大豆Glyma03g263000基因克隆、原核表达及纯化  

Cloning,Prokaryotic Expression and Purification of Glyma03g263000 Gene in Soybean

作　　者：刘鑫 李文辰 康越 齐泽铮 于璐 王芳[1] LIU Xin;LI Wen-chen;KANG Yue;QI Ze-zheng;YU Lu;WANG Fang(College of Life Sciences and Agroforestry,Qiqihar University/Heilongjiang Provincial Key Laboratory of Resistance Gene Engineering and Protection of Biodiversity in Cold Areas,Qiqihar 161006,China)

机构地区：[1]齐齐哈尔大学生命科学与农林学院/抗性基因工程与寒地生物多样性保护黑龙江省重点实验室,黑龙江齐齐哈尔161006

出　　处：《大豆科学》2023年第3期304-309,共6页Soybean Science

基　　金：国家自然科学基金(31801725);黑龙江省教育厅基本科研业务费科研项目(135309361);齐齐哈尔大学研究生创新科研项目。

摘　　要：大豆Glyma03g263000基因编码1个RING-H2 zinc finger蛋白,其表达水平受大豆胞囊线虫侵染影响。为促进该基因在抗线虫研究中的应用,利用生物信息学和PCR方法对该基因序列进行分析,并从抗病大豆品种中克隆基因CDS区。构建pET30a-Glyma03g263000原核表达载体,转化至大肠杆菌Rosetta(DE3)菌株中,并诱导Glyma03g263000蛋白表达,设置不同菌液浓度、IPTG浓度、温度及诱导时间,并采用SDS-PAGE电泳方法进行检测,分析诱导该蛋白表达的较好条件。结果显示:Glyma03g263000基因编码260个氨基酸,蛋白分子质量为27.88 kDa,在102~145个氨基酸区间具有1个RING保守结构域。温度是影响该蛋白表达的主要因素。该蛋白是不溶于水的包涵体蛋白,经变性裂解等试剂处理后可获得纯化蛋白。研究结果为Glyma03g263000蛋白的功能分析奠定了理论及材料基础。The soybean Glyma03g263000 gene encodes a RING-H2 zinc finger protein,its expression level is affected by soybean cyst nematode infection.In order to provide references for further clarifying its function in nematode resistance,this study performed a bioinformatic analysis and cloned CDS region of the gene from a disease-resistant soybean variety by PCR method.To obtain the purified target protein,prokaryotic expression vector pET30a-Glyma03g263000 was constructed and transformed into E.coli Rosetta(DE3)strain,we set different bacterial concentration,IPTG concentrations,temperatures and inducing times,and detected the target protein by SDS-PAGE electrophoresis to analyze the conditions for inducing the expression of this protein.Bioinformatic analysis showed that the gene encodes 260 amino acids with a molecular mass of 2788 kDa and a RING conserved domain in the 102-145 amino acid region.Through the analysis of induced expression conditions,it was found that temperature was the main factor affecting protein expression.Glyma03g263000 protein is a water-insoluble inclusion body protein,which can be obtained after treatment with reagents such as denaturation and cleavage.The results of this study laid a theoretical and material foundation for the functional analysis of Glyma03g263000 protein.

关 键 词：大豆 Glyma03g263000 原核表达 纯化蛋白 RING型E3泛素连接酶 

分 类 号：S565.1[农业科学—作物学]