CN113150099B - 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 - Google Patents

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 Download PDF

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CN113150099B
CN113150099B CN202110170633.7A CN202110170633A CN113150099B CN 113150099 B CN113150099 B CN 113150099B CN 202110170633 A CN202110170633 A CN 202110170633A CN 113150099 B CN113150099 B CN 113150099B
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吕志创
王晓迪
冀顺霞
刘万学
张桂芬
万方浩
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Abstract

本发明涉及农业生物技术领域，具体的涉及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，其编码氨基酸序列如SEQ ID NO:2所示的蛋白质。本发明首次从新发重要入侵害虫南美番茄潜叶蛾中克隆获得保幼激素信号通路转录因子Kr‑h1基因，上述入侵害虫幼虫取食该基因dsRNA后导致幼虫死亡。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

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
技术领域
本发明涉及农业生物技术领域，具体的涉及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。
背景技术
番茄潜叶蛾Tuta absoluta原产于南美洲西部的秘鲁，它能够在适宜的农业生态条件下迅速发展，在新的地区迅速扩散，并造成严重的经济损失，是一种典型的入侵物种。该世界检疫性害虫，于2006年入侵欧洲的西班牙，截至2017年5月，已在南美洲、欧洲、非洲、中美洲、亚洲等的85个国家/地区发生，并在22个国家/地区疑似发生，对番茄等作物造成毁灭性的灾害，可见其具有非常强的快速传播和适应能力。2017年8月在我国新疆伊犁地区首次发现番茄潜叶蛾，次年3月在云南临沧也相继发现其踪迹，目前已已扩散至云南、贵州、四川、广西、重庆、湖南等地。在新疆伊犁地区调查的番茄田中南美番茄潜叶蛾的危害程度达到了100％，在茄子田和马铃薯田的危害程度也分别达到了90％和100％。南美番茄潜叶蛾主要以幼虫进行危害，雌性成虫喜欢在刚刚展开的叶片上进行产卵，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。
因此对已发生区采取有效的防控措施，以及对未发生区进行检测监测和采取有效的预防措施是目前急需解决的关键问题。RNA干扰(RNA interference,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。将与靶基因的转录产物mRNA存在同源互补序列的双链RNA(double strand RNA,dsRNA)导入细胞后能特异性地降解该mRNA，从而产生相应的功能表型缺失。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。
保幼激素信号通路转录因子Kr-h1(Krüppel homolog 1)作为JH信号通路中下游关键应答基因，编码含有C2H2锌指结构的转录因子从而调控JH的合成。Kr-h1通过受体Met和Gce传递保幼激素信号，维持幼虫状态，调控幼虫的生长发育与变态。Kr-h1沉默出现幼虫的存活率显著降低、蛹畸形以及存活成虫的繁殖力降低等。因此，本发明基于RNA干扰技术，提供了对环境无毒无害、特异性强的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南美番茄潜叶蛾保幼激素信号通路转录因子。
本发明的再一目的是提供编码上述南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1基因。
本发明的再一目的是提供上述编码基因在南美番茄潜叶蛾防治中的应用。
根据本发明的具体实施方式，本发明首次克隆得到南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1，其cDNA全长核苷酸序列如SEQ ID No:1所示：
ATGATAGGTGAAGAGGAACGAGTACATCAGTGTGGAGAATGTGGCTTGACGCTTTCCACTCGGAGTGCATTAACTGCTCATGCACGCTCGCACCGTGCTACAGCCGATGCACACCGCTGCGACGTCTGTCACAAAACTTTCGCCGTGCCTGCACGTCTTGTGCGGCATTATAGGACCCATACTGGTGAACGACCTTTTGAATGTGAATATTGTCATAAGATGTTCGGTGTTAAAGAAAACCTTCAAGTCCATCGCCGTATTCATACTAAAGAAAGACCATACCGGTGTGGAGTATGCGGAGCGGCATTTGAGCATTCTGGGAAACTCCATCGTCACGCTCGGATTCATACCGGTGAACGGCCCCATGCGTGCCCGCATTGCCATAAGACTTTCATCCAGTCTGGCCAACTAGTTATTCATCTTCGTACACACACAGGAGAAAAACCATACCGTTGTCCAGCTGCTGGTTGCGGTAAAGGATTTACTTGCTCCAAACAACTTAAGGTTCATTCTCGAACACACACTGGAGAGCGACCTTACACTTGTGACATTTGCTTAAGAGACTTTGGGTATAATCACGTCCTAAAATTACACCGCTTCCAACACTACGGTGAACGTTGCTATCGCTGTACCGTATGTGATGGTACTTTCAACACTAAAAAGCAAATGGAAGCCCATATCTACAAAGAACATGGCGCTGAAACACCTCGTGCACCTTCATTGCAGTCAAATCTTCCTATGATTGTTAACGGGAACGTTATGTGTGAAATGGTTGAAGCGGCCTTACAACAGCTGCCACCTACGCCTCCAAGCTCGCCTCCATCGTCCCAGAGTGTGACCCCACCAGCTAGCAATAATGTAATTGAAACATCTTCACCAGCACCTTCACCATCTCCACCAACCACAACTCTGCAATATACATTTGCGCCGTCTTCCTTACCACCTAGAAAACGTAAACTTATTCCTCAGCCAGACCCTGTTACCCCACCTATCGTACGTCATACTTCAGTTATACAATTTGCACCAGCAGCTGTCGATGTATAAATTAGAAATAGTTACCTAGTACTTGTAA
南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1编码氨基酸序列如SEQ ID NO:2所示：
MIGEEERVHQCGECGLTLSTRSALTAHARSHRATADAHRCDVCHKTFAVPARLVRHYRTHTGERPFECEYCHKMFGVKENLQVHRRIHTKERPYRCGVCGAAFEHSGKLHRHARIHTGERPHACPHCHKTFIQSGQLVIHLRTHTGEKPYRCPAAGCGKGFTCSKQLKVHSRTHTGERPYTCDICLRDFGYNHVLKLHRFQHYGERCYRCTVCDGTFNTKKQMEAHIYKEHGAETPRAPSLQSNLPMIVNGNVMCEMVEAALQQLPPTPPSSPPSSQSVTPPASNNVIETSSPAPSPSPPTTTLQYTFAPSSLPPRKRKLIPQPDPVTPPIVRHT SVIQFAPAAV DV
上述氨基酸序列具有Kr-h1蛋白典型的结构特征：Kr-h1翻译后的氨基酸是一种含有2个Cys和2个His的锌指结构，经分析发现在南美番茄潜叶蛾中含有8个C2H2锌指结构(Zn1-Zn8)。
本发明提供了上述南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1的应用。对南美番茄潜叶蛾进行RNAi，结果显示，取食dsKr-h1的南美番茄潜叶蛾幼虫死亡率达到74.8％，说明Kr-h1基因在南美番茄潜叶蛾幼虫发育中起关键作用。本发明南美番茄潜叶蛾Kr-h1基因片段合成的dsRNA对南美番茄潜叶蛾幼虫具有高的死亡率，可以用于南美番茄潜叶蛾的防治，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南美番茄潜叶蛾中克隆获得保幼激素信号通路转录因子Kr-h1基因，上述入侵害虫幼虫取食该基因dsRNA后导致幼虫死亡。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南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1基因基酸序列与其他昆虫体内同源氨基酸序列的系统进化树；
图2显示饲喂Kr-h1基因dsRNA和饲喂dsEGFP条件下Kr-h1的表达量变化情况；
图3显示Kr-h1基因的dsRNA处理对南美番茄潜叶蛾幼虫死亡率的影响。
具体实施方式
实施例1：番茄潜叶蛾Tuta absoluta Kr-h1基因全长cDNA序列克隆
取南美番茄潜叶蛾幼虫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-One First-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Btbrm1基因全长cDNA的引物序列
引物名称 引物序列(5'-3')
Kr-h1-F79 TCAACAAATTCCGCATTCG
Kr-h1-R1347 TTTCAGGCAACATTCAACG
利用表1序列，通过PCR扩增，获得Kr-h1基因的cDNA序列全长为1069bp，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347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Kr-h1蛋白典型的结构特征：包括8个C2H2锌指结构。如图1所示，显示了南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1基因基酸序列与其他昆虫体内同源氨基酸序列的系统进化树
实施例2：分析Kr-h1基因对南美番茄潜叶蛾幼虫存活率的影响
3.1合成dsRNA
(1)设计合成加上T7启动子(下划线所示序列)的引物序列：
T7+dsKr-h1-F600：
5’-TAATACGACTCACTATAGGGTGGTTGCGGTAAAGGATT-3’
T7+dsKr-h1-R1117：
5’-TAATACGACTCACTATAGGGTAACAGGGTCTGGCTGAG-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。
3.2 dsRNA饲喂
(1)叶柄浸泡法
摘取新鲜叶片并将叶片干燥处理1小时，随后将叶柄浸泡在含有dsKr-h1的水溶液中，浓度为25μg/mL，待叶片充分吸收含有dsKr-h1的溶液后(约4小时)，将提前饥饿处理3小时的30头2龄幼虫轻轻挑回至浸泡处理后的叶片。保湿处理后将其放在人工气候箱(平均温度25±2℃，相对湿度50±10％，光周期为L14：D10)中饲养。每隔24小时观察幼虫状态，随机取4头幼虫用于表达量的检测，并记录非正常表型(提前化蛹或死亡等)，统计幼虫死亡率、幼虫期持续时间和蛹重等参数。以饲喂含有dsEGFP的水溶液(dsEGFP终浓度为25μg/mL)的南美番茄潜叶蛾为对照，各处理均设置4个生物学重复。
(2)叶面喷洒法
将配置好的dsKr-h1水溶液中均匀的喷洒在叶片表面，然后将幼虫转移至叶片上。其余试验方法同上。
通过2-ΔΔCT方法计算基因的相对表达量，结果如图2所示，饲喂dsKr-h1可以显著敲低Kr-h1基因的表达。利用SAS 9.4统计软件分析饲喂不同溶液后南美番茄潜叶蛾幼虫的死亡率，结果如图3所示，饲喂Kr-h1基因dsRNA的南美番茄潜叶蛾的死亡率显著高于饲喂dsEGFP组(P<0.05)，同时在NCBI(http://blast.ncbi.nlm.nih.gov/)BLAST显示，所饲喂的靶序列片度为Kr-h1基因特有的序列，由此确保了干扰效果为南美番茄潜叶蛾的Kr-h1基因所产生，说明Kr-h1基因在南美番茄潜叶蛾幼虫生长发育过程中起着关键作用。
本发明从南美番茄潜叶蛾中克隆得到Kr-h1基因的全长cDNA，荧光定量PCR显示饲喂靶基因双链RNA后Kr-h1基因的表达量显著降低；最后通过饲喂Kr-h1基因dsRNA，进而导致南美番茄潜叶蛾幼虫的死亡。根据本发明的具体实施方式，试验结果明确了Kr-h1基因在南美番茄潜叶蛾幼虫生长发育过程中起着关键作用。本发明为利用RNAi有效防治南美番茄潜叶蛾的研究奠定了基础，为日后通过保幼激素的相关研究降低南美番茄潜叶蛾的危害提供了方法依据。
序列表
<110> 中国农业科学院植物保护研究所
<120> 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
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1072
<212> DNA
<213> 番茄潜叶蛾(Tuta absoluta)
<400> 1
atgataggtg aagaggaacg agtacatcag tgtggagaat gtggcttgac gctttccact 60
cggagtgcat taactgctca tgcacgctcg caccgtgcta cagccgatgc acaccgctgc 120
gacgtctgtc acaaaacttt cgccgtgcct gcacgtcttg tgcggcatta taggacccat 180
actggtgaac gaccttttga atgtgaatat tgtcataaga tgttcggtgt taaagaaaac 240
cttcaagtcc atcgccgtat tcatactaaa gaaagaccat accggtgtgg agtatgcgga 300
gcggcatttg agcattctgg gaaactccat cgtcacgctc ggattcatac cggtgaacgg 360
ccccatgcgt gcccgcattg ccataagact ttcatccagt ctggccaact agttattcat 420
cttcgtacac acacaggaga aaaaccatac cgttgtccag ctgctggttg cggtaaagga 480
tttacttgct ccaaacaact taaggttcat tctcgaacac acactggaga gcgaccttac 540
acttgtgaca tttgcttaag agactttggg tataatcacg tcctaaaatt acaccgcttc 600
caacactacg gtgaacgttg ctatcgctgt accgtatgtg atggtacttt caacactaaa 660
aagcaaatgg aagcccatat ctacaaagaa catggcgctg aaacacctcg tgcaccttca 720
ttgcagtcaa atcttcctat gattgttaac gggaacgtta tgtgtgaaat ggttgaagcg 780
gccttacaac agctgccacc tacgcctcca agctcgcctc catcgtccca gagtgtgacc 840
ccaccagcta gcaataatgt aattgaaaca tcttcaccag caccttcacc atctccacca 900
accacaactc tgcaatatac atttgcgccg tcttccttac cacctagaaa acgtaaactt 960
attcctcagc cagaccctgt taccccacct atcgtacgtc atacttcagt tatacaattt 1020
gcaccagcag ctgtcgatgt ataaattaga aatagttacc tagtacttgt aa 1072
<210> 2
<211> 347
<212> PRT
<213> 番茄潜叶蛾(Tuta absoluta)
<400> 2
Met Ile Gly Glu Glu Glu Arg Val His Gln Cys Gly Glu Cys Gly Leu
1 5 10 15
Thr Leu Ser Thr Arg Ser Ala Leu Thr Ala His Ala Arg Ser His Arg
20 25 30
Ala Thr Ala Asp Ala His Arg Cys Asp Val Cys His Lys Thr Phe Ala
35 40 45
Val Pro Ala Arg Leu Val Arg His Tyr Arg Thr His Thr Gly Glu Arg
50 55 60
Pro Phe Glu Cys Glu Tyr Cys His Lys Met Phe Gly Val Lys Glu Asn
65 70 75 80
Leu Gln Val His Arg Arg Ile His Thr Lys Glu Arg Pro Tyr Arg Cys
85 90 95
Gly Val Cys Gly Ala Ala Phe Glu His Ser Gly Lys Leu His Arg His
100 105 110
Ala Arg Ile His Thr Gly Glu Arg Pro His Ala Cys Pro His Cys His
115 120 125
Lys Thr Phe Ile Gln Ser Gly Gln Leu Val Ile His Leu Arg Thr His
130 135 140
Thr Gly Glu Lys Pro Tyr Arg Cys Pro Ala Ala Gly Cys Gly Lys Gly
145 150 155 160
Phe Thr Cys Ser Lys Gln Leu Lys Val His Ser Arg Thr His Thr Gly
165 170 175
Glu Arg Pro Tyr Thr Cys Asp Ile Cys Leu Arg Asp Phe Gly Tyr Asn
180 185 190
His Val Leu Lys Leu His Arg Phe Gln His Tyr Gly Glu Arg Cys Tyr
195 200 205
Arg Cys Thr Val Cys Asp Gly Thr Phe Asn Thr Lys Lys Gln Met Glu
210 215 220
Ala His Ile Tyr Lys Glu His Gly Ala Glu Thr Pro Arg Ala Pro Ser
225 230 235 240
Leu Gln Ser Asn Leu Pro Met Ile Val Asn Gly Asn Val Met Cys Glu
245 250 255
Met Val Glu Ala Ala Leu Gln Gln Leu Pro Pro Thr Pro Pro Ser Ser
260 265 270
Pro Pro Ser Ser Gln Ser Val Thr Pro Pro Ala Ser Asn Asn Val Ile
275 280 285
Glu Thr Ser Ser Pro Ala Pro Ser Pro Ser Pro Pro Thr Thr Thr Leu
290 295 300
Gln Tyr Thr Phe Ala Pro Ser Ser Leu Pro Pro Arg Lys Arg Lys Leu
305 310 315 320
Ile Pro Gln Pro Asp Pro Val Thr Pro Pro Ile Val Arg His Thr Ser
325 330 335
Val Ile Gln Phe Ala Pro Ala Ala Val Asp Val
340 345

Claims (7)

1.南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1，其特征在于，编码氨基酸序列如SEQ ID NO:2所示的蛋白质。
2.根据权利要求1所述的南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1，其特征在于，所述保幼激素信号通路转录因子Kr-h1的核苷酸序列如SEQ ID NO:1所示。
3.包含权利要求1所述的南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1的dsRNA片段的重组载体。
4.一种防控南美番茄潜叶蛾的方法，其特征在于，所述方法包括向南美番茄潜叶蛾饲喂南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1的dsRNA片段的步骤，其中，所述南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1编码氨基酸序列如SEQ ID NO:2所示的蛋白质，通过以下引物扩增南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1的基因序列得到所述dsRNA片段，
5’-TAATACGACTCACTATAGGGTGGTTGCGGTAAAGGATT-3’；
5’-TAATACGACTCACTATAGGGTAACAGGGTCTGGCTGAG-3’。
5.根据权利要求4所述的防控南美番茄潜叶蛾的方法，其特征在于，所述保幼激素信号通路转录因子Kr-h1的核苷酸序列如SEQ ID NO:1所示。
6.一种保护植物免受南美番茄潜叶蛾侵害的方法，其特征在于，所述方法包括向植株喷施南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1的dsRNA片段的步骤，其中，所述南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1编码氨基酸序列如SEQ ID NO:2所示的蛋白质，通过以下引物扩增南美番茄潜叶蛾保幼激素信号通路转录因子Kr-h1的基因序列得到所述dsRNA片段，
5’-TAATACGACTCACTATAGGGTGGTTGCGGTAAAGGATT-3’；
5’-TAATACGACTCACTATAGGGTAACAGGGTCTGGCTGAG-3’。
7.根据权利要求6所述的保护植物免受南美番茄潜叶蛾侵害的方法，其特征在于，所述保幼激素信号通路转录因子Kr-h1的核苷酸序列如SEQ ID NO:1所示。
CN202110170633.7A 2021-02-08 2021-02-08 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 Active CN113150099B (zh)

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