抗虫/耐草甘膦双价融合基因表达载体的构建及烟草转化

Construction of Co-ordinated Expression Vectors with Insect Resistance/Glyphosate Tolerance and Transformation of Tobacco

随着植物基因工程的发展,将多个基因转化植株已成为研究的热点之一,利用连接肽进行多个基因融合的策略已引起广泛关注。利用连接多肽2A和LP4/2A分别将抗虫基因Bt(Bacillus thuringiensis,Bt)cry1Ah和耐草甘膦基因mG2-epsps连接起来,构建了4个融合基因表达载体pHAG、pHLAG、pGAH、pGLAH和两个单基因载体pSAh、pSmG2,其中pHAG、pHLAG是Bt cry1Ah基因在前,mG2-epsps基因在后,pGAH、pGLAH是mG2-epsps基因在前,Bt cry1Ah基因在后,分别用2A和LP4/2A作为连接肽,由CaMV35S启动子驱动。利用农杆菌介导法将6个植物表达载体转入烟草,得到再生植株529株。经PCR检测,有261株再生苗为阳性植株,转化率达到49.3%。获得的转基因烟草可用于分析连接肽2A和LP4/2A的剪切效率以及不同基因与连接肽连接位置差异对基因表达的影响。

With the development of plant genetic engineering,the research for introducing multiple heterologous proteins into plants becomes one of the hot spots and the polyprotein strategy for multiple genes transformation is paid growing attention.In this study,the Bt cry1Ah gene was connected with the mG2-epsps gene by the linker peptide 2A and LP4/2A.In order to compare the splicing efficiency between the 2A and the LP4/2A,and study the position effect to gene expression,four coordinated expression vectors were constructed,including pHAG,pHLAG,pGAH,pGLAH and two single expression vectors PSAh,pSmG2 were constructed as control.The six expression vectors were transferred into tobacco by Agrobacterium tumefaciens-mediated transformation and 529 transformants were obtained.PCR detection showed that there were 261 PCR-positive plants and the transformation efficiency was 49.3%.The transgenic plants were used for further splicing efficiency and positioneffect of target gene in polyprotein.