中华按蚊八个羧肽酶基因的鉴定及其与吸血餐相关的表达分析

摘要

[目的]本研究旨在鉴定中华按蚊Anopheles sinensis8个消化型羧肽酶基因,并分析这些羧肽酶基因在血餐前后的表达模式.[方法]通过生物信息学方法分析中华按蚊8个羧肽酶基因的特征,采用半定量PCR或定量PCR (qRT-PCR)方法分别分析这些基因在中华按蚊不同发育时期不同组织和在取食血液前后中华按蚊雌成虫中肠中的表达情况.[结果]通过生物信息学分析发现,8个羧肽酶基因中,6个编码羧肽酶A(AsCPA-Ⅰ～AsCPA-Ⅵ),2个编码羧肽酶B(AsCPB-Ⅰ和AsCPB-Ⅱ).表达分析发现,只有AsCPA-Ⅴ在中华按蚊4龄幼虫中肠和中肠外组织(仅去掉中肠的虫体)中表达,可能为幼虫特异性表达的基因,其余7个羧肽酶基因既在幼虫期表达又在成虫期表达.取食血液后,AsCPA-Ⅰ,sCPA-Ⅱ,AsCPA-Ⅲ,AsCPA-Ⅳ,AsCPA-Ⅵ,AsCPB-Ⅰ和AsCPB-Ⅱl在中华按蚊雌成虫中肠中的表达明显受到血液的影响,但表达模式不一样,说明血液蛋白的消化是由多个基因协同作用的复杂生理过程.其中,AsCPA-Ⅰ,AsCPA-Ⅲ,AsCPA-Ⅳ,As CPA-Ⅵ和AsCPB-Ⅱ在取食血液后表达上调,且都在吸血后的24h达到表达高峰.尤其是AsCPA-Ⅵ在吸血后24 h表达上调了约418倍,AsCPB-Ⅱ上调了40倍,可能与血液蛋白的消化有关.而AsCPA-Ⅱ和AsCPB-Ⅰ基因的表达水平在中华按蚊取食血液后显著下调.[结论]本研究对中华按蚊8个消化型羧肽酶基因进行了鉴定和表达分析.AsCPA-Ⅰ,AsCPA-Ⅲ,AsCPA-Ⅳ,AsCPA-Ⅵ和AsCPB-Ⅱ可能参与血液蛋白的消化,尤其是As CPA-Ⅵ和AsCPB-Ⅱ可能起着更为重要的作用.研究结果为深入剖析中华按蚊血液蛋白消化的分子机理奠定了基础.%[Aim] The objective of this study is to characterize eight digestive carboxypeptidase genes in Anopheles sinensis,and to analyze their expression patterns before and post blood meal feeding.[Methods] The characteristics of eight digestive carboxypeptidase genes of An.sinensis were analyzed using bioinformatics methods,and their expression levels in different tissues at different developmental stages and in the midguts of female adults at different time points post blood meal feeding (PBM) were compared by semi-quantitative reverse transcription PCR and quantitative real-time PCR (qRT-PCR),respectively.[Results] Bioinformatics analysis indicated that of the eight carboxypeptidase genes,six genes encode carboxypeptidases A (AsCPA-I-AsCPA-Ⅵ) and two encode carboxypeptidases B (AsCPB-Ⅰ and AsCPB-Ⅱ).The expression of AsCPA-Ⅴ was only detected in midguts and carcasses (whole mosquitoes minus midgut) of the 4th instar larvae,suggesting that it might have specific expression in larvae,while the other seven genes were simultaneously expressed in the 4th instar larvae and adults.After blood meal feeding,the expression levels of AsCPA-Ⅰ,AsCPA-Ⅱ,AsCPA-Ⅲ,AsCPA-Ⅳ,AsCPA-Ⅵ,AsCPB-Ⅰ and AsCPB-Ⅱ in the midguts of female adults significantly changed,but their expression patterns were completely different,suggesting that the blood protein digestion is a complex process involving the coordinative expressions of multiple genes.The expression levels of AsCPA-Ⅰ,AsCPA-Ⅲ,AsCPA-Ⅳ,AsCPA-Ⅵ and AsCPB-Ⅱ were all up-regulated and peaked at 24 h PBM.Especially,the expression level of AsCPA-Ⅵ rapidly increased at 3 h PBM,and peaked at 24 h PBM with ～418-fold induction,and that of AsCPB-Ⅱ also increased by more than 40-fold at 24 h PBM,suggesting that they might be involved in blood protein digestion.However,the expression levels of AsCPA-Ⅱ and AsCPB-Ⅰ in midguts of female adults were significantly down-regulated after blood meal feeding.[Conclusion] Eight carboxypeptidase genes from An.sinensis were characterized.Among them,AsCPA-Ⅰ,AsCPA-Ⅲ,AsCPA-Ⅳ,AsCPA-Ⅵ and AsCPB-Ⅱ are putatively involved in blood protein digestion;in particular,AsCPA-Ⅵ and AsCPB-Ⅱ might play more important roles.Our results provide a basis for further study on the molecular mechanism of blood protein digestion in mosquitos as well as in An.sinensis.