摘要
【目的】克隆茶树(Camellia sinensis)的一个类黄酮O-甲基转移酶基因,并对其进行生物信息学分析,构建该基因的原核表达载体,在大肠杆菌中诱导表达融合蛋白,为进一步研究其酶学特性和调控茶树中甲基化类黄酮物质的代谢机理奠定基础。【方法】采用同源克隆法,以从茶树叶片中提取的总RNA为模板,结合RT-PCR与RACE克隆技术获得该基因cDNA全长,测序正确后将该基因片段连接到原核表达载体pET-28a中,转化大肠杆菌BL21并诱导表达。【结果】该基因cDNA全长为1 246 bp,其开放阅读框为1 077 bp,编码358个氨基酸,推测的蛋白分子量为40.2 kD,理论等电点为5.62;其核苷酸编码序列与葡萄和毛果杨的类黄酮O-甲基转移酶基因相似性分别为80%和81%;经IPTG诱导和SDS-PAGE检测,所构建的原核表达载体表达的融合蛋白与预期蛋白相符合。【结论】利用RT-PCR与RACE克隆技术从茶树叶片中克隆得到了一个类黄酮O-甲基转移酶基因,成功构建了原核表达载体,并使其在大肠杆菌中得到了高效表达。
[ Objective ] Cloning, sequence analysis of the CsFOMT genes from Camellia sinensis and expression of recombinant proteins in Escherichia coli were conducted to further research the enzymatic characteristics and the metabolic mechanism of regulating the flavonoid in tea plant. [ Method ] The CsFOMT cDNA fragments were amplified from Camellia sinensis by reverse transcription PCR （RT-PCR） and RACE, and then the cloned genes of CsFOMT were inserted into vector pET-28a. The recombinant plasmids pET-28a-CsFOMT were expressed in the prokaryotic expression system after its transformation into E. coli BL21. [ Result] The whole cDNA sequence was 1 246 bp which contains an ORF of 1 077 bp and encodes 358 amino acids. The putative protein of the gene had an isoelectric point of 5.62 and a calculated molecular weight of 40.2 kD. The coding nucleotide sequence of tea showed 80% and 81% identity with that of Vitis vinifera and Populus trichocarpa, respectively. Results of SDS-PAGE showed that the specific fusion protein was successfully induced to express by IPTG. [Conclusion] In this study, a full length cDNA of flavonoid O-methyltransferase gene was obtained from Camellia sinensis by RT-PCR and RACE, and the prokaryotic expression vector for this gene was constructed, and then successfully induced to express by IPTG in BL21.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第2期325-333,共9页
Scientia Agricultura Sinica
基金
国家自然科学基金项目(30972404)
农业部国家现代农业产业技术体系建设专项资金(CARS-23)
中央级公益性科研院所基本科研业务费专项(2012zl053)
关键词
茶树
类黄酮O-甲基转移酶
克隆
原核表达
Camellia sinensis
flavonoid O-methyhransferase
cloning
prokaryotic expression
