CN109258681B - miR-71-5p在害虫防治中的应用 - Google Patents
miR-71-5p在害虫防治中的应用 Download PDFInfo
- Publication number
- CN109258681B CN109258681B CN201811128090.7A CN201811128090A CN109258681B CN 109258681 B CN109258681 B CN 109258681B CN 201811128090 A CN201811128090 A CN 201811128090A CN 109258681 B CN109258681 B CN 109258681B
- Authority
- CN
- China
- Prior art keywords
- mir
- prodenia litura
- expression
- insect
- mirna
- 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.)
- Expired - Fee Related
Links
- 108091078326 miR-71 stem-loop Proteins 0.000 title claims abstract description 53
- 108091074965 miR-71-1 stem-loop Proteins 0.000 title claims abstract description 49
- 108091050089 miR-71-2 stem-loop Proteins 0.000 title claims abstract description 49
- 241000607479 Yersinia pestis Species 0.000 title abstract description 23
- 241000985245 Spodoptera litura Species 0.000 claims abstract description 53
- 241000238631 Hexapoda Species 0.000 claims description 35
- 108091065376 miR-375 stem-loop Proteins 0.000 claims description 6
- 239000000575 pesticide Substances 0.000 claims description 5
- 239000003112 inhibitor Substances 0.000 claims description 4
- 239000002917 insecticide Substances 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 25
- 102100031701 Nuclear factor erythroid 2-related factor 2 Human genes 0.000 abstract description 20
- 101000588302 Homo sapiens Nuclear factor erythroid 2-related factor 2 Proteins 0.000 abstract description 18
- 241000196324 Embryophyta Species 0.000 abstract description 16
- 108090000623 proteins and genes Proteins 0.000 abstract description 13
- 230000001105 regulatory effect Effects 0.000 abstract description 13
- 241000219198 Brassica Species 0.000 abstract description 12
- 235000003351 Brassica cretica Nutrition 0.000 abstract description 12
- 235000003343 Brassica rupestris Nutrition 0.000 abstract description 12
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 abstract description 12
- 239000001963 growth medium Substances 0.000 abstract description 12
- 235000010460 mustard Nutrition 0.000 abstract description 12
- 230000001276 controlling effect Effects 0.000 abstract description 11
- 239000002028 Biomass Substances 0.000 abstract description 8
- 230000009471 action Effects 0.000 abstract description 5
- 230000007246 mechanism Effects 0.000 abstract description 4
- 230000009261 transgenic effect Effects 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 3
- 239000002679 microRNA Substances 0.000 description 25
- 108700011259 MicroRNAs Proteins 0.000 description 19
- 108091070501 miRNA Proteins 0.000 description 14
- IVYPNXXAYMYVSP-UHFFFAOYSA-N indole-3-methanol Chemical compound C1=CC=C2C(CO)=CNC2=C1 IVYPNXXAYMYVSP-UHFFFAOYSA-N 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 6
- 230000033228 biological regulation Effects 0.000 description 5
- 238000001784 detoxification Methods 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 241000124008 Mammalia Species 0.000 description 4
- 241000244206 Nematoda Species 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 230000035882 stress Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 241000255789 Bombyx mori Species 0.000 description 3
- 241000426497 Chilo suppressalis Species 0.000 description 3
- 241001046947 Ectropis obliqua Species 0.000 description 3
- 108010024636 Glutathione Proteins 0.000 description 3
- 241000255777 Lepidoptera Species 0.000 description 3
- 241000500437 Plutella xylostella Species 0.000 description 3
- 241000256251 Spodoptera frugiperda Species 0.000 description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 241000209140 Triticum Species 0.000 description 3
- 235000021307 Triticum Nutrition 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 206010017758 gastric cancer Diseases 0.000 description 3
- 229960003180 glutathione Drugs 0.000 description 3
- 108091031438 miR-375-3 stem-loop Proteins 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 201000011549 stomach cancer Diseases 0.000 description 3
- VFTRKSBEFQDZKX-UHFFFAOYSA-N 3,3'-diindolylmethane Chemical compound C1=CC=C2C(CC=3C4=CC=CC=C4NC=3)=CNC2=C1 VFTRKSBEFQDZKX-UHFFFAOYSA-N 0.000 description 2
- 108091032955 Bacterial small RNA Proteins 0.000 description 2
- 102000016938 Catalase Human genes 0.000 description 2
- 108010053835 Catalase Proteins 0.000 description 2
- YVGGHNCTFXOJCH-UHFFFAOYSA-N DDT Chemical compound C1=CC(Cl)=CC=C1C(C(Cl)(Cl)Cl)C1=CC=C(Cl)C=C1 YVGGHNCTFXOJCH-UHFFFAOYSA-N 0.000 description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 2
- 108010071382 NF-E2-Related Factor 2 Proteins 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide dismutase Proteins 0.000 description 2
- 230000004900 autophagic degradation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000021050 feed intake Nutrition 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 108091053735 lin-4 stem-loop Proteins 0.000 description 2
- 108091032363 lin-4-1 stem-loop Proteins 0.000 description 2
- 108091028008 lin-4-2 stem-loop Proteins 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 108091007428 primary miRNA Proteins 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000009211 stress pathway Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 229940093768 3,3'-diindolylmethane Drugs 0.000 description 1
- 235000010045 3,3'-diindolylmethane Nutrition 0.000 description 1
- 108010001572 Basic-Leucine Zipper Transcription Factors Proteins 0.000 description 1
- 102000000806 Basic-Leucine Zipper Transcription Factors Human genes 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 101710189994 Chitin synthase chs-1 Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 108091069308 Drosophila melanogaster miR-289 stem-loop Proteins 0.000 description 1
- 241000258955 Echinodermata Species 0.000 description 1
- 101001018064 Homo sapiens Lysosomal-trafficking regulator Proteins 0.000 description 1
- 101000962469 Homo sapiens Transcription factor MafF Proteins 0.000 description 1
- 101000962473 Homo sapiens Transcription factor MafG Proteins 0.000 description 1
- 101000667110 Homo sapiens Vacuolar protein sorting-associated protein 13B Proteins 0.000 description 1
- 241000012186 Litura Species 0.000 description 1
- 102100033472 Lysosomal-trafficking regulator Human genes 0.000 description 1
- 101150117406 Mafk gene Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108091093142 MiR-144 Proteins 0.000 description 1
- 101100144701 Mus musculus Drosha gene Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102000009572 RNA Polymerase II Human genes 0.000 description 1
- 108010009460 RNA Polymerase II Proteins 0.000 description 1
- 108091092508 RNA22 Proteins 0.000 description 1
- 108010057163 Ribonuclease III Proteins 0.000 description 1
- 102000003661 Ribonuclease III Human genes 0.000 description 1
- 102000004385 Sulfurtransferases Human genes 0.000 description 1
- 108090000984 Sulfurtransferases Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 102100039187 Transcription factor MafF Human genes 0.000 description 1
- 102100039188 Transcription factor MafG Human genes 0.000 description 1
- 102100039190 Transcription factor MafK Human genes 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 101150096483 atg5 gene Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000009702 cancer cell proliferation Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 101150072601 lin-14 gene Proteins 0.000 description 1
- 108091062895 miR-144 stem-loop Proteins 0.000 description 1
- 108091023108 miR-30e stem-loop Proteins 0.000 description 1
- 108091032902 miR-93 stem-loop Proteins 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000003992 organochlorine insecticide Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 208000007056 sickle cell anemia Diseases 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了miR‑71‑5p在害虫防治中的应用,属于害虫防治领域。本发明用农业害虫斜纹夜蛾进行实验,证实miR‑71‑5p影响斜纹夜蛾进食含芥菜次生物质的培养基,其作用机制为抑制SlNrf2的表达,进而影响了一系列受Nrf2调控的应对植物中次生物质的下游基因。综合上述的实验结果,可直接将miR‑71‑5p用于防治进食植物的害虫,或转基因植物进行害虫防治。
Description
技术领域
本发明属于害虫防治领域,具体涉及miR-71-5p在害虫防治中的应用。
背景技术
在哺乳类动物,已有报道证明了一系列转录因子参与调控解毒酶与谷胱甘肽代谢途径中酶的表达。其中NF-E2相关因子2(NF-E2-related factor2,Nrf2)是报道最多的一个转录因子,它是生物细胞氧化应激反应和抵抗外源有毒物质途径中的关键调控因子。在细胞核中,Nrf2可以与一类bZIP转录因子(MAFF,MAFG,MAFK)异源二聚化后,结合于下游基因的启动子上的调控元件,启动相关抗氧胁迫和解毒酶基因表达,包括谷胱甘肽代谢途径的γ-谷氨酸合成酶、抗氧胁迫途径的过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和谷胱甘肽硫转移酶(GST)等(Kwak et al.,2003;Min et al.,2008;Chen et al.,2018)。在昆虫中,相关的研究相对较少,并且较多的集中在果蝇抗药性。研究指出Nrf2在抗有机氯杀虫剂2,2-双(对氯苯基)-1,1,1-三氯乙烷(DDT)的果蝇抗性品系中的表达量显著高于敏感品系,通过促进解毒酶的表达而增强昆虫抗药性(Misra et al.,2013;Wan et al.,2014)。在农业害虫斜纹夜蛾中,证明了Nrf2参与昆虫抵抗多种植物次生物质和化学杀虫剂引起的氧胁迫伤害,其中一个机制为启动了解毒酶GST的表达(Chen et al.,2018)。
microRNA(miRNA)是生物中一类长度约为22个核苷酸的内源性非编码小分子RNA,在生物体内参与一系列生长和发育的调控。最早发现的miRNA家族成员是lin-4和lin-7。其中lin-4通过转录后调控lin-14基因,从而调控线虫发育时间(Lee et al.,1993)。miRNA在生物体内的合成受到严格的控制,miRNA和编码基因一样,由RNA polymeraseⅡ转录形成长度上千的初级转录本pri-miRNA,在Drosha酶作用下,pri-miRNA被切割形成大小约为70nt的茎环结构:pre-miRNA。当pre-miRNA从细胞核转运到细胞质时,在Dicer酶作用下,被剪切形成22nt左右miRNA。miRNA主要通过与靶基因结合后,降解mRNA或抑制翻译来调节靶基因的表达。miRNA和靶基因可以进行完全或不完全互补配对。通常认为,当进行完全互补时,miRNA可以启动对mRNA的切割,使靶基因表达下调,在这种机制中,miRNA的结合位点都在mRNA的编码区或者是开放阅读框中。当miRNA与mRNA不完全互补时,miRNA通过抑制靶基因的翻译而抑制表达,这种miRNA通常作用位点是在mRNA的3’非翻译区(UTR)(Bartel,2009)。
尽管已有大量miRNA的研究报道,但miRNA在昆虫与植物关系中的作用报道并不多,并且较多的研究报道为差异表达miRNA的分析。Khajuria等人分别用抗性和敏感品系的小麦种子喂食麦廮蝇,运用基因芯片技术分析,发现了dme-miR-2944-3p和dme-miR-289等miRNA的表达存在显著差异,暗示这些差异表达的miRNA可能参与麦廮蝇适应宿主植物(Khajuria et al.,2013)。在哺乳类动物,较多报道证明了植物次生物质治疗癌症中,miRNA参与了调控。在人胃癌细胞中,3,3'-二吲哚基甲烷处理通过miR-30e调节细胞自噬ATG5基因的表达,进而调节胃癌细胞自噬,达到抑制胃癌细胞增殖的效果(Yang et al.,2016)。在哺乳动物抗氧胁迫的研究中,报道了作用于Nrf2的miRNA(Narasimhan et al.,2012;Ren et al.,2015;Narasimhan et al.,2014;Yang et al.,2015;Wang et al.,2015)。在镰刀型细胞贫血症中,miR-144可以靶向Nrf2(Sangokoya et al.,2010)。在大鼠乳腺癌模型中,miR-93可以调控Nrf2活性(Singh et al.,2013)。在昆虫中还没有作用于Nrf2的miRNA的研究报道。研究调控Nrf2的重要miRNA对发展害虫防治新靶标有着重要的意义。
发明内容
本发明的目的在于提供昆虫类miR-71-5p在害虫防治中的应用。
本发明所采取的技术方案是:
目前的研究表明,miR-71-5p在原口动物和后口动物中的棘皮动物、半索动物和头索动物纲中存在,在脊椎动物和尾索动物中则没有miR-71(SGM De et al.,2013)。miR-71-5p的功能在线虫和昆虫中已经有研究报道。在蝗虫中,miR-71可以靶向几丁质合成酶CHS1的ORE区,负调控CHS1表达,参与调控蝗虫的蜕皮过程(Yang et al.,2016)。在线虫中,miR-71参与调控去除生殖细胞引发的寿命延长,超表达miR-71能增加去除生殖细胞的线虫寿命(Boulias et al.,2012)。虽然哺乳动物中已有miRNA调控Nrf2的报道(仅做调控研究,未做表征研究),但这些miRNA在昆虫中没有同源序列。而昆虫中miRNA对Nrf2的调控,目前未见有任何的报道。
发明人将5龄一天的斜纹夜蛾喂食其偏好的芥菜,在6h和48h提取中肠的总RNA,通过高通量测序分析,发现斜纹夜蛾进食芥菜后大量的解毒酶上调,Nrf2作为氧胁迫通路中的一个关键转录因子,在这个过程中也发生上调。同时发明人也进行了small RNA高通量测序分析,从测序所得到的所有miRNA中,通过预测分析可能作用于SlNrf2的miRNA。我们发现miR-71-5p可能作用于SlNrf2,因此我们将miR-71-5pmimics(模拟体)注射5龄1天的斜纹夜蛾,并喂食含有芥菜主要的次生物质的培养基,结果发现miR-71-5p能抑制斜纹夜蛾的生长,并且在注射一定时间后出现死亡。这个结果表明miR-71-5p对斜纹夜蛾防治的有效性。由于昆虫类miR-71-5p保守性很强,因此也可用于防治其他害虫。可直接将miR-71-5p用于防治进食植物的害虫,或转基因植物进行害虫防治。
昆虫类miR-71-5p在制备杀虫剂或昆虫抑制剂中的应用。
优选的,所述昆虫为鳞翅目昆虫。
优选的,所述鳞翅目昆虫包括斜纹夜蛾、蚕、茶尺蠖、小菜蛾、草地贪夜蛾、水稻二化螟。
优选的,所述鳞翅目昆虫为斜纹夜蛾。
优选的,所述斜纹夜蛾miR-375-3p的序列如5‘UGAAAGACAGGAGUAGUGAGAUG’3(SEQID NO:1)所示。
一种杀虫剂,所述杀虫剂中包含有昆虫类miR-71-5p。
优选的,所述昆虫为鳞翅目昆虫。
优选的,所述鳞翅目昆虫包括斜纹夜蛾、蚕、茶尺蠖、小菜蛾、草地贪夜蛾、水稻二化螟。
优选的,所述鳞翅目昆虫为斜纹夜蛾。
优选的,所述斜纹夜蛾miR-375-3p的序列如5‘UGAAAGACAGGAGUAGUGAGAUG’3(SEQID NO:1)所示。
一种昆虫抑制剂,所述昆虫抑制剂中包含昆虫类miR-71-5p。
优选的,所述昆虫为鳞翅目昆虫。
优选的,所述鳞翅目昆虫包括斜纹夜蛾、蚕、茶尺蠖、小菜蛾、草地贪夜蛾、水稻二化螟。
优选的,所述鳞翅目昆虫为斜纹夜蛾。
优选的,所述斜纹夜蛾miR-375-3p的序列如5‘UGAAAGACAGGAGUAGUGAGAUG’3(SEQID NO:1)所示。
本发明的有益效果是:
本发明发现斜纹夜蛾类miR-71-5p可有效防治斜纹夜蛾,该序列在别的昆虫内也存在,高度保守,因此也可用于防治其他害虫。
本发明用农业害虫斜纹夜蛾进行实验,证实miR-71-5p影响斜纹夜蛾进食含芥菜次生物质的培养基,其作用机制为抑制SlNrf2的表达,进而影响了一系列受Nrf2调控的应对植物中次生物质的下游基因。综合上述的实验结果,可直接将miR-71-5p用于防治进食植物的害虫,或转基因植物进行害虫防治。
附图说明
图1为miR-71-5p在不同昆虫中的序列比较;
图2为I3C处理斜纹夜蛾细胞后miR-71-5p(A)和SlNrf2(B)的表达谱分析;图中数据为平均值±SE,柱上星号表示差异显著(P<0.01;P<0.001)。CK:无I3C添加;
图3为miR-71-5pmimics处理斜纹夜蛾Spli-221细胞抑制SlNrf2的表达;
图4为miR-71-5pmimics处理斜纹夜蛾幼虫后,体重与进食量的变化;
图5为miR-71-5pmimics处理斜纹夜蛾幼虫后的表型。
具体实施方式
下面结合具体实施例对本发明作进一步的说明,但并不局限于此。
实施例1
1、靶向斜纹夜蛾和Nrf2的miRNA预测
结合转录组和small RNA测序的结果,并采用TargetScan、RNA22、RNAhybrid和PITA等4个网站预测可能靶向SlNrf2的miRNA,选择4个网站预测结果的交集miRNA。结果表明miR-71-5p可能靶向SlNrf2的3’末端,miR-71-5p与SlNrf2结合的预测自由能为-21.4kcal/mol。图1为miR-71-5p在不同昆虫中的序列比较。其中斜纹夜蛾的miR-71-5p序列为:5‘UGAAAGACAGGAGUAGUGAGAUG’3(SEQ ID No:1)。
核对我们的斜纹夜蛾中肠测序数据库,结果见表1。
表1 miRNA在进食芥菜的斜纹夜蛾中肠的表达
表1结果显示:斜纹夜蛾从培养基转食芥菜6h的中肠miR-71-5p的表达量下调(表1),表明miR-71-5p可能负调控基因的表达。
2、芥菜次生物质吲哚-3-甲醇(I3C)抑制miR-71-5p的表达
由于测序样品是斜纹夜蛾进食芥菜后的中肠组织,而13C为芥菜中的主要次生物质,Nrf2与植物次生物质产生的氧胁迫密切相关,为此,我们用I3C处理斜纹夜蛾,验证测序结果。结果表明了miR-71-5p表达量与I3C成反比,也暗示了miR-71-5p可能作用于SlNrf2。
进一步,在细胞株中,验证Nrf2和miRNA的可能关系。结果见图2。图2为I3C处理斜纹夜蛾细胞后miR-71-5p(A)和SlNrf2(B)的表达谱分析;图中数据为平均值±SE,柱上星号表示差异显著(P<0.01;P<0.001)。CK:无I3C添加。图2中显示,用I3C处理斜纹夜蛾细胞后,SlNrf2含量显著上调,miR-71-5p含量显著下降。因此它miR-71-5p可能是以其自身的降解,而解除了对靶基因的抑制作用,促进了靶基因的表达上调,从而影响斜纹夜蛾应对植物次生物质。
3、MicroRNA mimics处理斜纹夜蛾细胞株使预测靶基因表达发生改变
为了进一步研究SlNrf2与miR-71-5p之间调控关系,我们分别超表达miR-71-5pmimic于Spli-221细胞株,然后检测SlNrf2的表达情况。如图3所示,用0.1、0.5和1μg miR-71-5p的处理有浓度梯度效应,浓度越高,SlNrf2的表达受到的抑制越显著(图3)。
4、miR-71-5p mimics(模拟体)处理影响斜纹夜蛾生长
选取大小一致、健康状况一致的五龄第一天斜纹夜蛾幼虫,称量每头虫子的重量,随机分为miR-71-5p mimics(模拟体)处理组和NC mimics对照组。顺血液循环流动方向,用微量注射器从斜纹夜蛾幼虫的侧腹部第一和第二腹足之间,分别注射miR-71-5p mimics和NC mimics,注射量为每头虫2μg,注射后饲喂含有芥菜主要次生物质I3C的培养基。每天记录体重进食以及死亡率。
结果见表2和图4、图5。
表2注射miR-71-5p后进食含I3C培养基的斜纹夜蛾死亡率
图4表明:注射miR-71-5p并进食含I3C培养基120h时,斜纹夜蛾体重和进食量比进食正常培养基的斜纹夜蛾(对照)显著下降。表2和图5结果显示:注射MmiR-71-5p后,存活的虫子大部分都生长瘦小(见图5),并且有部分虫子死亡。
本发明用农业害虫斜纹夜蛾进行实验,证实miR-71-5p影响斜纹夜蛾进食含芥菜次生物质的培养基,其作用机制为抑制SlNrf2的表达,进而影响了一系列受Nrf2调控的应对植物中次生物质的下游基因。综合上述的实验结果,可直接将miR-71-5p用于防治进食植物的害虫,或转基因植物进行害虫防治。
为本领域的专业技术人员容易理解,以上所述仅为本发明专利的较佳实施例,并不用以限制本发明,凡本发明的精神和原则之内所作的任何修改、等同替换和改进等,均落在本发明要求的保护范围之内。
SEQUENCE LISTING
<110> 华南师范大学
<120> miR-71-5p在害虫防治中的应用
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> RNA
<213> Spodoptera litura (Fabricius)
<400> 1
ugaaagacag gaguagugag aug 23
Claims (3)
1.斜纹夜蛾miR-71-5p在制备杀虫剂或昆虫抑制剂中的应用,所述斜纹夜蛾miR-375-5p的序列如SEQ ID NO:1所示;所述昆虫为斜纹夜蛾。
2.一种杀虫剂,其特征在于:所述杀虫剂中包含有斜纹夜蛾miR-71-5p,所述斜纹夜蛾miR-375-5p的序列如SEQ ID NO:1所示;所述杀灭的昆虫为斜纹夜蛾。
3.一种昆虫抑制剂,其特征在于:所述昆虫抑制剂中包含斜纹夜蛾miR-71-5p,所述斜纹夜蛾miR-375-5p的序列如SEQ ID NO:1所示;所述昆虫为斜纹夜蛾。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811128090.7A CN109258681B (zh) | 2018-09-27 | 2018-09-27 | miR-71-5p在害虫防治中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811128090.7A CN109258681B (zh) | 2018-09-27 | 2018-09-27 | miR-71-5p在害虫防治中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109258681A CN109258681A (zh) | 2019-01-25 |
CN109258681B true CN109258681B (zh) | 2020-12-22 |
Family
ID=65198491
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201811128090.7A Expired - Fee Related CN109258681B (zh) | 2018-09-27 | 2018-09-27 | miR-71-5p在害虫防治中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109258681B (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112369428B (zh) * | 2020-11-19 | 2021-08-13 | 山东农业大学 | 一种调控昆虫翅发育的miR-318及其在害虫防治中的应用 |
CN112369429B (zh) * | 2020-11-19 | 2021-08-13 | 山东农业大学 | 一种调控昆虫翅发育的miR-927及其在害虫防治中的应用 |
CN114350661B (zh) * | 2021-12-08 | 2023-06-20 | 华南师范大学 | 一种小麦源miRNA在昆虫防治中的应用 |
CN117965545B (zh) * | 2024-03-29 | 2024-06-25 | 中国农业大学 | 一种防治鳞翅目害虫的miR-8492-3p及其应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103497950A (zh) * | 2013-07-10 | 2014-01-08 | 华南师范大学 | 一种小分子rna及其在害虫防治中的应用 |
CN104886117A (zh) * | 2015-05-27 | 2015-09-09 | 华南师范大学 | 鳞翅目昆虫抗菌肽Lebocin在害虫防治中的应用 |
CN105165885A (zh) * | 2015-09-16 | 2015-12-23 | 华南师范大学 | miR-375-3p在鳞翅目害虫防治中的应用 |
-
2018
- 2018-09-27 CN CN201811128090.7A patent/CN109258681B/zh not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103497950A (zh) * | 2013-07-10 | 2014-01-08 | 华南师范大学 | 一种小分子rna及其在害虫防治中的应用 |
CN104886117A (zh) * | 2015-05-27 | 2015-09-09 | 华南师范大学 | 鳞翅目昆虫抗菌肽Lebocin在害虫防治中的应用 |
CN105165885A (zh) * | 2015-09-16 | 2015-12-23 | 华南师范大学 | miR-375-3p在鳞翅目害虫防治中的应用 |
Non-Patent Citations (5)
Title |
---|
Characterization and expression profiling of microRNAs in response to plant feeding in two host-plant strains of the lepidopteran pest Spodoptera frugiperda;Yves Mone et al;《BMC Genomics》;20181106;1-15 * |
Computational Identification and Evolutionary Relationships of the MicroRNA Gene Cluster miR-71/2 in Protostomes;Matheus de Souza Gomes et al;《J Mol Evol》;20131231(第76期);353-358 * |
miR-305-3p和miR-71-5p通过调控谷胱甘肽代谢途径参与斜纹夜蛾应对植物次生物质;岑永杰等;《环境昆虫学报》;20191231;第41卷(第1期);33-41 * |
miR-71 and miR-263 Jointly Regulate Target Genes Chitin synthase and Chitinase to Control Locust Molting;Meiling Yang et al;《PLOS GENET》;20160817;第12卷(第8期);e1006257 * |
Sli-miR-34-5p 响应植物次生物质正调控斜纹夜蛾谷胱甘肽S-转移酶基因SlGSTe1的表达;马康等;《昆虫学报》;20190131;第62卷(第1期);1-8 * |
Also Published As
Publication number | Publication date |
---|---|
CN109258681A (zh) | 2019-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN109258681B (zh) | miR-71-5p在害虫防治中的应用 | |
Akbari et al. | Novel synthetic Medea selfish genetic elements drive population replacement in Drosophila; a theoretical exploration of Medea-dependent population suppression | |
Pitino et al. | Silencing of aphid genes by dsRNA feeding from plants | |
Fujii et al. | A miRNA involved in phosphate-starvation response in Arabidopsis | |
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 | |
CN103088023B (zh) | dsRNA及其组合在控制蚜虫危害中的应用 | |
Geiselhardt et al. | Egg laying of cabbage white butterfly (Pieris brassicae) on Arabidopsis thaliana affects subsequent performance of the larvae | |
Kumar et al. | Development of an RNAi based microalgal larvicide to control mosquitoes | |
CN103088024B (zh) | 两种dsRNA及其组合在控制蚜虫危害中的应用 | |
CN103201385A (zh) | 下调昆虫害虫中的基因表达 | |
CN104561018B (zh) | 舞毒蛾热激蛋白Hsp23基因及其dsRNA在无公害防治中的应用 | |
Heath et al. | RNA interference technology to control pest sea lampreys-a proof-of-concept | |
Mysore et al. | Preparation and use of a yeast shRNA delivery system for gene silencing in mosquito larvae | |
Romney et al. | Transcriptomic analysis of maternally provisioned cues for phenotypic plasticity in the annual killifish, Austrofundulus limnaeus | |
Sun et al. | A novel miRNA, miR-13664, targets CpCYP314A1 to regulate deltamethrin resistance in Culex pipiens pallens | |
Qiao et al. | MicroRNAs as potential biomarkers of insecticide exposure: a review | |
Wang et al. | The microRNA miR‐184 regulates the CYP303A1 transcript level to control molting of Locusta migratoria | |
CN113930424B (zh) | 一种调控红火蚁工蚁个体分工的小分子rna及其应用 | |
CN103497950B (zh) | 一种小分子rna及其在害虫防治中的应用 | |
CN104561006A (zh) | 舞毒蛾CYP6AN15v1基因dsRNA及其在无公害防治中的应用 | |
Kim et al. | Ingestion of antagomir or agomir of microRNA results in physiological changes and high mortality in Frankliniella occidentalis. | |
Zhang et al. | MicroRNA-190-5p confers chlorantraniliprole resistance by regulating CYP6K2 in Spodoptera frugiperda (Smith) | |
Wang et al. | Transgenic soybean plants expressing Spb18S dsRNA exhibit enhanced resistance to the soybean pod borer Leguminivora glycinivorella (Lepidoptera: Olethreutidae) | |
Deng et al. | A Dicer2 from Scylla paramamosain activates JAK/STAT signaling pathway to restrain mud crab reovirus | |
Ahmad et al. | Comparative transcriptomics of the irradiated melon fly (Zeugodacus cucurbitae) reveal key developmental genes |
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 | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20201222 |