上西早生柿ETR5基因RNAi植物表达载体构建及遗传转化研究

据已测序的上西早生柿ETR5基因序列,设计了2对带限制性内切酶位点的特异性引物,以测序质粒为模板,PCR扩增到2个ETR5-Uenishiwase基因片段。2个片段经单酶切消化后连接成反向互补的大片断。连接片断经双酶切消化后,定向连接到植物表达载体pSMAK321的35S启动子和NOS终止子之间,构建成Uenishiwase-ETR5基因的RNA干扰植物表达载体。载体质粒经酶切鉴定正确后通过冻融法转入根癌农杆菌EHA101中。以上西柿组培苗叶片为外植体,优化遗传转化体系,结果表明:农杆菌重悬液和共培养基中均添加200μmol/L的AS及Spc梯度筛选能有效提高转化率。转化植株经PCR检测得到7株阳性植株。

Uenishiwase Persimmon ETR5 Gene RNA Interference Expression Vector Construction and Genetic Transformation

Two pairs of primers containing restriction enzyme site were designed and used to amplify sequenced plasmid.Two PCR products were digested by the corresponding restricted enzymes respectively,and connect to be a long reverse complementary fragment.The fragment was double digested by corresponding enzymes and inserted between CaMv 35S promoter and NOS terminator of expression vector pSMAK311.Confirmed by restriction endonucleases,the RNAi expression vector was transformed into Agrobacterium tumefaciens strain EHA101 by freezing-thaw method.Using Uenishiwase′ persimmon in vitro plantlet leaves as explant,Agrobacterium-mediated transformation system was optimized.The results showed that adding 200 μmol/L of AS in both agrobacterium suspension liquid and culture medium and Spc concentration gradient screening could increase the frequency of transformation.7 strains of transformed Uenishiwase persimmon plantlets were verified by PCR.