甜瓜多聚半乳糖醛酸酶反义基因植物表达载体的构建及其转化烟草的研究

用限制性内切酶从目的基因供体质粒pBI-aPG上切下大小约2.3kb的目的基因,将它定向连接在受体质粒pCAMBIA2301载体上,构建成含有GUS基因和NPTⅡ基因的甜瓜多聚半乳糖醛酸酶反义基因植物表达载体pCB-aPG。采用直接转化法将pCB-aPG导入根癌农杆菌菌株LBA4404,采用该菌株对普通烟草进行了遗传转化研究。在Kanamycin选择压力下获得的烟草转化不定芽和完整植株,经过GUS基因组织化学法检测以及PCR方法鉴定,证实了该反义基因已导入烟草基因组中。此项研究为下一阶段用该反义基因转化甜瓜品种以改良甜瓜果实耐贮运性打下基础。

Construction of a vector expressing melon PG anti-sense gene and its transformation to Nicotiana tabacum

A donator plasmid pBI-aPG was digested with restriction endonucleases,then the acquired target gene was ligated to recipient plasmid pCAMBIA2301 which was digested with the same restriction endonucleases,and finally a plant expression vector named pCB-aPG expressing melon PG antisense gene,GUS gene and NPTⅡgene was constructed.Moreover,pCB-aPG was transferred into Agrobacterium tumefacien LBA4404 by direct DNA transfer.The transformation with the new engineering bacterium to Nictiana tabacum was studied.The adventitious shoots and complete plants obtained under the selection pressure of Kanamycin were determined by GUS gene with histochemical method and confirmed with PCR of genome of Nicotiana tabacum,which showed that the target gene was transferred into the plants.