CN112646817B - 一种RNA抑菌剂siR2及作物病菌抑制剂 - Google Patents
一种RNA抑菌剂siR2及作物病菌抑制剂 Download PDFInfo
- Publication number
- CN112646817B CN112646817B CN202110090041.4A CN202110090041A CN112646817B CN 112646817 B CN112646817 B CN 112646817B CN 202110090041 A CN202110090041 A CN 202110090041A CN 112646817 B CN112646817 B CN 112646817B
- Authority
- CN
- China
- Prior art keywords
- sir2
- botrytis cinerea
- rna
- tomato
- bacteriostatic agent
- 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
- 239000000022 bacteriostatic agent Substances 0.000 title claims abstract description 8
- 239000003112 inhibitor Substances 0.000 title abstract description 9
- 244000000037 crop pathogen Species 0.000 title abstract description 3
- 229920002477 rna polymer Polymers 0.000 title description 23
- 241000123650 Botrytis cinerea Species 0.000 claims abstract description 45
- 108091032973 (ribonucleotides)n+m Proteins 0.000 abstract description 11
- 230000035784 germination Effects 0.000 abstract description 8
- 208000015181 infectious disease Diseases 0.000 abstract description 7
- 238000005507 spraying Methods 0.000 abstract description 6
- 230000002265 prevention Effects 0.000 abstract description 3
- 235000013311 vegetables Nutrition 0.000 abstract description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 2
- 239000007921 spray Substances 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 34
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 33
- 241000196324 Embryophyta Species 0.000 description 19
- 230000003902 lesion Effects 0.000 description 15
- 239000003814 drug Substances 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 10
- 229940079593 drug Drugs 0.000 description 9
- 108091091209 miR-1001 stem-loop Proteins 0.000 description 9
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 230000009368 gene silencing by RNA Effects 0.000 description 8
- 239000003899 bactericide agent Substances 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 108020004459 Small interfering RNA Proteins 0.000 description 6
- 230000000844 anti-bacterial effect Effects 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 244000052616 bacterial pathogen Species 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 108091070501 miRNA Proteins 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241001465180 Botrytis Species 0.000 description 4
- 241000244203 Caenorhabditis elegans Species 0.000 description 4
- 244000061176 Nicotiana tabacum Species 0.000 description 4
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 239000002679 microRNA Substances 0.000 description 4
- 239000000575 pesticide Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000012227 spray-induced gene silencing Methods 0.000 description 4
- 108020004566 Transfer RNA Proteins 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 230000001018 virulence Effects 0.000 description 3
- YVLPJIGOMTXXLP-UHFFFAOYSA-N 15-cis-phytoene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=CC=CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C YVLPJIGOMTXXLP-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 108091032955 Bacterial small RNA Proteins 0.000 description 2
- 229920000298 Cellophane Polymers 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 241000244206 Nematoda Species 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
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000019113 chromatin silencing Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000009335 monocropping Methods 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000002924 silencing RNA Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000004763 spore germination Effects 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000012033 transcriptional gene silencing Methods 0.000 description 2
- BQPPJGMMIYJVBR-UHFFFAOYSA-N (10S)-3c-Acetoxy-4.4.10r.13c.14t-pentamethyl-17c-((R)-1.5-dimethyl-hexen-(4)-yl)-(5tH)-Delta8-tetradecahydro-1H-cyclopenta[a]phenanthren Natural products CC12CCC(OC(C)=O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C BQPPJGMMIYJVBR-UHFFFAOYSA-N 0.000 description 1
- CHGIKSSZNBCNDW-UHFFFAOYSA-N (3beta,5alpha)-4,4-Dimethylcholesta-8,24-dien-3-ol Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21 CHGIKSSZNBCNDW-UHFFFAOYSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- XYTLYKGXLMKYMV-UHFFFAOYSA-N 14alpha-methylzymosterol Natural products CC12CCC(O)CC1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C XYTLYKGXLMKYMV-UHFFFAOYSA-N 0.000 description 1
- YVLPJIGOMTXXLP-UUKUAVTLSA-N 15,15'-cis-Phytoene Natural products C(=C\C=C/C=C(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C)(\CC/C=C(\CC/C=C(\CC/C=C(\C)/C)/C)/C)/C YVLPJIGOMTXXLP-UUKUAVTLSA-N 0.000 description 1
- YVLPJIGOMTXXLP-BAHRDPFUSA-N 15Z-phytoene Natural products CC(=CCCC(=CCCC(=CCCC(=CC=C/C=C(C)/CCC=C(/C)CCC=C(/C)CCC=C(C)C)C)C)C)C YVLPJIGOMTXXLP-BAHRDPFUSA-N 0.000 description 1
- FPTJELQXIUUCEY-UHFFFAOYSA-N 3beta-Hydroxy-lanostan Natural products C1CC2C(C)(C)C(O)CCC2(C)C2C1C1(C)CCC(C(C)CCCC(C)C)C1(C)CC2 FPTJELQXIUUCEY-UHFFFAOYSA-N 0.000 description 1
- 102100025238 CD302 antigen Human genes 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 description 1
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 1
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 1
- 239000005759 Diethofencarb Substances 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 241000223195 Fusarium graminearum Species 0.000 description 1
- 241000726221 Gemma Species 0.000 description 1
- BKLIAINBCQPSOV-UHFFFAOYSA-N Gluanol Natural products CC(C)CC=CC(C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(O)C(C)(C)C4CC3 BKLIAINBCQPSOV-UHFFFAOYSA-N 0.000 description 1
- 101100273718 Homo sapiens CD302 gene Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- LOPKHWOTGJIQLC-UHFFFAOYSA-N Lanosterol Natural products CC(CCC=C(C)C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(C)(O)C(C)(C)C4CC3 LOPKHWOTGJIQLC-UHFFFAOYSA-N 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 description 1
- JEVVKJMRZMXFBT-XWDZUXABSA-N Lycophyll Natural products OC/C(=C/CC/C(=C\C=C\C(=C/C=C/C(=C\C=C\C=C(/C=C/C=C(\C=C\C=C(/CC/C=C(/CO)\C)\C)/C)\C)/C)\C)/C)/C JEVVKJMRZMXFBT-XWDZUXABSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100496109 Mus musculus Clec2i gene Proteins 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- CAHGCLMLTWQZNJ-UHFFFAOYSA-N Nerifoliol Natural products CC12CCC(O)C(C)(C)C1CCC1=C2CCC2(C)C(C(CCC=C(C)C)C)CCC21C CAHGCLMLTWQZNJ-UHFFFAOYSA-N 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 244000203593 Piper nigrum Species 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000005828 Pyrimethanil Substances 0.000 description 1
- 235000002560 Solanum lycopersicum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 239000005842 Thiophanate-methyl Substances 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 235000013734 beta-carotene Nutrition 0.000 description 1
- 239000011648 beta-carotene Substances 0.000 description 1
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 description 1
- 229960002747 betacarotene Drugs 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006013 carbendazim Substances 0.000 description 1
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 description 1
- 150000001746 carotenes Chemical class 0.000 description 1
- 235000005473 carotenes Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000000453 cell autonomous effect Effects 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 101150012655 dcl1 gene Proteins 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- LNJNFVJKDJYTEU-UHFFFAOYSA-N diethofencarb Chemical compound CCOC1=CC=C(NC(=O)OC(C)C)C=C1OCC LNJNFVJKDJYTEU-UHFFFAOYSA-N 0.000 description 1
- QBSJHOGDIUQWTH-UHFFFAOYSA-N dihydrolanosterol Natural products CC(C)CCCC(C)C1CCC2(C)C3=C(CCC12C)C4(C)CCC(C)(O)C(C)(C)C4CC3 QBSJHOGDIUQWTH-UHFFFAOYSA-N 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 235000012055 fruits and vegetables Nutrition 0.000 description 1
- 239000011491 glass wool Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000000383 hazardous chemical Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000004280 healthy diet Nutrition 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229940058690 lanosterol Drugs 0.000 description 1
- CAHGCLMLTWQZNJ-RGEKOYMOSA-N lanosterol Chemical compound C([C@]12C)C[C@@H](O)C(C)(C)[C@H]1CCC1=C2CC[C@]2(C)[C@H]([C@H](CCC=C(C)C)C)CC[C@@]21C CAHGCLMLTWQZNJ-RGEKOYMOSA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 235000004213 low-fat Nutrition 0.000 description 1
- 235000012661 lycopene Nutrition 0.000 description 1
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 description 1
- 239000001751 lycopene Substances 0.000 description 1
- 229960004999 lycopene Drugs 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 101150093826 par1 gene Proteins 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 235000011765 phytoene Nutrition 0.000 description 1
- 244000000003 plant pathogen Species 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 235000007686 potassium Nutrition 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229960003975 potassium Drugs 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- QXJKBPAVAHBARF-BETUJISGSA-N procymidone Chemical compound O=C([C@]1(C)C[C@@]1(C1=O)C)N1C1=CC(Cl)=CC(Cl)=C1 QXJKBPAVAHBARF-BETUJISGSA-N 0.000 description 1
- ZLIBICFPKPWGIZ-UHFFFAOYSA-N pyrimethanil Chemical compound CC1=CC(C)=NC(NC=2C=CC=CC=2)=N1 ZLIBICFPKPWGIZ-UHFFFAOYSA-N 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- QGHREAKMXXNCOA-UHFFFAOYSA-N thiophanate-methyl Chemical compound COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC QGHREAKMXXNCOA-UHFFFAOYSA-N 0.000 description 1
- ZCIHMQAPACOQHT-ZGMPDRQDSA-N trans-isorenieratene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/c1c(C)ccc(C)c1C)C=CC=C(/C)C=Cc2c(C)ccc(C)c2C ZCIHMQAPACOQHT-ZGMPDRQDSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 1
Images
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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
- A01N25/04—Dispersions, emulsions, suspoemulsions, suspension concentrates or gels
- A01N25/06—Aerosols
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Environmental Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Dispersion Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Dentistry (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明提供一种RNA抑菌剂siR2，其具有SEQ ID NO.1所示的RNA序列。本发明还提供一种作物病菌抑制剂，所述抑制剂中至少包含siR2。具体的，所述抑制剂可以为包含所述siR2的喷雾剂，通过喷雾来实现对灰霉病的有效预防和治疗。本发明施加siR2的溶液能显著抑制灰霉菌孢子的萌发以及灰霉菌对植物的感染毒力，可用于蔬菜作物的灰霉病的防治。
Description
技术领域
本发明涉及作物灰霉病菌的保护和治疗，具体涉及RNA抑菌剂siR2及一种作物病菌抑制剂。
背景技术
番茄(Solanum lycopersicum)是世界上仅次于马铃薯第二重要的茄科植物，在很多国家都有种植。番茄果实脂肪含量低、不含胆固醇、富含维生素A、抗坏血酸、钾和叶酸等营养成份；同时还含有大量非营养性化学物质，如胡萝卜素(番茄红素、八氢番茄红素和β-胡萝卜素)和多酚，由于其营养的健康性而广泛受到大众的喜爱。番茄的大面积推广种植，各种番茄病害也随之流行甚至爆发；尤其是随着设施农业的发展，番茄的连茬重茬种植，各种病害在番茄生产过程中造成了严重的经济损失，尤其是一些真菌性病害，其中番茄灰霉病是近10年来成为危害番茄的主要病害之一，常导致番茄减产20％～40％，严重时可达60％以上。所以，番茄灰霉病的危害日趋严重，已成为保护地番茄产业发展的主要限制因素。
引起番茄灰霉病的病原菌是灰葡萄孢(Botrytis cinerea)，属于葡萄孢属(Botrytis)半知菌类。该病原菌腐生性很强，寄主范围非常广泛，可侵染番茄、草莓、辣椒、葡萄等多种水果和蔬菜。番茄灰霉病通过气流传播，并且传播速度很快，使得该病在番茄的设施农业栽培过程中的防治非常困难。目前番茄灰霉病的防治还是主要依赖化学农药，如甲酰胺、嘧霉胺、乙霉威、多菌灵、腐霉利和甲基硫菌灵等。由于番茄灰霉病菌繁殖快、传播迅速和易变异等特性，伴随着大量杀菌剂的重复多年使用，番茄灰霉病菌逐渐对各种杀菌剂产生了抗药性，使得化学杀菌剂的防治效果大大降低。并且，随着人们生活水平的逐年提高，人类追求健康饮食的诉求也越来越高。番茄生产过程中大量化学杀菌剂的使用，农药生产和使用过程中对环境和水资源的污染等越来越受到人们的关注。因此迫切的需要一些替代性的防治措施去抵抗灰霉病，所以开发新的无公害、环保型的新型农药成为科学家们首要的研究目标。
小干扰RNA(Small interfering RNA；siRNA)也被称为短干扰RNA(shortinterfering RNA)、沉默RNA(silencing RNA)和非编码RNA，是一类双链RNA分子，长度为20∽25个核苷酸。siRNA可以以单链形式与侵入宿主的外源基因表达的mRNA相结合，并诱导其相应的mRNA降解的转录后基因沉默。RNAi现象首次在线虫中被发现。1995年，Guo等将par-1基因的正、反义链RNA注射到秀丽隐杆线虫(Caenorhabditis elegans)中，发现其能阻断该基因的表达，意外地观察到了双链RNA在动物细胞中特异地抑制基因的表达。随后Fire等将mex-3基因的dsRNA注射到秀丽隐杆线虫，发现dsRNA能够高效特异性地抑制线虫中目的基因表达，并将这种效应称之为RNA干涉(RNA interference，RNAi)。随后多个团队在植物、昆虫、水产动物等多种生物中也报道了类似的结果。
在植物和一些微生物中，RNAi通过转录后基因沉默(PTGS)或转录基因沉默(TGS)调节参与许多重要生理过程的基因表达，同时也调节参与复杂的植物---病原体相互作用和植物免疫应答基因的表达。RNAi信号具有移动的和非细胞自主等特性，其不仅在生物体内的细胞之间，也在不同物种或生物界中相互作用的生物之间。研究发现，秀丽隐杆线虫和昆虫可从环境中摄取能诱导RNAi的外部RNA，这种现象称为“环境RNAi”。在水果，蔬菜和花的表面上喷洒灰霉菌DCL1/2的dsRNA或sRNA显著抑制了灰霉病。喷雾诱导的基因沉默(Spray Induced Gene Silence,SIGS)对单子叶植物的病害控制也有效。科赫等人表明喷洒靶向禾谷镰刀菌细胞色素P450羊毛甾醇C-14a-去甲基化酶(CYP51)基因的dsRNA显著减少了大麦叶片的病害症状。
因此，这些靶向病原体基因RNA可以作为对双子叶植物和单子叶植物物种都有效的新一代杀菌剂。外源RNA可通过直接或间接机制转移到病原菌中。RNA可以直接被真菌细胞摄取或者先转移到植物细胞中，然后间接进入真菌细胞。有趣的是，局部喷洒的RNA也能抑制远端非喷雾叶片的病原菌毒力，这表明这些RNA能够在植物体内全身扩散。纳米粒子技术的最新进展改善了SIGS在植物保护中的潜在应用。裸露的dsRNA和sRNA处理可以在喷洒后达10天保护植物免受微生物病原体的侵害。以上研宄表明，RNAi可作为开发新型杀菌剂的研发方向用于真菌病害的防治，减少目前农业生产过程中由于大量使用化学杀菌剂所带来的环境危害。
发明内容
本发明的一个目的在于提供一种RNA抑菌剂siR2，其具有SEQ ID NO.1所示的RNA序列(AUAUACAAAUUUCUGCUCAUUU)。
本发明的另一个目的在于提供一种作物病菌抑制剂，所述抑制剂中至少包含siR2。
本发明所述抑制剂可抑制的病菌至少包括灰霉菌，及其同属病菌。
在某些实施例中，所述抑制剂为包含所述siR2的喷雾剂，通过喷雾来实现对灰霉病的有效预防和治疗。
本发明的有益效果主要体现在：
1、施加siR2的溶液能显著抑制灰霉菌孢子的萌发以及灰霉菌对植物的感染毒力，可用于蔬菜作物的灰霉病的防治。
2、siR2为植物体内产生，安全可靠。
3、基于SIGS技术防治作物病害的另一个优势在于RNA作为抑菌因子，具有环境友好、专一性强、抑菌效果良好等特性，是未来农药发展的主要方向之一。
附图说明
图1为siR2对灰霉病菌孢子萌发的抑制效果。
图2为siR2对灰霉病菌孢子侵染叶片的抑制效果(A)及病斑直径(B)。
图3为siR2对灰霉病菌菌丝侵染叶片的抑制效果(A)及病斑直径(B)。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
一、实验准备
(1)番茄siR2的发现
利用sRNA组学及实时定量RT-PCR验证，发现一个能够靶向灰霉病菌的编码基因的番茄内源性的小RNA，它与现在报道发现的所有番茄乃至所有的植物miRNA都没有同源性，也不具备植物miRNA所具备茎环前体结构特征，因此我们将它定义为siRNA，命名为siRNA——siR2。
(2)siR2药物的合成
在吉玛基因公司(中国上海)合成了siR2药物。每管siR2药物1OD，按照说明书要求加入无RNA酶的ddH2O，siR2药物的最终浓度为10μM。
(3)番茄灰霉菌孢子的培养
选取完整的无伤口的番茄，在超净工作台中用75％的无水乙醇反复擦洗5遍。如果番茄根部有蒂，最好把蒂去除。去除时注意不要造成伤口，伤口会使灰霉菌在培养的过程中容易污染。用灭过菌的手术刀在番茄上切出几个伤口，然后将灰霉菌块覆盖在番茄表面的伤口上。灰霉菌块是培养在固体PDA培养基上。将番茄放在架子上，然后置于盆中。放置前要将架子和盆用75％的无水乙醇多喷洒几遍，保证消毒彻底，可以减少灰霉菌培养过程中的污染。然后把灰霉菌放在22℃的潮湿的培养箱中进行培养。培养时一定要注意灰霉菌的保湿。2周后，番茄表面会长满灰霉菌菌丝及孢子，用作实验。采取灰霉菌孢子时，用刷子轻刷番茄的表面，溶于ddH2O。然后通过玻璃棉过滤得到灰霉菌的孢子。将过滤出来的灰霉菌孢子在无菌蒸馏水中洗涤两次。用细胞计数板进行计数，并调节至5×106分生孢子/mL的浓度。用于实验中的生物测定。
二、siR2对灰霉菌孢子萌发的影响
用Bilir等人描述的方法来进行分生孢子的萌发实验。简单来说，就是将玻璃纸剪成大小为1.0cm X 1.0cm大小，然后在高压灭菌锅下，115℃，灭菌15min，备用。灭菌时，将剪好的玻璃纸放入无菌水中在放进灭菌锅。然后在超净工作台中，将玻璃纸置于无添加抗生素的1/2MS培养基上。然后用移液枪在玻璃纸上滴加5μL的分生孢子的悬浮液，同时加入5μL的miRNA药物(终浓度为：10uM)；
对照组1以等量的NC RNA代替siR2药物，NC RNA是一条长度为21nt的RNA，但它不能靶向番茄和灰霉病菌的任何基因，也就是在番茄和灰霉病菌的RNA上没有结合位点。
对照组2：以等量的miR1001代替siR2药物，miR1001是一条已报道对灰霉病菌生长及植物感染有抑制作用的番茄miRNA。
将培养基放置于24℃的培养箱中进行培养。12h后在光照显微镜下观察分生孢子的萌发情况。结果如图1所示，siR2处理后绝大部分的灰霉菌孢子没有萌发，而未经siR2药物处理的对照组孢子萌发效率极高，长势良好；经miR1001处理的灰霉菌孢子虽有萌发，但萌发效率低，萌发后的菌丝生长慢。这些结果表明施加siR2对灰霉菌孢子的萌发具有明显的抑制效果。
三、siR2对灰霉病菌孢子感染植物叶片的影响
取上述浓度为5×106个/mL的孢子溶液5μL，加入siR2使终浓度达到10μM，混匀后将10μL的siR2-孢子混合液滴加到离体的烟草叶片上；
对照组1以等量的NC RNA代替siR2药物与孢子混合液后滴加到离体的烟草叶片上。
对照组2：以等量的miR1001代替siR2药物与孢子混合液后滴加到离体的烟草叶片上。
实验设3个重复。处理叶片在24℃光照培养箱中保温保湿培养3天后，对灰霉菌的侵染程度进行观测并拍照记录。并且进行了台盼蓝染色处理。侵染叶片时，为了方便拍照和后期的台盼蓝染色处理，我们会采用离体的番茄叶片及烟草叶片。培养时，要注意离体叶片的保湿。我们将离体叶片的根部用湿棉花包裹起来，培养期间小心地往棉花上喷无菌水。喷水时注意不要喷到植物叶片上，防止对实验结果造成影响。结果如图2所示，未经siR2处理，灰霉菌孢子在叶片上的病斑明显大于经miR1001处理的病斑,而miR001处理的病斑也明显大于siR2处理的病斑。经测量病斑直径，NC RNA处理的病斑平均直径约为17mm左右；miR001处理的病斑平均直径约为5mm，而经siR2处理的病斑平均直径非常小，小于2mm左右(图2)。这些结果表明，相比NC RNA和miR1001，施加siR2对灰霉病菌孢子侵染植物叶片的毒力抑制更好。
四、siR2对灰霉菌菌丝侵染番茄叶片的抑制效果
从PDA固体培养基上刮取约10mg灰霉菌菌丝，将其放置于植物的表面，然后往灰霉菌菌丝上添加5μL浓度为10μM的siR2药物，使药物完全覆盖灰霉菌的菌丝。
对照组1中以等量的NC RNA代替siR2药物。
对照组2：以等量的miR1001代替siR2药物。
将上述处理好的样品放在24℃光照培养箱中保温保湿培养3天后，观察叶片菌斑大小。结果如图3所示，经siR2处理后，灰霉病菌孢子在叶片上的病斑明显小于经NC RNA处理的对照1和经miR1001处理的对照2上的病斑。测量病斑直径，对照1组叶片的病斑平均直径约为15mm；对照2的平均直径约为6mm；而经siR2处理的病斑直径小于3mm(图3)，也证实施加siR2对灰霉病菌菌丝的侵染毒力抑制效果好于NC RNA和miR1001。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
序列表
<110> 浙江理工大学
<120> 一种RNA抑菌剂siR2及作物病菌抑制剂
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> 未知(Unknown)
<400> 1
auauacaaau uucugcucau uu 22
Claims (1)
1.一种RNA抑菌剂siR2在防治灰霉菌的应用，所述RNA抑菌剂siR2的序列如SEQ IDNO.1所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110090041.4A CN112646817B (zh) | 2021-01-22 | 2021-01-22 | 一种RNA抑菌剂siR2及作物病菌抑制剂 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110090041.4A CN112646817B (zh) | 2021-01-22 | 2021-01-22 | 一种RNA抑菌剂siR2及作物病菌抑制剂 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112646817A CN112646817A (zh) | 2021-04-13 |
| CN112646817B true CN112646817B (zh) | 2022-03-22 |
Family
ID=75370657
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110090041.4A Active CN112646817B (zh) | 2021-01-22 | 2021-01-22 | 一种RNA抑菌剂siR2及作物病菌抑制剂 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112646817B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114807133B (zh) * | 2022-04-11 | 2024-07-09 | 浙江理工大学 | 一种可用于灰霉病防治的RNAsqu纳米分子及一种作物病菌抑制剂 |
-
2021
- 2021-01-22 CN CN202110090041.4A patent/CN112646817B/zh active Active
Non-Patent Citations (3)
| Title |
|---|
| Characterization of miRNAs associated with Botrytis cinerea infection of tomato leaves;Weibo Jin等;《BMC Plant Biology》;20150116;第15卷(第1期);第1-14页 * |
| Comprehensive Transcriptome Analyses Reveal that Potato Spindle Tuber Viroid Triggers Genome-Wide Changes in Alternative Splicing, Inducible trans-Acting Activity of Phased Secondary Small Interfering RNAs, and Immune Responses;Yi Zheng等;《JOURNAL OF VIROLOGY》;20170322;第99卷(第11期);第1-18页 * |
| Identification and comparative analysis of the microRNA transcriptome in roots of two contrasting tobacco genotypes in response to cadmium stress;Xiaoyan He等;《scientific reports》;20160926;第6卷;第1-14页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112646817A (zh) | 2021-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ferraz et al. | Rhizobacteria induces resistance against Fusarium wilt of tomato by increasing the activity of defense enzymes | |
| CN109868225B (zh) | 棘孢木霉n-8-2及其应用 | |
| WO2010135893A1 (zh) | 防治作物土传病害的复合生防菌剂ps和psx及其制备方法和用途 | |
| CN109769535B (zh) | 内生真菌菌株r5-6-1在防治水稻白叶枯病中的应用 | |
| Guo et al. | Postharvest treatment with trans-2-hexenal induced resistance against Botrytis cinerea in tomato fruit | |
| CN112662675B (zh) | 一种RNA抑菌剂phasiRNA5及作物病菌抑制剂 | |
| Koc et al. | Influence of Phytophthora capsici L. inoculation on disease severity, necrosis length, peroxidase and catalase activity, and phenolic content of resistant and susceptible pepper (Capsicum annuum L.) plants | |
| Ahmad et al. | Decontamination of seed borne disease in pepper (Capsicum annuum L.) seed and the enhancement of seed quality by the emulated plasma technology | |
| CN112626071B (zh) | 一种RNA抑菌剂miRNA482a及作物病菌抑制剂 | |
| CN112646817B (zh) | 一种RNA抑菌剂siR2及作物病菌抑制剂 | |
| Liu et al. | Exogenous dopamine and MdTyDC overexpression enhance apple resistance to Fusarium solani | |
| Yadav et al. | Bacillus subtilis NBRI-W9 simultaneously activates SAR and ISR against Fusarium chlamydosporum NBRI-FOL7 to increase wilt resistance in tomato | |
| CN107517788A (zh) | 一种防治水稻稻瘟病的方法 | |
| CN112695035B (zh) | 一种RNA抑菌剂miRNA157d-3p及作物病菌抑制剂 | |
| CN113826621B (zh) | 硫代丁酸甲酯在防治番茄枯萎病中的应用 | |
| Lin et al. | Smoke-water controls Pythium damping-off in papaya seedling | |
| CN104711206B (zh) | 一种饲料类芽孢杆菌及其用途 | |
| CN112616847B (zh) | 氯唑沙宗在制备用于防治由植物病原菌引起的植物病害的杀菌剂中的应用 | |
| CN104513802A (zh) | 一种玉米内生枯草芽孢杆菌及其生防应用 | |
| Chu et al. | Effects of hexadecanoic acid on Fusarium oxysporum f. sp. niveum control and on growth of watermelon (Citrullus lanatus) | |
| CN103525708B (zh) | 落葵链格孢及其在苹果树腐烂病害防治中的应用 | |
| CN102613186A (zh) | 壳聚糖在防治植物细菌性果斑病中的应用 | |
| Khalil et al. | Effect of growing medium water content on the biological control of root pathogens in a closed soilless system | |
| CN113502227B (zh) | 藤仓镰刀菌、含有该菌的菌剂和除草剂及它们的应用 | |
| CN111937895B (zh) | 含吡唑醚菌酯及其组合物在防治桃枝枯病中的应用 |
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 |