摘要
从破色期番茄果实中提取了总RNA,用RT-PCR方法从中克隆到了627bp全长LeERF2基因。测序结果分析表明,该LeERF2基因序列与已发表序列的同源性为100%。将LeERF2基因亚克隆至载体pET-30a上,构建了原核表达载体pET-LeERF2,将其转化到大肠杆菌BL21中。蛋白质诱导表达的温度为37℃,诱导剂IPTG浓度为1mmol/L,诱导时间为3h,LeERF2表达蛋白在细菌沉淀中约占菌体总蛋白的58%。应用Ni-NTA亲和层纯化回收的LeERF2表达蛋白的纯度为99%。银染凝胶阻滞实验表明,LeERF2蛋白能与含有GCC盒的逆境相关的几丁质酶启动子基因TLBP1结合。
The 627bp entire coding sequence of LeERF2 was cloned by RT-PCR from breaking tomato fruit RNA. The sequencing result indicated that the identity between the cloned gene and the published gene was 100%. The PCR product was subcloned into the prokaryotic protein expression vector pET-30a to generate recombinant plasmid pET-LeERF2 in E. coli BL21. LeERF2 protein was expressed under 1.0mmol/L IPTG induction for 3 h at 37℃. The expression level of LeERF2 was about 58% of total cellular proteins. The purification level of LeERF2 purified by Ni-NTA agarose column was about 99%. The silver staining electrophoretic mobility shift assay showed that LeERF2 protein could bind to the promoter of the TLBP1 gene coding for pathogenesis-related chitinase with GCC box.
出处
《高技术通讯》
CAS
CSCD
北大核心
2007年第5期546-550,共5页
Chinese High Technology Letters
基金
国家自然科学基金（30270934）资助项目.
关键词
番茄
乙烯
LeERF2
原核蛋白表达
凝胶阻滞实验
tomato, ethylene, LeERF2, prokaryotic protein expression, electrophoretic mobility shift assay （EMSA）
