[1]陈金峰,韩艳霞,焦 婵.大豆GmCAT4基因在毕氏酵母中的表达和功能鉴定[J].大豆科学,2014,33(03):313-320.[doi:10.11861/j.issn.1000-9841.2014.03.0315]
　CHEN Jin-feng,HAN Yan-xia,JIAO Chan.Expression in Pichia pastoris and Functional Identification of Soybean GmCAT4 Gene[J].Soybean Science,2014,33(03):313-320.[doi:10.11861/j.issn.1000-9841.2014.03.0315]
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大豆GmCAT4基因在毕氏酵母中的表达和功能鉴定

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第33卷 期数: 2014年03期 页码: 313-320 栏目: 出版日期: 2014-06-25
Title:
Expression in Pichia pastoris and Functional Identification of Soybean GmCAT4 Gene
作者:
陈金峰; 韩艳霞; 焦 婵
开封大学 化学工程学院，河南 开封 475004
Author(s):
CHEN Jin-feng;  HAN Yan-xia; JIAO Chan
Chemical Engineering College of Kaifeng University,Kaifeng 475004,China
关键词:
大豆; 过氧化氢酶基因; 毕氏酵母; 表达
Keywords:
Soybean; Catalase gene; Pichia pastoris; Expression
分类号:
S565.1
DOI:
10.11861/j.issn.1000-9841.2014.03.0315
文献标志码:
A
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。
Abstract:
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 oybean 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.

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备注/Memo

收稿日期：2013-12-03

基金项目：河南省教育厅科技研究重点项目(12B180015）。
第一作者简介：陈金峰(1977-)，男，硕士，副教授，主要从事生物化学与分子生物学的教学及研究。E-mail:cjfchengssman@163.com。

更新日期/Last Update: 2014-08-01