两个大豆MYB转录因子在原核细胞中的高效表达  

High Expression of Two Soybean MYB Transcription Factor in Escherichia coli

作　　者：李晓薇[1] 苏连泰[1] 赵旭[2] 翟莹[1] 张海军[1] 张庆林[1] 李景文[1] 王庆钰[1] 

机构地区：[1]吉林大学植物科学学院,长春130062 [2]吉林省产品质量监督检验院,长春130022

出　　处：《生物技术通报》2011年第8期99-102,114,共5页Biotechnology Bulletin

基　　金：转基因生物新品种培育重大专项子课题(2008ZX08004-003);国家自然科学基金面上项目(30971808);吉林省科技发展计划重点项目(20080204);长春市科技局国际科技合作项目(08GH10);"211"三期建设项目

摘　　要：大豆(Glycine max)GmMYB12a与GmMYB12B2基因属于典型的植物R2R3-MYB转录因子。为进一步研究两个MYB12转录因子与相应顺式作用元件的相互作用,构建了两基因的原核表达载体,并在大肠杆菌Rosetta(DE3)中实现高效表达。利用PCR技术,将两端带有特异酶切位点的GmMYB12a与GmMYB12B2全长基因亚克隆于原核表达载体pET-28a(+),酶切、测序鉴定确认获得两基因的重组原核表达载体pET-28a-GmMYB12a与pET-28a-GmMYB12B2。然后把重组载体转化到大肠杆菌Rosetta(DE3)中,经IPTG诱导蛋白表达,提取细胞蛋白并采用SDS-PAGE检测目的蛋白的表达情况。结果表明,成功构建了两基因的原核表达载体,在IPTG浓度为1.2 mmol/L,诱导6 h后,目的蛋白能在Rosetta(DE3)中高效表达。Soybean（Glycine max）GmMYB12a and GmMYB12B2 are the typical plant R2R3-MYB transcription factors.For the further study of these two MYB12 transcription factors and their interactions between corresponding cis-regulatory elements,prokaryotic expression vectors of these two genes were constructed and achieved highly efficient expression in Escherichia coli Rosetta（DE3）,respectively.Full length of the two genes,GmMYB12a and GmMYB12B2,with specific restriction sites of both ends were subcloned in the prokaryotic expression vector pET-28a（＋）using PCR,restriction analysis and DNA sequencing confirmed that the two recombinant prokaryotic expression vectors pET-28a-GmMYB12a and pET-28a-GmMYB12B2 were gained.Then the recombinant vectors were transformed into E.coli Rosetta（DE3）,extracting cell proteins after induction by IPTG and tested expression of destined proteins using SDS-PAGE.The results showed that prokaryotic expression vectors of these two genes were successfully constructed,and after 6 h induction at the IPTG concentration of 1.2 mmol/L,target proteins could be highly expressed in E.coli Rosetta（DE3）.

关 键 词：大豆 MYB转录因子 载体构建 pET-28a(+) Rosetta(DE3) 原核表达 

分 类 号：S565.1[农业科学—作物学] Q943.2[生物学—植物学]