CN117247943A - 一种用于防控蛴螬的dsHpAGO1及其应用 - Google Patents
一种用于防控蛴螬的dsHpAGO1及其应用 Download PDFInfo
- Publication number
- CN117247943A CN117247943A CN202311342951.2A CN202311342951A CN117247943A CN 117247943 A CN117247943 A CN 117247943A CN 202311342951 A CN202311342951 A CN 202311342951A CN 117247943 A CN117247943 A CN 117247943A
- Authority
- CN
- China
- Prior art keywords
- dshpago1
- grubs
- seq
- gene
- hpago1
- 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 claims abstract description 36
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 22
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims abstract description 21
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 7
- 239000002917 insecticide Substances 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 230000030279 gene silencing Effects 0.000 abstract description 17
- 230000034994 death Effects 0.000 abstract description 5
- 230000012010 growth Effects 0.000 abstract description 5
- 230000002265 prevention Effects 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 4
- 230000005856 abnormality Effects 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 13
- 230000009368 gene silencing by RNA Effects 0.000 description 12
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 11
- 244000105624 Arachis hypogaea Species 0.000 description 9
- 235000020232 peanut Nutrition 0.000 description 9
- 238000002347 injection Methods 0.000 description 8
- 239000007924 injection Substances 0.000 description 8
- 239000003921 oil Substances 0.000 description 8
- 101000780643 Homo sapiens Protein argonaute-2 Proteins 0.000 description 6
- 241000500437 Plutella xylostella Species 0.000 description 6
- 102100034207 Protein argonaute-2 Human genes 0.000 description 6
- 235000017060 Arachis glabrata Nutrition 0.000 description 5
- 235000010777 Arachis hypogaea Nutrition 0.000 description 5
- 235000018262 Arachis monticola Nutrition 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 101000780650 Homo sapiens Protein argonaute-1 Proteins 0.000 description 4
- 102100034183 Protein argonaute-1 Human genes 0.000 description 4
- 108020004459 Small interfering RNA Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000012226 gene silencing method Methods 0.000 description 4
- 230000001418 larval effect Effects 0.000 description 4
- 239000004055 small Interfering RNA Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 102000008682 Argonaute Proteins Human genes 0.000 description 3
- 108010088141 Argonaute Proteins Proteins 0.000 description 3
- 241000346653 Holotrichia parallela Species 0.000 description 3
- 241000607479 Yersinia pestis Species 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 210000002615 epidermis Anatomy 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000003827 upregulation Effects 0.000 description 3
- 241001600407 Aphis <genus> Species 0.000 description 2
- 241000254173 Coleoptera Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 101150029131 ago1 gene Proteins 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000004624 confocal microscopy Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241000256182 Anopheles gambiae Species 0.000 description 1
- 241001124076 Aphididae Species 0.000 description 1
- 102000010637 Aquaporins Human genes 0.000 description 1
- 108010063290 Aquaporins Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 206010004716 Binge eating Diseases 0.000 description 1
- 208000032841 Bulimia Diseases 0.000 description 1
- 239000005946 Cypermethrin Substances 0.000 description 1
- 108700032337 Drosophila AGO2 Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 101150066002 GFP gene Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000346285 Ostrinia furnacalis Species 0.000 description 1
- 102000003661 Ribonuclease III Human genes 0.000 description 1
- 108010057163 Ribonuclease III Proteins 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 208000014679 binge eating disease Diseases 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- KAATUXNTWXVJKI-UHFFFAOYSA-N cypermethrin Chemical compound CC1(C)C(C=C(Cl)Cl)C1C(=O)OC(C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 KAATUXNTWXVJKI-UHFFFAOYSA-N 0.000 description 1
- 229960005424 cypermethrin Drugs 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 210000000087 hemolymph Anatomy 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 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
-
- 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.
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Microbiology (AREA)
- Insects & Arthropods (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明提供了一种用于防控蛴螬的dsHpAGO1及其应用，属于生物技术和农业应用领域。本发明中的dsHpAGO1由核苷酸序列如SEQ ID NO.1所示的HpAGO1基因和核苷酸序列如SEQ ID NO.3～SEQ ID NO.4所示的dsRNA引物合成。蛴螬注射了本发明中的dsHpAGO1后，与对照组相比，HpAGO1基因的表达量均显著降低，干扰效率为95.53％；蛴螬生长发育出现明显异常，且大量死亡，死亡率为91.7％；HpVAA基因的沉默效率降低了42.6％。所以本发明中的dsHpAGO1在蛴螬防控中的意义十分重大。
Description
技术领域
本发明属于生物技术和农业应用领域，尤其涉及一种用于防控蛴螬的dsHpAGO1及其应用。
背景技术
花生为豆科作物，优质食用油主要油料品种之一，出油率高达45％-50％，远高于其他油料作物，暗黑鳃金龟Holotrichiaparallela在我国分布较为广泛，是影响花生、大豆等各种经济作物和粮食作物的主要地下害虫之一，其幼虫蛴螬在土壤中栖息，危害隐蔽，造成的伤口增加了病原菌的侵染，预测困难，是国内外公认的难以防治的重大地下害虫。蛴螬对花生的危害主要分为两个阶段：第一阶段是在花生播种后，越冬幼虫取食花生种子或幼苗的根，导致缺苗断垄；第二阶段是在花生开花期和结荚期，二龄或三龄幼虫进入暴食期，取食花生嫩果，危害花生根系，导致花生颗粒无收甚至死亡。目前，针对于蛴螬的防治主要依赖于化学防治，辅以物理防治和生物防治等方法。化学防治中高毒农药的使用虽然有很好的防治效果，但也带来了严重的环境污染和水污染，对天敌昆虫、蜜蜂和人畜也存在严重的危害，长期不合理的施用化学农药也会带来抗药性和农药残留的问题。物理防治方法主要是人工捕虫、灯光诱杀等方法，该方法相比于化学方法，具有环境友好、人畜无毒等优点，但成本高、效率低。生物防治的方法主要是通过释放天敌生物和使用生物制剂等但是防治效率较低，生防产品较少。因此，寻找新型、有效的生物防治靶标，进而利用生物技术手段研发环保、可持续的新型药剂已迫在眉睫。
RNA干扰(RNAi)是由双链RNA(dsRNA)引发的内源特异性基因转录后高度保守的、序列特异性的RNA降解机制。dsRNA导入生物体内后，被细胞中称为Dicer的RNaseⅢ分解成21-23bp的小干扰RNA(siRNA)，siRNA在沉默复合体(RISC)的作用下与目标mRNA结合，序列特异性地降解靶mRNA，阻止相应蛋白产物的合成，造成靶标基因的功能丧失。自从这种快速且直接沉默特定基因的机制被发现以来，RNAi技术迅速在非模式动物的基因功能研究领域得到广泛应用。Bautista等以小菜蛾(Plutellaxylostella)P450家族基因CYP6BG1为靶标，体外合成其dsRNA并喂食幼虫，不仅实现了靶标基因CYP6BG1表达量下调，而且显著降低了小菜蛾对杀虫剂氯氰菊酯的抗性。喂食含有豌豆蚜水通道蛋白基因ApAQPI的dsRNA的人工饲料后引起靶标基因的下调表达，显著提高了豌豆蚜血淋巴的渗透压。使用细菌表达dsRNA及体外合成dsRNA喂食昆虫不仅普遍用于基因功能研究，而且应用于控害靶标基因的筛选。饲喂马铃薯甲虫5个靶基因的dsRNA，观察到处理组甲虫体重增加减缓和死亡率显著上升。
Argonaute(AGO)蛋白家族是RNAi的核心作用元件，其中AGO2是唯一在RISC复合物中具有催化活性和重要作用的成员，可以调控miRNA/siRNA诱导的基因沉默过程。诱导RNAi途径中核心基因之一的AGO2表达量上调可提高RNAi效率。向亚洲玉米螟导入dsGFP可以诱导AGO2的上调，进而提高靶基因沉默效率。在蚜虫体内先导入dsGFP诱导AGO2表达上调，再导入靶基因的dsRNA，发现其沉默效率被提高。AGO2介导的RNAi具有抗病毒作用，可以抵抗外来病原侵害和病毒感染以自我保护。果蝇AGO2是在昆虫的抗病毒RNA沉默途径中鉴定出来的。沉默冈比亚按蚊AGO2的表达将使其更容易感染病毒。
AGO1同样作为Argonaute(AGO)蛋白家族成员，但目前尚未有AGO1在RNAi效率提高方面的相关报道。
发明内容
为了解决上述技术问题，本发明提供了一种用于防控蛴螬的dsHpAGO1及其应用。本发明中的dsHpAGO1由核苷酸序列如SEQ ID NO.1所示的HpAGO1基因和核苷酸序列如SEQID NO.3～SEQ ID NO.4所示的dsRNA引物合成。蛴螬注射了本发明中的dsHpAGO1后，与对照组相比，HpAGO1基因的表达量均显著降低，干扰效率为95.53％；蛴螬生长发育出现明显异常，且大量死亡，死亡率为91.7％；HpVAA基因的沉默效率降低了42.6％。所以本发明中的dsHpAGO1在蛴螬防控中的意义十分重大。
为了实现上述目的，本发明采用了以下技术方案：
本发明的目的之一在于提供一种用于防控蛴螬的HpAGO1基因，其核苷酸序列如SEQ ID NO.1所示。
本发明的目的之二在于提供一种用于防控蛴螬的dsHpAGO1，该dsHpAGO1由核苷酸序列如SEQ ID NO.1所示的HpAGO1基因和核苷酸序列如SEQ ID NO.3～SEQ ID NO.4所示的dsRNA引物合成。
本发明的目的之三在于提供一种所述dsHpAGO1在蛴螬防控中的应用。
本发明的目的之四在于提供一种含有所述dsHpAGO1的生物制品。
进一步的，所述生物制品为注射式杀虫剂或饵剂。
本发明的目的之五在于提供一种所述生物制品在蛴螬防控中的应用。
与现有技术相比，本发明具有如下有益效果：
蛴螬注射本发明中的dsHpAGO172 h后，与对照组相比，HpAGO1表达量均显著降低，干扰效率为95.53％；进一步观察试虫生长发育的表型特征，与对照组相比，注射本发明中的dsHpAGO1后蛴螬生长发育出现明显异常，且大量死亡，死亡率为91.7％；注射本发明中的dsHpAGO1后，实时定量PCR方法检测目标基因HpVAA的沉默效率，发现HpVAA的沉默效率降低了42.6％；共聚焦显微结果显示，注射本发明中的dsHpAGO124 h后，cy3标记的dsRNA在蛴螬各组织的分布量减少，说明AGO1通过介导dsRNA的吸收转运影响干扰效率。
附图说明
图1为本发明实施例1中注射dsHpAGO1后HpAGO1基因的沉默效率及幼虫死亡率；
图2为本发明实施例1中注射dsHpAGO1后对蛴螬影响的照片；
图3为本发明实施例1中注射dsHpAGO1后对幼虫基因沉默的影响；
图4为本发明实施例1中注射dsHpAGO1后dsRNA在暗黑鳃金龟幼虫表皮中的分布情况。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和本质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。以下实施例中使用的试剂、试剂盒和仪器均可由市售获得，实施例中使用的方法如无特别说明，与常规使用的方法一致。
下面结合实施例，对本发明的技术方案进行进一步详细阐述。
实施例1
1.合成HpAGO1基因
蛴螬AGO1基因的开放阅读框为2514bp，编码837个氨基酸，预测蛋白分子量为95.4kDa。根据蛴螬AGO1基因合成HpAGO1基因，合成的HpAGO1基因核苷酸序列如SEQ IDNO.1所示，氨基酸序列如SEQ ID NO.2所示。
SEQ ID NO.1：
ATGTCAACAGTAACACGAGTAACTTCTCCACGGCCTATTACAGCAGGTGGCGATGCGCCAAGCCATATTTTGCCGCAAACACCGAGTGACGGAAGACGAGGCGGCAGGGAAGGAAGACTTGTAACAAGAGTAATAACAAACTTTTTCCCGCTGAACATAGGGAAGTTGCAAAATGCAATTCATTATGATGTCGCAATTGTGCCTGATAAACCTAAGAAACTGTTGCGCACAGTAATGGAAGAATTTAGGAGAAAGAAATACGGGAATAGGTATCCAGCATACGATGGAAGCAAAAACTTGTACAGTTCATCTCCTTTATTTCAAAGTGCAGAAATCTCTGATATGATAAGTGTTATGGAAGATAATAGAGAAAAAGAATTTCATGTTACTGTTAAGTTCGCTA
CTTACGTGGACCTGACATGTCTTCACAATTATACGAAGAATGTTGGCGGCAAATCCGCT
TTAGAATTGGAAAAACCTATGCAAGCGTTACAATGTGTCGATATAGTGTTGCGAAGTG
CCCCAGCACTTTCCTGTATACCCGTTGGTAGATCGTACTTTACACGACCGAACGAACCA
TTTAGACTTGGAGATGGAGCCGTACTATACAATGGTTTCTTTCAAGCTGCCATACTAGG
ATGGAAACCGTTTGTTAATATAGATGTCGCAAATAAAGCCTTTCCAGCCGCTCTTAATG
TGATTGACCTTGTAAAAGAAATGTGTAATGATGATTTAAATGGCGAACTATACAAGGC
ATCTGTGGACACGATCAATAAACATATGAGGACACTTAAGATTCAATATGAATTACCC
AGAAATCTAGCTAGTCGAAGAGTATACAGAGTAAATGAGCTTGGTGCTACGTCAAGCA
GGGCCACATTTAAGGAAGATGGTGGAAGAACTATGACTGTCACAGAGTATTTTAAAAC
AAAGAAGAATATCCAATTAAAATTCCCCAATCTACCAACTTTATGGGTCGGTAGTCGA
AGCCGGGAAAATAAAATTCTACTGCCCATGGAATTCTGTACAATTCTGCCCGATCAAG
TCGTGAACAAGAAAATGACAGAAGAACAGACTAGAAATATGATAAAACGTGCGGCGA
CCGATACGAAAACTCGATTACATAATATTTCGCAATCCGTTACGAAAGCTAGTTATAAC
GAAAGCCCTACAATGAGAGAATTTGGAATAACAGTTAATCCAACACCGCAAGAAGTTG
AAGCAAGAATATTACATCCGCCAAAGCTAGAATATAGCAATAAACAAGAAAATGTCTT
TAAAGGTACTTGGCGTGCCAGTCAGTTTGTACAAGGCGTATCATTAACCTCTTGGAGTG
TATTATGTCTAGACCGTCGCACTCGCGAAGAAAACATTCACCGTTTAGAACAAGAGTT
AATGAAGACTGCAAGGACACTGGGAATGAATATAGGTTCTCCCGCTCCCTTTACTCTCC
TTGGAGATAGAAATCTACGGCTAGATTTACGTAAGCATTTTGCAGAAAATAAAGGATT
ACAGATGATTATAGTTATATTGAACAGCAGAAGCGAATGTTACAACTGGGTGAAAGAA
GCATCCGAAATTGCTGTAGGTTGTCTAACGCAATGTTTAAAATCTAATACTGTCTACAA
ACTGTCAACATCAACAGTTGGTAATATCTTATTAAAAATAAACGCAAAGTTAAATGGG
GAGAATCATCATTTATACGGGCTAAAGAGCTTTTTAGTTCGGCCATGTATGATAATGGG
TGCAGATGTTACACATCCAGGTCCAGATTCCAAGGATTTTCCTAGTGTCGCAGCTGTTA
CTGCTTCGTTTGACGCGCATTTCTTCAAGTACGCATTCGAATGGCGTCTACAACAAGCT
CGACAAGAGATGATAGAAGATCTACAGGAGATCACAAAACAGCATCTTCTTAATTTCT
ATAGAAAAAATAATCAGGTGAAACCGGAAAAGCTAATATATGTCCGTGATGGCGTGTC
AGATGGGCAATTTGAACAGGTGCTAAATGTAGAATTGACGGCTATAAGAACGGCGTGC
GTGAGACTTCAACCTAATTACAATCCGCCTATTATTTTTGTTGTGGTGCAGAAACGACA
CCACACCAGATTCTTTCCGAAGGATCCGAGAATATCAGAAGACAGAAATTGTAATGTCCCAGCAGGTACGTGCGTAGACACCGAGATCACGCATCCGTCAATAATCGATTTTTACTTGGTCTCTCACGCCAGCATCCAGGGAGTTGCAAAACCCACAAAGTACGTCAAATTGTGGGACGATTGCAATATGTCAGAAGATGATCTAGAAAAGCTTATGTATTACTTGTGTCACATGTTTGCACGTTGCAACAGATCTGTCAGTTATCCTGCTCCAACATATTATGCCCATCTTGCTGCAGCGAGAGCACGAGTATATTGTGAAATTGATCGCATTGACATGAGGAATTTGCAGGCGGAGCAAAGACGTTTGACAATTAAAGAAAATATCTCAAAAAATCTACCCATGTTCTTTGTTTAA
SEQ ID NO.2：
MSTVTRVTSPRPITAGGDAPSHILPQTPSDGRRGGREGRLVTRVITNFFPLNIGKLQNAIHYDVAIVPDKPKKLLRTVMEEFRRKKYGNRYPAYDGSKNLYSSSPLFQSAEISDMISVMEDNREKEFHVTVKFATYVDLTCLHNYTKNVGGKSALELEKPMQALQCVDIVLRSAPALSCIPVGRSYFTRPNEPFRLGDGAVLYNGFFQAAILGWKPFVNIDVANKAFPAALNVIDLVKEMCNDDLNGELYKASVDTINKHMRTLKIQYELPRNLASRRVYRVNELGATSSRATFKEDGGRTMTVTEYFKTKKNIQLKFPNLPTLWVGSRSRENKILLPMEFCTILPDQVVNKKMTEEQTRNMIKRAATDTKTRLHNISQSVTKASYNESPTMREFGITVNPTPQEVEARILHPPKLEYSNKQENVFKGTWRASQFVQGVSLTSWSVLCLDRRTREENIHRLEQELMKTARTLGMNIGSPAPFTLLGDRNLRLDLRKHFAENKGLQMIIVILNSRSECYNWVKEASEIAVGCLTQCLKSNTVYKLSTSTVGNILLKINAKLNGENHHLYGLKSFLVRPCMIMGADVTHPGPDSKDFPSVAAVTASFDAHFFKYAFEWRLQQARQEMIEDLQEITKQHLLNFYRKNNQVKPEKLIYVRDGVSDGQFEQVLNVELTAIRTACVRLQPNYNPPIIFVVVQKRHHTRFFPKDPRISEDRNCNVPAGTCVDTEITHPSIIDFYLVSHASIQGVAKPTKYVKLWDDCNMSEDDLEKLMYYLCHMFARCNRSVSYPAPTYYAHLAAARARVYCEIDRIDMRNLQAEQRRLTIKENISKNLPMFFV
2.dsHpAGO1的化学合成
使用步骤1中合成的HpAGO1基因以及对照GFP基因的dsRNA特异引物，参照T7RiboMAXTMExpress RNAi System说明书合成dsRNA，用于RNAi试验。具体使用的引物序列如表1所示。
表1引物详情
3.RNAi试验
qRT-PCR法测定注射dsGFP、dsHpAGO1后72h的沉默效率，结果如图1～图2所示。
结果显示：幼虫HpAGO1基因沉默效率为95.53％，死亡率为91.7％，蛴螬虫体颜色变深，生长缓慢，动作迟缓，少动。
4.注射dsHpAGO1对幼虫基因沉默的影响
选取HpVAA为目标基因，注射dsHpAGO1后，检测HpVAA的的相对表达量，结果如图3所示。
结果显示：注射dsHpAGO1后，HpVAA沉默效率降低42.6％，表明HpAGO1抑制HpVAA的沉默。
5.HpAGO1对dsRNA在幼虫表皮中分布的影响
使用共聚焦显微镜观察dsHpAGO1在暗黑鳃金龟幼虫表皮中的分布情况，结果如图4所示。
结果显示：注射dsHpAGO124 h后，cy3标记的dsRNA在蛴螬各组织的分布量减少。说明AGO1通过介导dsRNA的吸收转运影响干扰效率。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (6)
1.一种用于防控蛴螬的HpAGO1基因，其特征在于，其核苷酸序列如SEQ ID NO.1所示。
2.一种用于防控蛴螬的dsHpAGO1，其特征在于，由权利要求1所述的HpAGO1基因和核苷酸序列如SEQ ID NO.3～SEQ ID NO.4所示的dsRNA引物合成。
3.权利要求2所述的dsHpAGO1在蛴螬防控中的应用。
4.一种生物制品，其特征在于，所述生物制品中含有权利要求2所述的dsHpAGO1。
5.根据权利要求4所述的生物制品，其特征在于，所述生物制品为注射式杀虫剂或饵剂。
6.权利要求4～5任一所述的生物制品在蛴螬防控中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311342951.2A CN117247943A (zh) | 2023-10-17 | 2023-10-17 | 一种用于防控蛴螬的dsHpAGO1及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311342951.2A CN117247943A (zh) | 2023-10-17 | 2023-10-17 | 一种用于防控蛴螬的dsHpAGO1及其应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117247943A true CN117247943A (zh) | 2023-12-19 |
Family
ID=89136783
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311342951.2A Pending CN117247943A (zh) | 2023-10-17 | 2023-10-17 | 一种用于防控蛴螬的dsHpAGO1及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117247943A (zh) |
-
2023
- 2023-10-17 CN CN202311342951.2A patent/CN117247943A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102776189B (zh) | 一种细胞色素P450基因dsRNA及其在抑制蚜虫生长中的应用 | |
| CN103201385A (zh) | 下调昆虫害虫中的基因表达 | |
| WO2017106171A1 (en) | Rna interference for control of insect pests | |
| CN113106108B (zh) | 一种增强生防菌杀灭白蚁效果的双链核酸Dicer-1 dsRNA | |
| US11795458B2 (en) | Antisense oligonucleotide and double stranded RNAS for control of hemipteran and lepidopteran pests | |
| CN109852617B (zh) | 一种豌豆蚜海藻糖酶基因的rna干扰序列的制备方法及应用 | |
| CN114478735A (zh) | 一种蚜虫高致死基因及其在蚜虫防治中的应用 | |
| CN104630275A (zh) | 一种通过灌溉防治害虫的RNAi技术及其应用 | |
| US10246710B2 (en) | Double strand RNA as molecular biopesticides for RNA interference through feeding in the hemipteran invasive insect pest, brown marmorated stink bug | |
| CN117247943A (zh) | 一种用于防控蛴螬的dsHpAGO1及其应用 | |
| CN110951730B (zh) | 青翅蚁形隐翅甲V-ATPase-A基因的dsRNA及其人工饲料和应用 | |
| CN105838727B (zh) | 用于控制昆虫侵袭的核苷酸序列及其方法 | |
| CN112695045B (zh) | 一种鳞翅目昆虫Hpx12基因及应用 | |
| Aryati et al. | Effect of soybean variety and pest management on the sustainable production | |
| CN106367428A (zh) | 一种绿盲蝽V‑ATPase‑D基因cDNA及其应用 | |
| CN103103191A (zh) | 用于防治昆虫和蜘蛛类动物的RNAi | |
| WO2018013801A1 (en) | Rnai insecticide materials and methods | |
| CN109880826A (zh) | dsRNA在马铃薯甲虫防治中的应用及产品 | |
| CN109907068A (zh) | dsRNA在马铃薯甲虫防治中的应用及产品 | |
| CN104263731A (zh) | 一种蚜虫共生菌基因的dsRNA及其在降低蚜虫存活率中的应用 | |
| CN111034736A (zh) | 一种杀虫组合物及其应用 | |
| CN104109671A (zh) | 一种蚜虫共生菌基因的dsRNA及其在抑制蚜虫生长中的应用 | |
| Mdellel et al. | Influence of compost fertilization on the biology and morphology of green peach aphid (Myzus persicae) on pepper. | |
| CN106318956A (zh) | 一种绿盲蝽V‑ATPase‑A基因cDNA及其应用 | |
| US20220248690A1 (en) | Sex-linked rnai insecticide materials and methods |
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 | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20240103 Address after: Yu Ling Temple Street 071000 No. 289 Hebei city of Baoding Province Applicant after: HEIBEI AGRICULTURAL UNIVERSITY Applicant after: GRADUATE SCHOOL OF CHINESE ACADEMY OF AGRICULTURAL SCIENCES Address before: Yu Ling Temple Street 071000 No. 289 Hebei city of Baoding Province Applicant before: HEIBEI AGRICULTURAL UNIVERSITY |