CN114437192B - 一种番茄潜叶蛾染色质重塑因子iswi及其编码基因和应用 - Google Patents

一种番茄潜叶蛾染色质重塑因子iswi及其编码基因和应用 Download PDF

Info

Publication number
CN114437192B
CN114437192B CN202210050393.1A CN202210050393A CN114437192B CN 114437192 B CN114437192 B CN 114437192B CN 202210050393 A CN202210050393 A CN 202210050393A CN 114437192 B CN114437192 B CN 114437192B
Authority
CN
China
Prior art keywords
leaf miner
tomato leaf
glu
iswi
lys
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
Application number
CN202210050393.1A
Other languages
English (en)
Other versions
CN114437192A (zh
Inventor
吕志创
冀顺霞
吴强文
王晓迪
唐艳红
毕思言
刘万学
张桂芬
万方浩
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Original Assignee
Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Institute of Plant Protection of Chinese Academy of Agricultural Sciences filed Critical Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority to CN202210050393.1A priority Critical patent/CN114437192B/zh
Publication of CN114437192A publication Critical patent/CN114437192A/zh
Application granted granted Critical
Publication of CN114437192B publication Critical patent/CN114437192B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • 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

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Insects & Arthropods (AREA)
  • Toxicology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

本发明涉及昆虫防治领域，具体涉及一种番茄潜叶蛾染色质重塑因子ISWI及其编码基因和应用。所述染色质重塑因子ISWI的氨基酸序列如SEQ ID NO：2所示。本发明首次从新发重要入侵害虫番茄潜叶蛾中克隆获得染色质重塑因子ISWI基因，上述入侵害虫卵注射基因dsRNA后导致卵巢畸形、产卵量显著降低和成虫寿命显著缩短。所获得的结果为利用RNAi防治新发入侵害虫提供新的途径。

Description

一种番茄潜叶蛾染色质重塑因子ISWI及其编码基因和应用
技术领域
本发明涉及昆虫防治领域，具体涉及一种番茄潜叶蛾染色质重塑因子ISWI及其编码基因和应用。
背景技术
番茄潜叶蛾Tuta absoluta原产于南美洲秘鲁，是一种世界检疫性害虫，对番茄等作物造成毁灭性的灾害。番茄潜叶蛾主要以幼虫进行危害，雌成虫在刚刚展开的叶片上产卵，幼虫一经孵化便潜入寄主植物的组织中，潜食叶片。如不采取防治措施，该害虫可导致番茄减产80-100％。与此同时，随着化学农药的持续使用，促使其抗药性不断增强，能迅速的对各种药剂靶标产生抗性。
RNA干扰(RNAinterference,RNAi)现象是一种进化上保守的抵御转基因或外来病毒侵犯的防御机制。RNAi广泛存在于生物界，通过RNAi技术沉默昆虫体内某些基因，使昆虫的某些能力增强或丧失，也能在特定时间抑制其功能基因表达使昆虫的发育停留在某个阶段。由于RNA干扰技术具有昆虫选择性和基因特异性，其不仅是基因功能研究的重要手段，也是至今为止最有可能应用于害虫防治的生物工程技术。利用RNAi技术进行害虫生物防治，其关键步骤之一是筛选靶标基因。
染色质重塑(chromatin remodeling)是表观遗传学重要的组成部分之一。在真核生物细胞内存在一系列特异的染色质重塑因子它们可以促进染色质中DNA与蛋白质的动态结合。这些因子是多亚基复合物，利用ATP水解产生的能量，通过滑动核小体等方式改变染色质的状态，从而调控特定基因的表达。染色质重塑在生物发育繁殖相关基因的表达调控中发挥着关键作用，染色质重塑因子ISWI(imitation SWI)能参与生物发育生殖的调控，在调节发育生殖相关基因的表达中起重要作用。ISWI沉默出现寿命缩短、卵巢畸形以及产卵量降低等。因此，本发明基于RNA干扰技术，提供了新型害虫防治分子靶标，期望为番茄潜叶蛾的防控提供新的思路，达到有效的防控的效果。
发明内容
本发明的目的是提供一种番茄潜叶蛾染色质重塑因子。
本发明的再一目的是提供上述番茄潜叶蛾染色质重塑因子ISWI的编码基因。
本发明的再一目的是提供上述编码基因的应用。
根据本发明的具体实施方式，首次克隆出番茄潜叶蛾染色质重塑因子ISWI，其cDNA全长核苷酸序列如SEQ ID NO:1所示：
ATGTCACAACCTGATGAAGCTATGGATGTAGGGGACGTGGAAGAAAACTCTAATGAGTCGTCCAGCGATACCACGTCCTCAAAGGGTAAAGAAGGAGACTTCGAAAGTAAAATTGAGACCGATCGTTCCAAGCGTTTTGATTTTTTGTTGAAACAAACTGAAATATTCTCGCATTTCATGACCAATGCGCCTAAAACCAGTAGTCCTCCTAAGGCTAAAGCCGGTCGACCTAAAAAGGTGAAAGAAGATCTGCCTGAGCAGTCTGAGGATGCTTCTGCAGCAGACCATCGGCACCGCAAGACTGAACAGGAAGAAGATGAGGAACTCCTTGCTGAGACAAACGCTAAGATTAAGCCAATATTTCGCTTTGAAGCATCACCTCCTTACATCAAGAATGGAGAAATGAGAGATTATCAAGTGAGAGGTCTCAACTGGATGATATCTCTCTATGAGAATGGTATTAATGGTATCTTGGCTGATGAGATGGGTCTGGGTAAAACATTACAAACCATTTCTCTTCTTGGCTACATGAAAAATTTCAGAAATATCCCTGGTCCACACATAGTAATTGTGCCAAAATCAACCCTAACCAACTGGATGAATGAGTTCAAAAAATGGTGTCCTTCCCTAAGAGCAGTGTGTCTTATTGGAGATCAGGAAACAAGGAACACATTCATCCGTGAAGTACTGATGCCTGGCAACTGGGATGTGTGCATCACCTCTTACGAGATGATTATCCGTGAGAGATCAGTATTTAAGAAGTTCACTTGGCGCTACATGGTGATTGACGAAGCCCATCGAATCAAGAATGAGAAGTCAAAGTTGTCAGAGCTTCTCCGGGAGTTCAAGAGTATGAACCGGCTGCTGCTCACTGGCACACCGTTGCAAAACAATCTTCACGAGCTGTGGGCACTTCTTAACTTCTTGTTACCAGATGTATTCAACAGCTCTGATGATTTTGACTCTTGGTTCAACACCAATGCAGCTCTCGGTGATAATCAGCTGGTGTCCCGGTTGCATGCTGTGTTGAGACCGTTCTTACTCAGACGTCTCAAATCTGAAGTAGAAAAGAAATTGAAACCCAAAAAGGAACTCAAAGTTTATGTTGGATTGAGTAAGATGCAAAGGGAGTGGTATACTAAAGTACTCATGAAGGATATTGATATTGTAAACGGCGCGGGCAAAGTCGAAAAGATGCGTCTCCAGAACATCCTGATGCAGCTCCGCAAGTGCTGCAACCACCCGTACCTGTTCGACGGCGCGGAGCCCGGCCCGCCCTACACCACGGACGAGCATCTCGTCTACAACTGCGGGAAACTCGCCATACTCGACAAACTGCTGCCCAAGCTGCAGCAGCAGGACTCCAGGGTGCTCATCTTCTCGCAGATGACCAGGATGCTGGATATTCTGGAGGATTATTGCCTGTGGAGGCAGTACAAGTACTGCCGCTTAGACGGTCAGACCCCACATGAAGACCGCAACCGACAGATCGAGGAGTATAACGCGGAAGGCTCCGAGAAGTTCGTGTTCATGTTGTCCACCCGCGCCGGCGGGCTGGGGATCAACCTCACCTCGGCAGATGTCGTCATCATATACGACTCCGACTGGAACCCGCAGATGGACCTGCAGGCGATGGACCGCGCGCATCGTATTGGGCAGATGAAACAAGTGCGCGTATTCAGACTGATCACGGAGAACACGGTGGAAGAGAAGATAGTGGAGCGCGCGGAAGTGAAACTACGCCTAGACAAGCTCGTAATCCAGTCCGGCCGGCTCGTGGACCAGAAGAACCAGCTCAACAAGGACGAAATGCTCAACATGATCAGGCACGGGGCCAACCACGTCTTCTCCTCGAAGGACTCCGAGATCACCGATGAGGATATTGACTCTATTCTAGCCAAAGGAGAGACCAAGACAGAAGAATTGAAACAGAAGTTGGAGAGCCTGGAGGAGTCATCTCTGAGAGCGTTCTCGATGGACACCCCCGGCGCGACCGACTCCGTCTACCAATTCGAAGGCGAGGACTACCGGGAGAAGCAGAAGTCGCACCCGCTGGGCAGCTGGATCGAGCCGCCGAAGCGCGAGCGCAAGGCCAACTACGCGGTGGACGCGTACTTCCGCGAGGCGCTGCGCGTCTCCGAGCCCAAGGCGCCGAAAGTGCAGGCGCCGCGCCCCCCCAAGCAGCCCATCGTGCAGGACTTCCAGTTCTTCCCGCCGAGGCTGTTCGAGCTGCTGGACCAGGAGATCTACCACTACAGAAAGACATTGGGGTACAAAGTGCCGCGTAACCCGGAGCTGGGCCCCGACGCGGCCAAGATACAGAGAGAGGAGCAGAGGAAGATCGACGACGCCGAAGCGCTCACCGAGGAGGAGGTTGCTGAGAAAGAAAACCTTCTCACTCAGGGTTTCACAAACTGGACAAAACGCGACTTCAACCAGTTCATCAAAGCTAATGAGAAGTATGGAAGGGATGACATCGAAAATATTGCAAAAGATGTCGAAGGAAAAACGCCCGAAGAGGTGATGGAATACTCGGCAGTGTTCTGGGAGCGCTGCCACGAGCTGCAGGACGTGGACCGGATCATGGGGCAGATCGAGCGCGGCGAGGCCAAGATACAGCGCCGCGCCTCCATCAAGAAGGCGCTCGACGCCAAGATGGCGCGCTACCGGGCGCCCTTCCACCAGCTCAGGATATCCTACGGGACTAACAAGGGGAAGAACTATGTCGAAGAAGAAGACAGGTTCCTGGTGTGCATGCTGCACAAGCTGGGCTTCGACAAGGAGAACGTGTACGAGGAGCTGCGCGCCGCCGTGCACGCCGCGCCGCAGTTCCGCTTCGACTGGTTCCTCAAGTCGCGCACGGCCGTGGAGCTGCAGCGCAGATGCAACACATTGATCACACTCATCGAAAGAGAGAACCAAGAGCTCGAAGAGAAGGAGCGCGCCGAGAAGAAAAAGAAGAGCGGCAGCGCCAACCAGAACACGCCCGGCGCCGCCAGCAAGGGCGCCGGCGCCGGCAAGCGCAAGGCAGACGCCGCGCAGGACTCCGCGCAGAAACAGAAGAAGAAGAAGAAATGA
番茄潜叶蛾染色质重塑因子ISW的氨基酸序列如SEQ ID NO:2所示：
MSQPDEAMDVGDVEENSNESSSDTTSSKGKEGDFESKIETDRSKRFDFLLKQTEIFSHFMTNAPKTSSPPKAKAGRPKKVKEDLPEQSEDASAADHRHRKTEQEEDEELLAETNAKIKPIFRFEASPPYIKNGEMRDYQVRGLNWMISLYENGINGILADEMGLGKTLQTISLLGYMKNFRNIPGPHIVIVPKSTLTNWMNEFKKWCPSLRAVCLIGDQETRNTFIREVLMPGNWDVCITSYEMIIRERSVFKKFTWRYMVIDEAHRIKNEKSKLSELLREFKSMNRLLLTGTPLQNNLHELWALLNFLLPDVFNSSDDFDSWFNTNAALGDNQLVSRLHAVLRPFLLRRLKSEVEKKLKPKKELKVYVGLSKMQREWYTKVLMKDIDIVNGAGKVEKMRLQNILMQLRKCCNHPYLFDGAEPGPPYTTDEHLVYNCGKLAILDKLLPKLQQQDSRVLIFSQMTRMLDILEDYCLWRQYKYCRLDGQTPHEDRNRQIEEYNAEGSEKFVFMLSTRAGGLGINLTSADVVIIYDSDWNPQMDLQAMDRAHRIGQMKQVRVFRLITENTVEEKIVERAEVKLRLDKLVIQSGRLVDQKNQLNKDEMLNMIRHGANHVFSSKDSEITDEDIDSILAKGETKTEELKQKLESLEESSLRAFSMDTPGATDSVYQFEGEDYREKQKSHPLGSWIEPPKRERKANYAVDAYFREALRVSEPKAPKVQAPRPPKQPIVQDFQFFPPRLFELLDQEIYHYRKTLGYKVPRNPELGPDAAKIQREEQRKIDDAEALTEEEVAEKENLLTQGFTNWTKRDFNQFIKANEKYGRDDIENIAKDVEGKTPEEVMEYSAVFWERCHELQDVDRIMGQIERGEAKIQRRASIKKALDAKMARYRAPFHQLRISYGTNKGKNYVEEEDRFLVCMLHKLGFDKENVYEELRAAVHAAPQFRFDWFLKSRTAVELQRRCNTLITLIERENQELEEKERAEKKKKSGSANQNTPGAASKGAGAGKRKADAAQDSAQKQKKKKK
上述氨基酸序列具有ISWI蛋白典型的结构特征，包含N-末端ATP酶结构域(DEXDc和HELICc)和C-末端HAND-SANT-SLIDE结构域。
本发明提供了上述番茄潜叶蛾染色质重塑因子ISWI的应用。对番茄潜叶蛾进行RNAi，结果显示，取食dsISWI的番茄潜叶蛾卵巢发生畸形、成虫产卵量显著性降低，说明ISWI基因在番茄潜叶蛾生殖发育中起关键作用。基于本发明的番茄潜叶蛾ISWI基因片段合成的dsRNA促使其卵巢畸形和显著降低成虫产卵量，以及显著缩短成虫寿命，可以作为番茄潜叶蛾RNAi防治的分子靶标，对于防治该入侵害虫具有重要的现实意义。
本发明首次从新发重要入侵害虫番茄潜叶蛾中克隆获得染色质重塑因子ISWI基因，上述入侵害虫卵注射基因dsRNA后导致卵巢畸形、产卵量显著降低和成虫寿命显著缩短。所获得的结果为利用RNAi防治新发入侵害虫提供新的途径。
附图说明
图1显示注射ISWI基因dsRNA和dsEGFP条件下雌成虫卵巢的变化情况；
图2不同发育阶段ISWI基因表达谱变化；
图3显示注射ISWI基因dsRNA和dsEGFP条件下ISWI的表达量变化情况；
图4显示注射ISWI基因dsRNA处理对番茄潜叶蛾成虫寿命的变化情况；
图5显示注射ISWI基因dsRNA处理对番茄潜叶蛾成虫产卵量的变化情况；
图6显示注射ISWI基因dsRNA处理对番茄潜叶蛾卵孵化率的变化情况。
具体实施方式
实施例1：番茄潜叶蛾ISWI基因全长cDNA序列克隆
取番茄潜叶蛾幼虫4头放入1.5mL的离心管，液氮冷冻后利用研磨棒将其研成粉末，然后提取RNA，-80℃保存以备用。根据全式金反转录试剂盒(TransScript All-in-OneFirst-Strand cDNA Synthesis SuperMix for PCR)说明，对提取的RNA进行反转录合成cDNA。以cDNA为模板，设计引物，进行PCR扩增。设计引物如表1所示：
表1克隆TaISWI基因全长cDNA的引物序列
利用表1的引物，通过PCR扩增，获得ISWI基因的cDNA序列全长为3078bp，所得的基因具有如SEQ ID NO:1所示的核苷酸序列，该基因编码1025个如SEQ ID NO:2所示的氨基酸序列。克隆获得的基因编码的氨基酸序列的保守结构域分析表明，其具有ISWI蛋白典型的结构特征：包含N-末端ATP酶结构域(DEXDc和HELICc)和C-末端HAND-SANT-SLIDE结构域。
实施例2：分析ISWI基因对番茄潜叶蛾生殖发育的影响
1、合成dsRNA
(1)设计合成加上T7启动子的引物序列：
T7+dsISWI-F：5’-TAATACGACTCACTATAGGGTATGGATGTAGGGGACG-3’
T7+dsISWI-R：5’-TAATACGACTCACTATAGGGCCAGTTGGTTAGGGTTG-3’。
由上海生工生物工程技术服务有限公司合成。
(2)总RNA提取及cDNA合成：同实施例1。
(3)T7引物PCR扩增、产物纯化，纯化所得的PCR产物即为合成dsRNA的模板。使用试剂盒合成和纯化dsRNA，按照试剂盒说明书进行操作。
2、dsRNA注射法
(1)收集番茄潜叶蛾羽化前的老熟雌蛹，排列并粘附在事先贴有透气双面胶的盖玻片表面上。使用Femtojet Express(Eppendorf)显微注射仪，在节间进行注射dsISWI(注射量为0.6μg dsRNA)、dsEGFP和ddH2O(0.1μl)。注射后，将雌蛹单头放置在透气的1.5mL离心管里，一旦羽化，将其与新羽化的野生型雄成虫配对单独放置在塑料盒，并提供足够的新鲜番茄叶供其取食，统计成虫的产卵量和寿命。同时，随机取羽化4天的成虫，解剖其卵巢并观察其形态特征。为检测RNAi干扰效率，将注射72h后的4头番茄潜叶蛾用于表达量的检测，各处理均设置4个生物学重复。如图1所示，显示注射ISWI基因dsRNA和dsEGFP条件下雌成虫卵巢的变化情况，与对照处理相比，注射ISWI基因dsRNA后雌成虫卵巢中成熟卵子的数量明显减少。
(2)通过2-ΔΔCT方法计算基因的相对表达量，结果如图2所示，显示不同发育阶段ISWI基因表达谱变化，ISWI基因在卵和蛹这两个发育阶段的表达量最高。图3显示注射ISWI基因dsRNA和dsEGFP条件下ISWI的表达量变化情况，结果表明注射dsISWI的dsRNA可以显著敲低ISWI基因的表达。
利用SAS 9.4统计软件分析注射不同溶液后番茄潜叶蛾成虫的产卵量(图5)和雌虫寿命(图4)，结果显示，饲喂dsISWI基因dsRNA的番茄潜叶蛾的总产卵量和寿命均显著低于注射dsEGFP组(P<0.05)。图6显示注射ISWI基因dsRNA后番茄潜叶蛾卵孵化率的变化情况，与对照相比，注射dsISWI可以显著抑制番茄潜叶蛾卵的孵化。
同时在NCBI(http://blast.ncbi.nlm.nih.gov/)BLAST显示，所注射的靶序列片度为ISWI基因特有的序列，由此确保了干扰效果为番茄潜叶蛾的ISWI基因所产生，因此，说明本发明的ISWI基因在番茄潜叶蛾发育生殖过程中起着关键作用。
本发明从番茄潜叶蛾中克隆得到ISWI基因的全长cDNA，荧光定量PCR显示注射靶基因双链RNA后ISWI基因的表达量显著降低；最后通过注射ISWI基因dsRNA，进而导致番茄潜叶蛾雌虫产卵量和寿命显著性降低。根据本发明的具体实施方式，试验结果明确了ISWI基因在南美番茄潜叶蛾发育生殖过程中起着关键作用。本发明为利用RNAi有效防治番茄潜叶蛾的研究奠定了基础，为日后通过染色质重塑因子的相关研究降低番茄潜叶蛾的危害提供了方法依据。
以上实施例仅用于解释本申请的技术方案，不限制本申请的保护范围。
序列表
<110> 中国农业科学院植物保护研究所
<120> 一种番茄潜叶蛾染色质重塑因子ISWI及其编码基因和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3078
<212> DNA
<213> 番茄潜叶蛾(Tuta absoluta)
<400> 1
atgtcacaac ctgatgaagc tatggatgta ggggacgtgg aagaaaactc taatgagtcg 60
tccagcgata ccacgtcctc aaagggtaaa gaaggagact tcgaaagtaa aattgagacc 120
gatcgttcca agcgttttga ttttttgttg aaacaaactg aaatattctc gcatttcatg 180
accaatgcgc ctaaaaccag tagtcctcct aaggctaaag ccggtcgacc taaaaaggtg 240
aaagaagatc tgcctgagca gtctgaggat gcttctgcag cagaccatcg gcaccgcaag 300
actgaacagg aagaagatga ggaactcctt gctgagacaa acgctaagat taagccaata 360
tttcgctttg aagcatcacc tccttacatc aagaatggag aaatgagaga ttatcaagtg 420
agaggtctca actggatgat atctctctat gagaatggta ttaatggtat cttggctgat 480
gagatgggtc tgggtaaaac attacaaacc atttctcttc ttggctacat gaaaaatttc 540
agaaatatcc ctggtccaca catagtaatt gtgccaaaat caaccctaac caactggatg 600
aatgagttca aaaaatggtg tccttcccta agagcagtgt gtcttattgg agatcaggaa 660
acaaggaaca cattcatccg tgaagtactg atgcctggca actgggatgt gtgcatcacc 720
tcttacgaga tgattatccg tgagagatca gtatttaaga agttcacttg gcgctacatg 780
gtgattgacg aagcccatcg aatcaagaat gagaagtcaa agttgtcaga gcttctccgg 840
gagttcaaga gtatgaaccg gctgctgctc actggcacac cgttgcaaaa caatcttcac 900
gagctgtggg cacttcttaa cttcttgtta ccagatgtat tcaacagctc tgatgatttt 960
gactcttggt tcaacaccaa tgcagctctc ggtgataatc agctggtgtc ccggttgcat 1020
gctgtgttga gaccgttctt actcagacgt ctcaaatctg aagtagaaaa gaaattgaaa 1080
cccaaaaagg aactcaaagt ttatgttgga ttgagtaaga tgcaaaggga gtggtatact 1140
aaagtactca tgaaggatat tgatattgta aacggcgcgg gcaaagtcga aaagatgcgt 1200
ctccagaaca tcctgatgca gctccgcaag tgctgcaacc acccgtacct gttcgacggc 1260
gcggagcccg gcccgcccta caccacggac gagcatctcg tctacaactg cgggaaactc 1320
gccatactcg acaaactgct gcccaagctg cagcagcagg actccagggt gctcatcttc 1380
tcgcagatga ccaggatgct ggatattctg gaggattatt gcctgtggag gcagtacaag 1440
tactgccgct tagacggtca gaccccacat gaagaccgca accgacagat cgaggagtat 1500
aacgcggaag gctccgagaa gttcgtgttc atgttgtcca cccgcgccgg cgggctgggg 1560
atcaacctca cctcggcaga tgtcgtcatc atatacgact ccgactggaa cccgcagatg 1620
gacctgcagg cgatggaccg cgcgcatcgt attgggcaga tgaaacaagt gcgcgtattc 1680
agactgatca cggagaacac ggtggaagag aagatagtgg agcgcgcgga agtgaaacta 1740
cgcctagaca agctcgtaat ccagtccggc cggctcgtgg accagaagaa ccagctcaac 1800
aaggacgaaa tgctcaacat gatcaggcac ggggccaacc acgtcttctc ctcgaaggac 1860
tccgagatca ccgatgagga tattgactct attctagcca aaggagagac caagacagaa 1920
gaattgaaac agaagttgga gagcctggag gagtcatctc tgagagcgtt ctcgatggac 1980
acccccggcg cgaccgactc cgtctaccaa ttcgaaggcg aggactaccg ggagaagcag 2040
aagtcgcacc cgctgggcag ctggatcgag ccgccgaagc gcgagcgcaa ggccaactac 2100
gcggtggacg cgtacttccg cgaggcgctg cgcgtctccg agcccaaggc gccgaaagtg 2160
caggcgccgc gcccccccaa gcagcccatc gtgcaggact tccagttctt cccgccgagg 2220
ctgttcgagc tgctggacca ggagatctac cactacagaa agacattggg gtacaaagtg 2280
ccgcgtaacc cggagctggg ccccgacgcg gccaagatac agagagagga gcagaggaag 2340
atcgacgacg ccgaagcgct caccgaggag gaggttgctg agaaagaaaa ccttctcact 2400
cagggtttca caaactggac aaaacgcgac ttcaaccagt tcatcaaagc taatgagaag 2460
tatggaaggg atgacatcga aaatattgca aaagatgtcg aaggaaaaac gcccgaagag 2520
gtgatggaat actcggcagt gttctgggag cgctgccacg agctgcagga cgtggaccgg 2580
atcatggggc agatcgagcg cggcgaggcc aagatacagc gccgcgcctc catcaagaag 2640
gcgctcgacg ccaagatggc gcgctaccgg gcgcccttcc accagctcag gatatcctac 2700
gggactaaca aggggaagaa ctatgtcgaa gaagaagaca ggttcctggt gtgcatgctg 2760
cacaagctgg gcttcgacaa ggagaacgtg tacgaggagc tgcgcgccgc cgtgcacgcc 2820
gcgccgcagt tccgcttcga ctggttcctc aagtcgcgca cggccgtgga gctgcagcgc 2880
agatgcaaca cattgatcac actcatcgaa agagagaacc aagagctcga agagaaggag 2940
cgcgccgaga agaaaaagaa gagcggcagc gccaaccaga acacgcccgg cgccgccagc 3000
aagggcgccg gcgccggcaa gcgcaaggca gacgccgcgc aggactccgc gcagaaacag 3060
aagaagaaga agaaatga 3078
<210> 2
<211> 1025
<212> PRT
<213> 番茄潜叶蛾(Tuta absoluta)
<400> 2
Met Ser Gln Pro Asp Glu Ala Met Asp Val Gly Asp Val Glu Glu Asn
1 5 10 15
Ser Asn Glu Ser Ser Ser Asp Thr Thr Ser Ser Lys Gly Lys Glu Gly
20 25 30
Asp Phe Glu Ser Lys Ile Glu Thr Asp Arg Ser Lys Arg Phe Asp Phe
35 40 45
Leu Leu Lys Gln Thr Glu Ile Phe Ser His Phe Met Thr Asn Ala Pro
50 55 60
Lys Thr Ser Ser Pro Pro Lys Ala Lys Ala Gly Arg Pro Lys Lys Val
65 70 75 80
Lys Glu Asp Leu Pro Glu Gln Ser Glu Asp Ala Ser Ala Ala Asp His
85 90 95
Arg His Arg Lys Thr Glu Gln Glu Glu Asp Glu Glu Leu Leu Ala Glu
100 105 110
Thr Asn Ala Lys Ile Lys Pro Ile Phe Arg Phe Glu Ala Ser Pro Pro
115 120 125
Tyr Ile Lys Asn Gly Glu Met Arg Asp Tyr Gln Val Arg Gly Leu Asn
130 135 140
Trp Met Ile Ser Leu Tyr Glu Asn Gly Ile Asn Gly Ile Leu Ala Asp
145 150 155 160
Glu Met Gly Leu Gly Lys Thr Leu Gln Thr Ile Ser Leu Leu Gly Tyr
165 170 175
Met Lys Asn Phe Arg Asn Ile Pro Gly Pro His Ile Val Ile Val Pro
180 185 190
Lys Ser Thr Leu Thr Asn Trp Met Asn Glu Phe Lys Lys Trp Cys Pro
195 200 205
Ser Leu Arg Ala Val Cys Leu Ile Gly Asp Gln Glu Thr Arg Asn Thr
210 215 220
Phe Ile Arg Glu Val Leu Met Pro Gly Asn Trp Asp Val Cys Ile Thr
225 230 235 240
Ser Tyr Glu Met Ile Ile Arg Glu Arg Ser Val Phe Lys Lys Phe Thr
245 250 255
Trp Arg Tyr Met Val Ile Asp Glu Ala His Arg Ile Lys Asn Glu Lys
260 265 270
Ser Lys Leu Ser Glu Leu Leu Arg Glu Phe Lys Ser Met Asn Arg Leu
275 280 285
Leu Leu Thr Gly Thr Pro Leu Gln Asn Asn Leu His Glu Leu Trp Ala
290 295 300
Leu Leu Asn Phe Leu Leu Pro Asp Val Phe Asn Ser Ser Asp Asp Phe
305 310 315 320
Asp Ser Trp Phe Asn Thr Asn Ala Ala Leu Gly Asp Asn Gln Leu Val
325 330 335
Ser Arg Leu His Ala Val Leu Arg Pro Phe Leu Leu Arg Arg Leu Lys
340 345 350
Ser Glu Val Glu Lys Lys Leu Lys Pro Lys Lys Glu Leu Lys Val Tyr
355 360 365
Val Gly Leu Ser Lys Met Gln Arg Glu Trp Tyr Thr Lys Val Leu Met
370 375 380
Lys Asp Ile Asp Ile Val Asn Gly Ala Gly Lys Val Glu Lys Met Arg
385 390 395 400
Leu Gln Asn Ile Leu Met Gln Leu Arg Lys Cys Cys Asn His Pro Tyr
405 410 415
Leu Phe Asp Gly Ala Glu Pro Gly Pro Pro Tyr Thr Thr Asp Glu His
420 425 430
Leu Val Tyr Asn Cys Gly Lys Leu Ala Ile Leu Asp Lys Leu Leu Pro
435 440 445
Lys Leu Gln Gln Gln Asp Ser Arg Val Leu Ile Phe Ser Gln Met Thr
450 455 460
Arg Met Leu Asp Ile Leu Glu Asp Tyr Cys Leu Trp Arg Gln Tyr Lys
465 470 475 480
Tyr Cys Arg Leu Asp Gly Gln Thr Pro His Glu Asp Arg Asn Arg Gln
485 490 495
Ile Glu Glu Tyr Asn Ala Glu Gly Ser Glu Lys Phe Val Phe Met Leu
500 505 510
Ser Thr Arg Ala Gly Gly Leu Gly Ile Asn Leu Thr Ser Ala Asp Val
515 520 525
Val Ile Ile Tyr Asp Ser Asp Trp Asn Pro Gln Met Asp Leu Gln Ala
530 535 540
Met Asp Arg Ala His Arg Ile Gly Gln Met Lys Gln Val Arg Val Phe
545 550 555 560
Arg Leu Ile Thr Glu Asn Thr Val Glu Glu Lys Ile Val Glu Arg Ala
565 570 575
Glu Val Lys Leu Arg Leu Asp Lys Leu Val Ile Gln Ser Gly Arg Leu
580 585 590
Val Asp Gln Lys Asn Gln Leu Asn Lys Asp Glu Met Leu Asn Met Ile
595 600 605
Arg His Gly Ala Asn His Val Phe Ser Ser Lys Asp Ser Glu Ile Thr
610 615 620
Asp Glu Asp Ile Asp Ser Ile Leu Ala Lys Gly Glu Thr Lys Thr Glu
625 630 635 640
Glu Leu Lys Gln Lys Leu Glu Ser Leu Glu Glu Ser Ser Leu Arg Ala
645 650 655
Phe Ser Met Asp Thr Pro Gly Ala Thr Asp Ser Val Tyr Gln Phe Glu
660 665 670
Gly Glu Asp Tyr Arg Glu Lys Gln Lys Ser His Pro Leu Gly Ser Trp
675 680 685
Ile Glu Pro Pro Lys Arg Glu Arg Lys Ala Asn Tyr Ala Val Asp Ala
690 695 700
Tyr Phe Arg Glu Ala Leu Arg Val Ser Glu Pro Lys Ala Pro Lys Val
705 710 715 720
Gln Ala Pro Arg Pro Pro Lys Gln Pro Ile Val Gln Asp Phe Gln Phe
725 730 735
Phe Pro Pro Arg Leu Phe Glu Leu Leu Asp Gln Glu Ile Tyr His Tyr
740 745 750
Arg Lys Thr Leu Gly Tyr Lys Val Pro Arg Asn Pro Glu Leu Gly Pro
755 760 765
Asp Ala Ala Lys Ile Gln Arg Glu Glu Gln Arg Lys Ile Asp Asp Ala
770 775 780
Glu Ala Leu Thr Glu Glu Glu Val Ala Glu Lys Glu Asn Leu Leu Thr
785 790 795 800
Gln Gly Phe Thr Asn Trp Thr Lys Arg Asp Phe Asn Gln Phe Ile Lys
805 810 815
Ala Asn Glu Lys Tyr Gly Arg Asp Asp Ile Glu Asn Ile Ala Lys Asp
820 825 830
Val Glu Gly Lys Thr Pro Glu Glu Val Met Glu Tyr Ser Ala Val Phe
835 840 845
Trp Glu Arg Cys His Glu Leu Gln Asp Val Asp Arg Ile Met Gly Gln
850 855 860
Ile Glu Arg Gly Glu Ala Lys Ile Gln Arg Arg Ala Ser Ile Lys Lys
865 870 875 880
Ala Leu Asp Ala Lys Met Ala Arg Tyr Arg Ala Pro Phe His Gln Leu
885 890 895
Arg Ile Ser Tyr Gly Thr Asn Lys Gly Lys Asn Tyr Val Glu Glu Glu
900 905 910
Asp Arg Phe Leu Val Cys Met Leu His Lys Leu Gly Phe Asp Lys Glu
915 920 925
Asn Val Tyr Glu Glu Leu Arg Ala Ala Val His Ala Ala Pro Gln Phe
930 935 940
Arg Phe Asp Trp Phe Leu Lys Ser Arg Thr Ala Val Glu Leu Gln Arg
945 950 955 960
Arg Cys Asn Thr Leu Ile Thr Leu Ile Glu Arg Glu Asn Gln Glu Leu
965 970 975
Glu Glu Lys Glu Arg Ala Glu Lys Lys Lys Lys Ser Gly Ser Ala Asn
980 985 990
Gln Asn Thr Pro Gly Ala Ala Ser Lys Gly Ala Gly Ala Gly Lys Arg
995 1000 1005
Lys Ala Asp Ala Ala Gln Asp Ser Ala Gln Lys Gln Lys Lys Lys Lys
1010 1015 1020
Lys
1025

Claims (6)

1.番茄潜叶蛾染色质重塑因子ISWI，其特征在于，其氨基酸序列如SEQ ID NO：2所示。
2.番茄潜叶蛾染色质重塑因子基因，其特征在于，所述基因编码权利要求1所述番茄潜叶蛾染色质重塑因子ISWI。
3.根据权利要求2所述的番茄潜叶蛾染色质重塑因子基因，其特征在于，所述番茄潜叶蛾染色质重塑因子基因的核苷酸序列如SEQ ID NO:1所示。
4.包含权利要求2所述的番茄潜叶蛾染色质重塑因子基因的重组表达载体。
5.包含权利要求2所述的番茄潜叶蛾染色质重塑因子基因的重组菌株。
6.权利要求2所述的番茄潜叶蛾染色质重塑因子基因作为干扰以下性状的RNAi干扰靶点的应用，通过给番茄潜叶蛾饲喂权利要求2所述的番茄潜叶蛾染色质重塑因子基因的dSRNA片段，
（1）降低番茄潜叶蛾雌性成虫的产卵量；
（2）缩短番茄潜叶蛾雌性成虫的寿命，
所述番茄潜叶蛾染色质重塑因子基因的dSRNA片段通过以下引物获得：
T7+dsISWI-F：5’-TAATACGACTCACTATAGGGTATGGATGTAGGGGACG-3’
T7+dsISWI-R：5’-TAATACGACTCACTATAGGGCCAGTTGGTTAGGGTTG-3’。
CN202210050393.1A 2022-01-17 2022-01-17 一种番茄潜叶蛾染色质重塑因子iswi及其编码基因和应用 Active CN114437192B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210050393.1A CN114437192B (zh) 2022-01-17 2022-01-17 一种番茄潜叶蛾染色质重塑因子iswi及其编码基因和应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210050393.1A CN114437192B (zh) 2022-01-17 2022-01-17 一种番茄潜叶蛾染色质重塑因子iswi及其编码基因和应用

Publications (2)

Publication Number Publication Date
CN114437192A CN114437192A (zh) 2022-05-06
CN114437192B true CN114437192B (zh) 2023-09-29

Family

ID=81367038

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210050393.1A Active CN114437192B (zh) 2022-01-17 2022-01-17 一种番茄潜叶蛾染色质重塑因子iswi及其编码基因和应用

Country Status (1)

Country Link
CN (1) CN114437192B (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107109426A (zh) * 2014-12-16 2017-08-29 美国陶氏益农公司 用于控制半翅目害虫的染色质重塑基因的亲代RNAi抑制
CN107208098A (zh) * 2014-12-16 2017-09-26 美国陶氏益农公司 用于控制鞘翅目害虫染色质重塑基因亲代rnai抑制
CN111763253A (zh) * 2020-07-29 2020-10-13 中国农业科学院植物保护研究所 染色质重塑因子iswi、编码基因和在烟粉虱med隐种温度耐受性中的作用
CN111763252A (zh) * 2020-07-29 2020-10-13 中国农业科学院植物保护研究所 烟粉虱MED隐种染色质重塑因子Btbrm2及其编码基因和应用
CN111925429A (zh) * 2020-07-29 2020-11-13 中国农业科学院植物保护研究所 烟粉虱MED隐种染色质重塑因子Btbrm1及其编码基因应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107109426A (zh) * 2014-12-16 2017-08-29 美国陶氏益农公司 用于控制半翅目害虫的染色质重塑基因的亲代RNAi抑制
CN107208098A (zh) * 2014-12-16 2017-09-26 美国陶氏益农公司 用于控制鞘翅目害虫染色质重塑基因亲代rnai抑制
CN111763253A (zh) * 2020-07-29 2020-10-13 中国农业科学院植物保护研究所 染色质重塑因子iswi、编码基因和在烟粉虱med隐种温度耐受性中的作用
CN111763252A (zh) * 2020-07-29 2020-10-13 中国农业科学院植物保护研究所 烟粉虱MED隐种染色质重塑因子Btbrm2及其编码基因和应用
CN111925429A (zh) * 2020-07-29 2020-11-13 中国农业科学院植物保护研究所 烟粉虱MED隐种染色质重塑因子Btbrm1及其编码基因应用

Also Published As

Publication number Publication date
CN114437192A (zh) 2022-05-06

Similar Documents

Publication Publication Date Title
Wang et al. Identification and characterization of transforming growth factor β-activated kinase 1 from Litopenaeus vannamei involved in anti-bacterial host defense
CN104593391B (zh) 褐飞虱生存及生长发育相关的NlPIK3R1基因、编码蛋白及其应用
Yue et al. Development of a novel‐type transgenic cotton plant for control of cotton bollworm
Zhang et al. Ectopic expression of a LEA protein gene TsLEA1 from Thellungiella salsuginea confers salt-tolerance in yeast and Arabidopsis
CN111944824B (zh) 美国白蛾速激肽受体基因及dsRNA和防治美国白蛾中应用
Zhang et al. A transferrin gene associated with development and 2‐tridecanone tolerance in H elicoverpa armigera
Ursic Bedoya et al. Molecular cloning and transcriptional activation of lysozyme‐encoding cDNAs in the mosquito Aedes aegypti
CN114437192B (zh) 一种番茄潜叶蛾染色质重塑因子iswi及其编码基因和应用
US7473769B2 (en) Insect desiccation resistance genes and uses thereof
CN111763253B (zh) 染色质重塑因子iswi、编码基因和在烟粉虱med隐种温度耐受性中的作用
Zou et al. Molecular cloning and characterization of two heat shock proteins in Thitarodes pui (Lepidoptera: Hepialidae)
CN108588082A (zh) 烟粉虱MED隐种高温耐受相关基因BtDnmt3及其应用
CN101525617A (zh) 中华绒螯蟹Crustin-1基因及体外重组表达
CN114380899B (zh) 番茄潜叶蛾染色质重塑因子brm及其编码基因和应用
CN112695045B (zh) 一种鳞翅目昆虫Hpx12基因及应用
Wang et al. Molecular characterization, expression analysis and RNAi knock-down of elongation factor 1α and 1γ from Nilaparvata lugens and its yeast-like symbiont
CN113150099B (zh) 南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1基因及其应用
Tang et al. MdRDH1, a HSP67B2-like rhodanese homologue plays a positive role in maintaining redox balance in Musca domestica
CN107506616B (zh) 象耳豆根转录组数据库、融合蛋白、浸泡体系、沉默体系
Zhang et al. Up‐regulation of lysozyme gene expression during metamorphosis and immune challenge of the cotton bollworm, Helicoverpa armigera
Zhou et al. A QM protein from Bombyx mori negatively regulates prophenoloxidase activation and melanization by interacting with Jun protein
CN111848768B (zh) BtISWI基因及其调控烟粉虱MEAM1隐种温度偏好性的应用
CN112175967B (zh) 一种增强植物抵抗鳞翅目害虫的pen1基因及其应用
CN111073895B (zh) 一种增强植物抵抗鳞翅目害虫的基因apx1及其应用
Zhou et al. Molecular analysis of the QM gene from Penaeus monodon and its expression on the different ovarian stages of development

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