棉铃虫羧酸酯酶基因HaCarE序列分析及其RNAi效应

Sequence analysis and RNAi of carboxylesterase gene HaCarE in cotton bollworm Helicoverpa armigera

为明确棉铃虫Helicoverpa armigera羧酸酯酶(carboxylesterase,CarE)编码基因的功能及其RNA干扰(RNA interfering,RNAi)效应,通过棉铃虫转录组数据筛选获得CarE基因,并克隆得到其全长cDNA序列,对其进行生物信息学分析,并利用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术检测该基因在棉铃虫不同发育时期和不同组织中的表达水平,同时通过RNAi技术检测其对棉铃虫的致死效果。结果表明,从棉铃虫中克隆获得的HaCarE基因全长cDNA序列为1677 bp,编码558个氨基酸,编码蛋白质由15个α螺旋和14个β折叠结构组成。qPCR检测结果显示该基因在棉铃虫不同发育时期均有表达,其中在1龄幼虫期的相对表达量最高,随着龄期的增长,相对表达量逐渐降低,在6龄幼虫期和蛹期的相对表达量最低;在5龄幼虫不同组织中,该基因在中肠中的相对表达量最高。注射dsHaCarE能够明显抑制靶标基因的表达,24 h抑制率为75.41%;饲喂dsHaCarE对棉铃虫2龄幼虫有显著致死作用,5 d时死亡率为64.57%,体长与对照组相比减少了23.29%。表明获得的HaCarE基因对棉铃虫生长发育有显著抑制作用,并有较好的致死效果。

To clarify the function of the carboxylesterase gene(CarE)and its RNAi effect,the CarE gene was obtained by screening cotton bollworm Helicoverpa armigera transcriptome data,and the fulllength cDNA sequence was cloned and analyzed by bioinformatics.The expression level of the gene was detected by quantitative real-time PCR(qPCR)in different developmental stages and tissues of H.armigera,and the lethality of the gene was detected by RNA interference(RNAi).The results showed that the full-length cDNA sequence of the HaCarE gene was 1677 bp,encoding 558 amino acids,and the encoded protein consisted of 15αhelices and 14βfolded structures.The qPCR results showed that the HaCarE gene was expressed in all developmental stages of H.armigera larvae,with the highest ex-pression in first instar larvae.The expression level gradually decreased with increasing instars,and the lowest expression was detected in 6th instar larvae and pupae.Tissue-specific expression analysis in the 5th instar larvae showed a relatively high expression in the midgut.Injection of dsRNA against the HaCarE gene could significantly inhibit the expression level of HaCarE in H.armigera,and the 24-h inhibition efficiency was 75.41%.It had a significant lethal effect on the 2nd instar larvae of H.armig-era after feeding dsRNA,and the mortality rate was 64.57%after 5 d.Moreover,the body length was reduced by 23.29%compared with the control group.It indicated that the obtained HaCarE gene had a significant inhibitory effect on the growth and development of H.armigera and had a good lethal effect.Further studies should be focused on the function and application of this gene.