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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。
The target gene of about 2.3 kb in size was cut off from the gene donor plasmid pBI-aPG by restriction enzyme and ligated to the acceptor plasmid pCAMBIA2301 vector to construct a melon-polymerized polyacrylamide gel containing the GUS gene and the NPTII gene Galacturonase antisense gene plant expression vector pCB-aPG. PCB-aPG was introduced into Agrobacterium tumefaciens strain LBA4404 by direct transformation method, and the genetic transformation of common tobacco was studied using this strain. Adventitious buds and whole plants transformed with Kanamycin under selective pressure were identified by GUS genomics and PCR, which confirmed that the antisense gene had been introduced into tobacco genome. This study laid the foundation for the transformation of melon varieties with this antisense gene in the next stage to improve the storage stability of melon fruits.