丹波黑大豆醛脱氢酶基因GmALDH3-1的克隆、原核表达和功能分析  

Cloning,Prokaryotic Expression and Function Analysis of GmALDH 3-1 from Danbo Black Soybean

作　　者：张乐[1] 陈宣钦[1] 王琳[1] 陈丽梅[1] 李昆志[1] 

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500

出　　处：《中国生物工程杂志》2013年第5期86-92,共7页China Biotechnology

基　　金：国家自然科学基金(31260297);云南省自然科学基金(KKSA201126058)资助项目

摘　　要：醛是一类具有高度反应活性的毒性物质,在生物体中主要由膜脂过氧化,氨基酸氧化和蛋白质的糖基化产生。醛脱氢酶(aldehyde dehydrogenase,ALDH)是一类以多种醛类为底物的酶类,醛脱氢酶基因是一类编码将醛脱氢氧化为相应羧基酸的基因,在植物,动物和微生物中均有发现。通过RT-PCR方法从丹波黑大豆根中扩增出基因GmALDH3-1。构建原核表达载体pGEX-4T-1-ALDH3-1,在E.coli BL21中表达,并研究不同浓度IPTG、时间和温度对ALDH3-1蛋白表达的影响,结果表明28℃下诱导6h ALDH蛋白表达量最大,而IPTG浓度对ALDH3-1的表达量影响不大。在添加有不同浓度Al、Cu和Cd的液体LB培养基中培养转化菌和对照菌,检测转化菌和对照菌的生长曲线,生长曲线实验结果表明ALDH3-1转化工程菌对上述金属离子具有一定的耐受性。这些结果为进一步研究其结构和生物学功能奠定了基础。Aldehydes were one kind of toxic substance with high activity of chemical reactions. In the body of organism, aldehydes were chiefly generated by the lipid peroxidation, amino acid oxidation, and protein glycation. ALDHs （ aldehyde dehydrogenase）, with a variety of aldehydes as the substrate , encoded one kind of proteins responsible for the oxidation of aldehyde to carboxylic acid, can be found in plants, animals, and microorganism. The ALDH eDNA sequence of ＇ Danbo black soybean＇ was amplified by RT-PCR. Prokaryotic expression vector pGEX-4T-1-ALDH3-1 was constructed and transformed into E. coli BL21 for expression. The effect of different concentrations of IPTG, time, and temperature on the ALDH protein expression was studied. The results showed that maximum recombinant protein was expressed in 6h at 28℃with induction of IPTG, while the concentration of IPTG has little effect on the expression level of ALDH3-1. The transformed bacteria and control were cultured in liquid LB medium containing different concentration of A1, Cu, and Cd ions The growth curve of them was detected. The results show that ALDH3-1 has a certain tolerance to the mental ions mentioned above in vivo. These results provided foundation for the further investigation on ALDH3-1 structure and biological function.

关 键 词：大豆 ALDH 序列分析 原核表达 金属抗性 

分 类 号：Q78[生物学—分子生物学]