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)

doi:10.16380/j.kcxb.2019.05.002

昆虫学报 ›› 2019, Vol. 62 ›› Issue (5): 535-546.doi: 10.16380/j.kcxb.2019.05.002

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)

陈春旭^1,2, 许抗抗^1,2, 杨洪^1,3,*, 赵凤¹, 严毅^1,2, 胡大鸣⁴, 杨文佳^1,2

(1. 贵州大学昆虫研究所, 贵州山地农业病虫害重点实验室, 贵阳550025; 2. 贵阳学院生物与环境工程学院, 贵州省山地珍稀动物与经济昆虫重点实验室, 贵阳 550005; 3. 贵州大学烟草学院, 贵阳 550025; 4. 贵州中烟工业有限责任公司, 贵阳 550025)

出版日期:2019-05-20 发布日期:2019-05-16

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

CHEN Chun-Xu^1,2, XU Kang-Kang^1,2, YANG Hong^1,3,*, ZHAO Feng¹, YAN Yi^1,2, HU Da-Ming⁴, YANG Wen-Jia^1,2

(1. Provincial Key Laboratory for Agricultural Pest Management of Mountainous Regions, Institute of Entomology, Guizhou University, Guiyang 550025, China; 2. Guizhou Provincial Key Laboratory for Rare Animal and Economic Insect of the Mountainous Region, College of Biology and Environmental Engineering, Guiyang University, Guiyang 550005, China; 3. College of Tobacco Science, Guizhou University, Guiyang 550025, China; 4. China Tobacco Guizhou Industrial Co., LTD, Guiyang 550005, China)

Online:2019-05-20 Published:2019-05-16

摘要/Abstract

摘要：

【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(LsCLIP1和LsCLIP2)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LsCLIP1和LsCLIP2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，LsCLIP1和LsCLIP2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LsCLIP1和LsCLIP2基因的表达量明显提高。【结论】推测LsCLIP1和LsCLIP2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。

关键词: 烟草甲, clip丝氨酸蛋白酶, 20羟基蜕皮甾酮, 免疫胁迫, 表达模式

Abstract:

【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 (LsCLIP1 and LsCLIP2) 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 LsCLIP1 and LsCLIP2 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 LsCLIP1 and LsCLIP2 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 LsCLIP1 and LsCLIP2 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.

Key words: Lasioderma serricorne, clip-domain serine proteases, 20-hydroxyecdysone, immune challenge, expression patterns

陈春旭, 许抗抗, 杨洪, 赵凤, 严毅, 胡大鸣, 杨文佳. 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)[J]. 昆虫学报, 2019, 62(5): 535-546.

CHEN Chun-Xu, XU Kang-Kang, YANG Hong, ZHAO Feng, YAN Yi, HU Da-Ming, YANG Wen-Jia. Cloning of two clip-domain serine protease genes and their expression in response to exogenous hormone and immune challenge in Lasioderma serricorne (Coleoptera: Anobiidae)(In English)[J]. Acta Entomologica Sinica, 2019, 62(5): 535-546.

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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)(In English)

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