CN108610427A - 飞蝗滞育激素基因及其在调控昆虫滞育中的应用 - Google Patents
飞蝗滞育激素基因及其在调控昆虫滞育中的应用 Download PDFInfo
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Abstract
本发明公开了飞蝗滞育激素基因及其在调控昆虫滞育中的应用。本发明克隆了滞育激素基因，并开发了滞育激素基因的特异性双链RNA干扰序列片段dsRNA，且运用注射法将dsRNA导入飞蝗对滞育激素基因进行RNAi。通过实验证明：利用滞育激素基因的特异性双链RNA干扰片段dsRNA成功实现干扰飞蝗相应基因的mRNA表达水平，且干扰效率显著，干扰后降低母代飞蝗生产的蝗卵的滞育率，说明滞育激素基因在昆虫滞育诱导过程中起着重要作用，本发明为开发新型杀虫剂、降低越冬后害虫种群数量提供基础。
Description
技术领域
本发明属于生物技术领域，具体涉及飞蝗滞育激素基因及其在调控昆虫滞育中的应用。
背景技术
蝗虫是我国历史上重大农业害虫，它具有种类多、影响大、并时常暴发成灾的特点，其发生范围和严重程度对我国国民经济、特别是农业生产常造成重大损失。近年来，随着全球气候变暖趋势增加，高密度蝗群时有发生，远距离迁飞风险加剧，严重威胁我国农业主产区的粮食生产，传统病虫害防治多以应急防治和化学防治为主，对病虫害防治过分依赖化学农药，急需前瞻性开发新型绿色无公害防控药剂。
滞育是昆虫长期适应环境周期性变化而进化出的遗传性生理反应，在不良环境来临之前发育受到抑制的生理状态。蝗虫卵滞育属于兼性滞育类型，为母代接受低温、短光照等环境信息诱导的卵滞育，胚胎发育停滞在胚体上升末期、胚转前。滞育激素(diapausehormone，dh)由昆虫脑和咽下神经节分泌，作用于靶器官卵巢上的G蛋白偶联受体，通过鸟苷酸环化酶使环鸟苷酸合成受阻，提高了卵巢膜上海藻糖酶活性，促进昆虫血淋巴海藻糖分解成葡萄糖，葡萄糖进入卵巢后合成糖原积累在卵中，最终在卵滞育准备阶段转化为山梨醇和甘油，作为防冻剂为越冬虫卵提供保护。滞育激素能够促进3-羟基犬尿氨酸从血淋巴转移到滞育卵浆膜中积累成眼色素。滞育激素能轻微提高昆虫卵中的脂含量，还可以抑制酯酶A，从而阻止卵黄细胞溶胞，确保胚胎发育。
发明内容
本发明要解决的技术问题是如何调控昆虫滞育。
为了解决上述技术问题，本发明首先提供了调控昆虫滞育的滞育激素蛋白，均来源于飞蝗，是如下a)或b)或c)或d)的蛋白质：
a)氨基酸序列是序列2或序列4或序列6或序列8或序列10所示的蛋白质；
b)在序列2或序列4或序列6或序列8或序列10所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质；
c)将序列2或序列4或序列6或序列8或序列10所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
d)与序列2或序列4或序列6或序列8或序列10所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的蛋白质。
为了使a)中的蛋白质便于纯化，可在序列表中序列2或序列4或序列6或序列8或序列10所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1、标签的序列
| 标签 | 残基 | 序列 |
| Poly-Arg | 5-6(通常为5个) | RRRRR |
| Poly-His | 2-10(通常为6个) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
上述c)中的蛋白质，所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述c)中的蛋白质可人工合成，也可先合成其编码基因，再进行生物表达得到。
上述c)中的蛋白质的编码基因可通过将序列1或序列3或序列5或序列7或序列9所示的DNA序列中缺失一个或几个氨基酸残基的密码子，和/或进行一个或几个碱基对的错义突变，和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
为了解决上述问题，本发明还提供了与上述蛋白质相关的生物材料。
本发明提供的与上述蛋白质相关的生物材料为下述A1)至A8)中的任一种：
A1)编码上述蛋白质的核酸分子；
A2)含有A1)所述核酸分子的表达盒；
A3)含有A1)所述核酸分子的重组载体；
A4)含有A2)所述表达盒的重组载体；
A5)含有A1)所述核酸分子的重组微生物；
A6)含有A2)所述表达盒的重组微生物；
A7)含有A3)所述重组载体的重组微生物；
A8)含有A4)所述重组载体的重组微生物。
上述生物材料中，A1)所述核酸分子为如下1)或2)或3)所示的基因：
1)其编码序列是序列1或序列3或序列5或序列7或序列9所示的cDNA分子或基因组DNA分子；
2)与1)限定的核苷酸序列具有75％或75％以上同一性，且编码上述蛋白质的cDNA分子或基因组DNA分子；
3)在严格条件下与1)或2)限定的核苷酸序列杂交，且编码上述蛋白质的cDNA分子或基因组DNA分子。
其中，所述核酸分子可以是DNA，如cDNA、基因组DNA或重组DNA；所述核酸分子也可以是RNA，如mRNA或hnRNA等。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明的编码上述蛋白质的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明分离得到的蛋白质的核苷酸序列75％或者更高同一性的核苷酸，只要编码上述蛋白质且具有相同功能，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列2或序列4或序列6或序列8或序列10所示的氨基酸序列组成的蛋白质的核苷酸序列具有75％或更高，或85％或更高，或90％或更高，或95％或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件，两个或多个序列之间的同一性可以用百分比(％)表示，其可以用来评价相关序列之间的同一性。
上述75％或75％以上同一性，可为80％、85％、90％或95％以上的同一性。
为了解决上述技术问题，本发明还提供了上述蛋白质或生物材料的新用途。
本发明提供了上述蛋白质或生物材料在如下a1)-a10)中任一种中的应用：
a1)调控昆虫滞育；
a2)制备调控昆虫滞育的产品；
a3)防治害虫；
a4)制备防治害虫的产品；
a5)降低昆虫滞育率；
a6)制备降低昆虫滞育率的产品；
a7)降低昆虫越冬存活率；
a8)制备降低昆虫越冬存活率的产品；
a9)降低昆虫越冬后种群数量；
a10)制备降低越冬后种群数量的产品。
为了解决上述技术问题，本发明还提供了一种降低昆虫滞育率的方法。
本发明提供的降低昆虫滞育率的方法包括降低昆虫中上述蛋白质的表达量和/或活性的步骤，从而实现降低昆虫滞育率。
上述方法中，所述降低昆虫中上述蛋白质的表达量和/或活性的方法是将抑制昆虫中上述蛋白质的编码基因表达的物质导入昆虫；
所述抑制昆虫中上述蛋白质的编码基因表达的物质为抑制昆虫中上述蛋白质的编码基因表达的dsRNA；
所述抑制昆虫中上述蛋白质的编码基因表达的dsRNA为由序列表中序列11或序列12或序列13所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA；
所述导入的方式可为注射。
上述方法在防治害虫中的应用也属于本发明的保护范围。
为了解决上述技术问题，本发明最后还提供了抑制上述蛋白质的编码基因表达的物质。
本发明提供的抑制上述蛋白质的编码基因表达的物质为由序列表中序列11或序列12或序列13所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA。
抑制上述蛋白质的编码基因表达的物质在防治害虫或降低昆虫滞育率中的应用也属于本发明的保护范围。
上述应用或方法中：所述昆虫或害虫为飞蝗。
本发明从飞蝗中克隆得到五个滞育激素基因，并制备得到相应基因的特异性双链RNA干扰序列片段dsRNA，且运用注射法将dsRNA导入飞蝗对飞蝗滞育激素基因进行RNAi。通过实验证明：利用滞育激素基因的特异性双链RNA干扰片段dsRNA成功实现干扰飞蝗相应基因的mRNA表达水平，干扰效率显著，且干扰后显著降低了母代飞蝗生产的蝗卵的滞育率，说明滞育激素基因在昆虫滞育诱导过程中起着重要作用，本发明为开发新型杀虫剂、降低越冬后害虫种群数量提供基础。
附图说明
图1为飞蝗dh基因全长的PCR产物琼脂糖凝胶电泳图。
图2为实验组和对照组中目的基因的mRNA相对表达水平。
图3为实验组和对照组的滞育率统计结果。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的供试虫源：在实验室内连续饲养20代的飞蝗纯化种群，没有接触过任何化学药剂。饲养过程如下：将采集于天津黄骅的飞蝗蝗卵在智能人工气候箱中孵化，孵化条件如下：湿度60％，光照时间:黑暗时间＝14h:10h。将同一时间孵化大小一致的蝗蝻转移到养虫笼(60cm×50cm×60cm)中用新鲜的麦苗进行饲养，直至成虫。饲养条件如下：光照时间:黑暗时间＝14h:10h，温度30±2℃，相对湿度60％。
下述实施例中的主要试剂：RNA分离试剂(invitrogen原装)，PrimeScriptTMII 1st Strand cDNA Synthesis Kit(takara)，DL2000DNA marker(takara)，RNA spin column(全式金)，GST Resin(全式金)，Trans1-T1PhageRsistant Chemically Competent Cell(全式金)，无核酸酶水(Ambion)，胶回收试剂盒(Axygen)，少量质粒提取试剂盒(Axygen)，EX Taq DNA聚合酶(takara)，SYBR Premix ExTaq II(Tli RNaseH Plus)(takara)，pGEM-T Easy Vector Systems(promega)，T7RiboMAXTMExpress RNAi System(promega)，无水乙醇、异丙醇、丙三醇等试剂为国产分析纯。
下述实施例中滞育激素基因的测序、制作滞育激素基因的dsRNA、以及滞育激素基因的rt-PCR引物均由上海生工生物工程股份有限公司合成。
下述实施例中所用到的特异性引物序列如表1所示。
表1、引物序列及其作用
下述实施例中的主要仪器：超净工作台(上海博讯实业有限公司)、东胜龙ETC-811PCR仪(北京东胜创新生物科技有限公司)、德国Sigma 3K15冷冻离心机(德国希格玛离心机有限公司)、NanoPhotometer微量分光光度计(德国IMPLEN公司)、HPX-9052MBE数显电热培养箱(上海博讯实业有限公司)、THZ-D台式恒温振荡器(华美生化仪器厂)、漩涡振荡器QL-901(海门市其林贝尔仪器制造)、高压灭菌锅YXQ-LS-50SII(上海博讯实业有限公司医疗设备厂)。
实施例1、飞蝗滞育激素基因的克隆
1、飞蝗总RNA的提取
用RNA分离试剂提取飞蝗组织样品的RNA。具体步骤如下：
1)2mL匀浆器放于烘箱中，160℃，3个小时，冷却至室温备用。
2)将匀浆器置于冰上，加入1mLRNA分离试剂和100-200mg飞蝗组织，研磨。
3)将匀浆液转移至1.5mL离心管中，室温静置5min。4℃，13000r离心5min。
4)将上清转移至干净的1.5mL离心管中，加入200μL氯仿，漩涡震荡15s。室温放置5min。4℃，13000r离心10min。
5)吸取400μL上清转移至新的1.5mL离心管中，加入200μL氯仿，漩涡震荡30s。室温放置5min。4℃，13000r离心10min。
6)吸取300μL上清，加入300μL异丙醇，漩涡震荡30s，转移至RNA spin column中，冰上静置10min。
7)4℃，13000r离心2min，弃滤液。
8)加入600μL RNA washsolution，吸打使沉降物洗涤，4℃，13000r离心2min，弃滤液。再次加入600μL RNA washsolution，吸打使沉降物洗涤，4℃，13000r离心2min，弃滤液。4℃，13000r空离3min，除去多余乙醇，空气吹干3min。
9)更换一个新的收集管，向RNA spin column中加入50μL 65℃预热的RNase-free-water，余热下热置5min，4℃，13000r离心3min。
10)收集滤出液，用NanoPhotometer微量分光光度计检测RNA浓度和OD260/280值，确认RNA质量。同时，取2μL提取的RNA，琼脂糖凝胶电泳检测，剩余RNA储存于-20℃备用。
2、反转录
用PrimeScriptTM 1st strand cDNA Synthesis Kit反转录试剂盒反转录获得cDNA。具体步骤如下：
1)配置10μL体系：Oligo dT Primer(50μM)1μL、dNTP Mixture(10mM each)1μL、total RNA<5μL、RNase Free dH2O补足体系至10μL。
2)65℃保温5min后，冰上迅速冷却。
3)配置20μL反应液：5×PrimeScript Buffer 4μL、RNase Inhibitor(400U/μL)0.5μL(20units)、PrimeScript RTase(200U/μL)1μL(200units)、RNase Free dH2O补足体系至20μL。
4)缓慢混匀。
5)42℃保温30-60min。
6)95℃保温5min，使酶失活，冰上放置，-20℃保存cDNA。
3、飞蝗滞育激素基因的PCR扩增
1)引物设计
根据前期获得的飞蝗转录组得到的飞蝗滞育激素基因的序列，利用DNAMAN8设计飞蝗滞育激素基因全长引物或片段引物。设计的引物如表1所示。
2)PCR反应
以飞蝗cDNA为模板，采用表1中的对应引物分别进行PCR扩增，得到PCR产物。
PCR反应体系如下(总体积50μL)：cDNA模板1μL、dNTP 4μL、10×Buffer 5μL、前引物1μL、后引物1μL、Taq酶0.25μL、ddH2O 38μL。
PCR反应条件如下：95℃3min；95℃30s，55℃30s，72℃1min 30s，35个循环；72℃10min；4℃保存。
3)PCR产物回收、克隆、测序
3-1)将PCR产物在TAE配制的1％的琼脂糖凝胶上进行电泳，当目标带分离较好时，用刀片切下目标带所在的胶块放入无菌的离心管中。再用胶回收试剂盒(Axygen)回收纯化目标带，回收纯化过程参照试剂盒说明书进行。飞蝗dh基因全长的PCR产物琼脂糖凝胶电泳图如图1所示。
3-2)PCR产物回收后连接pGEM-T Easy载体，得到重组载体。连接体系如下：T4DNAligase 1μL、2×buffer 5μL、pGEM-T Easy 1μL、PCR回收产物3μL。连接条件：室温下连接6个小时。
3-3)感受态细胞的制备与转化
在1.5mL离心管中，加入33.3μL Trans1-t1感受态细胞，冰上放置15min，42℃水浴90s，冰上放置10min。在每个1.5mL离心管中加入500μL的液体LB培养液，37℃，200rpm摇菌2h。摇菌后，吸取100μL菌液至1‰AMP LB固体培养基中，37℃培养过夜。挑选单菌落于2mL的离心管中，管中有1mL的1‰AMP LB液体培养基。37℃，200rpm摇菌3-6h，观察生长情况。
3-4)菌液PCR
对3-3)中的菌液进行PCR验证，菌液PCR反应体系(总体积50μL)如下：菌液1μL、dNTP 4μL、10×Buffer 5μL、前引物1μL、后引物1μL、Taq酶0.25μL、ddH2O 38μL。
反应条件如下：95℃3min；95℃30s，55℃30s，72℃1min 30s，35个循环；72℃10min；4℃保存。
将阳性克隆菌株送到上海生工生物工程技术服务有限公司进行序列测定并对测序结果进行分析。
测序结果表明：dh1F/dh1R引物PCR扩增得到大小为789bp的DNA片段，其核苷酸序列如序列1所示，将序列1所示的基因命名为飞蝗滞育激素基因dh1，飞蝗滞育激素基因dh1编码的蛋白的氨基酸序列如序列2所示。dh2/3F/dh2/3R引物PCR扩增得到大小为1512bp和1767bp的DNA片段，其核苷酸序列分别如序列3和序列5所示，将序列3所示的基因命名为飞蝗滞育激素基因dh2，飞蝗滞育激素基因dh2编码的蛋白的氨基酸序列如序列4所示；将序列5所示的基因命名为飞蝗滞育激素基因dh3，飞蝗滞育激素基因dh3编码的蛋白的氨基酸序列如序列6所示。dh4/5F/dh4/5R引物PCR扩增得到大小为732bp和1092bp的DNA片段，其核苷酸序列分别如序列7和序列9所示，将序列7所示的基因命名为飞蝗滞育激素基因dh4，飞蝗滞育激素基因dh4编码的蛋白的氨基酸序列如将序列8所示，将序列9所示的基因命名为飞蝗滞育激素基因dh5，飞蝗滞育激素基因dh5编码的蛋白的氨基酸序列如序列10。
实施例2、飞蝗滞育激素基因的dsRNA的制备及其应用
一、飞蝗滞育激素基因的dsRNA的制备
采用T7RiboMAXTM Express RNAi System试剂盒合成dsRNA。具体步骤如下：
1、dsRNA引物的合成
根据已克隆出的基因片段设计引物，扩增目的片段为800-1000bp左右，在引物的5'端引入T7启动子。引物序列如表1所示。
2、DNA模板的制备
用试剂盒分别提取菌液质粒，分别以含目的基因片段的质粒(实施例1中的重组载体)为模板，采用对应的引物进行PCR扩增，分别得到含有T7启动子序列的目的片段。
PCR反应体系如下(总体积50μL)：质粒1μL、dNTP 4μL、10×Buffer 5μL、前引物1μL、后引物1μL、Taq酶0.25μL、ddH2O 38μL。
PCR反应条件如下：95℃3min；95℃30s，55℃30s，72℃1min，35个循环；72℃10min；4℃保存。
回收PCR产物，并用NanoPhotometer微量分光光度计检测目标DNA浓度，回收浓度需大于150ng/μL。
3、dsRNA的合成
采用T7RiboMAX TM Express RNAi System试剂盒将回收的DNA体外转录合成飞蝗滞育激素基因的dsRNA，并用NanoPhotometer微量分光光度计检测dsRNA浓度，dsRNA浓度需大于1000ng/μL。再用RNase free ddH2O将dsRNA浓度调整至1μg/μL，得到dsRNA溶液。
本发明获得的飞蝗滞育激素基因的dsRNA均为双链RNA，由正义链和反义链组成。
飞蝗滞育激素基因dh1的dsRNA的正义链的核苷酸序列为序列11，其反义链的核苷酸序列为序列11的反向互补序列。将飞蝗滞育激素基因dh1的dsRNA命名为dsdh1。
飞蝗滞育激素基因dh2和dh3的dsRNA的正义链的核苷酸序列为序列12，其反义链的核苷酸序列为序列12的反向互补序列。将飞蝗滞育激素基因dh2和dh3的dsRNA命名为dsdh2/3。
飞蝗滞育激素基因dh4和dh5的dsRNA的正义链的核苷酸序列为序列13，其反义链的核苷酸序列为序列13的反向互补序列。将飞蝗滞育激素基因dh4和dh5的dsRNA命名为dsdh4/5。
上述飞蝗滞育激素基因dh1、dh2、dh3、dh4和dh5的dsRNA也可通过人工合成的方法获得。
4、对照GFP的dsRNA
按照上述方法合成对照GFP的dsRNA，将对照GFP的dsRNA命名为dsGFP，得到浓度为1μg/μL的dsGFP溶液。对照GFP的dsRNA为双链RNA，由正义链和反义链组成，其正义链的核苷酸序列为序列14，其反义链的核苷酸序列为序列14的反向互补序列。合成GFP dsRNA的引物如下：
GFP-1F：5’-TAATACGACTCACTATAGGTACGACTCACTATAGGAGTAAAGG-3’；
GFP-1R：5’-TAATACGACTCACTATAGGTAGGTTTGTATAGTTCATCCATACC-3’。
二、飞蝗滞育激素基因的dsRNA在调控昆虫滞育中的应用
1、实验方法
将dsRNA导入蝗虫体内。具体步骤如下：选取发育进度一致(羽化48h内)的飞蝗雌成虫进行微量注射，将相应的dsRNA用微量注射器从蝗虫腹部的第二和第三腹节之间的节间膜注射进入，注射器的针头和腹部平行，避免损伤蝗虫的内部器官组织。注射剂量为每头蝗虫注射10μL浓度为1μg/μL的dsRNA(dsdh1、dsdh2/3、dsdh4/5)或dsGFP或dsdhall(等摩尔比混合dsdh1、dsdh2/3和dsdh4/5)，同时以注射RNase free ddH2O(water)和不注射(blankcontrol)作为对照组。每个处理5个重复，每个重复5头飞蝗雌成虫。注射dsRNA后将所有处理的飞蝗雌成虫混入等量的雄成虫，放入恒温气候箱中饲养。饲养条件：光照时间:黑暗时间＝10h:14h，温度28±1℃，相对湿度60％。
2、实时荧光定量PCR检测干扰效率
注射处理48h后取样，提取总RNA并反转录，得到cDNA。利用荧光定量PCR检测各处理组飞蝗中相应滞育激素基因的相对mRNA水平，注射dsGFP处理为对照。荧光定量采用takara公司的SYBR Premix Ex Taq II(Tli RNaseH Plus)和ABI公司的ABI7500荧光定量PCR仪，结果采用2-ΔΔCT法进行分析。以actin基因作为内参基因。引物序列如表1所示。
荧光定量pcr对RNA干扰效率检测结果如图2所示。从图中可看出：注射dsdh1处理比注射dsGFP处理中dh1相对表达量降低了58.08％；注射dsdh2/3处理比注射dsGFP处理中dh2和dh3相对表达量降低了74.83％；注射dsdh4/5处理比注射dsGFP处理中dh4和dh5相对表达量降低了85.36％；注射dsdhall处理比注射dsGFP处理中dh1相对表达量降低了90.46％，dh2和dh3相对表达量降低了78.47％，dh4和dh5相对表达量降低了88.46％。mRNA相对水平差异的显著性均用Student's t-test检验，结果表明，所有注射了dsRNA的处理比注射RNase free ddH2O处理和注射dsGFP处理中相应基因的mRNA均显著下降(p<0.01)，干扰效果良好。
3、飞蝗滞育率统计
注射处理一周后飞蝗开始交配，再过一周后雌蝗开始产卵，在培养笼底垫10厘米厚的沙土供飞蝗产卵，沙土相对湿度20％。自产卵开始，每两天收集一次所产下的蝗卵，转移至用含有蛭石的孵化盒中，相对湿度20％。然后将孵化盒培养于另一个恒温气候箱中，孵化温度为28℃。约3-4周后，幼虫开始孵化，记录幼虫数量(A)。继续孵化约20天直到不再有幼虫孵化，再继续培养一周。将剩余未孵化的蝗卵从蛭石中挖出，用相对湿度20％的沙土埋入孵化盒中，转移至4℃冰箱保存30天。之后再次用相对湿度20％的蛭石埋入孵化盒，转移至恒温气候箱中孵化，孵化温度为28℃，直至所有幼虫孵化，记录幼虫数量(B)。计算蝗卵滞育率＝B/(A+B)。
RNA干扰后对飞蝗产的蝗卵滞育率的统计结果如图3所示。从图中可看出：注射RNase free ddH2O处理的平均滞育率为91.99％；未注射处理的平均滞育率为82.00％；注射dsGFP处理的平均滞育率为78.39％；注射dsdh1处理的平均滞育率为70.34％，比注射dsGFP处理的平均滞育率降低了8.05％；注射dsdh2/3处理的平均滞育率为35.84％，比注射dsGFP处理的平均滞育率降低了42.55％；注射dsdh4/5处理的平均滞育率为30.27％，比注射dsGFP处理的平均滞育率降低了48.12％；注射dsdhall处理的平均滞育率为42.59％，比注射dsGFP处理的平均滞育率降低了35.80％。通过单因素ANOVA的Duncan’s multiple-range test检验统计滞育率差异显著性，结果表明注射dsdh1、dsdh2/3、dsdh4/5和dsdhall处理，与对照注射dsGFP、注射RNase free ddH2O处理和未注射处理相比，滞育率均显著下降，p<0.001，F＝117.89。
以上结果表明，蝗虫有dh1、dh2、dh3、dh4和dh5五条滞育激素基因，RNA干扰这些基因后使蝗虫卵滞育率显著下降，说明利用滞育激素基因的dsRNA能有效的抑制蝗虫卵滞育率，降低其越冬存活率和越冬后种群数量，从而控制蝗灾爆发。
序列表
<110>中国农业科学院植物保护研究所
<120>飞蝗滞育激素基因及其在调控昆虫滞育中的应用
<160>14
<170>PatentIn version 3.5
<210>1
<211>789
<212>DNA
<213>人工序列(Artificial Sequence)
<400>1
atgccgagag gtgctcagct attcctgttg ctggcgctgg tcaccgctgc acgagtcctc 60
gacgcgaggg cggagtcctc acgggcggga agcgatgcgc agtcttcaca tcaggacggc 120
aggggagctt cacacgacag gaacgaagaa agcaacgagc tgaacgatga aaatcgttcc 180
gacggtgacg cccaagacgc aacgtttcgc agaggacgag agatgcaaga gttcgtttcg 240
catgctggtg gaccgattgg gaacggtgag gaagaccagt ggttggcact ggcggacgga 300
agttacatcc ctgccgaggc tgtgcgagaa ctcgtccagc ggggacctac tctaagcgag 360
aacacaggct tgtggtttgg acctcgttac ggacgacgaa cgagctgcgg cgagaacgtc 420
ccccttaagt ggttgtccca agtggagaag cgggcggcca aacagccagc gctgtggttc 480
ggtccgcgag tgggtcgcag tctcgacgaa gaacccaagg gcgaagaatg gagggacgac 540
gataagggac tcaaggacgg tagttcgcag cgccaagata ggagtgccca acctccgggt 600
ctgtggttcg gaccccgggt cggccggcgc agcgatgccc aggtcgacga catgctgtgg 660
ttcggacccc gacccggacg aagtgtggat actgacaagc aggatctgta cgacgatgag 720
gtcgctatgc gtgatcagcg gggtgccaaa catcctggtt tgtggttcgg gcctcgcttc 780
ggccgttaa 789
<210>2
<211>262
<212>PRT
<213>人工序列(Artificial Sequence)
<400>2
Met Pro Arg Gly Ala Gln Leu Phe Leu Leu Leu Ala Leu Val Thr Ala
1 5 10 15
Ala Arg Val Leu Asp Ala Arg Ala Glu Ser Ser Arg Ala Gly Ser Asp
20 25 30
Ala Gln Ser Ser His Gln Asp Gly Arg Gly Ala Ser His Asp Arg Asn
35 40 45
Glu Glu Ser Asn Glu Leu Asn Asp Glu Asn Arg Ser Asp Gly Asp Ala
50 55 60
Gln Asp Ala Thr Phe Arg Arg Gly Arg Glu Met Gln Glu Phe Val Ser
65 70 75 80
His Ala Gly Gly Pro Ile Gly Asn Gly Glu Glu Asp Gln Trp Leu Ala
85 90 95
Leu Ala Asp Gly Ser Tyr Ile Pro Ala Glu Ala Val Arg Glu Leu Val
100 105 110
Gln Arg Gly Pro Thr Leu Ser Glu Asn Thr Gly Leu Trp Phe Gly Pro
115 120 125
Arg Tyr Gly Arg Arg Thr Ser Cys Gly Glu Asn Val Pro Leu Lys Trp
130 135 140
Leu Ser Gln Val Glu Lys Arg Ala Ala Lys Gln Pro Ala Leu Trp Phe
145 150 155 160
Gly Pro Arg Val Gly Arg Ser Leu Asp Glu Glu Pro Lys Gly Glu Glu
165 170 175
Trp Arg Asp Asp Asp Lys Gly Leu Lys Asp Gly Ser Ser Gln Arg Gln
180 185 190
Asp Arg Ser Ala Gln Pro Pro Gly Leu Trp Phe Gly Pro Arg Val Gly
195 200 205
Arg Arg Ser Asp Ala Gln Val Asp Asp Met Leu Trp Phe Gly Pro Arg
210 215 220
Pro Gly Arg Ser Val Asp Thr Asp Lys Gln Asp Leu Tyr Asp Asp Glu
225 230 235 240
Val Ala Met Arg Asp Gln Arg Gly Ala Lys His Pro Gly Leu Trp Phe
245 250 255
Gly Pro Arg Phe Gly Arg
260
<210>3
<211>1512
<212>DNA
<213>人工序列(Artificial Sequence)
<400>3
atgctgagaa gaacacattc gtcgcttgca gttgctgtaa cagtggttgc catttccgcg 60
ttcgctgtat ccgcagcaaa accacgagaa atgcacagca agaatgtggt ggttcagcga 120
tgcatccaaa ctggtttttc tgatggcggc agcatccgca atgtacctga aacttctctt 180
tggttcggcc cccggatagg gcgcagcaac ttagaaactg ctttgttgtt tggacgttgt 240
gtggggtgta gccatccagg aaccagtcag tgttgtgaac cacttgcacg acgcagcggc 300
tctgaagata gtttgggata tagtagcccc gaaccaccgc acgacaacag aaagatcagt 360
cctgaatctg gtttgtggtt tgtagataga aatggtcgca gacaaccgga cactgctttg 420
tggtttggtc cacgtgtagg acgtcgtatg cagcacatcc aaccagaagc cagtgagtat 480
tttagaccag atgtacgacg cagtggtcct gaacatagtt tatggagtga tactcagcta 540
cgacagggcg atccagaacc accacttcat gttagaataa gcaatcctaa agaaaatttg 600
tggtttcgac ccgacactcg tcgcagacaa tcagaaaata tcctgcagtt tggtccacag 660
gtaggacgaa gaaactcaga aagtgccctg tggtttgggc ctcgtgtggg acgcagctat 720
ctagaaagta atctgtcaga tgagcgatat ggacagcata gaaatcttga acctggattg 780
tggtctaata atttgggaca aatccaaaaa acaccatcta tggatgtgac aaacagattt 840
cctggacgtg atttgtggtc tggaactgat ctcgaccgta cgaaacacga aaccgcgcta 900
tggtttgtac cacggatagg acgaagcaac ccagaaacca atttgtggtt tggacctcgt 960
gtgggacgta gtcatccaga aaccagtcag tcatttggac agtatgcacg acacggcaat 1020
gttgaaactg gtttatggcc tgatgatttt gttcaaagcc gcccaacaac gttggaggat 1080
gtcactaaaa gaaatcctga acgtaattgg tggtctgcaa atggtgttgg acgtacgaaa 1140
cacgaaacca cactatggtt cggaccacgg ataggacgca gcaatccaga aattaatttg 1200
tggtttggac ctcgtgtggg acgtagccat cctgaaaaca gtcagtcatt tggaccgtat 1260
tcacgccgca acaatgttga aagtggtttg tggcctgata atcttggtca aagacgccca 1320
acaacgtggg aggatgtcac taaaagatat cctggacaca attggtggtc tgttaatggt 1380
cttggacgta tgaaacacga aaccgcacta tggtttggac cacgaatagg acgcagcaac 1440
ccagatacgc atttatggtt tggacctcgt gtgggacgaa gccaatctct agcaagcgag 1500
cagttgcgct ag 1512
<210>4
<211>503
<212>PRT
<213>人工序列(Artificial Sequence)
<400>4
Met Leu Arg Arg Thr His Ser Ser Leu Ala Val Ala Val Thr Val Val
1 5 10 15
Ala Ile Ser Ala Phe Ala Val Ser Ala Ala Lys Pro Arg Glu Met His
20 25 30
Ser Lys Asn Val Val Val Gln Arg Cys Ile Gln Thr Gly Phe Ser Asp
35 40 45
Gly Gly Ser Ile Arg Asn Val Pro Glu Thr Ser Leu Trp Phe Gly Pro
50 55 60
Arg Ile Gly Arg Ser Asn Leu Glu Thr Ala Leu Leu Phe Gly Arg Cys
65 70 75 80
Val Gly Cys Ser His Pro Gly Thr Ser Gln Cys Cys Glu Pro Leu Ala
85 90 95
Arg Arg Ser Gly Ser Glu Asp Ser Leu Gly Tyr Ser Ser Pro Glu Pro
100 105 110
Pro His Asp Asn Arg Lys Ile Ser Pro Glu Ser Gly Leu Trp Phe Val
115 120 125
Asp Arg Asn Gly Arg Arg Gln Pro Asp Thr Ala Leu Trp Phe Gly Pro
130 135 140
Arg Val Gly Arg Arg Met Gln His Ile Gln Pro Glu Ala Ser Glu Tyr
145 150 155 160
Phe Arg Pro Asp Val Arg Arg Ser Gly Pro Glu His Ser Leu Trp Ser
165 170 175
Asp Thr Gln Leu Arg Gln Gly Asp Pro Glu Pro Pro Leu His Val Arg
180 185 190
Ile Ser Asn Pro Lys Glu Asn Leu Trp Phe Arg Pro Asp Thr Arg Arg
195 200 205
Arg Gln Ser Glu Asn Ile Leu Gln Phe Gly Pro Gln Val Gly Arg Arg
210 215 220
Asn Ser Glu Ser Ala Leu Trp Phe Gly Pro Arg Val Gly Arg Ser Tyr
225 230 235 240
Leu Glu Ser Asn Leu Ser Asp Glu Arg Tyr Gly Gln His Arg Asn Leu
245 250 255
Glu Pro Gly Leu Trp Ser Asn Asn Leu Gly Gln Ile Gln Lys Thr Pro
260 265 270
Ser Met Asp Val Thr Asn Arg Phe Pro Gly Arg Asp Leu Trp Ser Gly
275 280 285
Thr Asp Leu Asp Arg Thr Lys His Glu Thr Ala Leu Trp Phe Val Pro
290 295 300
Arg Ile Gly Arg Ser Asn Pro Glu Thr Asn Leu Trp Phe Gly Pro Arg
305 310 315 320
Val Gly Arg Ser His Pro Glu Thr Ser Gln Ser Phe Gly Gln Tyr Ala
325 330 335
Arg His Gly Asn Val Glu Thr Gly Leu Trp Pro Asp Asp Phe Val Gln
340 345 350
Ser Arg Pro Thr Thr Leu Glu Asp Val Thr Lys Arg Asn Pro Glu Arg
355 360 365
Asn Trp Trp Ser Ala Asn Gly Val Gly Arg Thr Lys His Glu Thr Thr
370 375 380
Leu Trp Phe Gly Pro Arg Ile Gly Arg Ser Asn Pro Glu Ile Asn Leu
385 390 395 400
Trp Phe Gly Pro Arg Val Gly Arg Ser His Pro Glu Asn Ser Gln Ser
405 410 415
Phe Gly Pro Tyr Ser Arg Arg Asn Asn Val Glu Ser Gly Leu Trp Pro
420 425 430
Asp Asn Leu Gly Gln Arg Arg Pro Thr Thr Trp Glu Asp Val Thr Lys
435 440 445
Arg Tyr Pro Gly His Asn Trp Trp Ser Val Asn Gly Leu Gly Arg Met
450 455 460
Lys His Glu Thr Ala Leu Trp Phe Gly Pro Arg Ile Gly Arg Ser Asn
465 470 475 480
Pro Asp Thr His Leu Trp Phe Gly Pro Arg Val Gly Arg Ser Gln Ser
485 490 495
Leu Ala Ser Glu Gln Leu Arg
500
<210>5
<211>1767
<212>DNA
<213>人工序列(Artificial Sequence)
<400>5
atgctgagaa gaacacattc gtcgcttgca gttgctgtaa cagtggttgc catttccgcg 60
ttcgctgtat ctgcagcaaa accacgagaa atgcacagca agaatgtggt ggttcagcga 120
tgcatccaaa ctggtttttc tgatggcggc aacatccgca gtgtacctga aacttctctt 180
tggttcggcc cccggatagg gcgcagcaac ttagaaactg ctttgttgtt tggacgttgt 240
gtggggtgta gccatccagg aaccagtcag tgttgtgaac cacttgcacg acgcagcggc 300
tctgaagata gtttgggata tagtagcccc gaaccaccgc acgacaacag aaagatcagt 360
cctgaatctg gtttgtggtt tgtagataga aatggtcgca gacaaccgga cactgctttg 420
tggtttggtc cacgtgtagg acgtcgtatg cagcacatcc aaccagaagc cagtgagtat 480
tttagaccag atgtacgacg cagtggtcct gaacatagtt tatggagtga tactcagcta 540
cgacagggcg atccagaacc accacttcat gttagaataa gcaatcctaa agaaaatttg 600
tggtttcgac ccgacactcg tcgcagacaa tcagaaaata tcctgcagtt tggtccacag 660
gtaggacgaa gaaactcaga aagtgccctg tggtttgggc ctcgtgtggg acgcagctat 720
ctagaaagta atctgtcaga tgagcgatat ggacagcata gaaatcttga acctggattg 780
tggtctaata atttgggaca aatccaaaca acaccatcta tggatgtgac aaacagattt 840
cctggacgtg atttgtggtc tggaactgat ctcgatcgta cgaaacacga aaccgcgcta 900
tggtttggac cacggatagg acgaagcaac ccagaaacca atttgtggtt tggtcctcgt 960
gtgggacgta gtcatccaga aaccagtcag tcatttggac agtatgcacg ccacgacaat 1020
gttgaaactg gtttatggcc tggtgatttt gttcaaagcc acccaaaaac attggaggat 1080
gtcactaaaa taaatcctga acgtaattgg tggtctgtta atggtgttgg acgtacgaaa 1140
cacgaaacta cactatggtt tggaccacgg ataggacgca gcaacccaga aaccaatttg 1200
tggtttggtc ctcgtgtggg acgtagtcat ccagaaacca gtcactcatt tggaccgtat 1260
gcacgccacg acagtgttga aattggtttg tggcctgatg attttggtca aagccgccca 1320
acaacgttgg aggatgtcac taaaagaaat cctgaacgta attggtggtc tgtaaatggt 1380
gttggacgta cgaaacacga aaccacacta tggtttggac cacggatagg acgcagcaac 1440
ccagaaacca atttgtggtt tggtcctcgt gtgggacgta gtcatccaga aaccagtgac 1500
tcgtttggac cgtacgcacg ccacgacaat gttgaaactg agttgtggcc tgatgatttt 1560
ggtcaaagca gcccaacaac gttggtggat gtcactaaaa gatatcctga acacaattgg 1620
tggtctgtaa atggtcttgg acgtacgaaa cacgaaaccg cactgtggtt tggaccaaga 1680
ataggacgca gcaacccaga aacgcattta tggtttggac ctcgtgtggg acgaagccaa 1740
tctctagcaa gcgagcagtt gcgctag 1767
<210>6
<211>588
<212>PRT
<213>人工序列(Artificial Sequence)
<400>6
Met Leu Arg Arg Thr His Ser Ser Leu Ala Val Ala Val Thr Val Val
1 5 10 15
Ala Ile Ser Ala Phe Ala Val Ser Ala Ala Lys Pro Arg Glu Met His
20 25 30
Ser Lys Asn Val Val Val Gln Arg Cys Ile Gln Thr Gly Phe Ser Asp
35 40 45
Gly Gly Asn Ile Arg Ser Val Pro Glu Thr Ser Leu Trp Phe Gly Pro
50 55 60
Arg Ile Gly Arg Ser Asn Leu Glu Thr Ala Leu Leu Phe Gly Arg Cys
65 70 75 80
Val Gly Cys Ser His Pro Gly Thr Ser Gln Cys Cys Glu Pro Leu Ala
85 90 95
Arg Arg Ser Gly Ser Glu Asp Ser Leu Gly Tyr Ser Ser Pro Glu Pro
100 105 110
Pro His Asp Asn Arg Lys Ile Ser Pro Glu Ser Gly Leu Trp Phe Val
115 120 125
Asp Arg Asn Gly Arg Arg Gln Pro Asp Thr Ala Leu Trp Phe Gly Pro
130 135 140
Arg Val Gly Arg Arg Met Gln His Ile Gln Pro Glu Ala Ser Glu Tyr
145 150 155 160
Phe Arg Pro Asp Val Arg Arg Ser Gly Pro Glu His Ser Leu Trp Ser
165 170 175
Asp Thr Gln Leu Arg Gln Gly Asp Pro Glu Pro Pro Leu His Val Arg
180 185 190
Ile Ser Asn Pro Lys Glu Asn Leu Trp Phe Arg Pro Asp Thr Arg Arg
195 200 205
Arg Gln Ser Glu Asn Ile Leu Gln Phe Gly Pro Gln Val Gly Arg Arg
210 215 220
Asn Ser Glu Ser Ala Leu Trp Phe Gly Pro Arg Val Gly Arg Ser Tyr
225 230 235 240
Leu Glu Ser Asn Leu Ser Asp Glu Arg Tyr Gly Gln His Arg Asn Leu
245 250 255
Glu Pro Gly Leu Trp Ser Asn Asn Leu Gly Gln Ile Gln Thr Thr Pro
260 265 270
Ser Met Asp Val Thr Asn Arg Phe Pro Gly Arg Asp Leu Trp Ser Gly
275 280 285
Thr Asp Leu Asp Arg Thr Lys His Glu Thr Ala Leu Trp Phe Gly Pro
290 295 300
Arg Ile Gly Arg Ser Asn Pro Glu Thr Asn Leu Trp Phe Gly Pro Arg
305 310 315 320
Val Gly Arg Ser His Pro Glu Thr Ser Gln Ser Phe Gly Gln Tyr Ala
325 330 335
Arg His Asp Asn Val Glu Thr Gly Leu Trp Pro Gly Asp Phe Val Gln
340 345 350
Ser His Pro Lys Thr Leu Glu Asp Val Thr Lys Ile Asn Pro Glu Arg
355 360 365
Asn Trp Trp Ser Val Asn Gly Val Gly Arg Thr Lys His Glu Thr Thr
370 375 380
Leu Trp Phe Gly Pro Arg Ile Gly Arg Ser Asn Pro Glu Thr Asn Leu
385 390 395 400
Trp Phe Gly Pro Arg Val Gly Arg Ser His Pro Glu Thr Ser His Ser
405 410 415
Phe Gly Pro Tyr Ala Arg His Asp Ser Val Glu Ile Gly Leu Trp Pro
420 425 430
Asp Asp Phe Gly Gln Ser Arg Pro Thr Thr Leu Glu Asp Val Thr Lys
435 440 445
Arg Asn Pro Glu Arg Asn Trp Trp Ser Val Asn Gly Val Gly Arg Thr
450 455 460
Lys His Glu Thr Thr Leu Trp Phe Gly Pro Arg Ile Gly Arg Ser Asn
465 470 475 480
Pro Glu Thr Asn Leu Trp Phe Gly Pro Arg Val Gly Arg Ser His Pro
485 490 495
Glu Thr Ser Asp Ser Phe Gly Pro Tyr Ala Arg His Asp Asn Val Glu
500 505 510
Thr Glu Leu Trp Pro Asp Asp Phe Gly Gln Ser Ser Pro Thr Thr Leu
515 520 525
Val Asp Val Thr Lys Arg Tyr Pro Glu His Asn Trp Trp Ser Val Asn
530 535 540
Gly Leu Gly Arg Thr Lys His Glu Thr Ala Leu Trp Phe Gly Pro Arg
545 550 555 560
Ile Gly Arg Ser Asn Pro Glu Thr His Leu Trp Phe Gly Pro Arg Val
565 570 575
Gly Arg Ser Gln Ser Leu Ala Ser Glu Gln Leu Arg
580 585
<210>7
<211>732
<212>DNA
<213>人工序列(Artificial Sequence)
<400>7
atggagagca gcatggacgt tttacctgta gctctcttga ctctggcagc tttgctcttt 60
tctgtggaaa cttcagagtt tttacacgag gtaaacgatc tcaatggcag caatcgggac 120
tctggagaac aaacaccttc tgcagttctt tcacgcagca aacggaacaa ctctggagca 180
catgttgata aatggtttcg accagatact ggagctggag aggactactg gaacgcgtta 240
tttcgagaag acgctgaaga tcatctctct aaaccggacg cgagtactgt agatcggctt 300
gagaatgttg ttcctacagc agaaacgcct cgtctcagtg tggcgcaaca tgaatctagg 360
cactcagaag gatcgcatga tgatcatagt gactccgaac ctgaagtttg gattggacaa 420
cactcaggaa gaagtatccc agagcctgga acttggttcg gtcctcgtat tggacgcagc 480
cacacagaac caggactgtg gtttggtcct agatatggac gcagctatcc ggaaccaggc 540
atgtggtttg gacctcgagt ggggcgcggc catccggaac cggaaatgtg gtttggacca 600
cgtgttggac gcaaccaagc ggaaccagga gtgtggtttg gggctcgcat tggtcggaac 660
cagcctgaac cagggacgtg gtttggtgct cgtattggac gcagccaccc agaaccaggg 720
atgtggtttt ga 732
<210>8
<211>243
<212>PRT
<213>人工序列(Artificial Sequence)
<400>8
Met Glu Ser Ser Met Asp Val Leu Pro Val Ala Leu Leu Thr Leu Ala
1 5 10 15
Ala Leu Leu Phe Ser Val Glu Thr Ser Glu Phe Leu His Glu Val Asn
20 25 30
Asp Leu Asn Gly Ser Asn Arg Asp Ser Gly Glu Gln Thr Pro Ser Ala
35 40 45
Val Leu Ser Arg Ser Lys Arg Asn Asn Ser Gly Ala His Val Asp Lys
50 55 60
Trp Phe Arg Pro Asp Thr Gly Ala Gly Glu Asp Tyr Trp Asn Ala Leu
65 70 75 80
Phe Arg Glu Asp Ala Glu Asp His Leu Ser Lys Pro Asp Ala Ser Thr
85 90 95
Val Asp Arg Leu Glu Asn Val Val Pro Thr Ala Glu Thr Pro Arg Leu
100 105 110
Ser Val Ala Gln His Glu Ser Arg His Ser Glu Gly Ser His Asp Asp
115 120 125
His Ser Asp Ser Glu Pro Glu Val Trp Ile Gly Gln His Ser Gly Arg
130 135 140
Ser Ile Pro Glu Pro Gly Thr Trp Phe Gly Pro Arg Ile Gly Arg Ser
145 150 155 160
His Thr Glu Pro Gly Leu Trp Phe Gly Pro Arg Tyr Gly Arg Ser Tyr
165 170 175
Pro Glu Pro Gly Met Trp Phe Gly Pro Arg Val Gly Arg Gly His Pro
180 185 190
Glu Pro Glu Met Trp Phe Gly Pro Arg Val Gly Arg Asn Gln Ala Glu
195 200 205
Pro Gly Val Trp Phe Gly Ala Arg Ile Gly Arg Asn Gln Pro Glu Pro
210 215 220
Gly Thr Trp Phe Gly Ala Arg Ile Gly Arg Ser His Pro Glu Pro Gly
225 230 235 240
Met Trp Phe
<210>9
<211>1092
<212>DNA
<213>人工序列(Artificial Sequence)
<400>9
atggagagca gcatggacgt tttacctgta gctctcttga ctctggcagc tttgctcttt 60
tctgtggaaa cttcagaggt tttacacgag gtaaacgatc tcaatggcag caatcgggac 120
tctggagaac aaacaccttc tgcagttctt tcacgcagca aacggaacaa ctctggagca 180
catgttgata aatggtttcg accagatact ggagctggag aggactactg gaacgcgtta 240
tttcgagaag acgctgaaga tcatctctct aaaccggacg cgagtactgt agatcggctt 300
gagaatgttg ttcctacagc agaaacgcct cgtctcagtg tggcgcaaca tgaatctagg 360
cactcagaag gatcgcatga tgatcatagt gactccgaac ctgaagtttg gattggacaa 420
cactcaggaa gaagtatccc agagcctgga acttggttcg gtcctcgtat tggacgcagc 480
cacacagaac caggactgtg gtttgttcct agatatggac gcagctatcc ggaaccaggc 540
atgtggtttg gacctcgagt gggacgcggc catccggaac cgggaatgtg gtttggacca 600
cgtgttggac gcaaccaagc ggaacctgga gtgtggtttg gagctcgcat tggtcggaac 660
cagcctgaac cagggacgtg gtttggtgct cgtattggac gcagccatct agaaccaggg 720
atgtggtttg gaccacgtgt tggacgcagc caagcggaac ctggagtgtg gtttggagct 780
cgcattggtc gaaatcagcc agaaccaggg acgtggtttg gtgctcgtat tggacgcagc 840
cacccagaac cagggatgtg gtttggacca cgtgttggac gcagtcaagc ggaacctgga 900
gcgtggtttg gagctcgcat tggtcggaac cagcaagaac cagggacgtg gtttggtgct 960
cgtattggac gcagccaccc agaaccaggg atgtggtttg gaccacgggt tggacgcagt 1020
cacccagaac cgggcacatg gtttggacct cgtatcggac gcagtcattc tgaacctgag 1080
cttagaatgt ag 1092
<210>10
<211>363
<212>PRT
<213>人工序列(Artificial Sequence)
<400>10
Met Glu Ser Ser Met Asp Val Leu Pro Val Ala Leu Leu Thr Leu Ala
1 5 10 15
Ala Leu Leu Phe Ser Val Glu Thr Ser Glu Val Leu His Glu Val Asn
20 25 30
Asp Leu Asn Gly Ser Asn Arg Asp Ser Gly Glu Gln Thr Pro Ser Ala
35 40 45
Val Leu Ser Arg Ser Lys Arg Asn Asn Ser Gly Ala His Val Asp Lys
50 55 60
Trp Phe Arg Pro Asp Thr Gly Ala Gly Glu Asp Tyr Trp Asn Ala Leu
65 70 75 80
Phe Arg Glu Asp Ala Glu Asp His Leu Ser Lys Pro Asp Ala Ser Thr
85 90 95
Val Asp Arg Leu Glu Asn Val Val Pro Thr Ala Glu Thr Pro Arg Leu
100 105 110
Ser Val Ala Gln His Glu Ser Arg His Ser Glu Gly Ser His Asp Asp
115 120 125
His Ser Asp Ser Glu Pro Glu Val Trp Ile Gly Gln His Ser Gly Arg
130 135 140
Ser Ile Pro Glu Pro Gly Thr Trp Phe Gly Pro Arg Ile Gly Arg Ser
145 150 155 160
His Thr Glu Pro Gly Leu Trp Phe Val Pro Arg Tyr Gly Arg Ser Tyr
165 170 175
Pro Glu Pro Gly Met Trp Phe Gly Pro Arg Val Gly Arg Gly His Pro
180 185 190
Glu Pro Gly Met Trp Phe Gly Pro Arg Val Gly Arg Asn Gln Ala Glu
195 200 205
Pro Gly Val Trp Phe Gly Ala Arg Ile Gly Arg Asn Gln Pro Glu Pro
210 215 220
Gly Thr Trp Phe Gly Ala Arg Ile Gly Arg Ser His Leu Glu Pro Gly
225 230 235 240
Met Trp Phe Gly Pro Arg Val Gly Arg Ser Gln Ala Glu Pro Gly Val
245 250 255
Trp Phe Gly Ala Arg Ile Gly Arg Asn Gln Pro Glu Pro Gly Thr Trp
260 265 270
Phe Gly Ala Arg Ile Gly Arg Ser His Pro Glu Pro Gly Met Trp Phe
275 280 285
Gly Pro Arg Val Gly Arg Ser Gln Ala Glu Pro Gly Ala Trp Phe Gly
290 295 300
Ala Arg Ile Gly Arg Asn Gln Gln Glu Pro Gly Thr Trp Phe Gly Ala
305 310 315 320
Arg Ile Gly Arg Ser His Pro Glu Pro Gly Met Trp Phe Gly Pro Arg
325 330 335
Val Gly Arg Ser His Pro Glu Pro Gly Thr Trp Phe Gly Pro Arg Ile
340 345 350
Gly Arg Ser His Ser Glu Pro Glu Leu Arg Met
355 360
<210>11
<211>801
<212>RNA
<213>人工序列(Artificial Sequence)
<400>11
augccgagag gugcucagcu auuccuguug cuggcgcugg ucaccgcugc acgaguccuc 60
gacgcgaggg cggaguccuc acgggcggga agcgaugcgc agucuucaca ucaggacggc 120
aggggagcuu cacacgacag gaacgaagaa agcaacgagc ugaacgauga aaaucguucc 180
gacggugacg cccaagacgc aacguuucgc agaggacgag agaugcaaga guucguuucg 240
caugcuggug gaccgauugg gaacggugag gaagaccagu gguuggcacu ggcggacgga 300
aguuacaucc cugccgaggc ugugcgagaa cucguccagc ggggaccuac ucuaagcgag 360
aacacaggcu ugugguuugg accucguuac ggacgacgaa cgagcugcgg cgagaacguc 420
ccccuuaagu gguuguccca aguggagaag cgggcggcca aacagccagc gcugugguuc 480
gguccgcgag ugggucgcag ucucgacgaa gaacccaagg gcgaagaaug gagggacgac 540
gauaagggac ucaaggacgg uaguucgcag cgccaagaua ggagugccca accuccgggu 600
cugugguucg gaccccgggu cggccggcgc agcgaugccc aggucgacga caugcugugg 660
uucggacccc gacccggacg aaguguggau acugacaagc aggaucugua cgacgaugag 720
gucgcuaugc gugaucagcg gggugccaaa cauccugguu ugugguucgg gccucgcuuc 780
ggccguuaaa caacagugcu a 801
<210>12
<211>1004
<212>RNA
<213>人工序列(Artificial Sequence)
<400>12
agaagaacac auucgucgcu ugcaguugcu guaacagugg uugccauuuc cgcguucgcu 60
guauccgcag caaaaccacg agaaaugcac agcaagaaug uggugguuca gcgaugcauc 120
caaacugguu uuucugaugg cggcagcauc cgcaauguac cugaaacuuc ucuuugguuc 180
ggcccccgga uagggcgcag caacuuagaa acugcuuugu uguuuggacg uugugugggg 240
uguagccauc caggaaccag ucaguguugu gaaccacuug cacgacgcag cggcucugaa 300
gauaguuugg gauauaguag ccccgaacca ccgcacgaca acagaaagau caguccugaa 360
ucugguuugu gguuuguaga uagaaauggu cgcagacaac cggacacugc uuugugguuu 420
gguccacgug uaggacgucg uaugcagcac auccaaccag aagccaguga guauuuuaga 480
ccagauguac gacgcagugg uccugaacau aguuuaugga gugauacuca gcuacgacag 540
ggcgauccag aaccaccacu ucauguuaga auaagcaauc cuaaagaaaa uuugugguuu 600
cgacccgaca cucgucgcag acaaucagaa aauauccugc aguuuggucc acagguagga 660
cgaagaaacu cagaaagugc ccugugguuu gggccucgug ugggacgcag cuaucuagaa 720
aguaaucugu cagaugagcg auauggacag cauagaaauc uugaaccugg auuguggucu 780
aauaauuugg gacaaaucca aaaaacacca ucuauggaug ugacaaacag auuuccugga 840
cgugauuugu ggucuggaac ugaucucgac cguacgaaac acgaaaccgc gcuaugguuu 900
guaccacgga uaggacgaag caacccagaa accaauuugu gguuuggacc ucguguggga 960
cguagucauc cagaaaccag ucagucauuu ggacaguaug cacg 1004
<210>13
<211>552
<212>RNA
<213>人工序列(Artificial Sequence)
<400>13
auggagagca gcauggacgu uuuaccugua gcucucuuga cucuggcagc uuugcucuuu 60
ucuguggaaa cuucagaguu uuuacacgag guaaacgauc ucaauggcag caaucgggac 120
ucuggagaac aaacaccuuc ugcaguucuu ucacgcagca aacggaacaa cucuggagca 180
cauguugaua aaugguuucg accagauacu ggagcuggag aggacuacug gaacgcguua 240
uuucgagaag acgcugaaga ucaucucucu aaaccggacg cgaguacugu agaucggcuu 300
gagaauguug uuccuacagc agaaacgccu cgucucagug uggcgcaaca ugaaucuagg 360
cacucagaag gaucgcauga ugaucauagu gacuccgaac cugaaguuug gauuggacaa 420
cacucaggaa gaaguauccc agagccugga acuugguucg guccucguau uggacgcagc 480
cacacagaac caggacugug guuugguccu agauauggac gcagcuaucc agaaccaggg 540
augugguuuu ga 552
<210>14
<211>769
<212>RNA
<213>人工序列(Artificial Sequence)
<400>14
uaauacgacu cacuauaggu acgacucacu auaggaguaa aggagaagaa cuuuucacug 60
gaguugugac aauucuuguu gaauuagaug gugauguuaa uggucacaaa uuuucuguua 120
guggagaggg ugaaggugau gcaacauacg gaaaacuuac ccuuaaauuu auuuguacua 180
cuggaaaacu accuguuccc uggccaacac uuguuacuac uuugacuuau gguguucaau 240
guuuuucaag auacccagau cacaugaaac ggcacgacuu uuucaagagu gcaaugcccg 300
aagguuaugu acaagaaaga acuauuuuuu ucaaagauga cgguaacuac aagacacgug 360
cugaaguuaa guuugaaggu gauacccuug uuaauagaau cgaguuaaaa gguauugauu 420
uuaaagaaga uggaaacauu cuuggacaca aauuggaaua caacuauaac ucacacaaug 480
uauacauuau ggcagacaaa caaaagaaug gaaucaaagu uaacuucaaa auuagacaca 540
acauugaaga uggaaguguu caacuagcag accauuauca acaaaauacu ccaauuggcg 600
auggcccugu ucuuuuacca gacaaccauu accuguccac acaaucugcu cuuucuaaag 660
aucccaacga aaagagagac cauauggugc uucuugaguu uguaacagcu gcugguauua 720
cacacgguau ggaugaacua uacaaaccua ccuauaguga gucguauua 769
Claims (10)
1.蛋白质，是如下a)或b)或c)或d)的蛋白质：
a)氨基酸序列是序列2或序列4或序列6或序列8或序列10所示的蛋白质；
b)在序列2或序列4或序列6或序列8或序列10所示的蛋白质的N端和/或C端连接标签得到的融合蛋白质；
c)将序列2或序列4或序列6或序列8或序列10所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的具有相同功能的蛋白质；
d)与序列2或序列4或序列6或序列8或序列10所示的氨基酸序列具有75％或75％以上的同源性且具有相同功能的蛋白质。
2.与权利要求1所述的蛋白质相关的生物材料，为下述A1)至A8)中的任一种：
A1)编码权利要求1所述的蛋白质的核酸分子；
A2)含有A1)所述核酸分子的表达盒；
A3)含有A1)所述核酸分子的重组载体；
A4)含有A2)所述表达盒的重组载体；
A5)含有A1)所述核酸分子的重组微生物；
A6)含有A2)所述表达盒的重组微生物；
A7)含有A3)所述重组载体的重组微生物；
A8)含有A4)所述重组载体的重组微生物。
3.根据权利要求2所述的相关生物材料，其特征在于：A1)所述核酸分子为如下1)或2)或3)所示的基因：
1)其编码序列是序列1或序列3或序列5或序列7或序列9所示的cDNA分子或基因组DNA分子；
2)与1)限定的核苷酸序列具有75％或75％以上同一性，且编码权利要求1所述的蛋白质的cDNA分子或基因组DNA分子；
3)在严格条件下与1)或2)限定的核苷酸序列杂交，且编码权利要求1所述的蛋白质的cDNA分子或基因组DNA分子。
4.权利要求1所述的蛋白质或权利要求2或3所述的生物材料在如下a1)-a10)中任一种中的应用：
a1)调控昆虫滞育；
a2)制备调控昆虫滞育的产品；
a3)防治害虫；
a4)制备防治害虫的产品；
a5)降低昆虫滞育率；
a6)制备降低昆虫滞育率的产品；
a7)降低昆虫越冬存活率；
a8)制备降低昆虫越冬存活率的产品；
a9)降低昆虫越冬后种群数量；
a10)制备降低越冬后种群数量的产品。
5.一种降低昆虫滞育率的方法，包括降低昆虫中权利要求1所述的蛋白质的表达量和/或活性的步骤，从而实现降低昆虫滞育率。
6.根据权利要求5所述的方法，其特征在于：所述降低昆虫中权利要求1所述的蛋白质的表达量和/或活性的方法是将抑制昆虫中权利要求1所述的蛋白质的编码基因表达的物质导入昆虫；
或，所述抑制昆虫中权利要求1所述的蛋白质的编码基因表达的物质为抑制昆虫中权利要求1所述的蛋白质的编码基因表达的dsRNA；
或，所述抑制昆虫中权利要求1所述的蛋白质的编码基因表达的dsRNA为由序列表中序列11或序列12或序列13所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA；
或，所述导入的方式为注射。
7.权利要求5或6所述的方法在防治害虫中的应用。
8.抑制权利要求1所述蛋白质的编码基因表达的物质，其为由序列表中序列11或序列12或序列13所示的核苷酸和其反向互补序列所示的核苷酸组成的双链RNA。
9.抑制权利要求1所述蛋白质的编码基因表达的物质在防治害虫中的应用；
或，抑制权利要求1所述蛋白质的编码基因表达的物质在降低昆虫滞育率中的应用。
10.根据权利要求4所述的应用或权利要求5或6所述的方法或权利要求7或9所述的应用，其特征在于：所述昆虫或害虫为飞蝗。
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| CN109734789A (zh) * | 2019-02-25 | 2019-05-10 | 中国农业科学院植物保护研究所 | 飞蝗fk506结合蛋白46及其编码基因与应用 |
| CN113433310A (zh) * | 2021-06-11 | 2021-09-24 | 安徽农业大学 | 一种提升昆虫药代动力学准确性的方法 |
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| CN109734789A (zh) * | 2019-02-25 | 2019-05-10 | 中国农业科学院植物保护研究所 | 飞蝗fk506结合蛋白46及其编码基因与应用 |
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| CN113433310A (zh) * | 2021-06-11 | 2021-09-24 | 安徽农业大学 | 一种提升昆虫药代动力学准确性的方法 |
| CN113481207A (zh) * | 2021-07-15 | 2021-10-08 | 西南大学 | 家蚕卵滞育的必需基因Bmtret1及其应用 |
| CN113621619A (zh) * | 2021-07-15 | 2021-11-09 | 西南大学 | 一个家蚕卵滞育的必需基因Bmpnd-2及其应用 |
| CN113621619B (zh) * | 2021-07-15 | 2023-09-12 | 西南大学 | 一个家蚕卵滞育的必需基因Bmpnd-2及其应用 |
| CN118684754A (zh) * | 2024-08-29 | 2024-09-24 | 中国农业科学院植物保护研究所 | 东亚飞蝗隐花色素基因及其编码蛋白和应用 |
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