Klenow大片段聚合酶及T_4DNA聚合酶在构建重组质粒中的应用

Application of Klenow Polymerase and T_4 DNA Polymerase in Construction Recombinant Plasmid

利用大肠杆菌Klenow大片段聚合酶5′→3′聚合酶活性和T4DNA聚合酶3′→5′外切酶特性构建植物高效表达载体pBLT35Svp4-ST。用pUC18-435S中的T35S启动子取代植物表达载体pBLG-VP4-ST中的双35S启动子,通过Klenow大片段的聚合酶活性对植物表达载体pBLG-VP4-ST中的HindⅢ3′凹端补平,再利用T4DNA聚合酶外切酶活性将质粒pUC18-435S的四倍35S启动子即T35S中的SacⅠ3′凸端削平。在T4DNA连接酶作用下将一端为平端,另一端为BamHⅠ的粘性末端的四倍35S启动子定向连接到植物表达载体PBLG-VP4-ST中取代原有的双35S启动子,转化并筛选阳性克隆。结果经PCR和酶切鉴定证实,四倍35S启动子成功连接到植物表达载体PBLG-VP4-ST中。

A plant high effective expression vector which named pBLT35Svp4-ST was constructed by using Klenow Polymerase＇ s 5＇→3＇ activation and T4DNA Polymerase＇ 3＇ →5＇ activation.We used the promoter T35S of the pUC18-T35S to re, place the promoter D35S of the plant expression vector pBLG-VP4-ST. We filled Hindm 3＇ of the pBLG-VP4-ST by using Klenow Polymemse＇s 5＇→3＇ activation, and cut Sac[3＇of the plasrnid pUC18-T35S＇ s T35S by using T4 DNA Polymerase＇ 3＇→5＇ activation. The promoter 435S was inserted into PBLG-VP4-STplant expression vector and replaced promoter D35S by ligating reaction, Then transformed the ligation preducts into the DH10B competent cells. Consequently, the recombinant plasmid was amplified and extracted from the transformants while the inserting was tested. The PCR and digestion with restrictive enzymes showed promoter T35S was inserted into PBLG-VP4-STplant expression vector successfully.