A CN116396972A (zh) | 2023-07-07 |
| CN116396972B true CN116396972B (zh) | 2024-03-22 |
Family
ID=87013490
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310292645.6A Active CN116396972B (zh) | 2023-03-23 | 2023-03-23 | 一种大豆甲基转移酶基因GmCCOMT及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116396972B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5728570A (en) * | 1991-05-30 | 1998-03-17 | Bayer Aktiengesellschaft | Caffeoyl-CoA 3-O-methyltransferase genes |
| CN109943579A (zh) * | 2019-04-18 | 2019-06-28 | 长江师范学院 | 一种岷江百合LrCCoAOMT基因及其应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6465229B2 (en) * | 1998-12-02 | 2002-10-15 | E. I. Du Pont De Nemours And Company | Plant caffeoyl-coa o-methyltransferase |
-
2023
- 2023-03-23 CN CN202310292645.6A patent/CN116396972B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5728570A (en) * | 1991-05-30 | 1998-03-17 | Bayer Aktiengesellschaft | Caffeoyl-CoA 3-O-methyltransferase genes |
| CN109943579A (zh) * | 2019-04-18 | 2019-06-28 | 长江师范学院 | 一种岷江百合LrCCoAOMT基因及其应用 |
Non-Patent Citations (6)
| Title |
|---|
| Identification of a novel salt tolerance gene in wild soybean by whole-genome sequencing;Xinpeng Qi等;Nat Commun.;第5卷;第4340篇 * |
| NCBI Reference Sequence: NM_001250549.2;无;GenBank;序列注释信息及序列表 * |
| Pathogen-induced autophagy regulates monolignol transport and lignin formation in plant immunity;Hwi Seong Jeon等;Autophagy;第19卷(第2期);第597-615页 * |
| 作物广谱抗病研究现状与关键科学问题;朱孝波等;中国科学基金;第34卷(第04期);第401-410页 * |
| 大豆抗大豆花叶病毒病基因的精细定位和候选基因分析;任锐;中国博士学位论文全文数据库 农业科技辑(第2021年第05期);第D046-44页 * |
| 大豆木质素相关基因Glyma06g28890基因功能预测及表达分析;李冬梅等;基因组学与应用生物学;第35卷(第10期);第2763-2767页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116396972A (zh) | 2023-07-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210246459A1 (en) | Methods for plant transformation using spectinomycin selection | |
| CN102803496A (zh) | 用于棉花中基因功能性分析的病毒诱导的基因沉默(vigs) | |
| CN116396972B (zh) | 一种大豆甲基转移酶基因GmCCOMT及其应用 | |
| Rao et al. | Pathogen-derived resistance using a viral nucleocapsid gene confers only partial non-durable protection in peanut against peanut bud necrosis virus | |
| CN111197035B (zh) | 抗白粉病的葡萄钙依赖蛋白激酶基因VpCDPK13及其应用 | |
| CN111118042B (zh) | 抗白粉病的葡萄钙依赖蛋白激酶基因VpCDPK9及其应用 | |
| AU2011255481B2 (en) | Altered leaf morphology and enhanced agronomic properties in plants | |
| Pak et al. | Hairy root induction in Oriental melon (Cucumis melo) by Agrobacterium rhizogenes and reproduction of the root-knot nematode (Meloidogyne incognita) | |
| CN104673803A (zh) | 基因甲基化在调控基因表达方面的应用 | |
| Dong et al. | Immunocytochemical localization of the kinetochore protein Nuf2p on the gametophyte chromosomes of a cultivar of Saccharina (Phaeophyta) | |
| CN116334036B (zh) | 一种从青枯菌中筛选抗青枯病的胞外核酸酶的方法及遗传改良应用 | |
| CN115786371B (zh) | 番茄基因SlLyk4在调控作物对土传病害抗性中的应用 | |
| CN117025636A (zh) | 烟草膨胀素基因NtEXPA11在抗马铃薯Y病毒侵染中的应用 | |
| CN115368450A (zh) | 一种人源α-乳清蛋白转基因表达载体、其制备方法及其应用 | |
| Stanislaus et al. | Functional characterization of the PHT1 family transporters of foxtail millet with a novel Agrobacterium-mediated transformation procedure | |
| CN116463373A (zh) | BpNAC9蛋白在调控白桦耐盐碱性中的应用 | |
| Hong et al. | A METHOD FOR SIMULTANEOUS ISOLATION OF CHLOROPLASTS AND MITOCHONDRIA FROM ICE PLANT | |
| Repka et al. | Immunohistochemical localization of the stress-related anionic peroxidase in germinating cucumber seeds | |
| CN117143872A (zh) | 一种用于抑制根结线虫病的RNAi靶标基因及其应用 | |
| CN117467669A (zh) | 香蕉MaFLA27基因及其编码蛋白在植物抗旱中的应用 | |
| CN114574492A (zh) | 一种来自甘蔗杆状病毒的组成型启动子pscbv-chn1及其应用 | |
| Alamin et al. | Characterization and Fine Mapping of SFL1, a Gene Controlling | |
| CN111172182A (zh) | 辣椒疫霉菌PcMPK12基因及其载体与应用 | |
| CN101663401A (zh) | 使用壮观霉素选择的植物转化方法 | |
| Cheng | Functional analysis of MAPK phosphatase AtMKP2 |
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 |