玉米象线粒体细胞色素C氧化酶亚基Ⅲ基因克隆与表达分析

doi:10.3864/j.issn.0578-1752.2016.23.007

摘要/Abstract

摘要： 【目的】克隆辣根素可能的作用位点——细胞色素C氧化酶亚基Ⅲ（COX Ⅲ）的全长cDNA，并对其进行序列分析及原核表达。【方法】在NCBI数据库中查找玉米象（Sitophilus zeamais）COX Ⅲ已知的部分序列，利用RT-PCR，结合RACE技术克隆获得玉米象COXⅢ全长序列；利用DNAMAN8.0、MEGA5.1、GENEDOC、ProtParam和TargetP 1.1 Server等软件对该基因全长cDNA序列、系统进化关系、基本理化性质及其三维结构进行分析；将玉米象COXⅢ基因分别与载体pEASY-Blunt E1、pET28a、pET30a、pET32a、pET42a连接，构建原核表达载体pEASY-Blunt E1-COXⅢ、pET28a-COXⅢ、pET30a-COXⅢ、pET32a-COXⅢ、pET42a-COXⅢ并分别转化至大肠杆菌BL21（DE3）中，在不同IPTG诱导浓度、温度以及时间内诱导融合蛋白表达，采用SDS-PAGE和Western blot（WB）对表达结果进行验证。【结果】获得的玉米象COXⅢ基因全长cDNA序列开放阅读框（ORF）为792 bp，编码263个氨基酸，编码的蛋白分子质量约为30 938.1 Da，理论等电点pI 6.51，预测分子式为C₁₄₉₂H₂₁₅₄N₃₃₈O₃₆₂S₁₀，不稳定系数为34.49，总平均亲水系数为0.492，为疏水的稳定蛋白。系统发育树显示玉米象与鞘翅目象鼻虫科昆虫米象进化关系最近。原核表达结果表明，该蛋白在18℃、1 mmol·L^-1 IPTG诱导24 h 的条件下即能实现融合蛋白的表达，可溶性检测表明该蛋白主要以包涵体的形式存在；Western blot印迹检测证实大肠杆菌BL21(DE3)中表达了一个分子量约为62 kD的融合蛋白。【结论】成功从玉米象昆虫克隆得到COXⅢ全长cDNA序列，并实现了其编码蛋白的原核表达，可为后续探究玉米象COXⅢ功能和异硫氰酸酯类化合物对玉米象的作用机理提供理论依据。

关键词: 玉米象, 细胞色素C氧化酶亚基Ⅲ, RACE技术, 生物信息学, 原核表达

Abstract: 【Objective】The objective of this study is to clone full-length cDNA of cytochrome C oxidase subunit Ⅲ (COXⅢ), which is a possible action site of allyl isothiocyanate (AITC), then analyze the sequence and prokaryotic expression．【Method】Firstly, finding the known part of the gene of COXⅢ in the NCBI, the full sequence of Sitophilus zeamais COXⅢ was cloned by RT-PCR and RACE techniques. The sequences of COXⅢ, phylogenetic relationship and properties of S. zeamais COXⅢ, the three-dimensional structure were analyzed by a variety of bioinformatics softwares such as DNAMAN8.0, MEGA5.1, GENEDOC, ProtParam and TargetP 1.1 Server et al. Using pEASY-Blunt E1, pET28a, pET30a, PET32a and pET42a as a fused expression vector, a recombinant plasmid pEASY-Blunt E1-COXⅢ, pET-28a-COXⅢ, pET30a-COXⅢ, pET32a-COXⅢ, pET42a-COXⅢ were constructed. Then inducing its expression in Escherichia coli BL21 (DE3) with IPTG at different concentrations, times and temperatures. SDS-PAGE was used to detect the fusion protein expression and the protein expression was verified by Western blot.【Result】The full-length sequences of COXⅢ from S. zeamais was obtained, and its ORF is 792 bp encoding 263 amino acid residues, the predicted molecular weight is 30 938.1 Da, isoelectric point pI is 6.51, the prediction formula is C₁₄₉₂H₂₁₅₄N₃₃₈O₃₆₂S₁₀, the unstable factor is 34.49, and the total average hydrophilic coefficient is 0.492. It is a stable hydrophobic protein. Phylogenetic tree showed that S. zeamais had the closest evolutionary relationship with S. oryzae at the amino acid level. Prokaryotic expression results showed that efficient expression of COXⅢ protein could be realized after induced with 1 mmol·L^-1 IPTG in E. coil BL21 (DE3) for 24 h at 18℃. Solubility analysis showed that the fused protein mainly existed as inclusion bodies. Western blot confirmed that the molecular weight of the recombinant COXⅢ is about 62 kD, consistent with the predicted result.【Conclusion】The full-length sequences of COXⅢ from S. zeamais was successfully obtained, and the prokaryotic expression of its encoding protein was achieved, which will lay a foundation for further function research of S. zeamais COXⅢ, and provide a theoretical basis for the research of the mechanism of the action on controlling S. zeamais by using isothiocyanates compound.

Key words: Sitophilus zeamais, cytochrome C oxidase subunit Ⅲ, RACE technique, bioinformatics analysis, prokaryotic expression

侯昌亮，王靖博，李永强，吴华，马志卿，冯俊涛，张兴. 玉米象线粒体细胞色素C氧化酶亚基Ⅲ基因克隆与表达分析[J]. 中国农业科学, 2016, 49(23): 4544-4554.

HOU Chang-liang, WANG Jing-bo, LI Yong-qiang, WU Hua, MA Zhi-qing, FENG Jun-tao, ZHANG Xing. Cloning and Expression Analysis of Cytochrome C Oxidase Subunit Ⅲ from Sitophilus zeamais[J]. Scientia Agricultura Sinica, 2016, 49(23): 4544-4554.

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