梭梭HabHLH74转录因子基因的克隆及在大肠杆菌中的表达  

Gene Cloning of Transcription Factor HabHLH74 from Haloxylon ammodendron and Expression in Escherichia coli

作　　者：王力伟 赵沛义 王朝 房永雨 刘红葵 郭慧琴[2] 王蕴华 何江峰 WANG Li-wei;ZHAO Pei-yi;WANG Chao;FANG Yong-yu;LIU Hong-kui;GUO Hui-qin;WANG Yun-hua;HE Jiang-feng(Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot010031,China;College of Life Sciences,Inner Mongolia Agricultural University,Hohhot010011,China)

机构地区：[1]内蒙古自治区农牧业科学院,内蒙古呼和浩特010031 [2]内蒙古农业大学生命科学学院,内蒙古呼和浩特010011

出　　处：《畜牧与饲料科学》2020年第5期13-18,共6页Animal Husbandry and Feed Science

基　　金：内蒙古自治区农牧业创新基金项目(2019CXJJM15);内蒙古自然科学基金项目(2018MS03006);内蒙古自治区科技重大专项(2020ZD0005-02)。

摘　　要：利用分子生物学技术从梭梭中克隆了HabHLH74转录因子基因,在大肠杆菌中表达HabHLH74蛋白,旨在为梭梭抗逆分子机理提供研究基础。研究结果表明:①利用RT-PCR技术成功扩增了HabHLH74的完整编码区cDNA序列,HabHLH74编码区cDNA序列长度为1218 bp。根据HabHLH74编码区序列预测出其编码蛋白的理化性质。②成功构建了HabHLH74基因的原核表达载体pET28a(+)-HabHLH74,并在大肠杆菌中成功表达了梭梭HabHLH74蛋白,其分子量为43.6 kDa。③对诱导条件进行单因素试验后获得的最佳诱导表达条件:诱导剂IPTG浓度为0.5 mmol/L,诱导温度为37℃,诱导时间为6 h。④对在优化条件下诱导后的菌体进行超声破碎后发现梭梭HabHLH74蛋白主要以包涵体形式存在;降低诱导温度(16℃、25℃诱导)未能得到可溶性表达的梭梭HabHLH74蛋白。In order to reveal the molecular mechanism underlining the resistant feature of Haloxylon ammodendron,the gene of transcription factor HabHLH74 was cloned from Haloxylon ammodendron and was subsequently expressed in Escherichia coli(E.coli)by using molecular biology technology in this study.The results showed that:①The cDNA sequence of complete coding region of HabHLH74 was successfully amplified using RT-PCR assay,and the length of the coding sequence was 1218 bp.According to the coding sequence,the physical and chemical properties of its encoding protein were predicted.②A prokaryotic expression vector of pET28a(+)-HabHLH74 was constructed,and the HabHLH74 protein was expressed successfully in E.coli.The molecular weight of the obtained protein was 43.6 kDa.③The induced expression condition was optimized by a single factor test.The optimal IPTG concentration,induction temperature and induction time were 0.5 mmol/L,37℃and 6 h,respectively.④Under the optimized induced expression condition,the HabHLH74 protein was expressed in inclusion bodies in E.coli.After decreasing the induction temperature(at 16℃or 25℃),the target protein was still expressed in inclusion form in E.coli.

关 键 词：梭梭 HabHLH74转录因子 克隆 原核表达 

分 类 号：Q943.2[生物学—植物学]