板栗过氧化氢酶基因CmCAT的克隆与原核表达

doi:10.16036/j.issn.1000-2650.2019.02.004

摘要/Abstract

摘要： 【目的】旨在克隆板栗CmCAT基因全长cDNA序列,分析其编码蛋白相关信息并进行原核表达研究。【方法】采用RT-PCR技术从板栗品种“石丰”中克隆获得CmCAT基因,用生物信息学方法对获得的cDNA序列及推定的氨基酸序列进行分析。构建原核表达载体pET28a-CmCAT,经PCR和酶切鉴定后转入大肠杆菌BL21（DE3）中进行诱导表达。【结果】板栗CmCAT基因开放阅读框（ORF）为1 479 bp,共编码492个氨基酸,推测蛋白分子质量为56.883 kDa,理论等电点pI为5.83,GenBank登录号为KY312851。其核苷酸序列与核桃（Juglans regia）CAT基因序列相似性最高,为85%。遗传进化分析表明,板栗CmCAT与核桃的亲缘关系最近。SDS-PAGE电泳分析表明,在30 ℃条件下,0.2 mmol/L IPTG诱导6 h表达量最佳,诱导的目的蛋白分子量约为60 kDa,其主要以包涵体的形式存在。【结论】成功克隆了板栗CmCAT基因,并对其编码蛋白的理化性质进行了预测分析,在大肠杆菌中获得了大量的表达,为其进一步的生物学功能研究奠定了基础。

关键词: 板栗, 过氧化氢酶, 基因克隆, 原核表达

Abstract: 【Objective】 The aim to clone the full-length cDNA of CmCAT gene from Castanea mollissima, and to analyze its protein-related information and to study its prokaryotic expression. 【Method】 The CmCAT gene was cloned from Castanea mollissima ‘Shifeng’ by RT-PCR, and the obtained cDNA sequence and the deduced amino acid sequence were analyzed by bioinformatics method. The prokaryotic expression vector pET28a-CmCAT was constructed and then transferred into E. coli BL21 (DE3) by PCR and restriction enzyme digestion. 【Result】 The open reading frame (ORF) of CmCAT gene was 1 479 bp and encoded 492 amino acids. The molecular weight of the protein was 56.883 kDa, the theoretical isoelectric point (pI) was 5.83, and the GenBank accession number was KY312 851. The CmCAT nucleotide sequence shared the highest homology( 85% ) with that in Juglans regia. Genetic evolution analysis showed that CmCAT had the closest genetic relationship with Juglans regia. SDS-PAGE analysis showed that the optimal expression conditions of gene were 6 h under 30 ℃ by 0.2 mmol/L IPTG inducing. The molecular weight of the target protein was about 60 kDa, which was mainly in the form of inclusion body. 【Conclusion】 The CmCAT gene from Castanea mollissima was successfully cloned, and its physicochemical properties were predicted and analyzed. CmCAT protein was expressed in BL21(DE3) largely. Which laid the foundation for further biological function research.

Key words: Castanea mollissima BL, catalase (CAT), gene cloning, prokaryotic expression

中图分类号:

S763

刘裕峰, 朱天辉, 刘应高, 谯天敏, 李姝江, 龙旭梅, 韩珊. 板栗过氧化氢酶基因CmCAT的克隆与原核表达[J]. 四川农业大学学报, 2019, 37(02): 168-176.

LIU Yufeng, ZHU Tianhui, LIU Yinggao, QIAO Tianmin, LI Shujiang, LONG Xumei, HAN Shan. Cloning and Prokaryotic Expression of Catalase Gene CmCAT from Castanea mollissima BL[J]. Journal of Sichuan Agricultural University, 2019, 37(02): 168-176.

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