枝状枝孢霉MD2的Unigene A03231的克隆及原核表达分析  

Cloning and Prokaryotic Expression Analysis of Unigene A03231 from Cladosporium cladosporioides MD2

作　　者：张鹏[1] 周兰 席晓圆[1] 德格 柯友胜 刘盼 宋发军[1] Zhang Peng;Zhou Lan;Xi Xiaoyuan;De Ge;Ke Yousheng;Liu Pan;Song Fajun;Zhang Peng;Zhou Lan;De Ge;Liu Pan(Key Laboratory for Biotechnology of the State Ethnic Affairs Commission/Hubei Provincial Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area of China,College of Life Sciences,South.Central University for Nationalities,Wuhan 430074,China)

机构地区：[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　　处：《中南民族大学学报（自然科学版）》2018年第1期35-40,共6页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：国家自然科学基金资助项目(31370118);湖北省本科高校“生物技术专业综合改革”试点资助项目(GJZ15006);中央高校基本科研业务费专项资金资助项目(CZW15019);校企合作资助项目(HZY14024);中南民族大学大学生创新创业资助项目(SCX1725,GCX1725,SKYCX17020)

摘　　要：为研究真菌紫杉醇合成相关基因的功能,从产紫杉醇真菌枝状枝孢霉MD2中克隆了Unigene A03231的DNA和c DNA全长序列(918 bp).结果表明:该基因不含内含子,其编码产物含305个氨基酸,与短链脱氢酶/还原酶一致性为64%,预测蛋白分子量为33071.5 Da,不存在信号肽,含有2个跨膜结构.Unigene A03231以单拷贝形式存在于基因组,在0~30 h内其表达水平随茉莉酸甲酯诱导表现为先升高后降低.构建了该基因的原核表达菌株,初步优化了其在大肠杆菌BL21中的诱导表达条件为:诱导温度28℃,IPTG浓度0.6 mmol/L,诱导时间8 h.为深入分析该基因的催化功能奠定了基础.To study the function of fungal paclitaxel-related genes,the DNA sequence and cDNA sequence(918bp)ofUnigene A03231were cloned from one paclitaxel-producing fungus of Cladosporium cladosporioides MD2.The resultsindicated that Unigene A03231sequence had no introns,whose encoded protein contained305amino acids,with apercentage of exact agreement of64%with short chain dehydrogenase/reductase.The gene was predicted to be of amolecular weight of33071.5Da,have no signal peptide,but have two transmembrane structures.Unigene A03231existedin the host genome with a single copy.The expression levels of Unigene A03231responding to methyl jasmonate inductionfirstly increased and then decreased within30h.Additionally,the prokaryotic expression strains of Unigene A03231wasconstructed,and its expression conditions in E.coli BL21were optimized as following:induced at28℃for8h with0.6mmol/L IPTG.These results would provide a basis for further analysis of the catalytic function of Unigene A03231.

关 键 词：枝状枝孢霉 紫杉醇 基因 克隆 原核表达 

分 类 号：Q36[生物学—遗传学]