钝齿棒杆菌<em>arg</em>R基因克隆、表达及其重组菌发酵产精氨酸研究

中国生物工程杂志

2011, Vol. 31

Issue (10): 57-62

研究报告

钝齿棒杆菌argR基因克隆、表达及其重组菌发酵产精氨酸研究

焦海涛¹, 袁永², 徐锋², 杨伟¹, 熊勇华², 陈雪岚¹

1. 江西师范大学生命科学学院南昌 330022;
2. 南昌大学生命科学与食品工程学院食品科学与技术国家重点实验室南昌 330047

Cloning and Expression of argR gene from Corynebacterium crenatum and Arginine Production with the Recombinant Strain

JIAO Hai-tao¹, YUAN Yong², XU Feng², YANG Wei¹, XIONG Yong-hua², CHEN Xue-lan¹

1. College of Life Science, JiangXi Normal University, Nanchang 330022, China;
2. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China

全文: PDF(1126 KB) HTML

摘要：

钝齿棒杆菌(Corynebacterium crenatum)AS.M7是筛选获得的一株高产精氨酸生产菌株。ArgR是精氨酸合成过程中的一种调控蛋白。为进一步验证其在钝齿棒杆菌中对精氨酸合成量的影响,利用特异性引物,分别扩增标准菌C. creantum AS 1.542和诱变菌C. creantum AS.M7的argR全长基因,测序后比较二者的差异;结果表明标准菌argR基因ORF全长516 bp,编码一个含172个氨基酸残基的蛋白;而诱变菌argR基因的109位碱基由C替换为T,导致ArgR蛋白在钝齿诱变菌中表达被提前终止。同时,将来源于标准菌的argR基因连接到穿梭表达载体pXMJ19中,电击转化至诱变菌C. crenatum AS.M7 得到重组菌株,用摇瓶发酵的方法观测重组菌产精氨酸量的变化。SDS-PAGE和Western blot分析证明标准菌的argR基因在诱变菌中得到了表达。对重组诱变菌产精氨酸量进行了测定,结果显示:产精氨酸能力由原来7.8 mg/ml下降至2.5 mg/ml,下降了约67.9%。

关键词： L-精氨酸; 钝齿棒杆菌; ArgR; 诱变菌

Abstract:

Corynebacterium crenatum AS. M7, mutated and stored is a high arginine producing strain. ArgR is one of the most important regulating proteins during arginine biosynthesis. To further confirm the effect of ArgR on arginine biosynthesis in C. crenatum, specific primers were designed and used for PCR amplification of full argR gene from wild strain C. crenatum AS 1.542 and a mutant strain C. creantum AS. M7. The amplified argR gene were aligned and compared for difference. Result showed that argR from C. crenatum AS 1.542 has ORF of 516 bp, encoding a protein consists of 172 amino acids. In comparison, argR from C. crenatum AS. M7 has a C instead of T on position 109, resulting termination of ArgR expression in C. crenatum AS. M7. Meanwhile, argR gene from C. crenatum AS 1.542 was cloned into pXMJ19 vector and transformed into C. crenatum AS. M7 to form a recombinant strain. Arginine production with recombinant strain was evaluated by fermentation in triangle flask. SDS-PAGE and Western blot were used for argR gene expression analysis, result showed a decrease of arginine production from 7.8 mg/ml to 2.5 mg/ml (appr. 67.9%).

Key words: L-Arginine Corynebacterium crenatum ArgR Mutated strain

收稿日期: 2011-06-29 出版日期: 2011-10-25

ZTFLH:

Q789

基金资助:

国家自然科学基金资助项目(30960012)

通讯作者: 熊勇华, 陈雪岚 E-mail: xuelanchen14@yahoo.com.cn; yixiongchen@163.com

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引用本文:

焦海涛, 袁永, 徐锋, 杨伟, 熊勇华, 陈雪岚. 钝齿棒杆菌argR基因克隆、表达及其重组菌发酵产精氨酸研究[J]. 中国生物工程杂志, 2011, 31(10): 57-62.

JIAO Hai-tao, YUAN Yong, XU Feng, YANG Wei, XIONG Yong-hua, CHEN Xue-lan. Cloning and Expression of argR gene from Corynebacterium crenatum and Arginine Production with the Recombinant Strain. China Biotechnology, 2011, 31(10): 57-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I10/57

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