金花茶查尔酮合成酶基因CnCHS的克隆及遗传转化研究

doi:10.7525/j.issn.1673-5102.2015.03.002

摘要/Abstract

摘要： 根据已发表的金花茶查尔酮合成酶（chalcone synthase，CHS）基因（CnCHS）序列设计全长扩增引物，以金花茶花瓣总cDNA为模板进行PCR扩增，成功获得了该基因cDNA全长。将扩增所得全长产物连接PMD18-T载体后转化大肠杆菌E.coli DH5α，提取质粒后经酶切、测序鉴定后，将其与双元表达载体pCAMBIA1300连接，成功构建了CnCHS基因的正义表达载体pCAM-CnCHS。将该重组表达载体转化农杆菌EHA105后，利用农杆菌介导法将CnCHS基因转入烟草，获得转基因烟草18株。利用PCR法及Southern blotting对所获得的转基因植株进行鉴定，结果显示CnCHS基因成功整合到烟草基因组中，阳性率达67%，并获得了单拷贝转基因植株。这些结果表明本研究成功构建了金花茶CnCHS基因对烟草的遗传转化体系，为深入研究CnCHS基因的功能及其对花色的调控效应奠定了基础。

关键词: 金花茶, 查尔酮合成酶, 表达载体, 转基因植株

Abstract: Using total cDNA from the petals of Camellia nitidissima as template, we amplified the full length chalcone synthase(CHS) cDNA of C.nitidissima successfully by the specific primers designed according to the CnCHS sequence in genbank. The amplified fragment of CnCHS was inserted into PMD18-T vector and then transformed into E.coli DH5α. After the compounded plasmid was identified by enzyme digestion and sequencing, it was digested and connected to the expression vector pCAMBIA1300. The compounded plasmid pCAM-CnCHS was identified and the sense expression vector of CnCHS was constructed successfully. Then, the pCAM-CnCHS was transformed into Agrobacterium tumefaciens EHA105 for plant infections, and 18 transgenic tobaccos containing CnCHS were obtained. The transgenic plants were then identified by PCR and Southern blotting, and the transgenic tobaccos which contain single copy of CnCHS gene were obtained with the positive rate of 67%. The genetic transformation system of CnCHS to tobaccos was constructed successfully, which would lay the foundation for further function research and regulation effects on flower color of CnCHS.

Key words: Camellia nitidissima, chalcone synthase, expression vector, transgenic plant

中图分类号:

周兴文；李纪元；朱宇林. 金花茶查尔酮合成酶基因CnCHS的克隆及遗传转化研究[J]. 植物研究, 2015, 35(3): 327-332.

ZHOU Xing-Wen；LI Ji-Yuan；ZHU Yu-Lin. Cloning and Genetic Transformation of CnCHS Gene from Camellia nitidissima[J]. Bulletin of Botanical Research, 2015, 35(3): 327-332.

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