甜瓜ACC氧化酶反义基因植物表达载体的构建及转化烟草的研究

Study on the Construction of a Vector Expressing Melon ACO1 Antisense Gene and Its Transference to Nicotiana tabacum

用限制性内切酶从目的基因供体质粒pBI-aACO1上切下大小约2.3kb的目的基因,将其定向连接在受体质粒pCAMBIA2301载体上,构建成含有GUS基因的甜瓜ACC氧化酶反义基因植物表达载体pCB-aACO1.采用直接转化法将pCB-aACO1导入根癌农杆菌菌株EHA105,并用新构建的工程菌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.

A donator plasmid pBI-aACO1 was digested with restriction endonucleases, then the acquired target genes was ligated to recipient plasmid pCAMBIA2301 which was digested with the same restriction endonucleases, and finally a plant expression vector named pCB-aACO1 expressing melon ACO1 antisense gene and GUS gene was constructed. Moreover, pCB-aACO1 was transferred into agrobacterium tumefacien EHA105 by direct DNA transfer. And the transference with the new engineering bacterium to Nicotiana tabacum was studied. The adventitious shoots and complete plants obtained under the selection pressure of Kanamycin were determinded 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.