粘虫EF-1a和AK基因的克隆及实时RT-PCR方法建立  

Cloning of EF-1a and AK Genes from Mythimna separata and Establishment of a Real Time RT-PCR Assay

作　　者：段云[1] 巩中军[1] 蒋月丽[1] 吴潇博 李彤[1] 苗进[1] 武予清[1] 

机构地区：[1]河南省农业科学院植物保护研究所,河南省农作物病虫害防治重点实验室/农业部华北南部有害生物治理重点实验室,郑州450002

出　　处：《中国农学通报》2016年第11期27-32,共6页Chinese Agricultural Science Bulletin

基　　金：国家公益性行业(农业)科研专项“粘虫监控技术研究与示范”(201403031);国家小麦产业技术体系“地下虫害防控岗位”(CARS-03)

摘　　要：为今后利用SYBR GreenⅠ荧光定量法研究粘虫EF-1a和AK基因的表达情况,设计合成用于PCR扩增的引物,运用RT-PCR方法克隆EF-1a和AK基因片段,并进行序列同源性分析;然后采用SYBR GreenⅠ荧光定量法设计特异性引物进行RT-PCR扩增,通过熔解曲线和标准曲线分析等建立实时RT-PCR方法。结果表明,从粘虫中克隆获得EF-1a和AK基因片段长度分别为622 bp和1020 bp,分别编码207个氨基酸和339个氨基酸;序列同源性分析结果表明,粘虫EF-1a与鳞翅目昆虫家蚕、桦尺蠖和柑桔凤蝶的氨基酸序列同源性为95%以上;AK与鳞翅目昆虫草地贪夜蛾、烟蚜夜蛾、棉铃虫、斜纹夜蛾和甜菜夜蛾的氨基酸序列同源性为98%以上。熔解曲线和标准曲线结果显示,设计的EF-1a和AK基因引物均可应用于实时RT-PCR扩增。In order to analyze the expression patterns of EF-1a and AK genes in Mythimna separata by realtime RT-PCR, the partial c DNAs of the two genes were cloned using the designed primers, and the sequencehomology was analyzed. Then specific primers were designed and the real time RT- PCR method wasestablished by analyzing melting curve and standard curve. Results showed the cloned c DNAs of EF-1a and AK genes were 622 bp and 1020 bp, and encoded peptides of 207 amino acids and 339 amino acids,respectively. The amino acid sequence of EF-1a shared homologies of above 95% with Bombyx mori, Biston betularia and Papilio xuthus, and the amino acid sequence of AK shared homologies of above 98% with Spodoptera frugiperda, Heliothis virescens, Helicoverpa armigera, Spodoptera litura and Spodoptera exigua.Analyses of melting curve and standard curve indicated that the designed primers of these two genes could beapplied to real time RT-PCR experiments. This study could provide a foundation for future gene expressionstudies in M. separata.

关 键 词：粘虫 延伸因子 精氨酸激酶 克隆 反转录实时定量PCR 

分 类 号：Q966[生物学—昆虫学]