松墨天牛漆酶基因MaLac1的克隆、原核表达及表达谱分析  

Molecular cloning, prokaryotic expression and expression profiling of the laccase gene MaLac1 in Monochamus alternatus (Coleoptera: Cerambycidae)

作　　者：陈昊 罗巧玉 马秋雨 初旭 袁超 刘颖[1] 余伟 吴松青[1,2] 王荣[1,2] 梁光红[1,2] 张飞萍[1,2] 胡霞[1,2] CHEN Hao;LUO Qiao-Yu;MA Qiu-Yu;CHU Xu;YUAN Chao;LIU Ying;YU Wei;WU Song-Qing;WANG Rong;LIANG Guang-Hong;ZHANG Fei-Ping;HU Xia(College of Forestry,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Key Laboratory of Integrated Pest Management in Ecological Forests,Fujian Province University,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学林学院,福州350002 [2]福建农林大学,生态公益林重大有害生物防控福建省高校重点实验室,福州350002

出　　处：《昆虫学报》2020年第4期381-389,共9页Acta Entomologica Sinica

基　　金：国家自然科学基金青年科学基金项目(31800548);国家重点研发项目(2017YFD0600105);福建省林业科技项目[闽林科(2019)16号]。

摘　　要：【目的】本研究旨在克隆并鉴定松墨天牛Monochamus alternatus内源漆酶基因MaLac1,分析其在松墨天牛不同发育阶段的表达水平,为进一步明确MaLac1功能提供依据。【方法】基于松墨天牛肠道转录组测序数据,通过RACE克隆松墨天牛MaLac1基因的全长cDNA序列,并对其进行生物信息学分析;将该基因与pET-32a载体链接构建表达载体pET-MaLac1,导入大肠杆菌Escherichia coli Rosetta (DE3)使其表达;使用qPCR检测MaLac1基因在松墨天牛不同发育阶段(低龄幼虫、老熟幼虫、蛹、雌成虫和雄成虫)肠道中的表达差异。【结果】克隆获得松墨天牛MaLac1的cDNA全长序列(GenBank登录号:KY073340)。MaLac1开放阅读框全长2 067 bp,编码一个含688个氨基酸的蛋白质,预测分子量为78.34 kD,等电点为5.30。SignalP 4.1 Server预测MaLac1在N端包含一个15个氨基酸的信号肽。序列比对分析表明,MaLac1具有典型的昆虫漆酶基因特征,与赤拟谷盗Tribolium castaneum漆酶基因的氨基酸序列一致性达93%。SDS-PAGE检测发现IPTG诱导表达了一条大约78 kD的特异蛋白条带,与推测大小一致。qPCR结果显示,MaLac1在不同发育阶段的松墨天牛肠道中均有表达,其中,在雌成虫肠道中表达量最高,在雄成虫肠道中的次之,在幼虫肠道中的最低。【结论】MaLac1在松墨天牛成虫中表达量显著高于其在幼虫中的,这一结果可能与幼虫和成虫的取食习性差异相关。MaLac1在松墨天牛体内的功能还有待进一步研究。【Aim】 This study aims to clone and identify the laccase gene MaLac1 from the Japanese pine sawyer, Monochamus alternatus, and to analyze its expression levels in different developmental stages of the beetle, so as to provide some clues for further study of the function of MaLac1. 【Methods】 Based on the transcriptome sequencing data of M. alternatus gut, the full-length cDNA of MaLac1 was cloned from M. alternatus using the RACE method and analyzed by bioinformatic software. MaLac1 sequence was then inserted into the prokaryotic expression vector pET-32 a(+) to construct a recombinant plasmid pET-MaLac1, which was then transformed into Escherichia coli Rosetta(DE3) to express MaLac1. The expression patterns of MaLac1 in the gut of M. alternatus at different developmental stages(early instar larva, mature larva, pupa, female adult, and male adult) were assayed by qPCR. 【Results】 The full-length cDNA of MaLac1 from M. alternatus(GenBank accession no.: KY073340) were obtained. Its open reading frame is 2 067 bp in length, encoding a protein of 688 amino acids, with an estimated molecular mass of 78.34 kD and an isoeletric point of 5.30. MaLac1 was predicted to contain an N-terminal signal peptide with 15 amino acids by SignalP 4.1 Server. Sequence alignment analysis showed that MaLac1 shows the typical characteristics of insect laccase genes, with 93% amino acid sequence identity with the laccase gene of Tribolium castaneum. SDS-PAGE results showed that IPTG induced a specific protein band of about 78 kD, which was consistent with the estimated size. The qPCR results showed that MaLac1 was expressed in the gut of M. alternatus at various developmental stages, with the highest expression level in the female adult gut, followed by that in the male adult gut, and the lowest expression level in the larval gut. 【Conclusion】 The expression level of MaLac1 in adults of M. alternatus is significantly higher than that in larvae, and this may be relevant to the feeding differences between adults and larvae. Fur

关 键 词：松墨天牛 肠道 漆酶 全长克隆 原核表达 表达谱 

分 类 号：Q966[生物学—昆虫学]