Molecular characterization and blood feeding-relative expression analysis of eight carboxypeptidase genes in Anopheles sinensis (Diptera: Culicidae)
Molecular characterization and blood feeding-relative expression analysis of eight carboxypeptidase genes in Anopheles sinensis (Diptera: Culicidae)
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中华按蚊（双翅目：蚊科）8个羧肽酶基因的分子特征及采血相关表达分析
DOI：
10.16380/j.kcxb.2017.06.002
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
何正波
中科院分区：
文献类型：
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作者：
史宗畔;支中婧;罗世惠;陈斌;何正波
【目的】本研究旨在对中华按蚊8个消化羧肽酶基因进行特性分析，并分析其在吸血前后的表达模式。【方法】运用生物信息学方法分析中华按蚊8个消化羧肽酶基因的特性，分别采用半定量逆转录PCR和实时荧光定量PCR（qRT-PCR）比较这些基因在不同发育阶段不同组织以及吸血后（PBM）不同时间点雌性成蚊中肠内的表达水平。【结果】生物信息学分析表明，8个羧肽酶基因中，6个基因编码羧肽酶A（AsCPA - I 至 AsCPA - VI），2个编码羧肽酶B（AsCPB - I和AsCPB - II）。AsCPA - V的表达仅在4龄幼虫的中肠和虫体（去除中肠的完整蚊虫）中检测到，表明其可能在幼虫中有特异性表达，而其他7个基因在4龄幼虫和成虫中同时表达。吸血后，雌性成蚊中肠内AsCPA - I、AsCPA - II、AsCPA - III、AsCPA - IV、AsCPA - VI、AsCPB - I和AsCPB - II的表达水平发生显著变化，但它们的表达模式完全不同，这表明血液蛋白消化是一个复杂的过程，涉及多个基因的协同表达。AsCPA - I、AsCPA - III、AsCPA - IV、AsCPA - VI和AsCPB - II的表达水平均上调，并在吸血后24小时达到峰值。特别是AsCPA - VI的表达水平在吸血后3小时迅速上升，在吸血后24小时达到峰值，诱导倍数约为418倍，AsCPB - II在吸血后24小时的表达水平也增加了40多倍，表明它们可能参与血液蛋白的消化。然而，吸血后雌性成蚊中肠内AsCPA - II和AsCPB - I的表达水平显著下调。【结论】对中华按蚊的8个羧肽酶基因进行了特性分析。其中，AsCPA - I、AsCPA - III、AsCPA - IV、AsCPA - VI和AsCPB - II可能参与血液蛋白的消化；特别是AsCPA - VI和AsCPB - II可能发挥更重要的作用。本研究结果为进一步研究蚊虫以及中华按蚊血液蛋白消化的分子机制奠定了基础。
【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.