CN116286818A - 靶向光肩星天牛嗅觉共表达受体基因的dsRNA及应用 - Google Patents
靶向光肩星天牛嗅觉共表达受体基因的dsRNA及应用 Download PDFInfo
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Abstract
本发明涉及分子生物技术领域,具体提供了一种靶向光肩星天牛嗅觉共表达受体基因的dsRNA,所述dsRNA以如SEQ ID No.1所示的核苷酸序列为模板,采用dsRNA合成试剂盒合成得到。本发明还提供了所述dsRNA或者包括SEQ ID No.1所示核苷酸序列的光肩星天牛嗅觉共表达受体AglaOrco基因在光肩星天牛防治中的应用。该dsRNA可干扰光肩星天牛雌雄成虫在两性间和寄主植物之间的嗅觉识别和信息交流过程,阻断其正常两性交配和寄主取食过程,进而有助于害虫防治。
Description
技术领域
本发明涉及分子生物技术领域,尤其涉及一种靶向光肩星天牛嗅觉共表达受体基因的dsRNA及应用。
背景技术
光肩星天牛Anoplophora glabripennis、属鞘翅目Coleoptera、天牛科Cerambycidae、沟胫天牛亚科Lamiinae、星天牛属Anoplophora。该虫源发于中国和朝鲜半岛,主要分布在我国北京、辽宁、江西、山西、内蒙古等20多个省市自治区。随着国际贸易的发展,光肩星天牛对林业生产造成了不可估量的损失,引起了世界各国的高度重视。
由于光肩星天牛主要以幼虫危害寄主植物内部,危害早期很难发现,自然控制作用较弱,加之其主要危害杨树等高大的阔叶树种,物理、化学和生物防治等措施较为困难。同时,雄性聚集信息素和寄主植物挥发物混合的引诱剂也已经用于林间的监测与防治,但是引诱效果并不理想(多个年份诱捕数量不超过100头,而其他近缘属如墨天牛属的引诱剂诱捕数量可达2000头以上)。因此,急需寻找新的途径筛选开发更为有效的活性化学物质用于林间防治。
发明内容
有鉴于背景技术中存在的技术问题,本发明的目的是提供一种靶向光肩星天牛嗅觉共表达受体基因的dsRNA及应用。
具体的,本发明的技术方案如下:
第一方面,本发明提供了一种靶向光肩星天牛嗅觉共表达受体基因的dsRNA,所述dsRNA以如SEQ ID No.1所示的核苷酸序列为模板,采用dsRNA合成试剂盒合成。光肩星天牛嗅觉共表达受体AglaOrco基因的核苷酸序列包括SEQ ID No.1,其编码的蛋白质氨基酸序列包括SEQ ID No.2。该氨基酸属于疏水、非分泌蛋白,在内质网上拥有7个跨膜螺旋结构,属于典型受体蛋白。在本发明中,所述dsRNA的核苷酸序列优选包括SEQ ID No.3,或,SEQID NO.3所示的核苷酸序列经过一个或多个核苷酸的替换、缺失或插入获得的具有相同功能的编码核苷酸序列。
第二方面,本发明提供了所述dsRNA,或者包括SEQ ID No.1所示核苷酸序列的光肩星天牛嗅觉共表达受体AglaOrco基因在防治害虫中的应用。目前,在鞘翅目上对于Orco基因的研究主要集中在基因鉴定、基因进化关系和基因表达的探索;关于其参与的嗅觉识别机制的功能研究以及针对靶标基因的产品开发未见报道。在本发明中,所述害虫优选包括光肩星天牛成虫。
具体的,防治优选包括如下至少一种:
(1)降低害虫对繁殖行为信息素的嗅觉响应;
(2)降低害虫对寄主植物挥发物的嗅觉响应。
其中,所述繁殖行为优选包括交配行为。所述繁殖行为信息素优选包括(Z)-9-二十三碳烯。所述寄主植物挥发物优选包括3-蒈烯和/或罗勒烯。
在本发明提供的具体实施方式中,通过向害虫导入dsRNA,可以诱导产生RNAi。本发明利用RNAi技术沉默光肩星天牛AglaOrco基因的表达,验证了AglaOrco的基因功能;同时,还可以干扰害虫两性间或与寄主植物间的信息交流,控制害虫种群数量,对科学绿色的害虫防治具有重要意义,也可以为鞘翅目同属昆虫的防治手段提供新的借鉴和参考。
第三方面,本发明提供了一种护林防虫制剂,所述护林防虫制剂包括所述dsRNA。
第四方面,本发明提供了一种护林防虫方法,所述护林防虫方法包括将所述dsRNA导入害虫体内的步骤,所述导入的方法优选包括饲喂法和/或注射法。
有益效果:
本发明提供了一种靶向光肩星天牛嗅觉共表达受体基因的dsRNA。实验验证:将5ug所述dsRNA(ds-Orco)注射雌雄成虫咽部72h后,经过荧光定量PCR检测可发现注射dsRNA(ds-Orco)处理组雌成虫相比未注射对照组的AglaOrco基因表达下调了68%,注射dsRNA(ds-Orco)处理组雄成虫下调了85%,有效的干扰了基因的表达。触角电位反应的结果显示,注射5ug/头dsRNA(ds-Orco)处理72h的雌成虫对寄主植物挥发物3-蒈烯和罗勒烯的电位反应显著下降;雄成虫对寄主植物挥发物3-蒈烯,罗勒烯和雌性接触性信息素成分之一(Z)-9-二十三碳烯的电位反应显著下降,说明本发明提供的dsRNA可干扰光肩星天牛雌雄成虫在两性间和寄主植物之间的嗅觉识别和信息交流过程,阻断其正常两性交配和寄主取食过程。
本发明还提供了一种环境友好、高效科学的害虫防治方法,为光肩星天牛的全面防控工作提供了基础。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图进行说明。
图1是本发明实施例1中光肩星天牛共表达受体基因AglaOrco的全长基因克隆电泳图;
图2是本发明实施例3中光肩星天牛共表达受体AglaOrco基因沉默效果的检测结果图;图2中A图为注射dsRNA光肩星天牛雌成虫AglaOrco基因表达水平图;图2中B图为注射dsRNA光肩星天牛雄成虫AglaOrco基因表达水平图;
图3是本发明实施例4中光肩星天牛雌雄成虫对气味分子的反应图;图3中A图为注射dsRNA光肩星天牛雌成虫对壬醛,乙酸乙酯,3-蒈烯,罗勒烯和4-庚氧基-1-丁醇的电位反应图;B图为注射dsRNA光肩星天牛雄成虫对α-蒎烯,乙酸乙酯,3-蒈烯,罗勒烯和(Z)-9-二十三碳烯的电位反应图。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细说明,但是不能把它们理解为对本发明保护范围的限定。若未特别指明,实施例中所使用的实验方法均为常规方法;所用的材料、试剂等均可从商业途径得到。
实施例1光肩星天牛共表达受体AglaOrco基因全长克隆
提取光肩星天牛雌雄成虫触角总RNA,使用反转录试剂盒PrimeScriptTM RTreagent Kit(TaKaRa)合成cDNA第一链,再以cDNA第一链为模板,根据光肩星天牛AglaOrco基因的编码区序列的两侧设计引物,正向引物:5’-ATGATGAAATTCAAGGTATC-3’(SEQ IDNo.4);反向引物:5’-TTATTTGAGTTGCACCAACAC-3’(SEQ ID No.5)。
PCR反应体系为2*primer STAR Max:25μL,上游引物:1.5μL,下游引物:1.5μL,cDNA:4μL,d2H2O:18μL。
PCR扩增程序为98℃ 10s;52℃ 15s;72℃ 10s;35个循环;4℃保存。
PCR产物用1%琼脂糖凝胶电泳检测,利用TIANgel Midi Purification Kit胶回收试剂盒回收1434bp的条带,其电泳结果图参见图1。
如图1所示:光肩星天牛AglaOrco基因开放阅读框(ORF)长为1434bp,其序列如SEQID No.1所示,编码477氨基酸。其编码的蛋白质氨基酸序列如SEQ ID No.2所示,蛋白分子量为53.81KDa,等电点为7.73,GRAVY为0.19,属于疏水蛋白;N端没有信号肽,属于非分泌蛋白;亚细胞定位显示蛋白在内质网上,拥有7个跨膜螺旋结构,属于典型受体蛋白。
将回收得到的条带与PEASY-Blunt1载体连接过夜,将连接产物转化至DH5α感受态细胞中。通过菌液PCR检测阳性克隆菌液送往北京睿博兴科生物技术有限公司提取质粒进行测序。
实施例2:光肩星天牛共表达受体AglaOrco基因dsRNA的合成
根据实施例1扩增得到的光肩星天牛AglaOrco基因序列,设计合成AglaOrco基因dsRNA的引物,正向引物:5’-GTTGACCAAGAAGCAGGAGC-3’(SEQ ID No.6);反向引物:5’-TTCATGGTGTTGGTGCAACT-3’(SEQ ID No.7)。扩增得到片段长度为480bp的序列。
选择外源基因GFP作为对照组,同样设计合成dsRNA引物,正向引物:5’-GCATCGACTTCAAGGAGGAC-3’(SEQ ID No.8),反向引物:5’-GAACTCCAGCAGGACCATGT-3’(SEQID No.9),扩增得到片段长度为290bp的序列。
上述每条引物前加20bp的T7启动子序列TAATACGACTCACTATAGGG(SEQ ID No.10),并通过Promega T7RiboMAXTM Express RNAi system试剂盒合成AglaOrco和GFP基因的dsRNA,并于微量分光光度计检测dsRNA浓度,保存于-80℃备用。
实施例3:光肩星天牛共表达受体AglaOrco基因沉默效果的检测
将实施例2合成的AglaOrco和GFP基因的dsRNA和d2H2O分别注射雌雄成虫各3头作为处理组,设置未做注射处理的为对照组,注射量均为5ug/头,分别于注射12h,24h,36h,48h和72h的处理时间后,提取试虫触角总RNA,反转录成cDNA第一链,利用RT-qPCR检测AglaOrco基因的表达情况。
所用的引物如表1所示,注射处理后的基因表达结果如图2所示。
表1本研究所用的引物
图2所示结果表明:注射水和注射ds-GFP组的雌雄成虫与未注射组的AglaOrco基因相对表达量并未有显著性差异,说明外源性的注射行为不会干扰AglaOrco基因的正常表达;但是注射ds-Orco的处理组在72h后AglaOrco基因的相对表达量相比于对照组均有显著的下调表达(p<0.05),分别是雌虫68%,雄虫85%。因此,注射5ug/头ds-Orco处理72h显著降低了雌雄成虫AglaOrco基因的mRNA转录水平,光肩星天牛AglaOrco基因得到了有效的沉默。
实施例4:注射AglaOrco基因dsRNA光肩星天牛雌雄成虫对气味分子的反应
利用触角电位EAG反应测定注射5ug/头ds-Orco处理72h的雌雄成虫对气味物质(α-蒎烯,乙酸乙酯,3-蒈烯,罗勒烯,壬醛,(Z)-9-二十三碳烯和4-庚氧基-1-丁醇)的反应变化,结果如图3所示。
图3所示结果表明:注射ds-Orco的光肩星天牛雌成虫对寄主植物挥发物3-蒈烯和罗勒烯的电位反应显著下降;雄成虫对寄主植物挥发物3-蒈烯,罗勒烯和雌性接触性信息素成分之一(Z)-9-二十三碳烯的电位反应显著下降(P<0.05),说明AglaOrco蛋白表达被干扰后,光肩星天牛对寄主植物挥发物和雌雄信息素成分等多种气味信号物质的嗅觉响应均大幅度降低,证实了AglaOrco蛋白功能的缺陷会引起光肩星天牛的嗅觉损伤。本发明提供的dsRNA可用于光肩星天牛成虫的防治。
以上所述实施例仅表达了本发明的几种实施方式,便于具体和详细地理解本发明的技术方案,但并不能因此而理解为对发明专利保护专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (10)
1.靶向光肩星天牛嗅觉共表达受体基因的dsRNA,其特征在于,所述dsRNA以如SEQ IDNo.1所示的核苷酸序列为模板,采用dsRNA合成试剂盒合成。
2.根据权利要求1所述靶向光肩星天牛嗅觉共表达受体基因的dsRNA,其特征在于,所述dsRNA的核苷酸序列包括SEQ ID No.3,或,SEQ ID NO.3所示的核苷酸序列经过一个或多个核苷酸的替换、缺失或插入获得的具有相同功能的编码核苷酸序列。
3.权利要求1或2所述dsRNA,或者包括SEQ ID No.1所示核苷酸序列的光肩星天牛嗅觉共表达受体AglaOrco基因在防治害虫中的应用。
4.根据权利要求3所述应用,其特征在于,所述害虫包括光肩星天牛成虫。
5.根据权利要求3或4所述应用,其特征在于,所述防治包括如下至少一种:
(1)降低害虫对繁殖行为信息素的嗅觉响应;
(2)降低害虫对寄主植物挥发物的嗅觉响应。
6.根据权利要求5所述应用,其特征在于,(1)中所述繁殖行为包括交配行为。
7.根据权利要求5所述应用,其特征在于,(1)中所述繁殖行为信息素包括(Z)-9-二十三碳烯。
8.根据权利要求5所述应用,其特征在于,(2)中所述寄主植物挥发物包括3-蒈烯和/或罗勒烯。
9.护林防虫制剂,其特征在于,所述护林防虫制剂包括权利要求1或2所述dsRNA。
10.护林防虫方法,其特征在于,包括如下步骤:将权利要求1或2所述dsRNA导入害虫体内;所述导入的方法包括饲喂法和/或注射法。
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