播娘蒿转录因子DsCBF与CRT/DRE元件的相互作用  

Interaction of DsCBF of Descurainia sophia with CRT/DRE Element

作　　者：王乐[1] 李晶[1] 曹瑜[1] 张玲[1] 巨艳[1] 乔代蓉[1] 曹毅[1] 

机构地区：[1]四川大学生命科学学院,微生物与代谢工程四川省重点实验室,成都610064

出　　处：《应用与环境生物学报》2010年第5期609-612,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(Nos.30771312;30871321;30971817)资助~~

摘　　要：CBF(C-repeat binding factor)是调控植物冷驯化相关基因表达的一种调控转录激活因子,CBF蛋白AP2结合domain可与COR基因启动子CBT/DRE元件特异性地结合,启动耐寒基因COR的表达.为研究播娘蒿DsCBF的功能,构建pET32a-DsCBF原核表达载体,通过冻融法转化入大肠杆菌BL21(DE3)中,进行原核表达,并通过镍亲和层析纯化CBF蛋白.利用EMSA(Electrophoretic mobility shift assay)分析DsCBF蛋白与播娘蒿DsCOR基因的启动子上CRT/DRE元件的相互作用.EMSA结果显示,DsCBF蛋白与含有CCGAC核心序列及TATA-box和AT-TA回文结构的50bp探针结合有滞后现象,表明与DsCOR启动子上的CRT/DRE元件有特异性结合;而与仅有CCGAC的CRT/DRE核心序列的40bp探针结合则无滞后现象,表明DsCBF与蛋白结合的识别可能与TATA-box和AT-TA回文结构有关.CBF,the DNA-binding proteins of AP2/EREBP family,recognizes the cold-and dehydration-responsive DNA regulatory element,which is called CRT （C-repeat）/DRE （Dehydration-responsive element） for short.A prokaryotic expression vector pET32a-DsCBF was constructed to study the function of DsCBF in Descurainia sophia.The recombinant plasmid was transformed into E.coli BL21,and the fusion protein was further purified through Ni-affinity chromatography.CBF had an AP2 domain which was bound with the element of CRT/DRE specially.The transaction of CBF and promoter of COR were studied through EMSA （Electrophoretic mobility shift assay）.Two probes were synthesized,and one of them had a TATA-box and AT-TA palindrome structure except core sequence CCGAC and the other only had the core sequence CCGAC.Our conclusion indicates that DsCBF bound with CRT/DRE element may be affected by TATA-box and AT-TA palindrome structure.

关 键 词：播娘蒿 CBF CRT/DRE EMSA 银染 

分 类 号：Q949.748.305[生物学—植物学]