CN112877336B - sfIMP-X1基因及其在草地贪夜蛾遗传防控中的应用 - Google Patents

sfIMP-X1基因及其在草地贪夜蛾遗传防控中的应用 Download PDF

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CN112877336B
CN112877336B CN202110254411.3A CN202110254411A CN112877336B CN 112877336 B CN112877336 B CN 112877336B CN 202110254411 A CN202110254411 A CN 202110254411A CN 112877336 B CN112877336 B CN 112877336B
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萧玉涛
程英
曾宇笑
靳明辉
廖重宇
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Agricultural Genomics Institute at Shenzhen of CAAS
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Abstract

本发明提供了一种sfIMP‑X1基因及其在草地贪夜蛾遗传防控中的应用，涉及基因工程技术领域。所述sfIMP‑X1基因的核苷酸序列如SEQ ID NO：1所示。本发明发现sfIMP‑X1基因缺失突变体幼虫蜕皮或体节发育过程异常，所有sfIMP‑X1基因缺失个体在蛹前期死亡，因此，sfIMP‑X1基因可以作为草地贪夜蛾幼虫期防控靶标基因，对草地贪夜蛾的防治具有重要的意义。

Description

sfIMP-X1基因及其在草地贪夜蛾遗传防控中的应用
技术领域
本发明涉及基因工程技术领域，尤其是涉及sfIMP基因及其在草地贪夜蛾遗传防控中的应用。
背景技术
草地贪夜蛾(Spodoptera frugiperda)(J.E.Smith)是2018年联合国粮农组织向全球发布预警的重要农业害虫，也是我国重要外来入侵物种。草地贪夜蛾的寄主极为广泛、适生范围极广、繁殖能力极强、迁飞扩散速度极快、突发危害重、防控难度较大。
昆虫生长发育及生殖等过程中一些重要的调控基因通常可以作为基因编辑的靶标，通过对其进行删除或突变，使昆虫生长发育异常，可以达到害虫防治的目的。在鳞翅目物种中，编码丝氨酸蛋白酶家族成员的卵巢丝氨酸蛋白酶(Osp)对卵子形成至关重要。利用CRISPR/Cas9技术敲除家蚕和斜纹夜蛾中Osp基因后，雌性突变体不育，虽然能正常交配但与野生型相比产卵量减少，且卵不能孵化。Ser2(serine protease 2)基因编码精液蛋白丝氨酸蛋白酶2，是昆虫精液重要组成蛋白，发现Ser2基因缺失导致家蚕雄性不育，其精子无法完成受精，但是雌性生殖不受影响。Osp与Ser2基因可以作为潜在的靶标应用于害虫的遗传防控。
然而，上述Osp基因、Ser2基因主要作用于害虫成虫的生殖系统，而害虫对农作物的危害主要是在幼虫期。害虫防治过程中，往往需要在幼虫时期控制害虫生长发育，以减轻对农作物的危害。草地贪夜蛾在我国危害发生历史较短，遗传防控相关研究刚刚起步。目前针对草地贪夜蛾幼虫期遗传防控的靶标研究较少，因此亟待发现作用于草地贪夜蛾幼虫期的靶标基因。
基于此，特提出本发明。
发明内容
本发明的目的在于提供sfIMP-X1基因及其在草地贪夜蛾遗传防控中的应用。本发明首次发现sfIMP-X1基因缺失突变体幼虫蜕皮或体节发育过程异常，所有sfIMP-X1基因缺失个体在蛹前期死亡，因此，sfIMP-X1基因可以作为草地贪夜蛾幼虫期防控靶标基因。
本发明提供的技术方案如下：
在第一方面，本发明提供了sfIMP-X1基因，所述sfIMP基因的核苷酸序列如SEQ IDNO：1。IMP基因是指编码IGF2 mRNA结合蛋白(IGF2 mRNA-binding protein)的基因。草地贪夜蛾IMP基因可简写为sfIMP基因。
本发明首次克隆了sfIMP基因，鉴定到sfIMP基因2个转录本IMP-X1与IMP-X2。经测序获得sfIMP基因2个转录本编码区序列，分别如sfIMP-X1(全长1959bp，其中CDS长1614bp，位于102-1715，编码537个氨基酸)，即SEQ ID NO：1；和Sfimp-X2(全长1788bp，其中CDS长1443bp，位于102-1544，编码480个氨基酸)，即SEQ ID NO：3。
在第二方面，本发明还提供了所述sfIMP-X1基因编码的蛋白，所述蛋白的氨基酸序列分别如SEQ ID NO：2所示。
在第三方面，本发明提供了所述sfIMP-X1基因或所述基因编码的蛋白作为靶点在草地贪夜蛾遗传防控中的应用。
在一个实施方案中，所述应用为通过作用于所述靶点影响草地贪夜蛾幼虫正常蜕皮或体节发育过程。sfIMP-X1基因缺失突变体幼虫蜕皮或体节发育过程异常，所有sfIMP-X1基因缺失个体在蛹前期死亡。因此，IMP-X1基因可以作为草地贪夜蛾遗传防控重要靶点。
在一个实施方案中，所述应用包括采用基因敲除、基因敲减或者化学药物抑制所述基因的表达或使其功能缺失。
在一个实施方案中，所述基因敲除使用ZFNs、TALENs、CRISPR/Cas9系统或siRNA进行；优选使用CRISPR/Cas9系统进行。
在第四方面，本发明还提供了一种敲除sfIMP-X1基因的CRISPR/Cas9系统，所述CRISPR/Cas9系统包括Cas9和特异性靶向sfIMP-X1基因的sgRNA。
在一个实施方案中，特异性靶向sfIMP-X1基因的sgRNA的核苷酸引物序列如ACCGCTGCTCTCACAGTATGG(SEQ ID NO：4)和AAGTGATGAACACCGCTTGGG(SEQ ID NO：5)所示。
在一个具体的实施方案中，将所述sgRNA与Cas9蛋白通过显微注射的方式导入草地贪夜蛾受精卵中进行基因敲除。
一种防治草地贪夜蛾的方法，所述方法包括：抑制或下调所述草地贪夜蛾体内sfIMP-X1基因的表达或活性。
有益效果：
本发明研究提供了一种草地贪夜蛾幼虫期防控靶标基因。本发明增加了现有草地贪夜蛾遗传防控候选靶标基因，同时为新的生物农药研发提供参考。具有良好的应用前景。
本发明提供了一种基于抑制或下调基因表达而增强草地贪夜蛾防治的方法，为害虫的有效控制提供了新的途径，为进一步制定有效的防控措施提供重要基础。通过CRISPR/Cas9系统敲除sfIMP-X1基因，使得基因缺失突变体出现蜕皮或体节发育困难而最终死亡。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例提供的sgRNA靶位点，其序列以下划线标示；
图2为sfIMP-X1基因敲除个体表型示意图；
图3为sfIMP-X1突变个体基因型与野生型(WT)序列比对；
图4为sfIMP-X1基因敲除后sfIMP-X1 mRNA表达量示意图。
具体实施方式
下面将结合实施例对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
1、sfIMP基因克隆
本发明通过草地贪夜蛾全基因组测序及鳞翅目昆虫IMP基因(IGF2 mRNA-bindingprotein)同源比对获得草地贪夜蛾sfIMP基因cds序列。
设计上下游扩增引物如下：
引物名称 序列(5’-3’) 编号
SF-IMP-F ACGCTTCGAAATAAGCCACC SEQ ID NO：6
SF-IMP-R GACAGCCTGCCTGCACTAAA SEQ ID NO：7
取草地贪夜蛾成虫雌雄各3头混合，提取RNA反转录获得雌雄成虫cDNA，以此cDNA为模板，利用上述引物扩增，扩增体系及程序如下：
2×Phanta Master Mix：25μL
引物SF-IMP-F：1μL
引物SF-IMP-R：1μL
cDNA：1μL
水：22μL
PCR扩增程序：95℃，3min；95℃，10sec；64℃，10sec；72℃，90sec；72℃/5min，扩增34个循环。
回收上述条带TA克隆并测序，获得sfIMP基因2个转录本编码区序列如下(5’-3’)：
sfIMP-X1：(全长1959bp，其中CDS长1614bp，位于102-1715，编码537个氨基酸)
ACGCTTCGAAATAAGCCACCAGTCTTAAAAGACCATTCCAATTTTAACCAGTATTTAAAAATCGTTATTGCCGCGGTAAAGGAAACCATAAAAAAGTGAAAATGTCTAATTCTGTGGAACAACAATTTAGCGAGCTTAGTTTGTCACAAGAGGACCATGATCAATATGAACCAGAAGATCACCAAGACCAAAGCCGGTCATCAAAAGTTCTCATCAGTGGGCTACCTCTACATATCCGCTTCGACAACATCGAACCGCTGCTCTCACAGTATGGCAACGTACAACACTGCGACAAAGCCAACTCGCGCGATGCCAACACCCAAGCGGTGTTCATCACTTTCGAGACTCCGGAGCAGGCGCAGCAAGCCATCAATGGTTTGAATGGATGCGAGTTAGAGGGCAGTCGGATGAAGGTAGAGGCAGCTGAGCAAAACACCCGTGGCGTCAGGCGCGGACGCCCTGGTGGCGGCCGCGGCGGCGGCGGTGCCACCGGTGGCGGCTCGCGACCCACCGACTTCCCATTGCGGCTCCTCGTCCAGAGCGACATGGTCGGCGCCATCATCGGCCGCCAGGGCAGCACCATCCGCCTCATTACACAACAGAGCCGTGCACGCGTTGATGTCCACCGCAAGGACAACGTTGGTTCTCTTGAGAAAGCTATTACCATATATGGTAATCCTGAGAACTGCACTAATGCATGCAAAAGAATATTGGAAGTCATGCAACAGGAAGCCAACAACACCAACAAGGGTGAGATTAGCCTCAAAATTTTGGCTCACAATAACCTAATCGGCCGTATTATTGGCAAAGGCGGTAACACTATTAAAAGAATCATGCAAGAAACTGATACGAAGATTACCGTCTCCTCCATCAATGATATCAACAGTTTCAATTTGGAGCGCATCATCACAGTTAAGGGCACTATTGAAAATATGGCTAAAGCGGAATCGCAGATTTCAGCCAAGCTTCGCCAGAGCTACGAAAGTGATTTGCAGATGCTGGCACCGCAAAGCATTATGTTCCCAGGGCTGCACCCCATGGCCATGATGTCCACCGGACGTGGATTCTGCGGCGCTCCTCCACCATTCCCGCCTCCAATATACGCTCCTCTGCCGGGCCAGGGCGGGGCACAGCAGGGAGCTGGCGACTCACAGGAAACAACTTACTTGTACATCCCAAACAATGCCGTGGGCGCCATCATCGGTACGAAGGGCCTGCACATTCGTAACATCATAAGATTCAGCAACGCATCTGTGAAGATAGCTCCTCTGGAGCAAGACAAGCAAGGCGAGAACCAAAGCAATCCCCAACAGGAGCGTAAGGTCACCATTGTTGGCAGCCCTGAGGCTCAATGGAAGGCTCAGTATCTCATCTTCGAGAAGATGCGAGAAGAAGGTTTCATGTCAGGCTCTGATGACGTGCGGCTTACAGTGGAGATAGTGGTGGCTTCATCCCAGGTCGGCCGTATCATCGGCAAAGGCGGGCAAAACGTACGGGAGCTGCAACGCGTAACAGGATCGCTAATTAAGCTTCCCGAACAACCACAGCAGCAGCAGGGCGGCGGGCAGCAACAGGATCACGATACAACAGTGCATATCGTCGGCCCCTTCTACAGCGTGCAGTCGGCGCAGCGACGCATTCGTGCAATGGTTGCGCAGGCGAGCGCACCGGGCCGCCGCCGCGCTGCCCAACCGCCGCCTCCCGTGCAGCAGTAAACTGCCTCCCACATGTTCGAGATCCCCGCGCGCCCTCAAGCACCTCTTCACCCTCAAGCCCTCGCAACACCCCGTCAGCTCGGGCAGCCTAGGCCGCCTGCGGTCGCAGTCACCCAATGCTAGCGTCGTCGCAATGATGCGCTCCATCCACTAAACTGGCTTCATATATGCACATAAGTGTTACACAAAGGATAGACGCTGTCGTAAGTAACGCTTCACTTGAATTTAGTGCAGGCAGGCTGTC(SEQ IDNO：1)；
上述CDS对应编码氨基酸预测序列如下：
MSNSVEQQFSELSLSQEDHDQYEPEDHQDQSRSSKVLISGLPLHIRFDNIEPLLSQYGNVQHCDKANSRDANTQAVFITFETPEQAQQAINGLNGCELEGSRMKVEAAEQNTRGVRRGRPGGGRGGGGATGGGSRPTDFPLRLLVQSDMVGAIIGRQGSTIRLITQQSRARVDVHRKDNVGSLEKAITIYGNPENCTNACKRILEVMQQEANNTNKGEISLKILAHNNLIGRIIGKGGNTIKRIMQETDTKITVSSINDINSFNLERIITVKGTIENMAKAESQISAKLRQSYESDLQMLAPQSIMFPGLHPMAMMSTGRGFCGAPPPFPPPIYAPLPGQGGAQQGAGDSQETTYLYIPNNAVGAIIGTKGLHIRNIIRFSNASVKIAPLEQDKQGENQSNPQQERKVTIVGSPEAQWKAQYLIFEKMREEGFMSGSDDVRLTVEIVVASSQVGRIIGKGGQNVRELQRVTGSLIKLPEQPQQQQGGGQQQDHDTTVHIVGPFYSVQSAQRRIRAMVAQASAPGRRRAAQPPPPVQQ*(SEQ ID NO：2)；
sfIMP-X2：(全长1788bp，其中CDS长1443bp，位于102-1544，编码480个氨基酸)
ACGCTTCGAAATAAGCCACCAGTCTTAAAAGACCATTCCAATTTTAACCAGTATTTAAAAATCGTTATTGCCGCGGTAAAGGAAACCATAAAAAAGTGAAAATGTCTAATTCTGTGGAACAACAATTTAGCGAGCTTAGTTTGTCACAAGAGGACCATGATCAATATGAACCAGAAGATCACCAAGACCAAAGAGCCATCAATGGTTTGAATGGATGCGAGTTAGAGGGCAGTCGGATGAAGGTAGAGGCAGCTGAGCAAAACACCCGTGGCGTCAGGCGCGGACGCCCTGGTGGCGGCCGCGGCGGCGGCGGTGCCACCGGTGGCGGCTCGCGACCCACCGACTTCCCATTGCGGCTCCTCGTCCAGAGCGACATGGTCGGCGCCATCATCGGCCGCCAGGGCAGCACCATCCGCCTCATTACACAACAGAGTCGTGCACGCGTTGATGTCCACCGCAAGGACAACGTTGGTTCTCTTGAGAAAGCTATTACCATATATGGTAATCCTGAGAACTGCACTAATGCATGCAAAAGAATATTGGAAGTCATGCAACAGGAAGCCAACAACACCAACAAGGGTGAGATTAGCCTCAAAATTTTGGCTCACAATAACCTAATCGGCCGTATTATTGGCAAAGGCGGTAACACTATTAAAAGAATCATGCAAGAAACTGATACGAAGATTACCGTCTCCTCCATCAATGATATCAACAGTTTCAATTTGGAGCGCATCATCACAGTTAAGGGCACTATTGAAAATATGGCTAAAGCGGAATCGCAGATTTCAGCCAAGCTTCGCCAGAGCTACGAAAGTGATTTGCAGATGCTGGCACCGCAAAGCATTATGTTCCCAGGGCTGCACCCCATGGCCATGATGTCCACCGGACGTGGATTCTGCGGCGCTCCTCCACCATTCCCGCCTCCAATATACGCTCCTCTGCCGGGCCAGGGCGGGGCACAGCAGGGAGCTGGCGACTCACAGGAAACAACTTACTTGTACATCCCAAACAATGCCGTGGGCGCCATCATCGGTACGAAGGGCCTGCACATTCGTAACATCATAAGATTCAGCAACGCATCTGTGAAGATAGCTCCTCTGGAGCAAGACAAGCAAGGCGAGAACCAAAGCAATCCCCAACAGGAGCGTAAGGTCACCATTGTTGGCAGCCCTGAGGCTCAATGGAAGGCTCAGTATCTCATCTTCGAGAAGATGCGAGAAGAAGGTTTCATGTCAGGCTCTGATGACGTGCGGCTTACAGTGGAGATAGTGGTGGCTTCATCCCAGGTCGGCCGTATCATCGGCAAAGGCGGGCAAAACGTACGGGAGCTGCAACGCGTAACAGGATCGCTAATTAAGCTTCCCGAACAACCACAGCAGCAGCAGGGCGGCGGGCAGCAACAGGATCACGATACAACAGTGCATATCGTCGGCCCCTTCTACAGCGTGCAGTCGGCGCAGCGACGCATTCGTGCAATGGTTGCGCAGGCGAGCGCACCGGGCCGCCGCCGCGCTGCCCAACCGCCGCCTCCCGTGCAGCAGTAAACTGCCTCCCACATGTTCGAGATCCCCGCGCGCCCTCAAGCACCTCTTCACCCTCAAGCCCTCGCAACACCCCGTCAGCTCGGGCAGCCTAGGCCGCCTGCGGTCGCAGTCACCCAATGCTAGCGTCGTCGCAATGATGCGCTCCATCCACTAAACTGGCTTCATATATGCACATAAGTGTTACACAAAGGATAGACGCTGTCGTAAGTAACGCTTCACTTGAATTTAGTGCAGGCAGGCTGTC(SEQ ID NO：3)。
2、利用CRISPR/Cas9技术敲除sfIMP-X1
采用sgRNAcas9设计软件对sfIMP-X1基因进行sgRNA靶位点扫描，以全基因组为参考序列进行脱靶风险评估，选取得分最高且无脱靶风险且sfIMP-X1特异靶向的N18NGG序列(图1下划线所示，其中Cas9蛋白切割位点以^表示)，位点1序列为：ACCGCTGCTCTCACAGTATGG(PAM序列以粗体表示)(SEQ ID NO：4)，位点2序列为：AAGTGATGAACACCGCTTGGG(PAM序列以粗体表示)(SEQ ID NO：5)；根据上述序列设计2对特异性引物：
IMP-1F:TAATACGACTCACTATAGGACCGCTGCTCTCACAGTA(SEQ ID NO：8)；
IMP-1R:TTCTAGCTCTAAAACTACTGTGAGAGCAGCGGTCC(SEQ ID NO：9)；
IMP-2F:TAATACGACTCACTATAGGAAGTGATGAACACCGCTT(SEQ ID NO：10)；
IMP-2R:TTCTAGCTCTAAAACAAGCGGTGTTCATCACTTCC(SEQ ID NO：11)。
利用Precision gRNA Synthesis Kit(Invitrogen，A29377)试剂盒体外转录成sgRNA。将sgRNA与Cas9蛋白(TrueCut Cas9 Protein v2，Invitrogen，A36498)混合通过显微注射的方式导入草地贪夜蛾受精卵中，sgRNA终浓度为250ng/uL，Cas9蛋白的终浓度为500ng/uL，正常饲养，至出现发育停滞后记录表型，并提取DNA及RNA进行检测。
3、sfIMP-X1敲除个体表型鉴定
观察sfIMP-X1基因敲除个体发育情况，并拍照记录，结果如图2所示。结果显示sfIMP-X1基因敲除个体在幼虫发育过程出现蜕皮或体节发育障碍，所有sfIMP-X1基因缺失个体在蛹前期死亡。
4、sfIMP-X1敲除个体基因型及RNA表达水平检测
根据上述DNA表型观测结果，利用AxyPrep基因组DNA小量制备试剂盒(Axygen，AP-MN-MS-GDNA-250)提取基因组，对sfIMP-X1靶位点进行PCR扩增，PCR检测引物序列如下：
SfIMP-E2F：GAACCCCCTCCCCCTAAAAA(SEQ ID NO：12)；
SfIMP-E2R：CCCCTCGGTACATCTTTGGC(SEQ ID NO：13)。
扩增体系及程序如下：
DreamTaq Green PCR Master Mix(2X)：12.5uL
引物F：1uL
引物R：1uL
DNA：4uL
水：6.5uL
程序：95℃/3min；95℃/30sec，58℃/30sec，72℃/30sec；72℃/5min，扩增34个循环。
对上述片段进行测序，结果如图3所示，在Cas9蛋白切割位点附近检测到片段缺失及插入突变，下划线所示为sgRNA靶位点序列。
利用Trizol法分别提取野生型(WT)与敲除(KO)组幼虫RNA(每组各4头混合，2个重复)，使用反转录试剂盒(TransScript One-step gDNA Removal and cDNA SynthesisSuperMix)，按照其操作说明书对上述总RNA逆转录合成cDNA。并利用qPCR技术检测sfIMP基因表达量变化，所用引物序列如下：
sfimp-QF：CCGCTGCTCTCACAGTATGG(SEQ ID NO：14)；
sfimp-QR：ACTGCCCTCTAACTCGCATC(SEQ ID NO：15)。
qPCR结果如图所示(图4)，sfIMP-X1敲除后与野生型相比其RNA水平表达量显著降低。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 中国农业科学院农业基因组研究所
<120> sfIMP-X1基因及其在草地贪夜蛾遗传防控中的应用
<130> PA20048259
<160> 15
<170> PatentIn version 3.3
<210> 1
<211> 1959
<212> DNA
<213> sfIMP-X1
<400> 1
acgcttcgaa ataagccacc agtcttaaaa gaccattcca attttaacca gtatttaaaa 60
atcgttattg ccgcggtaaa ggaaaccata aaaaagtgaa aatgtctaat tctgtggaac 120
aacaatttag cgagcttagt ttgtcacaag aggaccatga tcaatatgaa ccagaagatc 180
accaagacca aagccggtca tcaaaagttc tcatcagtgg gctacctcta catatccgct 240
tcgacaacat cgaaccgctg ctctcacagt atggcaacgt acaacactgc gacaaagcca 300
actcgcgcga tgccaacacc caagcggtgt tcatcacttt cgagactccg gagcaggcgc 360
agcaagccat caatggtttg aatggatgcg agttagaggg cagtcggatg aaggtagagg 420
cagctgagca aaacacccgt ggcgtcaggc gcggacgccc tggtggcggc cgcggcggcg 480
gcggtgccac cggtggcggc tcgcgaccca ccgacttccc attgcggctc ctcgtccaga 540
gcgacatggt cggcgccatc atcggccgcc agggcagcac catccgcctc attacacaac 600
agagccgtgc acgcgttgat gtccaccgca aggacaacgt tggttctctt gagaaagcta 660
ttaccatata tggtaatcct gagaactgca ctaatgcatg caaaagaata ttggaagtca 720
tgcaacagga agccaacaac accaacaagg gtgagattag cctcaaaatt ttggctcaca 780
ataacctaat cggccgtatt attggcaaag gcggtaacac tattaaaaga atcatgcaag 840
aaactgatac gaagattacc gtctcctcca tcaatgatat caacagtttc aatttggagc 900
gcatcatcac agttaagggc actattgaaa atatggctaa agcggaatcg cagatttcag 960
ccaagcttcg ccagagctac gaaagtgatt tgcagatgct ggcaccgcaa agcattatgt 1020
tcccagggct gcaccccatg gccatgatgt ccaccggacg tggattctgc ggcgctcctc 1080
caccattccc gcctccaata tacgctcctc tgccgggcca gggcggggca cagcagggag 1140
ctggcgactc acaggaaaca acttacttgt acatcccaaa caatgccgtg ggcgccatca 1200
tcggtacgaa gggcctgcac attcgtaaca tcataagatt cagcaacgca tctgtgaaga 1260
tagctcctct ggagcaagac aagcaaggcg agaaccaaag caatccccaa caggagcgta 1320
aggtcaccat tgttggcagc cctgaggctc aatggaaggc tcagtatctc atcttcgaga 1380
agatgcgaga agaaggtttc atgtcaggct ctgatgacgt gcggcttaca gtggagatag 1440
tggtggcttc atcccaggtc ggccgtatca tcggcaaagg cgggcaaaac gtacgggagc 1500
tgcaacgcgt aacaggatcg ctaattaagc ttcccgaaca accacagcag cagcagggcg 1560
gcgggcagca acaggatcac gatacaacag tgcatatcgt cggccccttc tacagcgtgc 1620
agtcggcgca gcgacgcatt cgtgcaatgg ttgcgcaggc gagcgcaccg ggccgccgcc 1680
gcgctgccca accgccgcct cccgtgcagc agtaaactgc ctcccacatg ttcgagatcc 1740
ccgcgcgccc tcaagcacct cttcaccctc aagccctcgc aacaccccgt cagctcgggc 1800
agcctaggcc gcctgcggtc gcagtcaccc aatgctagcg tcgtcgcaat gatgcgctcc 1860
atccactaaa ctggcttcat atatgcacat aagtgttaca caaaggatag acgctgtcgt 1920
aagtaacgct tcacttgaat ttagtgcagg caggctgtc 1959
<210> 2
<211> 537
<212> PRT
<213> sfIMP-X1 氨基酸
<400> 2
Met Ser Asn Ser Val Glu Gln Gln Phe Ser Glu Leu Ser Leu Ser Gln
1 5 10 15
Glu Asp His Asp Gln Tyr Glu Pro Glu Asp His Gln Asp Gln Ser Arg
20 25 30
Ser Ser Lys Val Leu Ile Ser Gly Leu Pro Leu His Ile Arg Phe Asp
35 40 45
Asn Ile Glu Pro Leu Leu Ser Gln Tyr Gly Asn Val Gln His Cys Asp
50 55 60
Lys Ala Asn Ser Arg Asp Ala Asn Thr Gln Ala Val Phe Ile Thr Phe
65 70 75 80
Glu Thr Pro Glu Gln Ala Gln Gln Ala Ile Asn Gly Leu Asn Gly Cys
85 90 95
Glu Leu Glu Gly Ser Arg Met Lys Val Glu Ala Ala Glu Gln Asn Thr
100 105 110
Arg Gly Val Arg Arg Gly Arg Pro Gly Gly Gly Arg Gly Gly Gly Gly
115 120 125
Ala Thr Gly Gly Gly Ser Arg Pro Thr Asp Phe Pro Leu Arg Leu Leu
130 135 140
Val Gln Ser Asp Met Val Gly Ala Ile Ile Gly Arg Gln Gly Ser Thr
145 150 155 160
Ile Arg Leu Ile Thr Gln Gln Ser Arg Ala Arg Val Asp Val His Arg
165 170 175
Lys Asp Asn Val Gly Ser Leu Glu Lys Ala Ile Thr Ile Tyr Gly Asn
180 185 190
Pro Glu Asn Cys Thr Asn Ala Cys Lys Arg Ile Leu Glu Val Met Gln
195 200 205
Gln Glu Ala Asn Asn Thr Asn Lys Gly Glu Ile Ser Leu Lys Ile Leu
210 215 220
Ala His Asn Asn Leu Ile Gly Arg Ile Ile Gly Lys Gly Gly Asn Thr
225 230 235 240
Ile Lys Arg Ile Met Gln Glu Thr Asp Thr Lys Ile Thr Val Ser Ser
245 250 255
Ile Asn Asp Ile Asn Ser Phe Asn Leu Glu Arg Ile Ile Thr Val Lys
260 265 270
Gly Thr Ile Glu Asn Met Ala Lys Ala Glu Ser Gln Ile Ser Ala Lys
275 280 285
Leu Arg Gln Ser Tyr Glu Ser Asp Leu Gln Met Leu Ala Pro Gln Ser
290 295 300
Ile Met Phe Pro Gly Leu His Pro Met Ala Met Met Ser Thr Gly Arg
305 310 315 320
Gly Phe Cys Gly Ala Pro Pro Pro Phe Pro Pro Pro Ile Tyr Ala Pro
325 330 335
Leu Pro Gly Gln Gly Gly Ala Gln Gln Gly Ala Gly Asp Ser Gln Glu
340 345 350
Thr Thr Tyr Leu Tyr Ile Pro Asn Asn Ala Val Gly Ala Ile Ile Gly
355 360 365
Thr Lys Gly Leu His Ile Arg Asn Ile Ile Arg Phe Ser Asn Ala Ser
370 375 380
Val Lys Ile Ala Pro Leu Glu Gln Asp Lys Gln Gly Glu Asn Gln Ser
385 390 395 400
Asn Pro Gln Gln Glu Arg Lys Val Thr Ile Val Gly Ser Pro Glu Ala
405 410 415
Gln Trp Lys Ala Gln Tyr Leu Ile Phe Glu Lys Met Arg Glu Glu Gly
420 425 430
Phe Met Ser Gly Ser Asp Asp Val Arg Leu Thr Val Glu Ile Val Val
435 440 445
Ala Ser Ser Gln Val Gly Arg Ile Ile Gly Lys Gly Gly Gln Asn Val
450 455 460
Arg Glu Leu Gln Arg Val Thr Gly Ser Leu Ile Lys Leu Pro Glu Gln
465 470 475 480
Pro Gln Gln Gln Gln Gly Gly Gly Gln Gln Gln Asp His Asp Thr Thr
485 490 495
Val His Ile Val Gly Pro Phe Tyr Ser Val Gln Ser Ala Gln Arg Arg
500 505 510
Ile Arg Ala Met Val Ala Gln Ala Ser Ala Pro Gly Arg Arg Arg Ala
515 520 525
Ala Gln Pro Pro Pro Pro Val Gln Gln
530 535
<210> 3
<211> 1788
<212> DNA
<213> sfIMP-X2
<400> 3
acgcttcgaa ataagccacc agtcttaaaa gaccattcca attttaacca gtatttaaaa 60
atcgttattg ccgcggtaaa ggaaaccata aaaaagtgaa aatgtctaat tctgtggaac 120
aacaatttag cgagcttagt ttgtcacaag aggaccatga tcaatatgaa ccagaagatc 180
accaagacca aagagccatc aatggtttga atggatgcga gttagagggc agtcggatga 240
aggtagaggc agctgagcaa aacacccgtg gcgtcaggcg cggacgccct ggtggcggcc 300
gcggcggcgg cggtgccacc ggtggcggct cgcgacccac cgacttccca ttgcggctcc 360
tcgtccagag cgacatggtc ggcgccatca tcggccgcca gggcagcacc atccgcctca 420
ttacacaaca gagtcgtgca cgcgttgatg tccaccgcaa ggacaacgtt ggttctcttg 480
agaaagctat taccatatat ggtaatcctg agaactgcac taatgcatgc aaaagaatat 540
tggaagtcat gcaacaggaa gccaacaaca ccaacaaggg tgagattagc ctcaaaattt 600
tggctcacaa taacctaatc ggccgtatta ttggcaaagg cggtaacact attaaaagaa 660
tcatgcaaga aactgatacg aagattaccg tctcctccat caatgatatc aacagtttca 720
atttggagcg catcatcaca gttaagggca ctattgaaaa tatggctaaa gcggaatcgc 780
agatttcagc caagcttcgc cagagctacg aaagtgattt gcagatgctg gcaccgcaaa 840
gcattatgtt cccagggctg caccccatgg ccatgatgtc caccggacgt ggattctgcg 900
gcgctcctcc accattcccg cctccaatat acgctcctct gccgggccag ggcggggcac 960
agcagggagc tggcgactca caggaaacaa cttacttgta catcccaaac aatgccgtgg 1020
gcgccatcat cggtacgaag ggcctgcaca ttcgtaacat cataagattc agcaacgcat 1080
ctgtgaagat agctcctctg gagcaagaca agcaaggcga gaaccaaagc aatccccaac 1140
aggagcgtaa ggtcaccatt gttggcagcc ctgaggctca atggaaggct cagtatctca 1200
tcttcgagaa gatgcgagaa gaaggtttca tgtcaggctc tgatgacgtg cggcttacag 1260
tggagatagt ggtggcttca tcccaggtcg gccgtatcat cggcaaaggc gggcaaaacg 1320
tacgggagct gcaacgcgta acaggatcgc taattaagct tcccgaacaa ccacagcagc 1380
agcagggcgg cgggcagcaa caggatcacg atacaacagt gcatatcgtc ggccccttct 1440
acagcgtgca gtcggcgcag cgacgcattc gtgcaatggt tgcgcaggcg agcgcaccgg 1500
gccgccgccg cgctgcccaa ccgccgcctc ccgtgcagca gtaaactgcc tcccacatgt 1560
tcgagatccc cgcgcgccct caagcacctc ttcaccctca agccctcgca acaccccgtc 1620
agctcgggca gcctaggccg cctgcggtcg cagtcaccca atgctagcgt cgtcgcaatg 1680
atgcgctcca tccactaaac tggcttcata tatgcacata agtgttacac aaaggataga 1740
cgctgtcgta agtaacgctt cacttgaatt tagtgcaggc aggctgtc 1788
<210> 4
<211> 21
<212> DNA
<213> 人工序列
<400> 4
accgctgctc tcacagtatg g 21
<210> 5
<211> 21
<212> DNA
<213> 人工序列
<400> 5
aagtgatgaa caccgcttgg g 21
<210> 6
<211> 20
<212> DNA
<213> 人工序列
<400> 6
acgcttcgaa ataagccacc 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列
<400> 7
gacagcctgc ctgcactaaa 20
<210> 8
<211> 37
<212> DNA
<213> 人工序列
<400> 8
taatacgact cactatagga ccgctgctct cacagta 37
<210> 9
<211> 35
<212> DNA
<213> 人工序列
<400> 9
ttctagctct aaaactactg tgagagcagc ggtcc 35
<210> 10
<211> 37
<212> DNA
<213> 人工序列
<400> 10
taatacgact cactatagga agtgatgaac accgctt 37
<210> 11
<211> 35
<212> DNA
<213> 人工序列
<400> 11
ttctagctct aaaacaagcg gtgttcatca cttcc 35
<210> 12
<211> 20
<212> DNA
<213> 人工序列
<400> 12
gaaccccctc cccctaaaaa 20
<210> 13
<211> 20
<212> DNA
<213> 人工序列
<400> 13
cccctcggta catctttggc 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列
<400> 14
ccgctgctct cacagtatgg 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列
<400> 15
actgccctct aactcgcatc 20

Claims (6)

1.sfIMP-X1基因或其编码的蛋白在草地贪夜蛾遗传防控中的应用，其特征在于，所述应用为采用基因敲除抑制所述基因的表达或使其功能缺失来影响草地贪夜蛾幼虫正常蜕皮或体节发育；
所述sfIMP-X1基因的核苷酸序列如SEQ ID NO：1所示。
2.根据权利要求1所述的应用，其特征在于，所述基因敲除使用ZFNs、TALENs或CRISPR/Cas9系统进行。
3.根据权利要求2所述的应用，其特征在于，所述CRISPR/Cas9系统包括Cas9和特异性靶向sfIMP-X1基因的sgRNA。
4.根据权利要求3所述的应用，其特征在于，特异性靶向sfIMP-X1基因的sgRNA的核苷酸引物序列如SEQ ID NO：4和SEQ ID NO：5所示。
5.根据权利要求4所述的应用，其特征在于，将所述sgRNA与Cas9蛋白通过显微注射的方式导入草地贪夜蛾受精卵中进行基因敲除。
6.一种防治草地贪夜蛾的方法，其特征在于，所述方法为采用基因敲除抑制所述草地贪夜蛾体内sfIMP-X1基因的表达或活性；所述sfIMP-X1基因的核苷酸序列如SEQ ID NO：1所示。
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