变形链球菌乳酸脱氢酶与霍乱毒素B亚单位嵌合基因植物表达质粒的构建及转化烟草研究

Construction and tobacco transformation of the plant expression vector of ldh and ctx B fused gene

目的构建含变链菌乳酸脱氢酶(LDH)和霍乱毒素B亚单位(CTB)嵌合基因的植物表达质粒并以根癌农杆菌介导法转化烟草,获得转基因烟草。方法应用PCR技术扩增嵌合基因,并与植物中间表达载体p2355双酶切连接,构建植物表达载体p2355-ldh-ctxB,电击法导入根癌农杆菌EHA105,根癌农杆菌叶盘转化法转化烟草,对抗性植株进行PCR、GUS组织染色、RT-PCR检测。结果载体p2355-ldh-ctxB经双酶切及PCR证实均能得到与预期相符的片段,农杆菌介导转化烟草得到的抗性植株GUS组织染色呈阳性,PCR扩增nptⅡ基因和目的基因部分片段,均获得了与预计相符的片段,部分样品RT-PCR能扩增出与预期一致的片段。结论成功构建植物表达质粒p2355-ldh-ctxB,经根癌农杆菌介导转化烟草后获得了转基因烟草植株。

Objective To construct the vector of fused gene of ldh and ctxB and obtain transgenic tobacco by Agrobacterium tumefaciens.Methods Both the fused gene and plasmid p2355 were digested and linked to construct the plant expression vector p2355-ldh-ctxB.The vector was transformed into Agrobacterium tumefaciens EHA105 by electroporation.Transforming the fused gene into tobacco induced by leaf discs method of Agrobacterium tumefaciens.the resistant plants were detected by GUS Histochemical Assay,PCR and RT-PCR.Results The fused gene were obtained by the plasmid p2355-ldh-ctxB restriction endonuclease digestion and PCR.Agrobacterium-mediated transformation of tobacco plants to be resistant GUS staining was positive.The target gene npt-Ⅱand some fragments were obtained by PCR which were consistent with the expected.In part of the sample which was amplified by RT-PCR,the expected fragments were obtained.Conclusion The recombinant expression vector p2355-ldh-ctxB was successfully constructed.The transgenic tobacco plants were obtained and the fused gene were integrated into the tobacco genome successfully.