生物学杂志

生物学杂志

• 研究报告 •    下一篇

枝状枝孢霉MD2的Unigene A09801克隆与原核表达分析

  

  1. 中南民族大学生命科学学院 生物技术国家民委重点实验室 武陵山区特色资源植物种质保护与利用湖北省重点实验室， 武汉 430074
  • 出版日期:2016-08-18 发布日期:2016-08-18
  • 通讯作者: 张 鹏，博士，副教授，研究方向为微生物生物技术，E-mail: zhangpenghust@126.com
  • 作者简介:席晓圆，硕士研究生，研究方向为生物化学与分子生物学，E-mail: orchidgrape@163.com
  • 基金资助:
    国家自然科学基金(No.31370118);中南民族大学硕士研究生学术创新基金项目(No.2015sycxjj119); 中南民族大学大学生创新创业项目(GCX15006; SCX15004)

Cloning and prokaryotic expression analysis of Unigene A09801from Cladosporium cladosporioides MD2

  1. College of Life Science, South-Central University for Nationalities/Key Laboratory for/Hubei Provincial Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area of China, Wuhan 430074， China
  • Online:2016-08-18 Published:2016-08-18

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摘要： 真菌紫杉醇合成酶类基因的功能研究是构建紫杉醇高产工程菌株的前提。首次从产紫杉醇真菌——枝状枝孢霉MD2中克隆了Unigene A09801的DNA和cDNA全长序列（1674 bp），该基因不含有内含子，其编码产物含557个氨基酸，与GMC (glucose-methanol-choline) oxidoreductase一致性为56%，蛋白分子质量为61 291.8 u，等电点为5.47，不存在跨膜结构和信号肽。Unigene A09801以单拷贝方式存在于基因组中，其表达量受茉莉酸甲酯诱导表现出先降低后升高再降低的趋势，最高表达量出现在24 h。构建了Unigene A09801的原核表达载体，并初步优化其表达条件为：诱导温度32 ℃，IPTG 浓度1.2 mmol/L，诱导时间4 h。结果为体外（in vitro）分析该基因编码蛋白的催化功能奠定了基础。

关键词: 枝状枝孢霉, 紫杉醇, 合成酶, 原核表达

Abstract: Molecular cloning and functional research on genes related to fungal taxol biosynthesis is a key for the genetic engineering improvement of taxol-producing fungi. In this study, the full length sequences (1674 bp) of DNA and cDNA of Unigene A09801 were cloned from the taxol-producing fungus, Cladosporium cladosporioides MD2, respectively. Unigene A09801 sequence had no introns. Unigene A09801 encoding protein contained 557 amino acids, had 56% identity with GMC (glucose-methanol-choline) oxidoreductase, possessed 61 291.8 u molecular weight and 5.47 isoelectric point value, and had no trans-membrane structure and signal peptide. Unigene A09801 existed in the fungal genome with a single copy. The expression levels of Unigene A09801 responded to Methyl Jasmonate were showed to decrease firstly, increase subsequently and then decrease, and the highest expression level appeared at 24 h. The prokaryotic expression vector of Unigene A09801 was constructed, and the optimized expression conditions of Unigene A09801 in E. coli BL 21 were: induced at 32℃ for 4 h, with 1.2 mmol/L TPTG. These results would lay a foundation for further in vitro analyzing catalytic function of Unigene A09801 encoding protein.

Key words: Cladosporium cladosporioides, taxol, synthase, prokaryotic expression