CN109762839A - 抑制CCE001a蛋白表达和/或活性的物质在降低棉铃虫对棉酚的耐受性中的应用 - Google Patents
抑制CCE001a蛋白表达和/或活性的物质在降低棉铃虫对棉酚的耐受性中的应用 Download PDFInfo
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- CN109762839A CN109762839A CN201910067767.9A CN201910067767A CN109762839A CN 109762839 A CN109762839 A CN 109762839A CN 201910067767 A CN201910067767 A CN 201910067767A CN 109762839 A CN109762839 A CN 109762839A
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Abstract
本发明公开了抑制CCE001a蛋白表达和/或活性的物质在降低棉铃虫对棉酚的耐受性中的应用。将序列表中的序列1所示的双链RNA或序列表中的序列6所示的双链RNA注射至棉铃虫幼虫的腹部倒数第二与第三足节间处，然后先用正常饲料饲喂24h，测量体重并记为M1；再用含0.1％(m/m)棉酚的正常饲料饲喂72h，测量体重并记为M2；计算净增重；净增重＝M2‑M1。结果表明，与注射序列表中的序列6所示的双链RNA的棉铃虫相比，注射序列表中的序列1所示的双链RNA的棉铃虫的净增重显著降低(p＜0.05)。由此可见，序列表中的序列1所示的双链RNA可以降低棉铃虫对棉酚的耐受性。本发明具有重大的应用价值。
Description
技术领域
本发明属于生物技术领域，具体涉及抑制CCE001a蛋白表达和/或活性的物质在降低棉铃虫对棉酚的耐受性中的应用。
背景技术
棉铃虫(Helicoverpa armigera Hübner)隶属鳞翅目夜蛾科，广泛分布在我国及世界各地。棉铃虫为杂食性害虫，寄主植物有棉花、玉米、烟草、番茄等30多科200余种，给我国农业生产造成了巨大的经济损失。目前防治棉铃虫害的主要手段是大量使用化学杀虫剂，但伴随产生的一些问题逐渐引起关注，如环境污染，边际效应对非靶标昆虫产生的危害，自然平衡被扰乱引起的害虫猖獗和害虫抗药性的增加等问题。为了解决这些问题，科学家们在寻找更有效的可替代化学杀虫剂的抗虫技术方面做了很多尝试，包括天敌的利用，基于天然产物的杀虫剂的使用，抗虫转基因作物的研发等等。其中，以表达苏云金芽孢杆菌(Bacillus thuringiensis，Bt)毒蛋白的转基因作物应用最为广泛。Bt毒蛋白可以杀死一些主要以作物为食的害虫种群，而对脊椎动物和其它生物无害。近年来，在实验室或者种植Bt转基因作物的大田中都发现对Bt蛋白产生抗性的害虫。另一方面，由于长期种植Bt转基因作物，农田的害虫种群结构产生了变化，由单一害虫占主导的种群结构逐渐发展成多个害虫占主导的种群结构，这为控制害虫带来了更多困难。综上所述，亟待一种经济有效、环境友好的方法控制棉铃虫具有重要意义。
RNA干扰(RNA interference，RNAi)是由双链RNA引发的基因沉默现象，通过体外合成一段与靶基因同源的dsRNA或siRNA，使内源性靶基因mRNA降解并阻断转录，抑制翻译。由于RNAi具有高特异的沉默特性，已经作为一种新的方法来防治农业害虫。主要途径有以下三种：一是通过转基因手段将害虫靶基因dsRNA转入植物体内，当害虫取食转基因植物时，植物源表达的dsRNA引发害虫体内的RNAi现象，使得害虫取食能力下降或杀死害虫；二是将害虫靶基因dsRNA直接注射进昆虫的卵、血腔或局部组织，可引发远距离靶基因的特异性沉默；三是可以通过化学合成具有致死效应的siRNA，做成新型生物农药，对害虫进行防治。
棉酚是一种黄色多酚羟基双萘醛类化合物，属于棉花的主要次生物质之一，主要存在于锦葵植物棉花的根、茎、叶和种子内，对许多生物体具有毒性，是植物源杀虫物质。
发明内容
本发明的目的是防治棉铃虫。
本发明首先保护抑制CCE001a蛋白表达和/或活性的物质在降低棉铃虫对棉酚的耐受性中的应用。
本发明还保护抑制CCE001a蛋白表达和/或活性的物质在制备棉铃虫杀虫剂中的应用。
本发明还保护抑制CCE001a蛋白表达和/或活性的物质在培育抗棉铃虫棉花中的应用。
上述任一所述的应用中，所述“抑制CCE001a蛋白表达和/或活性的物质”可为特异RNA；所述特异RNA可为序列表中的序列1所示的双链RNA。
本发明还保护一种降低棉铃虫对棉酚的耐受性的方法，可包括如下步骤：向棉铃虫注射抑制CCE001a蛋白表达和/或活性的物质；与未注射抑制CCE001a蛋白表达和/或活性的物质的棉铃虫相比，注射了抑制CCE001a蛋白表达和/或活性的物质的棉铃虫对棉酚的耐受性降低。
上述方法中，所述棉铃虫可为棉铃虫幼虫。
上述方法中，所述注射的部位可为棉铃虫幼虫腹部倒数第二与第三足节间处。
上述方法中，当棉铃虫为棉铃虫幼虫时，注射剂量可为每只棉铃虫幼虫注射2.0μg以上特异RNA(如2.0-3.0μg特异RNA、2.0μg特异RNA、2.5μg特异RNA或3.0μg特异RNA)。
上述任一所述的方法中，所述“抑制CCE001a蛋白表达和/或活性的物质”可为特异RNA；所述特异RNA可为序列表中的序列1所示的双链RNA。
本发明还保护一种棉铃虫杀虫剂，其可含有抑制CCE001a蛋白表达和/或活性的物质。
所述棉铃虫杀虫剂具体可由含有抑制CCE001a蛋白表达和/或活性的物质组成。
上述棉铃虫杀虫剂还可含有棉酚。棉酚和“含有抑制CCE001a蛋白表达和/或活性的物质”分别独立包装。
所述棉铃虫杀虫剂具体可由“含有抑制CCE001a蛋白表达和/或活性的物质”和棉酚组成。
上述棉铃虫杀虫剂中，所述“抑制CCE001a蛋白表达和/或活性的物质”可为特异RNA；所述特异RNA可为序列表中的序列1所示的双链RNA。
上述任一所述特异RNA也属于本发明的保护范围。
本发明还保护CCE001a蛋白在调控棉花抗虫性中的应用。
上述任一所述CCE001a蛋白可为a1)或a2)或a3)：
a1)氨基酸序列是序列表中序列2所示的蛋白质；
a2)在序列表中序列2所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质；
a3)将序列表中序列2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/或添加得到的与棉花抗虫性相关的蛋白质。
本发明还保护编码CCE001a蛋白的核酸分子在调控棉花抗虫性中的应用。
上述任一所述编码CCE001a蛋白的核酸分子可为如下b1)或b2)或b3)或b4)所示的DNA分子：
b1)编码区是序列表中序列3所示的DNA分子；
b2)核苷酸序列是序列表中序列3所示的DNA分子；
b3)与b1)或b2)限定的核苷酸序列具有75％或75％以上同一性，且编码所述CCE001a蛋白的DNA分子；
b4)在严格条件下与b1)或b2)限定的核苷酸序列杂交，且编码所述CCE001a蛋白的DNA分子。
上述任一所述抗虫可为抗棉铃虫。
上述任一所述棉铃虫可为棉铃虫室内饲养品系(96S)。
将特异RNA(序列表中的序列1所示的双链RNA)或对照RNA(序列表中的序列6所示的双链RNA)注射至棉铃虫幼虫的腹部倒数第二与第三足节间处(注射剂量可为2.5μg特异RNA/只)，然后先用正常饲料饲喂24h，测量体重并记为M1；再用含0.1％(m/m)棉酚的正常饲料饲喂72h，测量体重并记为M2；计算净增重，净增重＝M2-M1。结果表明，与注射对照RNA的棉铃虫相比，注射所述特异RNA的棉铃虫的净增重显著降低(p＜0.05)。由此可见，序列表中的序列1所示的双链RNA可以降低棉铃虫对棉酚的耐受性。本发明具有重大的应用价值。
附图说明
图1为dsCCE001a对其靶标基因的干扰效率。
图2为注射dsRNA后棉铃虫幼虫取食棉酚饲料体重的测定。
图3为CCE001a基因对棉铃虫幼虫生长发育的影响。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的实验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量实验，均设置三次重复实验，结果取平均值。
棉铃虫室内饲养品系(96S)1996年采集自中国农业科学院新乡实验基地，人工饲料饲养至今，饲养条件为温度27±2℃，相对湿度75％±10％，光周期14L：10D，成虫羽化后饲喂配制的10％糖水。在下文中，棉铃虫室内饲养品系(96S)简称棉铃虫。
Wizard SV Gel and PCR Clean-Up System试剂盒为Promega公司的产品。
T7 High Yield RNA Transcription Kit为南京诺唯赞生物科技有限公司的产品。10×Reaction Buffer、ATP Solution、GTP Solution、UTP Solution、CTP Solution和T7 RNA Polymerase Mix均为T7 High Yield RNA Transcription Kit中的组件。
TransScript One-step gDNA Removal and cDNA Synthesis SuperMix为北京全式金生物技术有限公司的产品。Anchored Oligo(dT)18primer、2×TS Reaction Mix、TransScript RT Enzyme Mix和gDNA Remover均为TransScript One-step gDNA Removaland cDNA Synthesis SuperMix中的组件。
实施例1、棉铃虫幼虫注射dsRNA试验
本发明的发明人利用生物信息学技术，分析了棉铃虫不同寄主处理下的转录组，获得了棉铃虫CCE001a蛋白的编码基因(即CCE001a基因)。CCE001a基因的核苷酸序列如序列表中的序列2所示。CCE001a蛋白的氨基酸序列如序列表中的序列3所示。
一、dsCCE001a的制备
1、采用Trizol试剂提取棉铃虫(由棉铃虫幼虫、蛹和成虫混合而成)的总RNA。
2、完成步骤1后，取棉铃虫的总RNA，采用TransScript One-step gDNA Removaland cDNA Synthesis SuperMix合成棉铃虫的第一链cDNA(以下简称棉铃虫的cDNA)。
反应体系为20μL，由XμL棉铃虫的总RNA(约1μg)、1μL Anchored Oligo(dT)18primer、10μL 2×TS Reaction Mix、1μL TransScript RT Enzyme Mix、1μL gDNARemover和(7-X)μL RNase-free Water组成。
反应条件：42℃孵育15min；85℃5sec(目的为使TransScript RT Enzyme Mix失活)。
3、由生工生物工程(上海)股份有限公司合成dsCCE001a-Forward：5′-TAATACGAC TCACTATAGGGATTCACGGATTCCTATGT-3′(下划线为T7启动子的核苷酸序列)和dsCCE001a-Reverse：5′-TAATACGACTCACTATAGGGCTGTAGTTGCCAGTCTTTA-3′(下划线为T7启动子的核苷酸序列)。
4、以步骤2得到的棉铃虫的cDNA为模板，以步骤3合成的dsCCE001a-Forward和dsCCE001a-Reverse为引物进行PCR扩增。
反应体系为50μL，由1μL棉铃虫的cDNA(约0.5μg)、1μL dsCCE001a-Forward水溶液(浓度为10μM)、1μL dsCCE001a-Reverse水溶液(浓度为10μM)、10μL 5×TransStartFastPfu Buffer、4μL dNTPs(dATP、dTTP、dGTP和dCTP的浓度均为2.5mM)、1μL FastPfu DNAPolymerase和32μL ddH2O组成。
反应条件：95℃预变性2min；95℃变性20s，60℃退火20s，72℃延伸1min，35个循环；72℃延伸5min。
5、取完成步骤4后的反应体系，采用Wizard SV Gel and PCR Clean-Up System试剂盒回收约500bp的PCR扩增产物。
6、取步骤5回收的PCR扩增产物，参考T7 High Yield RNA Transcription Kit的说明书合成dsRNA，即dsCCE001a。具体步骤如下：
(1)制备反应体系。反应体系为20μL，由2μL 10×Reaction Buffer、2μL ATPSolution、2μL GTP Solution、2μL UTP Solution、2μL CTP Solution、XμL步骤5回收的PCR扩增产物(约1.5μg)、2μL T7 RNA Polymerase Mix和(8-X)μL RNase-free H2O组成。
(2)完成步骤(1)后，取反应体系，37℃孵育4h。
(3)取完成步骤(2)后的反应体系，加入1μL DNase I，37℃孵育15min(目的为消化DNA模板)，得到dsCCE001a。
两个反向的T7启动子双向启动序列表中序列2自5’末端起第466-956位，这样在RNA水平上就可以形成如序列表序列1所示的双链RNA，引发RNAi，从而可用于抑制CCE001a基因的表达。
二、dsGFP的制备(dsGFP作为对照)
1、人工合成序列表中序列4所示的双链DNA分子(即GFP基因)。GFP基因编码GFP蛋白(GFP蛋白的氨基酸序列如序列表中的序列5所示)，且在棉铃虫体内并不存在。
2、由生工生物工程(上海)股份有限公司合成dsGFP-Forward：5′-TAATACGACTCAC TATAGGGCGATTTCAAGGAGGACGG-3′(下划线为T7启动子的核苷酸序列)和dsGFP-Reverse：5′-TAATACGACTCACTATAGGGCCATGCCATGTGTAATCCC-3′(下划线为T7启动子的核苷酸序列)。
3、以步骤1合成的双链DNA分子为模板，以步骤2合成的dsGFP-Forward和dsGFP-Reverse为引物进行PCR扩增。
反应体系为50μL，由1μL棉铃虫的cDNA(约0.5μg)、1μL dsGFP-Forward水溶液(浓度为10μM)、1μL dsGFP-Reverse水溶液(浓度为10μM)、10μL 5×TransStart FastPfuBuffer、4μL dNTPs(dATP、dTTP、dGTP和dCTP的浓度均为2.5mM)、1μL FastPfu DNAPolymerase和32μL ddH2O组成。
反应条件：95℃预变性2min；95℃变性20s，60℃退火20s，72℃延伸1min，35个循环；72℃延伸5min。
4、取完成步骤3后的反应体系，采用Wizard SV Gel and PCR Clean-Up System试剂盒回收约300bp的PCR扩增产物。
5、取步骤4回收的PCR扩增产物，参考T7 High Yield RNA Transcription Kit的说明书合成dsRNA，即dsGFP。具体步骤如下：
(1)制备反应体系。反应体系为20μL，由2μL 10×Reaction Buffer、2μL ATPSolution、2μL GTP Solution、2μL UTP Solution、2μL CTP Solution、XμL步骤4回收的PCR扩增产物(约1.5μg)、2μL T7 RNA Polymerase Mix和(8-X)μL RNase-free H2O组成。
(2)完成步骤(1)后，取反应体系，37℃孵育4h。
(3)取完成步骤(2)后的反应体系，加入1μL DNase I，37℃孵育15min(目的为消化DNA模板)，得到dsGFP。
两个反向的T7启动子双向启动序列表中序列4自5’末端起第390-706位，这样在RNA水平上就可以形成如序列表序列6所示的双链RNA，引发RNAi，从而可用于抑制GFP基因的表达。
三、沉默效率的检测
实验重复三次取平均值，每次重复的步骤如下：
1、取10头3龄棉铃虫幼虫，随机分成dsCCE001a组和dsGFP组两组(每组5头)，分别进行如下处理：
dsCCE001a组：通过拉针仪拉出的毛细管将dsCCE001a注射至棉铃虫幼虫的腹部倒数第二与第三足节间处。注射剂量为2.5μg dsCCE001a/只；
dsGFP组：通过拉针仪拉出的毛细管将dsGFP注射至棉铃虫幼虫的腹部倒数第二与第三足节间处。注射剂量为2.5μg dsGFP/只。
2、将完成步骤1的棉铃虫幼虫置于装有正常饲料的24孔板，24h后，实时定量检测dsCCE001a组和dsGFP组的各个幼虫的cDNA中CCE001a基因的相对表达量(以β-actin基因为内参)，然后分别取平均值。
3、将完成步骤1的棉铃虫幼虫置于装有正常饲料的24孔板，72h后，实时定量检测dsCCE001a组和dsGFP组的各个幼虫的cDNA中CCE001a基因的相对表达量(以β-actin基因为内参)，然后分别取平均值。
检测CCE001a基因的引物为5’-AGCCTTCCTGACTGAATC-3’和5’-AGCGAATCCATACAACACT-3’。检测β-actin基因(内参基因)的引物为5’-CCTGGTATTGCTGACCGTATGC-3’和5’-CTGTTGGAAGGTGGAGAGGGAA-3’。
4、完成步骤2和3后，利用SPSS软件的独立样本t测验进行差异显著性分析。
实验结果见图1(dsCCE001a为dsCCE001a组，dsGFP为dsGFP组)。结果表明，注射dsCCE001a 24h后，dsCCE001a组CCE001a基因的表达量受到抑制，沉默效率达到50％。
四、注射dsRNA后棉铃虫幼虫取食棉酚饲料体重的测定
实验重复三次取平均值，每次重复的步骤如下：
1、取48头3龄棉铃虫幼虫，随机分成dsCCE001a组和dsGFP组两组(每组24头)，分别进行如下处理：
dsCCE001a组：通过拉针仪拉出的毛细管将dsCCE001a注射至棉铃虫幼虫的腹部倒数第二与第三足节间处。注射剂量为2.5μg dsCCE001a/只；
dsGFP组：通过拉针仪拉出的毛细管将dsGFP注射至棉铃虫幼虫的腹部倒数第二与第三足节间处。注射剂量为2.5μg dsGFP/只。
2、将完成步骤1的棉铃虫幼虫置于装有正常饲料的24孔板，24h后，测量体重并取各组体重平均值，记为M1。
3、将完成步骤2的棉铃虫幼虫置于装有含0.1％(m/m)棉酚的正常饲料的24孔板，72h后，测量体重并取各组体重平均值，记为M2。
4、完成步骤3后，计算净增重；净增重＝M2-M1。
实验结果见图2(dsCCE001a为dsCCE001a组，dsGFP为dsGFP组)。结果表明，当饲喂含棉酚的饲料时，与dsGFP组相比，dsCCE001a组的净增重显著降低(p＜0.05)。
五、CCE001a基因对棉铃虫幼虫生长发育的影响
实验重复三次取平均值，每次重复的步骤如下：
1、取48头3龄棉铃虫幼虫，随机分成dsCCE001a组和dsGFP组两组(每组24头)，测量体重并取各组体重平均值，记为N1。
2、完成步骤1后，分别进行如下处理：
dsCCE001a组：通过拉针仪拉出的毛细管将dsCCE001a注射至棉铃虫幼虫的腹部倒数第二与第三足节间处。注射剂量为2.5μg dsCCE001a/只；
dsGFP组：通过拉针仪拉出的毛细管将dsGFP注射至棉铃虫幼虫的腹部倒数第二与第三足节间处。注射剂量为2.5μg dsGFP/只。
3、将完成步骤2的棉铃虫幼虫置于装有正常饲料的24孔板，72h后，测量体重取各组体重平均值，记为N2。
4、完成步骤3后，计算净增重；净增重＝N2-N1。
实验结果见图3(dsCCE001a为dsCCE001a组，dsGFP为dsGFP组)。结果表明，当饲喂不含棉酚的饲料时，dsCCE001a组和dsGFP组的净增重无显著差异。
上述结果表明，CCE001a基因并没有参与调节棉铃虫幼虫生长发育，很可能是CCE001a基因参与了棉酚的解毒代谢过程。抑制棉铃虫中CCE001a基因的表达量，可降低棉铃虫对棉酚的耐受性。
<110> 中国农业科学院深圳农业基因组研究所
<120> 抑制CCE001a蛋白表达和/或活性的物质在降低棉铃虫对棉酚的耐受性中的应用
<160> 6
<170> PatentIn version 3.5
<210> 1
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<212> RNA
<213> 人工序列
<220>
<223>
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auucacggau uccuaugucu cggcacugag gacgcgccag gcaaugcugg caugaaggac 60
cagguggcgc ugcugcgcug ggugcagaag aacaucgcca gcuucggcgg uaacccugau 120
gauguuacua uugcagggua uagugcaggu uccgcaucag uagaucuguu aaugcuuuca 180
aaaucagccg aaggguuauu ucaccgaguu auacccgaaa guggcggaaa ucuugccgca 240
uuuucaauuc aacgggaucc ugucgagauu gcuaaaucau acgccagcaa auuaggcuuc 300
gauaacggag acgauauuua ugcguuaggg aaguuuuaua ugacagcucc aauugaaaag 360
uugacguccg auccauuuuu ugacagaacu gauucuacau uuuuguucgc accaugugua 420
gaacgugaaa caggggacgg agccuuccug acugaaucac cucuaacaau acuaaagacu 480
ggcaacuaca g 491
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<220>
<223>
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atgaagtggt ggacgtgtgt ggtgttcgcg tgcgcggccg tgctggctga cgacgagtgg 60
cgcgaggtga ggactgcgca agggccggtg cgcgggcgca agcaccccac tgcagacatg 120
tacgccttct acaacatacc ctacgccacc gcgcctacgg gccaggataa gttcaaggca 180
cctcttcctc caccagtgtg gttagaacca tttgacgcaa tcgacgagca cgttatatgc 240
ccacagccaa tgtttcctgg tgatctcatg cccaaaaatg tagtgaccaa agaaaattgt 300
ctcatcgcca acgtatttat gcctaataca aaagaaaaga acctttcagt tgtcgtatat 360
gtacatggag gagcttttat tatgggctgg ggggaaatgt ttaaggcaag acaattcatg 420
aagacaaaag attttattgt ggtgacgttt aattaccgcc ttggaattca cggattccta 480
tgtctcggca ctgatgacgc gccaggcaat gctggcatga aggaccaggt ggcgttgctg 540
cgctgggtgc agaagaacat cgccagcttc ggcggtaacc ctgatgatgt tactattgca 600
gggtatagtg caggttccgc atcagtagat ctgttaatgc tttcaaaatc agccgaaggg 660
ttatttcacc gagttatacc cgaaagtggc ggaaatcttg ccgcattttc aattcaacgg 720
gatcctgtcg agattgctaa atcatacgcc agcaaattag gcttcgataa cggagacgat 780
atttatgcgt tagggaagtt ttatatgaca gctccaattg aaaagttgac gtccgatcca 840
tttttcgaca gaactgattc tacatttttg ttcgcaccat gcgtagaacg tgaaacaggg 900
gacggagcct tcctgactga atcacctcta acaatactaa agactggcaa ctacagaaag 960
ctgccagtgt tgtatggatt cgctgaaatg gaaggattaa tacgtattga tttctttgaa 1020
ctttggaagc ataaaatgaa tgaaaagttt tctgatttct tgccagctga cttgaaattt 1080
gattcagaag aagaaagaga agaagtggca aataagataa aggagtttta ctttggcgac 1140
aagccagtcg gcaatgaaaa tattttaaaa tacgttgatt tcttttcgga tgttatattt 1200
gcttacccca tgctttgggc tgtgaagcta cacgtcgaag ctggtaacaa tcaagtatat 1260
ttgtatgaat atagctttgt ggacgaagat gttcctgtgg tacctcatac taatatacgt 1320
ggagctaacc actgtgccca gactatggct ttgagtgatg ggaaaaactt cacacaccat 1380
gatgacaccc tcgcaacacc acagtttaga gaaatgaaaa agactattcg tgaaatatgg 1440
cacaattttg tgaaaactgg agtgccagta ccagagggct catggctgcc ggcgtggccg 1500
gcggcgggcg cggaccgggc gccgcacatg tcgctgggcg agcggctgga gctgcgcggc 1560
gcgctgctgc cggagcgcac gcgcttctgg gatgacatct accagagata ctaccgggac 1620
gcggtgccgc cgccgaaacc cccgcccaga ccacgcaacg agttgtag 1668
<210> 3
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Met Lys Trp Trp Thr Cys Val Val Phe Ala Cys Ala Ala Val Leu Ala
1 5 10 15
Asp Asp Glu Trp Arg Glu Val Arg Thr Ala Gln Gly Pro Val Arg Gly
20 25 30
Arg Lys His Pro Thr Ala Asp Met Tyr Ala Phe Tyr Asn Ile Pro Tyr
35 40 45
Ala Thr Ala Pro Thr Gly Gln Asp Lys Phe Lys Ala Pro Leu Pro Pro
50 55 60
Pro Val Trp Leu Glu Pro Phe Asp Ala Ile Asp Glu His Val Ile Cys
65 70 75 80
Pro Gln Pro Met Phe Pro Gly Asp Leu Met Pro Lys Asn Val Val Thr
85 90 95
Lys Glu Asn Cys Leu Ile Ala Asn Val Phe Met Pro Asn Thr Lys Glu
100 105 110
Lys Asn Leu Ser Val Val Val Tyr Val His Gly Gly Ala Phe Ile Met
115 120 125
Gly Trp Gly Glu Met Phe Lys Ala Arg Gln Phe Met Lys Thr Lys Asp
130 135 140
Phe Ile Val Val Thr Phe Asn Tyr Arg Leu Gly Ile His Gly Phe Leu
145 150 155 160
Cys Leu Gly Thr Asp Asp Ala Pro Gly Asn Ala Gly Met Lys Asp Gln
165 170 175
Val Ala Leu Leu Arg Trp Val Gln Lys Asn Ile Ala Ser Phe Gly Gly
180 185 190
Asn Pro Asp Asp Val Thr Ile Ala Gly Tyr Ser Ala Gly Ser Ala Ser
195 200 205
Val Asp Leu Leu Met Leu Ser Lys Ser Ala Glu Gly Leu Phe His Arg
210 215 220
Val Ile Pro Glu Ser Gly Gly Asn Leu Ala Ala Phe Ser Ile Gln Arg
225 230 235 240
Asp Pro Val Glu Ile Ala Lys Ser Tyr Ala Ser Lys Leu Gly Phe Asp
245 250 255
Asn Gly Asp Asp Ile Tyr Ala Leu Gly Lys Phe Tyr Met Thr Ala Pro
260 265 270
Ile Glu Lys Leu Thr Ser Asp Pro Phe Phe Asp Arg Thr Asp Ser Thr
275 280 285
Phe Leu Phe Ala Pro Cys Val Glu Arg Glu Thr Gly Asp Gly Ala Phe
290 295 300
Leu Thr Glu Ser Pro Leu Thr Ile Leu Lys Thr Gly Asn Tyr Arg Lys
305 310 315 320
Leu Pro Val Leu Tyr Gly Phe Ala Glu Met Glu Gly Leu Ile Arg Ile
325 330 335
Asp Phe Phe Glu Leu Trp Lys His Lys Met Asn Glu Lys Phe Ser Asp
340 345 350
Phe Leu Pro Ala Asp Leu Lys Phe Asp Ser Glu Glu Glu Arg Glu Glu
355 360 365
Val Ala Asn Lys Ile Lys Glu Phe Tyr Phe Gly Asp Lys Pro Val Gly
370 375 380
Asn Glu Asn Ile Leu Lys Tyr Val Asp Phe Phe Ser Asp Val Ile Phe
385 390 395 400
Ala Tyr Pro Met Leu Trp Ala Val Lys Leu His Val Glu Ala Gly Asn
405 410 415
Asn Gln Val Tyr Leu Tyr Glu Tyr Ser Phe Val Asp Glu Asp Val Pro
420 425 430
Val Val Pro His Thr Asn Ile Arg Gly Ala Asn His Cys Ala Gln Thr
435 440 445
Met Ala Leu Ser Asp Gly Lys Asn Phe Thr His His Asp Asp Thr Leu
450 455 460
Ala Thr Pro Gln Phe Arg Glu Met Lys Lys Thr Ile Arg Glu Ile Trp
465 470 475 480
His Asn Phe Val Lys Thr Gly Val Pro Val Pro Glu Gly Ser Trp Leu
485 490 495
Pro Ala Trp Pro Ala Ala Gly Ala Asp Arg Ala Pro His Met Ser Leu
500 505 510
Gly Glu Arg Leu Glu Leu Arg Gly Ala Leu Leu Pro Glu Arg Thr Arg
515 520 525
Phe Trp Asp Asp Ile Tyr Gln Arg Tyr Tyr Arg Asp Ala Val Pro Pro
530 535 540
Pro Lys Pro Pro Pro Arg Pro Arg Asn Glu Leu
545 550 555
<210> 4
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<212> DNA
<213> 人工序列
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<223>
<400> 4
atggctagca gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta 60
gatggtgatg ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca 120
tacggaaaac ttacccttaa atttatttgc actactggaa aactacctgt tccttggcca 180
acacttgtca ctactttctc ttatggtgtt caatgctttt caagataccc agatcatatg 240
aagcggcacg acttcttcaa gagcgccatg cctgagggat acgtgcagga gaggaccatc 300
tctttcaagg acgacgggaa ctacaagaca cgtgctgaag tcaagtttga gggagacacc 360
ctcgtcaaca ggatcgagct taagggaatc gatttcaagg aggacggaaa catcctcggc 420
cacaagttgg aatacaacta caactcccac aacgtataca tcacggcaga caaacaaaag 480
aatggaatca aagctaactt caaaattaga cacaacattg aagatggaag cgttcaacta 540
gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac 600
cattacctgt ccacacaatc tgccctttcg aaagatccca acgaaaagag agaccacatg 660
gtccttcttg agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa 720
taa 723
<210> 5
<211> 240
<212> PRT
<213> 人工序列
<220>
<223>
<400> 5
Met Ala Ser Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile
1 5 10 15
Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
20 25 30
Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe
35 40 45
Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr
50 55 60
Thr Phe Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
65 70 75 80
Lys Arg His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln
85 90 95
Glu Arg Thr Ile Ser Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala
100 105 110
Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys
115 120 125
Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu
130 135 140
Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys
145 150 155 160
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
165 170 175
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
180 185 190
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
195 200 205
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
210 215 220
Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys
225 230 235 240
<210> 6
<211> 317
<212> RNA
<213> 人工序列
<220>
<223>
<400> 6
cgauuucaag gaggacggaa acauccucgg ccacaaguug gaauacaacu acaacuccca 60
caacguauac aucacggcag acaaacaaaa gaauggaauc aaagcuaacu ucaaaauuag 120
acacaacauu gaagauggaa gcguucaacu agcagaccau uaucaacaaa auacuccaau 180
uggcgauggc ccuguccuuu uaccagacaa ccauuaccug uccacacaau cugcccuuuc 240
gaaagauccc aacgaaaaga gagaccacau gguccuucuu gaguuuguaa cagcugcugg 300
gauuacacau ggcaugg 317
Claims (10)
1.抑制CCE001a蛋白表达和/或活性的物质在降低棉铃虫对棉酚的耐受性中的应用。
2.抑制CCE001a蛋白表达和/或活性的物质在制备棉铃虫杀虫剂中的应用。
3.抑制CCE001a蛋白表达和/或活性的物质在培育抗棉铃虫棉花中的应用。
4.一种降低棉铃虫对棉酚的耐受性的方法，包括如下步骤：向棉铃虫注射抑制CCE001a蛋白表达和/或活性的物质；
与未注射抑制CCE001a蛋白表达和/或活性的物质的棉铃虫相比，注射了抑制CCE001a蛋白表达和/或活性的物质的棉铃虫对棉酚的耐受性降低。
5.如权利要求4所述的方法，其特征在于：所述棉铃虫为棉铃虫幼虫；所述注射的剂量为每只棉铃虫幼虫注射2.0μg以上特异RNA。
6.一种棉铃虫杀虫剂，其含有抑制CCE001a蛋白表达和/或活性的物质。
7.如权利要求1至3任一所述的应用、或、权利要求4或5所述的方法、或、权利要求6所述的棉铃虫杀虫剂，其特征在于：所述“抑制CCE001a蛋白表达和/或活性的物质”为特异RNA；
所述特异RNA为序列表中的序列1所示的双链RNA。
8.特异RNA；所述特异RNA为序列表中的序列1所示的双链RNA。
9.CCE001a蛋白在调控棉花抗虫性中的应用。
10.编码CCE001a蛋白的核酸分子在调控棉花抗虫性中的应用。
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| CN114717216A (zh) * | 2022-03-31 | 2022-07-08 | 石河子大学 | 一种棉酚降解酶CCE001a基因、氨基酸序列及其应用 |
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