番茄NP24基因超表达载体的构建

Construction of Over-expression Vector NP24 Gene in Tomato

根据NCBI上提供的NP24基因(Gen Bank登录号为543979)的序列,设计全长引物,扩增编码区c DNA,通过克隆至中间载体p MD18-T,将类甜蛋白基因NP24插入植物表达载体PBI121,构建超表达载体PBI121-NP24,采用Ca Cl2冻融法将其转入农杆菌EHA105菌株中。酶切鉴定表明,目的基因已经正确地插入到PBI121载体中,超表达载体构建成功。菌液PCR鉴定,超表达载体已转入农杆菌中。

According to the sequence of NP24 gene（Gen Bank registration number 543979） in NCBI, wedesigned primers and amplified c DNA encoding area. Then the c DNA was cloned to intermediate carrierp MD18- T. We built the over- expression vector PBI121- NP24 by inserting the gene NP24 into plantexpression vector PBI121. The over- expression vector PBI121- NP24 was transferred into agrobacteriumEHA105 by the Ca Cl2 freeze-thaw method. Enzyme identification showed that the gene had been correctlyinserted into the PBI121 carrier and the over- expression vector was built successfully. Microbial PCRidentification proved that the over-expression vector had been transferred into agrobacterium.