棉铃虫四个丝氨酸蛋白酶抑制剂基因的克隆与表达分析

Cloning and expression analysis of four serine protease inhibitor genes in cotton bollworm Helicoverpa armigera

为明确棉铃虫Helicoverpa armigera丝氨酸蛋白酶抑制剂(serine protease inhibitor,serpin)的种类及其表达特性,利用PCR技术克隆棉铃虫的serpin基因,使用生物信息学软件预测其结构并进行系统进化分析,采用实时荧光定量PCR(real time quantitative PCR,RT-qPCR)技术比较serpin基因在棉铃虫不同发育阶段和组织中的表达量及取食Cry1Ac后其表达量的变化。结果表明,共获得serpin-a、serpin-b、serpin-c、serpin-e四个棉铃虫serpin基因,全长为1119~1254 bp,编码373~418个氨基酸,均包含一段具有反应中心环的保守结构域,且与斜纹夜蛾Spodoptera litura、草地贪夜蛾S.frugiperda等鳞翅目昆虫serpin的同源性较高。serpin-a和serpin-e在棉铃虫4龄幼虫期的表达量最高,serpin-b和serpin-c分别在成虫期和蛹期表达量最高。serpin-a在中肠和围食膜中表达量最高,serpin-b在头、中肠和表皮中表达量最高,serpin-c在头部表达量最高,serpin-e在中肠和血淋巴中的表达量显著高于其他组织。棉铃虫取食低浓度Cry1Ac后,中肠的serpin-b和serpin-e的表达量显著增加。推测不同serpin基因在棉铃虫不同发育时期和组织中可能发挥不同的作用,其中serpin-b和serpin-e可能参与棉铃虫对Cry1Ac的解毒过程。

To clarify the species and expression characteristics of serine protease inhibitor(serpin)in the cotton bollworm Helicoverpa armigera,serpin genes in H.armigera were cloned by using PCR method,and their structure and phylogenesis were predicted and analyzed with bioinformatics.Realtime quantitative polymerase chain reaction(RT-qPCR)was used to compare the relative expression levels of serpin genes in the different stages and different tissues,and the changes in the expression level of serpins in the larvae fed with Cry1Ac were also compared.The results showed that the full-length sequences of four serpin genes(serpin-a,serpin-b,serpin-c,serpin-e)were 1119-1254 bp,encoding 373-418 amino acids.All four serpin genes contained reactive center loop domains,with a high homology with those of Lepidoptera,such as Spodoptera litura and S.frugiperda.The highest expression levels of serpin-a and serpin-e occurred in the 4th-instar larvae,and the highest expression of serpin-b and serpin-c was observed during the adult and pupal stages.The highest expression of serpin-a was in midgut and the Malpighian tube.The expression of serpin-b was the highest in head,midgut and cuticle.The highest relative expression level of serpin-c was in head,and the expression of serpin-e in midgut and hemolymph was significantly higher than that in other tissues.The expression levels of serpin-b and serpin-e in the midgut of H.armigera were significantly up-regulated after its larvae were fed with low concentrations of Cry1Ac.The results indicated that different serpin genes might play different roles in different developmental stages and tissues in H.armigera,and serpin-b and serpin-e might be involve in the toxicity of Cry1Ac to H.armigera.