大豆LEA蛋白Em的表达可提高大肠杆菌和烟草耐盐性  

Expression of Em gene (LEA1) from soybean immature seeds confers salt tolerance to Escherichia coli and tobacco plants

作　　者：蔡丹[1,2] 郑易之[1,2] 兰英[1,2] 

机构地区：[1]深圳大学生命科学学院 [2]深圳市微生物基因工程重点实验室,深圳518060

出　　处：《深圳大学学报（理工版）》2006年第3期230-236,共7页Journal of Shenzhen University(Science and Engineering)

基　　金：深圳市科技计划资助项目(200441);科技部国家转基因植物研究与产业化开发资助项目(JY03A18)

摘　　要：将大豆Em(LEA1)基因构建到原核表达载体pET28-Em中.转化大肠杆菌,经SDS-PAGE电泳及电喷雾质谱鉴定.结果显示,经IPTG诱导的重组菌可表达约18kDa的特异蛋白.这种蛋白具有良好的可溶性和热稳定性.在含800 m mol/L NaC l培养基中,含空载体的对照菌生长迟滞期约为50 h,含Em基因的重组菌生长迟滞期为32 h,表明大豆Em蛋白的表达可提高大肠杆菌对盐胁迫的适应能力及耐盐性.在此基础上,构建了含Em基因的真核表达载体pB I-Em.再利用根癌农杆菌介导法将该基因转入烟草.PCR及RT-PCR结果显示,大豆Em基因已整合到转基因烟草的基因组DNA中,并能正常转录.转化率为53%.转Em基因烟草植株在含有质量分数1.5%NaC l的培养基中生长状况好于对照植株,叶片的叶绿素含量高于对照植株的叶片.结果表明,大豆Em基因的表达可提高大肠杆菌和转基因烟草的耐盐性.A gene of Em （ LEA1 ） was cloned from soybean immature seeds. The expression vector containing Em gene （pET28-Em） was constructed and then transferred into Escherichia coli strain. The protein pattern of SDS-PAGE and mass spectrometry showed that the specific protein Em of 18kDa was expressed in the E. coli recombinants induced by IPTG. The protein has characteristics of high hydrophilicity and heat-stabilization. Compared with control strain containing the empty vector without Em gene, the E. coli recombinants expressing Em protein showed shorter lag period after transferred into liquid medium supplementary with 800 mmol/L NaC1. This result indicated the over-expression Em protein directly contributed to enhancing salt tolerance of E. coli recombinant under stress condition of high salt. Then, the eukaryotic expression vector containing Em gene （pBI-Em） was constructed and transferred into tobacco leaf discs by Agrobacterium tumefaciens transformation. The pattern analysis of PCR and RT-PCR amplified reactions indicated that the soybean Em gene was integrated into the genomic DNA of transgenic tobacco plants and could be transcripted at RNA level. The transformation efficiency was up to 53%. On MS growing media supplimented with 1.5% NaCl, the transgenic tobacco seedlings showed better growth performance and higher chlorophyll content of their leaves, comparing to the non-transformants as control. In summary, the expression of soybean Em gene （ LEA1 ） can enhance salt tolerance of E. coli recombinants and transgenic tobacco plants as well.

关 键 词：大豆 LEA1基因 Em蛋白 大肠杆菌 根癌农杆菌转化 转基因烟草 盐胁迫 

分 类 号：Q943.2[生物学—植物学] Q786