象草4CL基因片段的克隆及RNAi表达载体构建  

Cloning of a 4CL gene fragment and construction of a RNAi expression vector of Pennisetum purpureum

作　　者：霍松[1] 陈慧[1] 朱琼华[1] 解新明[1] 

机构地区：[1]华南农业大学农学院,广东广州510642

出　　处：《草业学报》2012年第1期296-301,共6页Acta Prataculturae Sinica

基　　金：国家自然科学基金项目(30972138和30671489)资助

摘　　要：象草是热带和亚热带地区广泛栽培的多年生资源植物。为探讨华南象草4-香豆酸:CoA连接酶(4CL)基因下调表达对木质素合成的影响,本研究设计1对特异引物,PCR扩增获得一段长为374bp的干扰片段。通过TO-PO克隆,将该片段克隆到载体pCR/GW/TOPO,构建了入门克隆载体pENTR-4CL;再经过LR反应使pE-NTR-4CL上的干扰片段进入目的载体pCB2004B上,形成表达载体pCB2004B-4CL,最后通过冻融法将其转入到根癌农杆菌EHA105中。菌液PCR结果表明RNAi质粒已成功转入农杆菌,为进一步利用RNAi技术研究4CL基因的功能奠定了基础。Pennisetum purpureum is a perennial resource plant and widely cultivated in tropical and sub-tropical regions.A pair of specific primers was designed to assess the effect of down-regulated expression of the 4-coumarate： CoA ligase（4CL） gene on lignin composition of P.purpureum cv.Huanan,using the 4CL gene sequence of P.purpureum cv.Mott（accession No： GU997597.1）.An interference fragment of 374 bp was obtained by PCR amplification with the above primers and it was cloned into the pCR/GW/TOPO vector by TOPO cloning to obtain pENTER-4CL.The corresponding fragment on destination vector pCB2004B was then replaced by the interference fragment of pENTR-4CL by LR reaction to form the RNAi（RNA interference） expression vector pCB2004B-4CL.The Agrobacterium-mediated transformation of the expression vector was carried out using the freeze-thaw method,and the expected fragments appeared in the PCR products using bacterial liquid as a template.This study was a basis for research on the 4CL function of P.purpureum by RNAi technology.

关 键 词：象草 GATEWAY技术 4CL基因 RNAI 载体构建 

分 类 号：S543.9[农业科学—作物学] S561