飞蝗发育相关基因Omb的克隆、 原核表达及时空表达分析

doi:10.16380/j.kcxb.2019.01.004

摘要/Abstract

摘要：

【目的】本研究克隆飞蝗Locusta migratoria发育相关的optomotor-blind(Omb)基因，并对其进行序列和表达分析，旨在更好地了解Omb基因在飞蝗翅发育中的作用及为进一步蝗灾的治理和防治提供新的理论依据。【方法】利用RT-RCR扩增飞蝗Omb基因cDNA序列，采用生物信息学软件分析该基因的核苷酸和氨基酸序列，利用 MEGA 6.0构建昆虫纲分子系统进化树；构建重组表达载体pET-30a/LmOmb，转化到大肠杆菌Escherichia coli BL21(DE3)中，SDS-PAGE及Western blot鉴定重组表达蛋白；基于qPCR技术分析Omb基因在飞蝗不同发育时期及成虫不同组织中的表达谱。【结果】克隆获得飞蝗Omb基因部分cDNA序列，命名为LmOmb(GenBank登录号: MG867658)，其长792 bp，编码264个氨基酸，在第37-219位氨基酸之间存在一个T-box superfamily保守结构域。同源序列比对分析表明LmOmb与褐飞虱Nilaparvata lugens NlOmb氨基酸序列一致性为93%。在IPTG诱导下目标蛋白以6×His标签融合蛋白的形式在宿主菌中得到稳定表达。荧光定量PCR结果显示LmOmb基因在飞蝗不同发育时期均有表达，其中胚胎期的表达量最高，进入若虫期后表达量下降，且各龄若虫之间表达量相对平稳。LmOmb基因在雌性和雄性成虫的胸部、足、腹部和翅中都有表达，且雌性和雄性之间表达量明显不同。【结论】LmOmb基因可能参与了飞蝗的胚胎发育。研究结果为进一步研究飞蝗LmOmb的功能提供了依据。

关键词: 飞蝗, Omb, 基因克隆, 原核表达, 表达谱

Abstract: 【Aim】 The objective of this study is to clone the coding sequence of the developmental gene optomotor-blind (omb) in Locus migratoria and to analyze its structure properties and expression profiles, so as to further understand the role of omb in the wing development of L. migratoria and to provide a new fundamental evidence for the pest management and control. 【Methods】 The cDNA sequence of omb was amplified from L. migratoria with RT-PCR, and the nucleotide and deduced amino acid sequences of the gene were analyzed using different bioinformatics software. The phylogenetic tree was constructed using neighbor-joining method of MEGA 6.0. The recombinant expression vector pET-30a/LmOmb was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein was identified by SDS-PAGE and Western blotting. The expression profiles of omb in different developmental stages and adult tissues of L. migratoria was detected by qPCR. 【Results】 We cloned the partial cDNA sequence of omb from L. migratoria, which is named LmOmb (GenBank accession no.: MG867658) and 792 bp in length encoding 264 amino acids, with a conserved T-box superfamily domain between 37-219 amino acids. Homologous sequence alignment analyses showed that LmOmb has 93% amino acid sequence identity with NlOmb of Nilaparvata lugens. The recombinant expression vector pET-30a/LmOmb was constructed and the target protein was stably expressed in host bacteria in the form of 6×His tag fusion protein after IPTG induction. The qPCR result revealed that LmOmb was expressed in L. migratoria at different developmental stages, and the expression level was the highest in the embryonic stage. The expression level of LmOmb decreased in the nymphal stage, and was relatively stable among nymphs of different instars. LmOmb was expressed in the thorax, leg, abdomen and wing of adults, and showed obviously different expression level between male and female. 【Conclusion】 LmOmb may be involved in the embryonic development of L. migratoria. The results provide a basis for further studying the function of LmOmb in L. migratoria.

Key words: Locusta migratoria, Omb; gene cloning, prokaryotic expression, expression profile

张晓红, 冯莉, 刘亚超, 乔宁, 印红. 飞蝗发育相关基因Omb的克隆、原核表达及时空表达分析[J]. 昆虫学报, 2019, 62(1): 33-40.

ZHANG Xiao-Hong, FENG Li, LIU Ya-Chao, QIAO Ning, YIN Hong. Cloning, prokaryotic expression and spatio-temporal expression profiling of the developmental gene optomotor-blind (omb) in Locusta migratoria (Orthoptera: Acrididae)[J]. Acta Entomologica Sinica, 2019, 62(1): 33-40.

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