CN114380899B - 番茄潜叶蛾染色质重塑因子brm及其编码基因和应用 - Google Patents

番茄潜叶蛾染色质重塑因子brm及其编码基因和应用 Download PDF

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CN114380899B
CN114380899B CN202210050927.0A CN202210050927A CN114380899B CN 114380899 B CN114380899 B CN 114380899B CN 202210050927 A CN202210050927 A CN 202210050927A CN 114380899 B CN114380899 B CN 114380899B
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吕志创
冀顺霞
吴强文
王晓迪
毕思言
唐艳红
刘万学
张桂芬
万方浩
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Abstract

本发明涉及昆虫防治领域，具体涉及番茄潜叶蛾染色质重塑因子BRM及其编码基因和应用。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BRM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

番茄潜叶蛾染色质重塑因子BRM及其编码基因和应用
技术领域
本发明涉及昆虫防治领域，具体涉及番茄潜叶蛾染色质重塑因子BRM及其编码基因和应用。
背景技术
番茄潜叶蛾Tuta absoluta原产于南美洲的秘鲁，严重危害多种茄科作物，是最具毁灭性的世界检疫性害虫，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，该害虫可导致番茄田、茄子田和马铃薯田的危害达到90%以上。目前防治该虫，使用化学农药是最主要措施只要，随着化学农药的持续使用，促使其抗药性不断增强，并迅速的对各种药剂靶标产生抗性。
RNA干扰（RNAi，RNA interference）广泛存在于生物界，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。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，将DNA包装到染色质对于其基因组的组织和表达至关重要，染色质构型整体和局部的动态改变时基因功能调控的重要因素。细胞内进化产生一系列特异的染色质重塑因子，进而保证染色质中的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BRM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。BRM沉默出现卵巢畸形、产卵量和卵孵化率降低、以及雌成虫寿命缩短等。因此，本发明基于RNA干扰技术，明确了BRM基因在番茄潜叶蛾发育繁殖中的功能作用，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BRM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BRM，其cDNA全长核苷酸序列如SEQ ID NO:1所示：
ATGCGCCGCCTGATGGCGGAGGACGAGGAGGGTTACCGGAAGCTCATCGACCAGAAGAAGGACAAGCGGCTGGCGTTCCTGTTGTCGCAGACGGACGAGTACATCGCCAGCCTCACCGAGATGGTGAAGCAGCACAAACAGGAGCAGCGCAAGAAACAAGTCGAGGAGGAGAAACGGAAGCGGAAATCCCGGAAGAAGAAGTTGCTGGAGGGCGGGGAGATCGACGCGATGGACGACAGCTCGCAGACCTCCGACTCGCGTGTCACCGTCATGGACCCCAAGACGGGCGAAGTGTTGAAGGGCGAGGAAGCCCCGCTGCTGTCTCAGCTGAAGGGCTGGATGGAGCAGCACCCCGGCTGGGAGGTGGTGTCGGACTCCGACGACTCGGGCGACGACAGCCAGGACGACGACGGACACTACAAGAGACACAAACACAGGGACGACAAGAATGAGAACAGGGAGAAGACCGAGGAGGAGAAGGCGCGCGAGATGATCAAGAAAGCCAAGGTGGAGGACGACGAGTACAAGACAGAGGAGCAGACATACTACAGCATTGCTCATACGGTCCACGAATCCGTCACAGAACAAGCCAGCATTTTGGTCAACGGAAAACTCAAGGAATACCAGATCAAGGGTCTGGAATGGCTGGTGTCTTTGTTCAACAACAACCTGAACGGTATCCTGGCGGACGAGATGGGTCTCGGCAAGACCATCCAGACGATAGCGCTGGTCACATACCTCATGGAGAAGAAGAAAGTCAACGGACCATTCCTCATCATTGTACCGCTCAGTACGCTATCAAACTGGGTGCTGGAGTTCGAGAAGTGGGCGCCGACCGTGTGCGTAGTGTCGTACAAGGGCTCGCCCGCGTCGCGCCGCCTCGTGCAGAACCAGATGAGGTCCACCAAGTTCAACGTGCTGCTCACCACCTACGAGTACGTCATCAAGGACAAGGGAGTGCTCGCTAAGGTGCAATGGAAGTACATGATAATCGACGAGGGCCACCGCATGAAGAACCACCACTGCAAGCTGACGCAGGTGCTGAACACGCACTACATAGCGCCGCACCGCCTGCTGCTGACCGGCACGCCGCTGCAGAACAAGCTGCCCGAACTCTGGGCGCTGCTGAACTTCCTCCTGCCGTCCATATTCAAGAGTTGCTCCACCTTCGAACAGTGGTTCAATGCGCCCTTCGCTACCACTGGAGAAAAGGTGGAACTAAACGAGGAAGAAACGATCCTGATCATCCGTCGTCTACACAAAGTGCTGCGTCCGTTCTTACTGCGGCGCCTGAAGAAGGAAGTAGAGAGCCAGCTGCCCGACAAAGTGGAGTACATCATCAAGTGCGACATGAGCGGGCTGCAGCGAGTGCTCTACAAACACATGCAGTCGAAGGGCGTGCTGCTGACGGACGGCTCGGAGAAGGGCACGAAGGGCAAGGGCGGGGCGAAGGCGCTCATGAACACCATCGTGCAGCTGCGCAAGCTCTGCAACCATCCCTTCATGTTCCAGCACATCGAGGAGAAGTTCTGCGACCACGTCGGCTCTGGCAGCACCGTCGTTTCTGGTCCGGACCTGTACCGCGTGTCGGGCAAGTTCGAGCTGCTGGACCGCATCCTGCCCAAGCTGAAGCGCACCGGCCACCGCGTGCTCGTGTTCTGCCAGATGACGCAGTGCATGACCATCATCGAGGACTACCTCTCCTGGAGGGGCTTCCAATACTTGCGGCTGGACGGTATGACGAAGGCGGAGGACCGTGGCGAGCTGCTCAAGAAGTTCAACTGCAAGGACTCGGAATATTTCCTCTTCTTGCTGTCTACGAGAGCTGGTGGACTTGGACTCAATCTCCAGTCGGCTGACACTGTCATCATCTTCGATTCCGATTGGAATCCGCATCAGGATCTGCAAGCGCAAGACCGCGCGCATCGTATCGGACAACGTAACGAAGTGCGCGTGTTGCGACTGATGACCGTCAACTCCGTCGAGGAACGGATTCTTGCCGCCGCCAGGTACAAATTGAACATGGACGAGAAGGTTATCCAAGCCGGTATGTTCGATCAAAAGTCCACCGGCTCAGAGCGACAGCAGTTCCTGCAGAGCATTCTGCATCAAGACGGCGACGATGAAGAAGAAGAAAATGAACTCCCGGACGACGACCTCATCAACGAGATGATCGCGCGCTCGGAGGAGGAGCTCGAGATCTTCAAACAGATCGACATCGAGCGGAAGAAAACCGAAACCACCTCCCGTCTCATAGAGGAGTGCGAGCTGCCCGACTGGCTCACCAAGAACGAGGACGAAGTTGTGTGCAACAAGGGCCAAGGGTGGGGCAACTTCCTGGACGCTGAGGAGACGTTCGGGCGCGGCTCCCGGCAGCGCAAAGAGGTTGACTACACGGACTCGCTCACGGAGAAGGAGTGGCTCAAGGCCATCGACGACGAGTTCGACGACGAGGAGGAGGAGGACGACGACGACGAAGTGCTCGACAAGAAGCGCAAGAAGGGCCGCAAGCGACGCCGCCAAGAAGAGTCGGATGAGGAAGAAGTGGCCAGCTCATCTAAGAAGAAGAATAAGACGGAAGCCAACCTACTGAAGAAAAGGCTCAAGAGCATTATGAAGAAAGTCGTGGACTACGCTGACGAGGGCGGTAGAGTCCTGTCGGAGCCGTTCATGAAGCTGCCGTCGCGGCGCGAGCTGCCCGACTACTACGACGTCATCAAGAAACCGCTCGATATCAAGAAAATCATGACGCGCATCGAAGACGGGAAGTACAACGACATTACGGACTTGGAGCGCGATTTCTTCACTCTGTGTCAGAACGCACAAACCTACAACGAGGAGGCGTCGCTCATCTACGCGGACTCCGTGCGACTGAGGAATGTTTTCATTGAGTTCCGACGCAACTACGAGGCGGGCAACAATTCCGACAACTCCGACGATGACAAAGATAAAGAAGAAGAAGACTCGGACGGCGAGTCGAACCGCTCGGTCAAGATGAAGATCAAGCTGAAAGGCAAAGGCAAGGGCACGCCCTCGAGGAAAAAGAAACAAAAGAAGTATATCTCCGACGACGAGGACTATGAGGAGGATTGA
番茄潜叶蛾染色质重塑因子BRM编码氨基酸序列如SEQ ID NO:2所示：
MRRLMAEDEEGYRKLIDQKKDKRLAFLLSQTDEYIASLTEMVKQHKQEQRKKQVEEEKRKRKSRKKKLLEGGEIDAMDDSSQTSDSRVTVMDPKTGEVLKGEEAPLLSQLKGWMEQHPGWEVVSDSDDSGDDSQDDDGHYKRHKHRDDKNENREKTEEEKAREMIKKAKVEDDEYKTEEQTYYSIAHTVHESVTEQASILVNGKLKEYQIKGLEWLVSLFNNNLNGILADEMGLGKTIQTIALVTYLMEKKKVNGPFLIIVPLSTLSNWVLEFEKWAPTVCVVSYKGSPASRRLVQNQMRSTKFNVLLTTYEYVIKDKGVLAKVQWKYMIIDEGHRMKNHHCKLTQVLNTHYIAPHRLLLTGTPLQNKLPELWALLNFLLPSIFKSCSTFEQWFNAPFATTGEKVELNEEETILIIRRLHKVLRPFLLRRLKKEVESQLPDKVEYIIKCDMSGLQRVLYKHMQSKGVLLTDGSEKGTKGKGGAKALMNTIVQLRKLCNHPFMFQHIEEKFCDHVGSGSTVVSGPDLYRVSGKFELLDRILPKLKRTGHRVLVFCQMTQCMTIIEDYLSWRGFQYLRLDGMTKAEDRGELLKKFNCKDSEYFLFLLSTRAGGLGLNLQSADTVIIFDSDWNPHQDLQAQDRAHRIGQRNEVRVLRLMTVNSVEERILAAARYKLNMDEKVIQAGMFDQKSTGSERQQFLQSILHQDGDDEEEENELPDDDLINEMIARSEEELEIFKQIDIERKKTETTSRLIEECELPDWLTKNEDEVVCNKGQGWGNFLDAEETFGRGSRQRKEVDYTDSLTEKEWLKAIDDEFDDEEEEDDDDEVLDKKRKKGRKRRRQEESDEEEVASSSKKKNKTEANLLKKRLKSIMKKVVDYADEGGRVLSEPFMKLPSRRELPDYYDVIKKPLDIKKIMTRIEDGKYNDITDLERDFFTLCQNAQTYNEEASLIYADSVRLRNVFIEFRRNYEAGNNSDNSDDDKDKEEEDSDGESNRSVKMKIKLKGKGKGTPSRKKKQKKYISDDEDYEED。
上述氨基酸序列具有BRM蛋白典型的结构特征，包含BRK结构域、ATP酶结构域（DEXDc和HELICc）、SnAC结构域和BROMO结构域。
本发明提供了上述番茄潜叶蛾染色质重塑因子BRM的应用。对番茄潜叶蛾进行RNAi，结果显示，取食dsIBRM的番茄潜叶蛾出现卵巢畸形、产卵量和卵孵化率降低、以及雌成虫寿命缩短，说明BRM基因在番茄潜叶蛾发育繁殖中起关键作用。本发明番茄潜叶蛾BRM基因片段合成的dsRNA促使其卵巢畸形和成虫产卵量显著降低，以及成虫寿命显著缩短，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BRM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BRM基因dsRNA和dsEGFP条件下雌成虫卵巢的变化情况；
图2 显示不同发育阶段BRM基因表达谱变化；
图3 显示注射BRM基因dsRNA和dsEGFP条件下ISWI的表达量变化情况；
图4 显示注射BRM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BRM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BRM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 BRM基因全长cDNA序列克隆
取番茄潜叶蛾幼虫4头放入1.5 mL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TaBRM基因全长cDNA的引物序列
引物名称 引物序列 (5'-3')
BRM-F1 AACCATCACGCTAACGC
BRM-R1 GCTGGAACATGAAGGGAT
BRM-F2 ATCATCAAGTGCGACAT
BRM-R2 CCGACAGGACTCTACCG
BRM-F3 CGGAAGCCAACCTACT
BRM-R3 CCTGCCCTTTCACTAA
利用表1引物，通过PCR扩增，获得BRM基因的cDNA序列全长为3093bp，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1030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BRM蛋白典型的结构特征：包含BRK结构域、ATP酶结构域（DEXDc和HELICc）、SnAC结构域和BROMO结构域。
实施例2 分析BRM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+dsBRM-F：5’- TAATACGACTCACTATAGGGGAACCATCACGCTAACG-3’
T7+dsBRM-R：5’- TAATACGACTCACTATAGGGGGATTCGTGGACCGTA-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dsIBRM（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。
（2）图1 显示注射BRM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BRM基因dsRNA后雌成虫卵巢中成熟卵子的数量明显减少。图2 显示不同发育阶段BRM基因表达谱变化，通过2-ΔΔCT方法计算基因的相对表达量，结果如图2所示，BRM基因在卵中的表达量最高。图3 显示注射dsBRM和dsEGFP后BRM基因的表达情况，结果表明注射dsBRM可以显著敲低BRM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dsBRM基因dsRNA 的番茄潜叶蛾的产卵量和寿命显著低于注射dsEGFP组（P<0.05）。
图6 显示注射BRM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dsBRM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BRM基因特有的序列，由此确保了干扰效果为番茄潜叶蛾的BRM基因所产生，因此，本发明说明BRM基因在番茄潜叶蛾发育繁殖过程中起着关键作用。
本发明从番茄潜叶蛾中克隆得到BRM基因的全长cDNA，荧光定量PCR显示注射靶基因双链RNA后BRM基因的表达量显著降低；最后通过注射BRM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BRM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。
上述实施例仅用于解释本申请的技术方案，不限定本申请的保护范围。
序列表
<110> 中国农业科学院植物保护研究所
<120> 番茄潜叶蛾染色质重塑因子BRM及其编码基因和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3093
<212> DNA
<213> 番茄潜叶蛾(Tuta absoluta)
<400> 1
atgcgccgcc tgatggcgga ggacgaggag ggttaccgga agctcatcga ccagaagaag 60
gacaagcggc tggcgttcct gttgtcgcag acggacgagt acatcgccag cctcaccgag 120
atggtgaagc agcacaaaca ggagcagcgc aagaaacaag tcgaggagga gaaacggaag 180
cggaaatccc ggaagaagaa gttgctggag ggcggggaga tcgacgcgat ggacgacagc 240
tcgcagacct ccgactcgcg tgtcaccgtc atggacccca agacgggcga agtgttgaag 300
ggcgaggaag ccccgctgct gtctcagctg aagggctgga tggagcagca ccccggctgg 360
gaggtggtgt cggactccga cgactcgggc gacgacagcc aggacgacga cggacactac 420
aagagacaca aacacaggga cgacaagaat gagaacaggg agaagaccga ggaggagaag 480
gcgcgcgaga tgatcaagaa agccaaggtg gaggacgacg agtacaagac agaggagcag 540
acatactaca gcattgctca tacggtccac gaatccgtca cagaacaagc cagcattttg 600
gtcaacggaa aactcaagga ataccagatc aagggtctgg aatggctggt gtctttgttc 660
aacaacaacc tgaacggtat cctggcggac gagatgggtc tcggcaagac catccagacg 720
atagcgctgg tcacatacct catggagaag aagaaagtca acggaccatt cctcatcatt 780
gtaccgctca gtacgctatc aaactgggtg ctggagttcg agaagtgggc gccgaccgtg 840
tgcgtagtgt cgtacaaggg ctcgcccgcg tcgcgccgcc tcgtgcagaa ccagatgagg 900
tccaccaagt tcaacgtgct gctcaccacc tacgagtacg tcatcaagga caagggagtg 960
ctcgctaagg tgcaatggaa gtacatgata atcgacgagg gccaccgcat gaagaaccac 1020
cactgcaagc tgacgcaggt gctgaacacg cactacatag cgccgcaccg cctgctgctg 1080
accggcacgc cgctgcagaa caagctgccc gaactctggg cgctgctgaa cttcctcctg 1140
ccgtccatat tcaagagttg ctccaccttc gaacagtggt tcaatgcgcc cttcgctacc 1200
actggagaaa aggtggaact aaacgaggaa gaaacgatcc tgatcatccg tcgtctacac 1260
aaagtgctgc gtccgttctt actgcggcgc ctgaagaagg aagtagagag ccagctgccc 1320
gacaaagtgg agtacatcat caagtgcgac atgagcgggc tgcagcgagt gctctacaaa 1380
cacatgcagt cgaagggcgt gctgctgacg gacggctcgg agaagggcac gaagggcaag 1440
ggcggggcga aggcgctcat gaacaccatc gtgcagctgc gcaagctctg caaccatccc 1500
ttcatgttcc agcacatcga ggagaagttc tgcgaccacg tcggctctgg cagcaccgtc 1560
gtttctggtc cggacctgta ccgcgtgtcg ggcaagttcg agctgctgga ccgcatcctg 1620
cccaagctga agcgcaccgg ccaccgcgtg ctcgtgttct gccagatgac gcagtgcatg 1680
accatcatcg aggactacct ctcctggagg ggcttccaat acttgcggct ggacggtatg 1740
acgaaggcgg aggaccgtgg cgagctgctc aagaagttca actgcaagga ctcggaatat 1800
ttcctcttct tgctgtctac gagagctggt ggacttggac tcaatctcca gtcggctgac 1860
actgtcatca tcttcgattc cgattggaat ccgcatcagg atctgcaagc gcaagaccgc 1920
gcgcatcgta tcggacaacg taacgaagtg cgcgtgttgc gactgatgac cgtcaactcc 1980
gtcgaggaac ggattcttgc cgccgccagg tacaaattga acatggacga gaaggttatc 2040
caagccggta tgttcgatca aaagtccacc ggctcagagc gacagcagtt cctgcagagc 2100
attctgcatc aagacggcga cgatgaagaa gaagaaaatg aactcccgga cgacgacctc 2160
atcaacgaga tgatcgcgcg ctcggaggag gagctcgaga tcttcaaaca gatcgacatc 2220
gagcggaaga aaaccgaaac cacctcccgt ctcatagagg agtgcgagct gcccgactgg 2280
ctcaccaaga acgaggacga agttgtgtgc aacaagggcc aagggtgggg caacttcctg 2340
gacgctgagg agacgttcgg gcgcggctcc cggcagcgca aagaggttga ctacacggac 2400
tcgctcacgg agaaggagtg gctcaaggcc atcgacgacg agttcgacga cgaggaggag 2460
gaggacgacg acgacgaagt gctcgacaag aagcgcaaga agggccgcaa gcgacgccgc 2520
caagaagagt cggatgagga agaagtggcc agctcatcta agaagaagaa taagacggaa 2580
gccaacctac tgaagaaaag gctcaagagc attatgaaga aagtcgtgga ctacgctgac 2640
gagggcggta gagtcctgtc ggagccgttc atgaagctgc cgtcgcggcg cgagctgccc 2700
gactactacg acgtcatcaa gaaaccgctc gatatcaaga aaatcatgac gcgcatcgaa 2760
gacgggaagt acaacgacat tacggacttg gagcgcgatt tcttcactct gtgtcagaac 2820
gcacaaacct acaacgagga ggcgtcgctc atctacgcgg actccgtgcg actgaggaat 2880
gttttcattg agttccgacg caactacgag gcgggcaaca attccgacaa ctccgacgat 2940
gacaaagata aagaagaaga agactcggac ggcgagtcga accgctcggt caagatgaag 3000
atcaagctga aaggcaaagg caagggcacg ccctcgagga aaaagaaaca aaagaagtat 3060
atctccgacg acgaggacta tgaggaggat tga 3093
<210> 2
<211> 1030
<212> PRT
<213> 番茄潜叶蛾(Tuta absoluta)
<400> 2
Met Arg Arg Leu Met Ala Glu Asp Glu Glu Gly Tyr Arg Lys Leu Ile
1 5 10 15
Asp Gln Lys Lys Asp Lys Arg Leu Ala Phe Leu Leu Ser Gln Thr Asp
20 25 30
Glu Tyr Ile Ala Ser Leu Thr Glu Met Val Lys Gln His Lys Gln Glu
35 40 45
Gln Arg Lys Lys Gln Val Glu Glu Glu Lys Arg Lys Arg Lys Ser Arg
50 55 60
Lys Lys Lys Leu Leu Glu Gly Gly Glu Ile Asp Ala Met Asp Asp Ser
65 70 75 80
Ser Gln Thr Ser Asp Ser Arg Val Thr Val Met Asp Pro Lys Thr Gly
85 90 95
Glu Val Leu Lys Gly Glu Glu Ala Pro Leu Leu Ser Gln Leu Lys Gly
100 105 110
Trp Met Glu Gln His Pro Gly Trp Glu Val Val Ser Asp Ser Asp Asp
115 120 125
Ser Gly Asp Asp Ser Gln Asp Asp Asp Gly His Tyr Lys Arg His Lys
130 135 140
His Arg Asp Asp Lys Asn Glu Asn Arg Glu Lys Thr Glu Glu Glu Lys
145 150 155 160
Ala Arg Glu Met Ile Lys Lys Ala Lys Val Glu Asp Asp Glu Tyr Lys
165 170 175
Thr Glu Glu Gln Thr Tyr Tyr Ser Ile Ala His Thr Val His Glu Ser
180 185 190
Val Thr Glu Gln Ala Ser Ile Leu Val Asn Gly Lys Leu Lys Glu Tyr
195 200 205
Gln Ile Lys Gly Leu Glu Trp Leu Val Ser Leu Phe Asn Asn Asn Leu
210 215 220
Asn Gly Ile Leu Ala Asp Glu Met Gly Leu Gly Lys Thr Ile Gln Thr
225 230 235 240
Ile Ala Leu Val Thr Tyr Leu Met Glu Lys Lys Lys Val Asn Gly Pro
245 250 255
Phe Leu Ile Ile Val Pro Leu Ser Thr Leu Ser Asn Trp Val Leu Glu
260 265 270
Phe Glu Lys Trp Ala Pro Thr Val Cys Val Val Ser Tyr Lys Gly Ser
275 280 285
Pro Ala Ser Arg Arg Leu Val Gln Asn Gln Met Arg Ser Thr Lys Phe
290 295 300
Asn Val Leu Leu Thr Thr Tyr Glu Tyr Val Ile Lys Asp Lys Gly Val
305 310 315 320
Leu Ala Lys Val Gln Trp Lys Tyr Met Ile Ile Asp Glu Gly His Arg
325 330 335
Met Lys Asn His His Cys Lys Leu Thr Gln Val Leu Asn Thr His Tyr
340 345 350
Ile Ala Pro His Arg Leu Leu Leu Thr Gly Thr Pro Leu Gln Asn Lys
355 360 365
Leu Pro Glu Leu Trp Ala Leu Leu Asn Phe Leu Leu Pro Ser Ile Phe
370 375 380
Lys Ser Cys Ser Thr Phe Glu Gln Trp Phe Asn Ala Pro Phe Ala Thr
385 390 395 400
Thr Gly Glu Lys Val Glu Leu Asn Glu Glu Glu Thr Ile Leu Ile Ile
405 410 415
Arg Arg Leu His Lys Val Leu Arg Pro Phe Leu Leu Arg Arg Leu Lys
420 425 430
Lys Glu Val Glu Ser Gln Leu Pro Asp Lys Val Glu Tyr Ile Ile Lys
435 440 445
Cys Asp Met Ser Gly Leu Gln Arg Val Leu Tyr Lys His Met Gln Ser
450 455 460
Lys Gly Val Leu Leu Thr Asp Gly Ser Glu Lys Gly Thr Lys Gly Lys
465 470 475 480
Gly Gly Ala Lys Ala Leu Met Asn Thr Ile Val Gln Leu Arg Lys Leu
485 490 495
Cys Asn His Pro Phe Met Phe Gln His Ile Glu Glu Lys Phe Cys Asp
500 505 510
His Val Gly Ser Gly Ser Thr Val Val Ser Gly Pro Asp Leu Tyr Arg
515 520 525
Val Ser Gly Lys Phe Glu Leu Leu Asp Arg Ile Leu Pro Lys Leu Lys
530 535 540
Arg Thr Gly His Arg Val Leu Val Phe Cys Gln Met Thr Gln Cys Met
545 550 555 560
Thr Ile Ile Glu Asp Tyr Leu Ser Trp Arg Gly Phe Gln Tyr Leu Arg
565 570 575
Leu Asp Gly Met Thr Lys Ala Glu Asp Arg Gly Glu Leu Leu Lys Lys
580 585 590
Phe Asn Cys Lys Asp Ser Glu Tyr Phe Leu Phe Leu Leu Ser Thr Arg
595 600 605
Ala Gly Gly Leu Gly Leu Asn Leu Gln Ser Ala Asp Thr Val Ile Ile
610 615 620
Phe Asp Ser Asp Trp Asn Pro His Gln Asp Leu Gln Ala Gln Asp Arg
625 630 635 640
Ala His Arg Ile Gly Gln Arg Asn Glu Val Arg Val Leu Arg Leu Met
645 650 655
Thr Val Asn Ser Val Glu Glu Arg Ile Leu Ala Ala Ala Arg Tyr Lys
660 665 670
Leu Asn Met Asp Glu Lys Val Ile Gln Ala Gly Met Phe Asp Gln Lys
675 680 685
Ser Thr Gly Ser Glu Arg Gln Gln Phe Leu Gln Ser Ile Leu His Gln
690 695 700
Asp Gly Asp Asp Glu Glu Glu Glu Asn Glu Leu Pro Asp Asp Asp Leu
705 710 715 720
Ile Asn Glu Met Ile Ala Arg Ser Glu Glu Glu Leu Glu Ile Phe Lys
725 730 735
Gln Ile Asp Ile Glu Arg Lys Lys Thr Glu Thr Thr Ser Arg Leu Ile
740 745 750
Glu Glu Cys Glu Leu Pro Asp Trp Leu Thr Lys Asn Glu Asp Glu Val
755 760 765
Val Cys Asn Lys Gly Gln Gly Trp Gly Asn Phe Leu Asp Ala Glu Glu
770 775 780
Thr Phe Gly Arg Gly Ser Arg Gln Arg Lys Glu Val Asp Tyr Thr Asp
785 790 795 800
Ser Leu Thr Glu Lys Glu Trp Leu Lys Ala Ile Asp Asp Glu Phe Asp
805 810 815
Asp Glu Glu Glu Glu Asp Asp Asp Asp Glu Val Leu Asp Lys Lys Arg
820 825 830
Lys Lys Gly Arg Lys Arg Arg Arg Gln Glu Glu Ser Asp Glu Glu Glu
835 840 845
Val Ala Ser Ser Ser Lys Lys Lys Asn Lys Thr Glu Ala Asn Leu Leu
850 855 860
Lys Lys Arg Leu Lys Ser Ile Met Lys Lys Val Val Asp Tyr Ala Asp
865 870 875 880
Glu Gly Gly Arg Val Leu Ser Glu Pro Phe Met Lys Leu Pro Ser Arg
885 890 895
Arg Glu Leu Pro Asp Tyr Tyr Asp Val Ile Lys Lys Pro Leu Asp Ile
900 905 910
Lys Lys Ile Met Thr Arg Ile Glu Asp Gly Lys Tyr Asn Asp Ile Thr
915 920 925
Asp Leu Glu Arg Asp Phe Phe Thr Leu Cys Gln Asn Ala Gln Thr Tyr
930 935 940
Asn Glu Glu Ala Ser Leu Ile Tyr Ala Asp Ser Val Arg Leu Arg Asn
945 950 955 960
Val Phe Ile Glu Phe Arg Arg Asn Tyr Glu Ala Gly Asn Asn Ser Asp
965 970 975
Asn Ser Asp Asp Asp Lys Asp Lys Glu Glu Glu Asp Ser Asp Gly Glu
980 985 990
Ser Asn Arg Ser Val Lys Met Lys Ile Lys Leu Lys Gly Lys Gly Lys
995 1000 1005
Gly Thr Pro Ser Arg Lys Lys Lys Gln Lys Lys Tyr Ile Ser Asp Asp
1010 1015 1020
Glu Asp Tyr Glu Glu Asp
1025 1030

Claims (6)

1.番茄潜叶蛾染色质重塑因子BRM，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所述的番茄潜叶蛾染色质重塑因子BRM。
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的在以下方面的应用，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）降低番茄潜叶蛾卵的孵化率；
（3）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+dsBRM-F：5’- TAATACGACTCACTATAGGGGAACCATCACGCTAACG-3’
T7+dsBRM-R：5’- TAATACGACTCACTATAGGGGGATTCGTGGACCGTA-3’。
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