拟南芥AtMYB50和AtMYB61蛋白的原核表达研究  

Prokaryotic expression of Arabidopsis AtMYB50 and AtMYB61 fusion proteins

作　　者：杜李继 顾菊 陈光朗[1] 孙成磊[1] 阳立波 曹树青[1] 

机构地区：[1]合肥工业大学生物与食品工程学院,安徽合肥230009

出　　处：《合肥工业大学学报（自然科学版）》2014年第1期105-109,共5页Journal of Hefei University of Technology：Natural Science

基　　金：高等学校博士学科点专项科研基金资助项目(20120111110009);中国博士后基金资助项目(2012M521213);安徽省青年科学基金资助项目(1308085QC56)

摘　　要：文章提取拟南芥植株总RNA,通过RT-PCR扩增出AtMYB50与AtMYB61基因cDNA片段,连接到pEASY-Blunt载体上,转化到Trans1-T1感受态细胞内,筛选阳性单克隆进行菌落PCR鉴定并测序验证;利用限制性内切酶双酶切重组载体获得含有黏性末端的目的基因片段,并对原核表达载体pET-32a+和pGEX-4T-1进行完全双酶切,连接目的基因片段后获得重组原核表达载体,将重组原核表达载体转化至大肠杆菌原核表达菌株BL21(DE3);IPTG诱导表达获得目的蛋白,并对蛋白表达条件(诱导时间、诱导温度及IPTG浓度等)进行优化,鉴定表达蛋白水溶性。Total RNA was extracted from Arabidopsis seedlings, and cDNA fragments of AtMYB50 and AtMYB61 genes were amplified by RT-PCR. eDNA fragments were subsequently cloned into pEASY-Blunt vectors, and then were transformed into Trans-T1 phage resistant chemically competent cells. The analysis of bacterial colony PCR and cDNA sequencing were performed to confirm that eD- NA of the Arabidopsis thaliana AtMYB50 and AtMYB61 genes was successfully cloned. Their eDNA fragments with sticky ends were isolated by using restriction enzyme digested. The eDNA fragments were cloned into prokaryotic expression vectors pET-32a＋ and pGEX-4T-1. In addition, the recombi- nant plasmids were transformed into BL21（DE3）plysS chemically competent cells. The expression of MYB61 and MYB50 was induced to generate their fusion proteins by IPTG. The protein expression conditions such as the inducing time and temperature and the IPTG concentration were optimized, and the water solubility of the protein was tested.

关 键 词：拟南芥 转录因子 AtMYB50 AtMYB61 原核表达 

分 类 号：Q751[生物学—分子生物学]