稻瘟菌蛋白激发子基因pemG1植物表达载体的构建及其转化烟草的研究

Construction of Plant Expression Vector for Elicitor-encoding Gene pemG1 from Magnaporthe grisea and its Transformation to Tabacco

设计含MluⅠ酶切位点的接头,将其连接入经KpnⅠ和BamHⅠ酶切后的质粒pCAMBIA2300-U-bi-Ocs,得到pCAMBIA2300-Ubi-adaptor-Ocs。设计含有KpnⅠ和MluⅠ酶切位点的引物,以稻瘟菌基因组DNA为模板扩增得到pemG1序列。将其连接入pMD18-T得到pMD18-T-pemG1。最后,用KpnⅠ和MluⅠ从pMD18-T-pemG1切下pemG1基因片段,将其连接入pCAMBIA2300-Ubi-adaptor-Ocs的KpnⅠ-MluⅠ酶切位点,构建成稻瘟菌蛋白激发子基因pemG1的植物表达载体体pCAMBIA2300-Ubi-pemG1-Ocs。用冻融法将pCAMBIA2300-Ubi-pemG1-Ocs导入根癌农杆菌菌株AGL-1,再通过根癌农杆菌介导转化获得了转基因烟草株系。用PCR,Northern和Western杂交验证了pemG1基因在受体烟草基因组中的整合,转录和表达。此研究为下一步通过稻瘟菌蛋白激发子基因pemG1的表达来提高转基因烟草的抗病性打下了基础。

An adaptor harboring Mlu I restriction site was designed and cloned into Kpn I -BamH I site ofpCAM- BIA2300-Ubi-Ocs plasmid to get pCAMBIA2300-Ubi-adaptor-Ocs. Primer pair harboring Kpn I and Mlu I sites was also designed to amplify Mognaporthe grisea DNA template to get pemG1 sequence, which was cloned into pMD 18-T vector to form pMD 18-T- pemG1. Finally, the Kpn I -Mlu I fragment ofpemG1 from pMD 18-T-pemG1 was cloned at Kpn I -Mlu I site of pCAMBIA2300-Ubi-adaptor-Ocs to form plant expression vector pCAMBIA2300-Ubi-pemGl-Ocs. The expression vector construct was transfered to Agrobacterium tumefaciens AGL-I strain by freeze-thaw method. Transgenic tobacco lines were produced by A grobacterium-mediated transformation. Integration, transcription and expression ofpemG1 in tobacco were respectively confirmed by PCR, Northern blot and Western blot. This work layed a good foundation for enhancement of disease resistance conferred by expression ofpemG1 gene in tobacco.