柞蚕RR-1亚族表皮蛋白基因ApCP12与ApCP23的表达特征与功能分析

摘要

[目的]表皮蛋白(cuticular proteins,CPs)种类丰富,在昆虫生长发育中起着重要作用.本研究旨在丰富柞蚕Antheraea pernyi体内所含表皮蛋白的类型,探讨不同发育阶段表皮蛋白基因的表达规律.[方法]利用PCR及3'RACE技术从柞蚕幼虫表皮组织中克隆得到两个表皮蛋白基因,并进行生物信息学分析及系统进化分析;半定量RT-PCR检测该基因在柞蚕胚胎发育期的表达规律及在幼虫各组织中的表达分布;实时荧光定量PCR(qPCR)分析该基因在柞蚕不同发育时期及3龄幼虫RNAi后的表达量变化.[结果]克隆获得两个柞蚕表皮蛋白基因并分别命名为ApCP12和ApCP23,GenBank登录号分别为MF318874和MF318875.生物信息学分析表明,ApCP12基因长度为690 bp,开放阅读框(open reading frame,ORF)长336 bp,编码111个氨基酸,推测得到的蛋白质分子量为12 kD;ApCP23长度为1 243 bp,ORF长度为594 bp,编码197个氨基酸,相对分子质量为23 kD.两种蛋白都具有RR-1亚族的保守基序,与RR-2亚族表皮蛋白聚类分析位于不同分支.组织特异性表明,ApCP12基因在柞蚕幼虫各组织中的分布比ApCP23更为广泛.柞蚕不同发育时期中,ApCP12在胚胎发育期表达量逐渐升高;幼虫眠起3d内相对于眠期,ApCP12的表达量最高增加了约3倍,而ApCP23的表达量增加了13倍;蛹黑化时期,ApCP12的表达量高于ApCP23;羽化前期,基因的表达量无明显变化,直至羽化前ld,ApCP12和ApCP23的表达量相对于注射蜕皮激素第1天分别增加了3.5和3倍.RNAi处理后,3龄幼虫体内ApCP12的表达量下降了5倍,ApCP23的表达量降低了3倍.[结论]结果提示,RR-1亚族的这两种柞蚕表皮蛋白参与了柞蚕不同发育阶段幼虫表皮、蛹皮、成虫表皮的构建,与柞蚕生长发育的整个生命周期关系密切.%[Aim] Cuticular proteins (CPs) are abundant in insects,playing important roles in insect growth and development.This study aims to enrich different types of CPs in Antheraea pernyi and to determine the expression patterns of CPs during different developmental stages.[Methods] Two CP genes were cloned from cuticle of A.pernyi larvae using PCR and 3'RACE technology,and the phylogenetic relationship was analyzed by bioinformatics methods.The expression patterns of the two genes in different tissues of larvae and during embryonic development of A.pernyi were analyzed by semi-quantitative RTPCR,and the relative expression levels of the two genes in A.pernyi at different developmental stages and in the 3rd instar larvae after RNAi treatment were measured using real-time quantitative PCR (qPCR) technology.[Results] Two CP genes were cloned from A.pernyi and named ApCP12 (GenBank accession number:MF318874) and ApCP23 (GenBank accession number:MF318875),respectively.Bioinformatic analysis indicated that ApCP12 is 690 bp in length with an open reading frame (ORF) of 336 bp,encoding a protein of 111 amino acid residues with the calculated molecular weight of 12 kD,while ApCP23 is 1 243 bp in length with an ORF of 594 bp,encoding a protein of 197 amino acid residues with the calculated molecular weight of 23 kD.Both proteins contain RR-1 consensus and are classified in different clusters with RR-2 cuticular proteins by phylogenetic analysis.Tissue-specific mRNA expression profiling showed that ApCP12 was more widely distributed than ApCP23.In the progress of embryonic development,the expression level of ApCP12 increased gradually.During larval ecdysis stage,the expression level of ApCP12 increased by 3-fold,and that of ApCP23 increased by 13-fold in 3 d after ecdysis than in molting stage.During pupal melanization,the expression level of ApCP12 was higher than that of ApCP23.The expression levels of the two genes had no significant changes during the earlier stage of pupal eclosion,while on the last day before eclosion,the expression levels of ApCP12 and ApCP23 increased by 3.5-and 3-fold,respectively,as compared with that on the first day of 20E injection.After RNAi treatment,the expression levels of ApCP12 and ApCP23 in the 3rd instar larvae were down-regulated 5-and 3-fold,respectively.[Conclusion] The results suggest that these two CPs of subtribe RR-1 participate in the cuticle building of larva,pupa and adult and have a close relationship with the whole life cycle of A.pernyi development.