张 鹏,周 兰,席晓圆,德 格,柯友胜,刘 盼,宋发军.枝状枝孢霉MD2的Unigene A03231的克隆及原核表达分析[J].中南民族大学学报自然科学版,2018,(1):35-40
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
  
DOI:10.12130/znmdzk.20180108
中文关键词: 枝状枝孢霉  紫杉醇  基因  克隆  原核表达
英文关键词: Cladosporium cladosporioides  taxol  gene  clone  prokaryotic expression
基金项目: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);中南民族大学基本科研业务费专项基金(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)
作者单位
张 鹏,周 兰,席晓圆,德 格,柯友胜,刘 盼,宋发军* 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 
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中文摘要:
      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cDNA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.
英文摘要:
      To study the function of fungal paclitaxel-related genes, the DNA sequence and cDNA sequence (918 bp) of Unigene A03231 were cloned from one paclitaxel-producing fungus of Cladosporium cladosporioides MD2. The results indicated that Unigene A03231 sequence had no introns, whose encoded protein contained 305 amino acids, with a percentage of exact agreement of 64% with short chain dehydrogenase/reductase. The gene was predicted to be of a molecular weight of 33071.5 Da, have no signal peptide, but have two transmembrane structures. Unigene A03231 existed in the host genome with a single copy. The expression levels of Unigene A03231 responding to methyl jasmonate induction firstly increased and then decreased within 30 h. Additionally, the prokaryotic expression strains of Unigene A03231 was constructed, and its expression conditions in E. coli BL21 were optimized as following: induced at 28 ℃ for 8 h with 0.6 mmol/L IPTG. These results would provide a basis for further analysis of the catalytic function of Unigene A03231.
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