农杆枪法基因遗传转化技术初报

Primary Study on Genetic Transformation Technique Basing on Agrolistic

为提高遗传转化的效率,克服受体范围的限制,采用农杆枪法进行基因的遗传转化。根据已报导的VirD1和VirD2基因的序列设计特异引物,以农杆菌C58菌株的质粒为模板进行PCR扩增,对VirD1和VirD2基因进行克隆。将序列正确的VirD1和VirD2基因连接到植物表达载体PBI121启动子CaMV35S和终止子nos之间,构建VirD1和VirD2基因的表达载体pB-VirD1及pB-VirD2。经PCR及酶切鉴定,基因已成功构建到表达载体上,为农杆枪法进行目的基因转化的研究奠定了基础。

Agrolistic was introduced in genetic transformation, because it can improve the efficiency of transformation and overcome acceptor range obviously. Basing on the reported sequences of gene VirD1 and VirD2, we designed two pairs of special primers, according to PCR, amplified VirD1 and VirD2 using A- grobacterium tumefacions C58 as template. The appropriate sequences were linked with plant expression vector PBI121 between the promotor CaMV 35S and terminator nos, expression vectors of gene VirD1 and VirD2（pB-VirD1 and pB-VirD2） was constructed. The identification of PCR and restriction enzyme digestion showed gene had been constructed into expression vector successfully which contributed to transforming the objective gene using Agrolistic.