烟草甲clip丝氨酸蛋白酶基因的克隆及其对外源激素和免疫胁迫的表达响应（英文）

Cloning of two clip-domain serine protease genes and their expression in response to exogenous hormone and immune challenge in Lasioderma serricorne (Coleoptera:Anobiidae)

【目的】作为细胞外信号级联通路的重要组成部分,含有clip结构域的丝氨酸蛋白酶(clip-domain serine proteases, CLIPs)在昆虫发育和先天免疫过程中起着重要作用。本研究旨在克隆烟草甲 Lasioderma serricorne CLIP基因,解析其在烟草甲不同发育阶段和幼虫不同组织中的表达模式,分析其在外源激素20-羟基蜕皮酮(20E)和免疫胁迫后的表达特征,为进一步研究其生理功能奠定基础。【方法】采用RT-PCR技术克隆获得烟草甲两个 CLIPs 基因( LsCLIP 1和 LsCLIP 2)全长cDNA序列,并利用生物信息学软件预测其编码蛋白的结构和特征,利用MEGA 6.06构建昆虫CLIPs系统发育树;利用实时荧光定量PCR(quantitative real-time PCR, qPCR)研究这两个基因在不同发育阶段[低龄幼虫(卵孵化后24 h内)、高龄幼虫(4龄以上)、蛹(化蛹后48 h以上)、早期成虫(化蛹后24 h内)和晚期成虫(化蛹后7 d)]、5龄幼虫不同组织(表皮、脂肪体、肠道和剩余组织)中以及注射20E(120 ng/幼虫)和来源于大肠杆菌 Escherichia coli 和金黄色葡萄球菌 Staphylococcus aureus 的肽聚糖(0.2 μL)后4龄幼虫中的表达模式。【结果】克隆获得烟草甲 LsCLIP 1和 LsCLIP 2基因的cDNA全序列,其开放阅读框长度均为1 194 bp,编码397个氨基酸。序列分析显示,其氨基酸序列各自具有一个clip结构域和胰蛋白酶结构域。系统发育分析表明,CLIP1和CLIP2都属于subfamily C CLIPs。qPCR结果表明, LsCLIP 1和 LsCLIP 2基因在所检测的各发育阶段和幼虫各组织中均有表达,分别尤以蛹期和表皮中表达量最高;经20E和肽聚糖诱导后,烟草甲幼虫体内 LsCLIP 1和 LsCLIP 2基因的表达量明显提高。【结论】推测 LsCLIP 1和 LsCLIP 2可能参与了烟草甲的蜕皮发育和对免疫胁迫的应激响应。本研究将为后续研究昆虫CLIPs的分子调控提供参考。

【Aim】 As essential components of extracellular signaling cascades, clip-domain serine proteases (CLIPs) play important roles in insect development and innate immunity. This study aims to clone two CLIP genes from the cigarette beetle, Lasioderma serricorne , to clarify their developmental stage- and tissue-specific expression profiles, and to analyze their expression changes in response to exogenous hormone 20-hydroxyecdysone (20E) and immune challenge, so as to lay the foundation for further understanding the physiological functions of CLIPs in L. serricorne .【Methods】 The full-length cDNAs of two CLIP-encoding genes ( LsCLIP 1 and LsCLIP 2) were cloned using RT-PCR, and the deduced protein structure was predicted using different bioinformatics software, and the phylogenetic tree of insect CLIPs was constructed using the MEGA 6.06 program. The Mrna expression profiles of these two genes in different developmental stages [early larva (<24 h post egg hatching), late larva (older than the 4th instar), pupa (>48 h post pupation), early adult (<24 h post eclosion), and late adult (7 d post eclosion)] and different tissues (cuticle, fat body, gut, and carcass) of the 5th instar larvae, and the 4th instar larvae of L. serricorne injected with 20E (120 ng/larva) and peptidoglycan (0.2 Μl) from Escherichia coli or Staphylococcus aureus were detected by quantitative real-time PCR (Qpcr).【Results】 The full-length Cdna sequences of LsCLIP 1 and LsCLIP 2 were cloned from L. serricorne , and both genes have an open reading frame of 1 194 bp encoding 397 amino acid residues. The predicted CLIP1 and CLIP2 proteins both contain a typical clip domain and a trypsin-like serine protease domain. Phylogenetic analyses revealed that CLIP1 and CLIP2 belong to subfamily C CLIPs. The Qpcr analyses confirmed that both LsCLIP 1 and LsCLIP 2 were expressed throughout the developmental stages and in all the assayed larval tissues of L. serricorne , with particularly high expression level in the pupal stage and the cuticle, respectively. Expression of both genes was significantly up-regulated in larvae exposed to 20E and following challenge with peptidoglycan from E. coli or S. aureus .【Conclusion】 It is suggested that LsCLIP 1 and LsCLIP 2 might be involved in insect molting and innate immune response. These results provide valuable information for further study on the regulation of CLIPs in insects.