CN117551652A - 沙葱萤叶甲GdBR-C基因及其在RNAi介导的害虫防治方面的应用 - Google Patents
沙葱萤叶甲GdBR-C基因及其在RNAi介导的害虫防治方面的应用 Download PDFInfo
- Publication number
- CN117551652A CN117551652A CN202311546264.2A CN202311546264A CN117551652A CN 117551652 A CN117551652 A CN 117551652A CN 202311546264 A CN202311546264 A CN 202311546264A CN 117551652 A CN117551652 A CN 117551652A
- Authority
- CN
- China
- Prior art keywords
- gene
- gdbr
- allium mongolicum
- rnai
- fluorescent
- 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.)
- Pending
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 47
- 230000009368 gene silencing by RNA Effects 0.000 title claims abstract description 17
- 241000607479 Yersinia pestis Species 0.000 title claims abstract description 11
- 241001124134 Chrysomelidae Species 0.000 title claims abstract description 9
- 230000001404 mediated effect Effects 0.000 title claims abstract description 8
- 108091030071 RNAI Proteins 0.000 title claims abstract 4
- 244000257727 Allium fistulosum Species 0.000 title description 4
- 235000008553 Allium fistulosum Nutrition 0.000 title description 4
- 241000383620 Allium mongolicum Species 0.000 claims abstract description 37
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 36
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims abstract description 21
- 230000003321 amplification Effects 0.000 claims description 15
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 14
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 239000002299 complementary DNA Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 241000234282 Allium Species 0.000 claims description 4
- 238000011084 recovery Methods 0.000 claims description 4
- 239000004576 sand Substances 0.000 claims description 4
- 238000001962 electrophoresis Methods 0.000 claims description 3
- 235000002732 Allium cepa var. cepa Nutrition 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 18
- 230000019617 pupation Effects 0.000 abstract description 15
- 238000011161 development Methods 0.000 abstract description 14
- 230000018109 developmental process Effects 0.000 abstract description 14
- 238000002347 injection Methods 0.000 abstract description 13
- 239000007924 injection Substances 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 11
- 239000000575 pesticide Substances 0.000 abstract description 11
- 230000029052 metamorphosis Effects 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 5
- 230000001105 regulatory effect Effects 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 3
- 238000001228 spectrum Methods 0.000 abstract description 3
- 230000002159 abnormal effect Effects 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 238000012217 deletion Methods 0.000 abstract description 2
- 230000037430 deletion Effects 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 16
- 241000238631 Hexapoda Species 0.000 description 14
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 13
- UPEZCKBFRMILAV-JNEQICEOSA-N Ecdysone Natural products O=C1[C@H]2[C@@](C)([C@@H]3C([C@@]4(O)[C@@](C)([C@H]([C@H]([C@@H](O)CCC(O)(C)C)C)CC4)CC3)=C1)C[C@H](O)[C@H](O)C2 UPEZCKBFRMILAV-JNEQICEOSA-N 0.000 description 11
- UPEZCKBFRMILAV-UHFFFAOYSA-N alpha-Ecdysone Natural products C1C(O)C(O)CC2(C)C(CCC3(C(C(C(O)CCC(C)(C)O)C)CCC33O)C)C3=CC(=O)C21 UPEZCKBFRMILAV-UHFFFAOYSA-N 0.000 description 11
- UPEZCKBFRMILAV-JMZLNJERSA-N ecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@H]([C@H](O)CCC(C)(C)O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 UPEZCKBFRMILAV-JMZLNJERSA-N 0.000 description 11
- 239000000047 product Substances 0.000 description 9
- 230000014509 gene expression Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 108091023040 Transcription factor Proteins 0.000 description 5
- 102000040945 Transcription factor Human genes 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 241000254158 Lampyridae Species 0.000 description 4
- 108091092724 Noncoding DNA Proteins 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000012257 pre-denaturation Methods 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 230000019491 signal transduction Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000005058 diapause Effects 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 230000035611 feeding Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- NKDFYOWSKOHCCO-YPVLXUMRSA-N 20-hydroxyecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@](C)(O)[C@H](O)CCC(C)(O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 NKDFYOWSKOHCCO-YPVLXUMRSA-N 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 2
- 108010082399 Autophagy-Related Proteins Proteins 0.000 description 2
- 102000003954 Autophagy-Related Proteins Human genes 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 241000040599 Prunus mongolica Species 0.000 description 2
- 241000382353 Pupa Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 108010057988 ecdysone receptor Proteins 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HXWZQRICWSADMH-SEHXZECUSA-N 20-hydroxyecdysone Natural products CC(C)(C)CC[C@@H](O)[C@@](C)(O)[C@H]1CC[C@@]2(O)C3=CC(=O)[C@@H]4C[C@@H](O)[C@@H](O)C[C@]4(C)[C@H]3CC[C@]12C HXWZQRICWSADMH-SEHXZECUSA-N 0.000 description 1
- 244000295724 Allium chinense Species 0.000 description 1
- 235000016790 Allium chinense Nutrition 0.000 description 1
- 240000000966 Allium tricoccum Species 0.000 description 1
- 235000006905 Allium tricoccum Nutrition 0.000 description 1
- 102000016614 Autophagy-Related Protein 5 Human genes 0.000 description 1
- 108010092776 Autophagy-Related Protein 5 Proteins 0.000 description 1
- 239000005878 Azadirachtin Substances 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 235000011777 Corchorus aestuans Nutrition 0.000 description 1
- 240000004792 Corchorus capsularis Species 0.000 description 1
- 235000010862 Corchorus capsularis Nutrition 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 241000489975 Diabrotica Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010011145 Fushi Tarazu Transcription Factors Proteins 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 241001617472 Galeruca daurica Species 0.000 description 1
- 241000894800 Galerucinae Species 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 108050002220 Green fluorescent protein, GFP Proteins 0.000 description 1
- ZSBXGIUJOOQZMP-UHFFFAOYSA-N Isomatrine Natural products C1CCC2CN3C(=O)CCCC3C3C2N1CCC3 ZSBXGIUJOOQZMP-UHFFFAOYSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- ZSBXGIUJOOQZMP-JLNYLFASSA-N Matrine Chemical compound C1CC[C@H]2CN3C(=O)CCC[C@@H]3[C@@H]3[C@H]2N1CCC3 ZSBXGIUJOOQZMP-JLNYLFASSA-N 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 240000001341 Reynoutria japonica Species 0.000 description 1
- 235000018167 Reynoutria japonica Nutrition 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 235000008950 Sommerlauch Nutrition 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- VEHPJKVTJQSSKL-UHFFFAOYSA-N azadirachtin Natural products O1C2(C)C(C3(C=COC3O3)O)CC3C21C1(C)C(O)C(OCC2(OC(C)=O)C(CC3OC(=O)C(C)=CC)OC(C)=O)C2C32COC(C(=O)OC)(O)C12 VEHPJKVTJQSSKL-UHFFFAOYSA-N 0.000 description 1
- FTNJWQUOZFUQQJ-IRYYUVNJSA-N azadirachtin A Natural products C([C@@H]([C@]1(C=CO[C@H]1O1)O)[C@]2(C)O3)[C@H]1[C@]23[C@]1(C)[C@H](O)[C@H](OC[C@@]2([C@@H](C[C@@H]3OC(=O)C(\C)=C/C)OC(C)=O)C(=O)OC)[C@@H]2[C@]32CO[C@@](C(=O)OC)(O)[C@@H]12 FTNJWQUOZFUQQJ-IRYYUVNJSA-N 0.000 description 1
- FTNJWQUOZFUQQJ-NDAWSKJSSA-N azadirachtin A Chemical compound C([C@@H]([C@]1(C=CO[C@H]1O1)O)[C@]2(C)O3)[C@H]1[C@]23[C@]1(C)[C@H](O)[C@H](OC[C@@]2([C@@H](C[C@@H]3OC(=O)C(\C)=C\C)OC(C)=O)C(=O)OC)[C@@H]2[C@]32CO[C@@](C(=O)OC)(O)[C@@H]12 FTNJWQUOZFUQQJ-NDAWSKJSSA-N 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- NKDFYOWSKOHCCO-UHFFFAOYSA-N beta-ecdysone Natural products C1C(O)C(O)CC2(C)C(CCC3(C(C(C)(O)C(O)CCC(C)(O)C)CCC33O)C)C3=CC(=O)C21 NKDFYOWSKOHCCO-UHFFFAOYSA-N 0.000 description 1
- 238000010523 cascade reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000027326 copulation Effects 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000001418 larval effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 229930014456 matrine Natural products 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 238000009304 pastoral farming Methods 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 230000008560 physiological behavior Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000007862 touchdown PCR Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/04—Insecticides
-
- 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
-
- 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)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Insects & Arthropods (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Agronomy & Crop Science (AREA)
- Toxicology (AREA)
- Dentistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了沙葱萤叶甲GdBR‑C基因及其在RNAi介导的害虫防治方面的应用。属于基因工程技术领域。本发明从沙葱萤叶甲中克隆获得了沙葱萤叶甲广谱复合物基因GdBR‑C,通过双链RNA注射法实现对GdBR‑C基因的干扰,确定GdBR‑C基因在沙葱萤叶甲变态发育中的重要作用。经表型观察,发现GdBR‑C基因的缺失导致沙葱萤叶甲三龄幼虫化蛹异常,目前已经证明GdBR‑C基因能够调控沙葱萤叶甲的变态发育过程。这一研究结果可以以GdBR‑C基因为靶标,为开发利用新型RNA农药来防治沙葱萤叶甲奠定必要的基础。
Description
技术领域
本发明涉及基因工程技术领域,更具体的说是涉及沙葱萤叶甲GdBR-C基因及其在RNAi介导的害虫防治方面的应用。
背景技术
沙葱萤叶甲Galeruca daurica是近些年来肆虐危害草原植被的一种新害虫,属鞘翅目Coleoptera、叶甲科Chrysomelidae、萤叶甲亚科Galerucinae,该虫分布广泛,在国内和国外均有发生,在国内主要分布在北部及西北部(内蒙古、新疆、甘肃等),在国外分布在俄罗斯西伯利亚、蒙古国和朝鲜、韩国这些国家。该虫一年发生一代,属完全变态昆虫,一生要经历卵、幼虫、蛹及成虫四个生命阶段,属寡食类害虫,以百合科葱属类植物为食,幼虫和成虫皆可取食为害。该虫于四月中上旬以越冬卵的状态孵化,四月下旬至五月中为幼虫期,呈带状分布为害植被;于六月上旬羽化为成虫,取食约一周后躲藏在石块、牛粪及杂草下不食不动,从而抵御不利环境进入滞育期,在8月下旬滞育期解除,成虫开始活动取食,并开始进行交配产卵,后又以滞育卵开始越冬。该虫自09年在内蒙古锡林郭勒草原突然大爆发后,危害趋势逐年加重。草场植被遭受取食后,草地破坏严重,植被紧缺,土壤结构受损,致使牧草数量和质量急剧下降,草原生态受到恶劣影响,降低了农牧民的经济效益,严重威胁了草原的可持续发展。
沙葱萤叶甲的发生危害已经严重影响到草原的生态环境质量,目前对于沙葱萤叶甲的防治主要以印楝素、苦参碱等植物源农药掺杂化学农药为主,但此种方法靶向性较差,在防治该虫的同时杀伤天敌昆虫和中性昆虫,严重破坏草原生态系统的生物多样性。此外,农药残留会对草原牲畜放牧安全造成一定威胁,给牧民带来巨大经济损失。
因此,迫切需要开发一种沙葱萤叶甲绿色防控新技术。核酸农药具备靶向性强、环境兼容性好等诸多优势,开发绿色环保的新型RNA农药将在沙葱萤叶甲绿色防控技术中具有巨大的应用前景。但是目前关于沙葱萤叶甲的RNA农药未见报道。
综上,如何提供一种沙葱萤叶甲的RNA农药是本领域技术人员亟需解决的技术问题。
发明内容
有鉴于此,本发明提供了沙葱萤叶甲GdBR-C基因及其在RNAi介导的害虫防治方面的应用。靶基因的筛选和挖掘是一项费时费力的工作,本发明以转录组测序为基础,并查阅大量相关文献,寻找沙葱萤叶甲生长发育相关基因。
本发明从沙葱萤叶甲中克隆获得了沙葱萤叶甲广谱复合物基因GdBR-C,通过双链RNA注射法实现对GdBR-C基因的干扰,确定GdBR-C基因在沙葱萤叶甲变态发育中的重要作用。经表型观察,发现GdBR-C基因的缺失导致沙葱萤叶甲三龄幼虫化蛹异常,目前已经证明GdBR-C基因能够调控沙葱萤叶甲的变态发育过程。这一研究结果可以以GdBR-C基因为靶标,为开发利用新型RNA农药来防治沙葱萤叶甲奠定必要的基础。
为了实现上述目的,本发明采用如下技术方案:
沙葱萤叶甲GdBR-C基因,其核苷酸序列如SEQ ID NO:1所示。
ATTTTTTTGCGCGCTTCAATTTTTAATATACGCCCGAAACTCGTGATGTTCTGACGAACGGTCCACAATAGCACGATTGCATTTGTAATATTCGCGAAGTGTGACTCATTTATATTTTAACTGGAAACAGTTTTTAAATTTACCGCGTTTTTTGTGTGTTTTCAAAACATCTGTGTATCAAGGCGCGAACTCATCACACGATGTTATTCTAACGTAATATGGATTCTGCAGAGAAAATACAAACATTTCAATGATTATTTTTAATCTCAGGTGAATTTTATTTACAATGGTAGATACACAACATTTTTGTCTGCGTTGGAACAACTATCAGAGCAGCATAACATCAGCTTTCGAAAACCTCCGTGACGATGAAGATTTTGTAGATGTTACGTTAGCCTGTGATGGAAAAAGTTTGAAGGCTCATCGAGTGGTTTTATCAGCTTGTAGTCCATATTTTAGAGAATTACTCAAGAGTACACCTTGTAAACATCCAGTGATAGTACTTCAAGATGTCGCTTGGACGGACTTGCACGCATTGGTCGAGTTCATTTATCACGGAGAAGTTAATGTACATCAACGTTCCCTCTCGTCATTCCTTAAAACGGCAGAAGTCCTCAGGGTTAGCGGTCTCACACAACAACACGGAGATGGTCGAGAACAGTTGGCACAAGTACAAAGTATGGTCAGATCACAGCAACATTCGACGCCAGTCTCCAACCACCATTCAACGTTTACGGATAAGCTAGTAGAAGATAGTTTGTTCACGTCTCCTTCCTCCCCTCCCCATGGCGCTACTGTCAATCAATTGCTCAGGAGAGCCGCATTGCAATACAGGAGAGAGAGAAGGATATCAATGGATCCAGATAATGAACATAAAAGGACAAGAATTGATCACATTATGGGCAATAACAATAATAATAACAACAATAATAATGAATTAAATTTATCGACGACGCATTCGCAACACCTCCCACCTCAGACGCAACCAGCAGACTTTTCCCCTAGCGCTATGAAAAATAACGTGCTCAATCTCAATCTTAATTCAATAAAAACCGATACAGAAGTTAATAATGGTATAAGTAGTACGGATCGAGAAGCTTCACCGTGTTCGCCGTCCCCCACTCTCCTCCATTCCCGATGTAATAACAACGACAACGATAACAACAACATTAAAAATGAACCCATAGAATTGCTCTGTAATACCAACCAAGACGACAACAGCAACGACTCAGCCGACATGACCAACGATAATGGGGGCAGTGGGCCTCATGGCGGACCTCTTTCGGGTAGCTCGGGGGGCGGCGGGGACCACGACGATCATGATAGCGCTATAAGTCCTTATTTAACGCCGACGGAAAGCAAGTTATTTGCTACGGCGGCGGGAAGTTTTAATTTTAGTATGGCGGCGCTAGCGGGTGATACTTCTATGTTAGGAGGATTGAATCAGTCTCTACAGGGAAACGACAGTTTGGCTGGGACTTCCCAAGCCGAACGTCGTCTCAGTTTCCCCCTCCCTTTGAACGCCAGCCACCGATGTGACGTTTGCGGCAAGCTACTCAGCACCAAACTAACCCTCAAACGGCATAAGGAGCAGCAGCATCTCCAGCCGTTGAACAGTGCAGTCTGCTGTCTTTGCCGGAAAGTGTTTCGAACTTTAAATTCGCTGAATAACCACAGAAGCATTTATCATAGGAGGCAGAAAGACATTCATAATAATAATCCGCCTGTTAAGCCCCCGCTCTGATTCTTTTTAGTTTCTATTTTAGACTAGACGATGTTTTAGAAATGTTTTTCTTAAACATAAACAGGTAGCAAAAGGACATTATGACGGTGAATCCAGAGAATAATAGTTTTTGTAACATTGAATCATATGAGTAAAAACAACAAACTGTTCGGCATCTTCATGTTTAGTGCGAAATGTCCGTAAATAATTATTATAGAATTATACTTCAATATTTGTCAACAAGAAGTAGTTAAATGTAAACAAACACAAATTCTTAAAGGAAATTAAAACATATCCTTCGAAATTGTAATCCAATTTTAGGAAATTACAATTCTTTCGACAAGATTTTGAAAATTATTCATTAGTTTGTCTTCAAAGTTAAATTTTACTGTCGTACTCAAATAAAATGAAGAGATTCATTCTGTTCATTATTGGATTCTTACTCTCGAAAATAAGCTCCAAAAAAAAAAAAAAAAAAAAAAAAAAA,SEQ ID NO:1。
变态是昆虫生长发育过程中重要的生理行为。Broad-Complex(Br-C)作为蜕皮激素信号通路的主要应答基因,在昆虫的生长发育中发挥重要的调控作用。20-羟基蜕皮酮(20E)信号通路会通过级联反应促使基因的表达从而调节蜕皮过程,而BR-C基因则位于信号转导的初级应答阶段,在20E信号转导中占据重要地位,并且受到20E的调控。因此,广谱复合物BR-C基因作为蜕皮激素通路中的关键调控因子,在昆虫的变态发育和生长发育过程中都发挥着极其重要的作用。
上述的沙葱萤叶甲GdBR-C基因在RNAi介导的害虫防治方面的应用。
RNA干扰技术(RNA interference,RNAi)首次被发现是1990年Naploli等人在研究矮牵牛花的颜色中,对其进行了基因诱导,得到了与预想中完全相反的结果,自此,RNAi技术开始被人们所研究。RNAi是在生物内进行内源或外源的双链RNA的诱导,使其体内的mRNA与其进行特异性结合后,在转录后水平致使mRNA降解,达到基因沉默的作用。RNAi技术在生物体的基因功能研究和害虫防治等方面发挥着重大的作用。
进一步的,所述RNAi介导中,合成的dsRNA通过注射进入沙葱萤叶甲体内。
进一步的,所述dsRNA的核苷酸序列如SEQ ID NO:2所示。
AGACGCAACCAGCAGACUUUUCCCCUAGCGCUAUGAAAAAUAACGUGCUCAAUCUCAAUCUUAAUUCAAUAAAAACCGAUACAGAAGUUAAUAAUGGUAUAAGUAGUACGGAUCGAGAAGCUUCACCGUGUUCGCCGUCCCCCACUCUCCUCCAUUCCCGAUGUAAUAACAACGACAACGAUAACAACAACAUUAAAAAUGAACCCAUAGAAUUGCUCUGUAAUACCAACCAAGACGACAACAGCAACGACUCAGCCGACAUGACCAACGAUAAUGGGGGCAGUGGGCCUCAUGGCGGACCUCUUUCGGGUAGCUCGGGGGGCGGCGGGGACCACGACGAUCAUGAUAGCGCUAUAAGUCCUUAUUUAACGCCGACGGAAAGCAAGUUAUUUGCUACGGCGGCGGGAAGUUUUAAUUUUAGUAUGGCGGCGCUAGCGGGUGAUACUUCUAUGUUAGGAGGAUUGAAUCAGUCUCUACAGGGAAACGACAGUUUGG,SEQ ID NO:2。
进一步的,所述dsRNA的合成步骤如下:
提取沙葱萤叶甲的总RNA,反转录成为cDNA作为扩增模板,以序列为SEQ ID NO:9的上游引物和以序列为SEQ ID NO:10的下游引物进行PCR扩增,PCR扩增产物进行电泳后回收产物,以胶回收产物为模板合成得到沙葱萤叶甲GdBR-C基因的dsRNA。
经由上述的技术方案可知,与现有技术相比,本发明取得的有益效果为:核酸农药具备靶向性强、环境兼容性好等诸多优势,开发绿色环保的新型RNA农药将在沙葱萤叶甲绿色防控技术中具有巨大的应用前景。本发明从沙葱萤叶甲中克隆获得了沙葱萤叶甲广谱复合物基因GdBR-C,通过双链RNA注射法实现对GdBR-C基因的干扰,确定GdBR-C基因在沙葱萤叶甲变态发育中的重要作用,为开发利用新型RNA农药来防治沙葱萤叶甲奠定必要的基础。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明实施例2中阳性克隆鉴定结果;
图2附图为本发明实施例2中沙葱萤叶甲3龄幼虫注射dsBR-C48h后对BR-C基因表达水平的影响;
图3附图为本发明实施例2中沙葱萤叶甲3龄幼虫注射dsBR-C48h后对蜕皮激素相关基因表达水平的影响;
图4附图为本发明实施例3中沙葱萤叶甲3龄幼虫注射dsBR-C后的对预蛹率的影响;
图5附图为本发明实施例3中沙葱萤叶甲3龄幼虫注射dsBr-C后的对化蛹率和羽化率的影响;
图6附图为本发明实施例3中沙葱萤叶甲3龄幼虫注射dsBr-C后对幼虫化蛹表型的影响。
具体实施方式
下面将结合本发明实施例中的附图,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。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。
本发明所需药剂为常规实验药剂,采购自市售渠道;未提及的实验方法为常规实验方法,在此不再一一赘述。
下面实施例中所用引物如表1所示。
表1克隆基因的引物信息
实施例1
沙葱萤叶甲GdBR-C基因的全长克隆
GdBR-C基因中间片段扩增:
2023年4月于内蒙古锡林郭勒盟镶黄旗草原采集沙葱萤叶甲幼虫,置于实验室自然条件下饲养,提取总RNA,参照PrimeScriptTM RT reagent Kit with gDNA Eraser试剂盒(TaKaRa)说明书将其反转录为cDNA。
以cDNA为模板,用PCR特异性引物进行扩增,引物序列为SEQ ID NO:3和SEQ IDNO:4。
PCR扩增体系如表2所示:
表2PCR扩增体系
PCR扩增程序为:94℃预变性5min;94℃30s,72℃30s,72℃1min,35个循环;最后72℃延伸10min。
PCR反应程序完成后,经1.5%琼脂糖凝胶电泳检测目的条带,按照EastepTM Geland PCR Cleanup Kit试剂盒进行PCR产物回收纯化,目的条带纯化后用1.5%琼脂糖凝胶电泳检测回收准确性。
连接pMD19-T克隆载体(TaKaRa),具体过程如下:
所用试剂如表3所示。
表3连接pMD19-T克隆载体试剂
上述反应液于16℃反应30分钟,加入到含有大肠杆菌Escherichia coli感受态细胞DH5α(TIANGEN)的1.5ml的离心管中,于冰上放置30分钟后,迅速置于42℃热激45s,再于冰上1分钟;加入LB液体培养基445μL后于37℃恒温下振荡培养60min。高温灭菌后的固体培养基在55℃时加入AMP(20ml培养基加入20μL 100μg/μl的氨苄Ampicillin(Amp)),并倒入培养皿中等待凝固;凝固后的固体培养基中依次加入40μL X-gal,20μLIPTG和200μL培养完成的菌液进行均匀涂布;于37℃培养12h,挑取白色单菌落进行PCR验证。
PCR反应程序及体系如下:
表4反应体系
扩增程序:94℃预变性5min;94℃30s,72℃30s,72℃1min,35个循环;最后72℃延伸10min。
然后将验证正确的菌液于LB液体培养基中过夜培养,再将菌液进行送样测序。
GdBR-C基因3′-和5′-末端序列扩增:
根据上一步测序验证结果,设计3′-和5′-特异性引物(SEQ ID NO:5~SEQ ID NO:8),按照RACE 5′/3′Kit(TaKaRa)试剂盒说明书进行末端扩增,采用降落式PCR,反应体系如下:
表5反应体系
程序如下:94℃30s,72℃2min,5个循环;94℃30s,70℃30s,72℃2min,5个循环;94℃30s,68℃30s,72℃2min,25个循环。
再以降落式PCR产物为模板进行巢氏PCR(引物序列如SEQ ID NO:5~SEQ ID NO:8),反应体系如下:
表6反应体系
程序如下:94℃30s,68℃30s,72℃2min,25个循环。得到扩增产物处理方法同上(GdBR-C基因中间片段扩增后续步骤)。
测序得到的3′-、5′-目的片段序列与中间片段进行拼接,利用DNAMAN软件进行序列一致性比对分析,最终获得该基因cDNA全长序列如SEQ ID NO:1所示。GdBR-C基因cDNA全长为2210bp,其中5′非编码区286bp,3′非编码区466bp,开放阅读框ORF全长1458bp,编码485个氨基酸,氨基酸序列如SEQ ID NO:13所示。
在上述序列中,下划线部分为5′非编码区,波浪线部分为3′非编码区,中间部分为开放阅读框ORF。
MVDTQHFCLRWNNYQSSITSAFENLRDDEDFVDVTLACDGKSLKAHRVVLSACSPYFRELLKSTPCKHPVIVLQDVAWTDLHALVEFIYHGEVNVHQRSLSSFLKTAEVLRVSGLTQQHGDGREQLAQVQSMVRSQQHSTPVSNHHSTFTDKLVEDSLFTSPSSPPHGATVNQLLRRAALQYRRERRISMDPDNEHKRTRIDHIMGNNNNNNNNNNELNLSTTHSQHLPPQTQPADFSPSAMKNNVLNLNLNSIKTDTEVNNGISSTDREASPCSPSPTLLHSRCNNNDNDNNNIKNEPIELLCNTNQDDNSNDSADMTNDNGGSGPHGGPLSGSSGGGGDHDDHDSAISPYLTPTESKLFATAAGSFNFSMAALAGDTSMLGGLNQSLQGNDSLAGTSQAERRLSFPLPLNASHRCDVCGKLLSTKLTLKRHKEQQHLQPLNSAVCCLCRKVFRTLNSLNNHRSIYHRRQKDIHNNNPPVKPPL,SEQ ID NO:13。
实施例2
沙葱萤叶甲GdBR-C基因的RNA干扰
(1)dsRNA引物设计
根据获得的GdBR-C基因的ORF序列,通过在线软件(https://www.flyrnai.org/cgi-bin/RNAi_find_primers.pl)进行dsRNA引物的设计,设计完成后,在两段引物的5'端分别加入T7启动子序列,以绿色荧光蛋白基因(Green Fluorescent Protein,GFP)作为对照。具体引物序列见SEQ ID NO:9~SEQ ID NO:12,其中下划线部分为T7启动子序列。
(2)dsRNA模板制备
以沙葱萤叶甲幼虫cDNA为模板,扩增含有T7启动子的BR-C基因序列,PCR扩增体系和程序如下:
表7扩增体系
扩增程序:94℃预变性5min;94℃30s,72℃30s,72℃1min,35个循环;最后72℃延伸10min。
获得的PCR产物纯化后,连接至-T Easy载体中,组分如下:
表8组分
反应液混合均匀后置于PCR仪中25℃反应2h。加入到预先解冻的DH5α感受态细胞(50μL)中,冰浴30min;42℃热激45s后迅速置于冰上1min;加入LB培养基500μL,37℃恒温震荡1h;培养结束后,15000rpm,离心5min进行菌体复苏,弃300μL上清,剩余液体用移液枪吸打混匀,在固体培养基中依次加入10μL Amp、10μL IPTG、20μL X-gal(避光)、剩余菌液,使用涂布棒依次涂匀,置于37℃培养箱中正置15min后倒置培养12~16h。阳性克隆鉴定及测序步骤同上(实施例1)。克隆鉴定结果如图1。
测序验证正确后,菌液与LB培养基按照1:100的比例混合后,加入抗生素后在37℃恒温振荡培养箱中培养12~16h。根据天根质粒小提试剂盒说明书进行质粒提取。回收产物后再次进行电泳。将获得正确的质粒为模板,进行PCR扩增,反应扩增体系及程序如下:
表9扩增体系
扩增程序:94℃预变性5min;94℃30s,72℃30s,72℃1min,35个循环;最后72℃延伸10min。
产物参照EastepTM Gel and PCR Cleanup Kit试剂盒进行胶回收,并进行浓度的测定,浓度测定值为106ng/μL。
(3)dsRNA合成
以纯化的质粒作为模板,参照T7 RiboMAXTM Express RNAi System试剂盒合成dsRNA,反应液均在冰上配置,组分如下:
表3组分
在0.2ml离心管中将上述组分轻柔混合,置于PCR仪中37℃反应4h;72℃10min,反应结束后晾至25℃,加入1μLRNase Free H2O稀释后的RNase和1μL DNase,于37℃温育30min(消除单链RNA),将全量22μL的反应液转移至1.5mL无酶离心管中,加入2μL的3M醋酸钠和20μL的异丙醇,均匀混合后置于冰上5min;在4℃离心机中、12000rpm离心20min,弃上清,用500μL 70%的乙醇洗涤沉淀4℃离心机中、12000rpm离心15min,重复洗涤离心;25℃无菌条件下静置15min;加入无酶水溶解沉淀,用超微量分光光度计检测浓度并稀释至1000ng/μL,分装后置于-80℃冰箱备用。dsRNA的核苷酸序列如SEQ ID NO:2所示。
(4)dsRNA注射
选择第3天的沙葱萤叶甲3龄幼虫,利用显微注射器分别将0.8μL dsBR-C溶液注射到沙葱萤叶甲幼虫体内,等量的dsGFP作为对照,每组处理3个生物学重复,每个重复5只幼虫。
注射48h后,提取幼虫总RNA后反转录进行荧光定量检测干扰效率(图2)。结果表明,对3龄幼虫进行干扰后,48h的检测结果发现,BR-C的表达水平显著降低了67.02%。表明dsRNA注射48h后对GdBR-C的干扰效率较高,可以满足后续试验。
选择蜕皮激素通路相关基因蜕皮激素受体(Ecdysone receptor,ECR)、蜕皮激素诱导转录因子74(Ecdysone inducible transcription factor 74,E74)、蜕皮激素诱导转录因子75(Ecdysone inducible transcription factor 75,E75)、蜕皮酮诱导的转录因子(Fushi-tarazu factor 1,FTZ-F1)、激素受体(Hormine receptor 3,HR3)和自噬相关蛋白(autophagy relatedprotein5,ATG5)进行表达量变化的检测(图3)。结果显示,与对照组相比,GdBR-C基因被干扰后,蜕皮激素通路相关基因的表达水平全部呈现下降趋势。表明GdBR-C基因表达水平的降低可以调控蜕皮激素通路相关基因的表达水平下调。
实施例3
观察实验(dsRNA注射处理同实施例2)
完全变态昆虫在变态过程中要经历幼虫-蛹的转变,在这个过程中,昆虫要进行预蛹的过渡,因此对试虫进行RNA干扰处理后,每只幼虫单独饲养,每日以新鲜野韭喂食,每日观察其取食及活动变化,当幼虫停止活动变为蜷曲时被视为进入预蛹状态;当预蛹蜕皮变为黄色蛹时被视为化蛹状态,记录幼虫每天的活动情况并进行统计。
结果发现,当GdBR-C被干扰后,幼虫需要再经过4.92天预蛹率才能达到50%,而对照组仅需再经过3.72天即有50%的幼虫进入预蛹期,处理组的预蛹时间要晚于对照组1.2天(图4);并且幼虫在注射dsBR-C后的化蛹率为13.33%,而对照组为86.67%,对照组化蛹率为处理组的6.5倍(图5),大部分幼虫表现为化蛹失败的现象(图6);并且处理组的羽化率为6.67%与对照组羽化率为61.13%相比降低了9.17倍(图4)。总之,GdBR-C基因被干扰后会使沙葱萤叶甲预蛹时间延迟,导致幼虫不能正常化蛹,化蛹率极度降低。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (5)
1.沙葱萤叶甲GdBR-C基因,其特征在于,其核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述的沙葱萤叶甲GdBR-C基因在RNAi介导的害虫防治方面的应用。
3.根据权利要求2所述的应用,其特征在于,所述RNAi介导中,合成的dsRNA通过注射进入沙葱萤叶甲体内。
4.根据权利要求3所述的应用,其特征在于,所述dsRNA的核苷酸序列如SEQ ID NO:2所示。
5.如权利要求4所述的应用,其特征在于,所述dsRNA的合成步骤如下:
提取沙葱萤叶甲的总RNA,反转录成为cDNA作为扩增模板,以序列为SEQ ID NO:9的上游引物和以序列为SEQ ID NO:10的下游引物进行PCR扩增,PCR扩增产物进行电泳后回收产物,以胶回收产物为模板合成得到沙葱萤叶甲GdBR-C基因的dsRNA。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311546264.2A CN117551652A (zh) | 2023-11-20 | 2023-11-20 | 沙葱萤叶甲GdBR-C基因及其在RNAi介导的害虫防治方面的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311546264.2A CN117551652A (zh) | 2023-11-20 | 2023-11-20 | 沙葱萤叶甲GdBR-C基因及其在RNAi介导的害虫防治方面的应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN117551652A true CN117551652A (zh) | 2024-02-13 |
Family
ID=89816276
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311546264.2A Pending CN117551652A (zh) | 2023-11-20 | 2023-11-20 | 沙葱萤叶甲GdBR-C基因及其在RNAi介导的害虫防治方面的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117551652A (zh) |
-
2023
- 2023-11-20 CN CN202311546264.2A patent/CN117551652A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106754948B (zh) | 褐飞虱NlMLP基因、编码蛋白及其应用 | |
CN103201385A (zh) | 下调昆虫害虫中的基因表达 | |
CN110468137B (zh) | 一种茄二十八星瓢虫高致死基因及其在防治瓢虫的应用 | |
CN110592117B (zh) | 高致死基因vATPase B及其防瓢虫应用 | |
CN110616223B (zh) | 一种防治茄二十八星瓢虫的靶基因及其应用 | |
CN110551730B (zh) | 茄二十八星瓢虫rps18基因及其在防虫害中的应用 | |
CN113248586B (zh) | 褐飞虱pib14蛋白及其编码基因在调控植物抗褐飞虱中的应用 | |
Islam et al. | Transcription of biotic stress associated genes in white clover (Trifolium repens L.) differs in response to cyst and root-knot nematode infection | |
CN109837291A (zh) | 一种利用rna干扰技术提高植物抗虫性的方法及其专用dna片段 | |
CN106957358B (zh) | 禾谷孢囊线虫Ha34609蛋白、编码基因及其应用 | |
CN107208097A (zh) | 用于控制鞘翅目害虫kruppel基因亲代rnai抑制 | |
CN109402133B (zh) | 舞毒蛾FTZ-F1基因、其编码蛋白及其dsRNA在害虫防治中的应用 | |
CN110616222A (zh) | 防治茄二十八星瓢虫的靶基因及其应用 | |
CN117551652A (zh) | 沙葱萤叶甲GdBR-C基因及其在RNAi介导的害虫防治方面的应用 | |
CN114058618B (zh) | 谷氨酸脱氢酶作为靶点在防治害虫中的应用 | |
CN110511937B (zh) | Snf7基因及其防治瓢虫的应用 | |
CN110511936B (zh) | 茄二十八星瓢虫生长发育相关基因chs1及其应用 | |
CN110628774B (zh) | 基因deltaCOPI及其在防治酸浆瓢虫中的应用 | |
CN110669768B (zh) | 一种防治酸浆瓢虫的方法 | |
CN110628773B (zh) | 一种用于防治酸浆瓢虫的靶基因和方法 | |
CN116751769B (zh) | 咖啡短体线虫Pc-CL蛋白、编码基因及其应用 | |
CN110628771B (zh) | 一种防治酸浆瓢虫的试剂盒 | |
CN110628772B (zh) | 基因gammaCOPI及其在防治酸浆瓢虫中的应用 | |
CN117551674A (zh) | 沙葱萤叶甲微管亲和调节激酶GdMARK2基因的克隆及其功能 | |
CN113881683B (zh) | 一种飞蝗zen基因dsRNA及其应用 |
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 |