大豆GmCAT4基因在毕氏酵母中的表达和功能鉴定  

Expression in Pichia pastoris and Functional Identification of Soybean GmCAT4 Gene

作　　者：陈金峰[1] 韩艳霞[1] 焦婵[1] 

机构地区：[1]开封大学化学工程学院,河南开封475004

出　　处：《大豆科学》2014年第3期315-320,共6页Soybean Science

基　　金：河南省教育厅科技研究重点项目(12B180015)

摘　　要：利用生物信息学分析了大豆GmCAT4蛋白的结构,其目的蛋白编码492个氨基酸,分子量为56.7 kD,等电点约为6.80,同时预测了蛋白质的二级和高级结构。利用双酶切的方法从克隆载体pMD19-T-GmCAT4上双酶切下目的基因的cDNA,然后将其和表达载体pPIC9K进行连接,构建重组载体pPIC9K-GmCAT4,经电击转化入毕氏酵母,PCR证实了GmCAT4基因已整合进酵母基因组。目的蛋白经甲醇诱导后,在上清和细胞内均有表达,发酵液上清粗酶活性为124.3 kU·L-1,诱导后的酵母对NaCl和H2O2表现出一定的抗性。Bioinformatics analysis were conducted on the structure of soybean GmCAT4 protein, which encoded 492 amino acids. The molecular weight of the enzyme was 56.7 kD, and the pI was 6.80, the secondary and tertiary structures of the protein were predicted. After digested by both enzymes from pMD19-T-GmCAT4, the target gene was inserted into expression vector pPIC9K, and the yease strain Pichia pastoris was transformed by electrophoration with pPIC9K-GmCAT4. It was confirmed that soybean GmCAT4 gene was integrated into the yeast genome by PCR. After 96 h induction by methanol, the proteins in supernatant and cell lysates were measured by SDS-PAGE, and the activity of the crude liquid enzyme from the supernatant reached 124.3 kU ·L^-1. The transgene yeast strengthened its ability of salt and hydrogen peroxide tolerance.

关 键 词：大豆 过氧化氢酶基因 毕氏酵母 表达 

分 类 号：S565.1[农业科学—作物学]