磷酸烯醇式丙酮酸羧化酶cDNA在T7 RNA多聚酶/启动子系统中专一性表达的研究

用PT7和pGP1—2质粒偶联表达系统,通过温度诱导(30～42℃),使温度敏感基因CI875失活;加入利福霉素选择性抑制E.coliRNA多聚酶的表达,从而使外源PEP羧化酶cDNA得到专一性的表达。产物经SDS—PAGE和Wesern杂交分析,表明该系统表达出两条PEP羧化酶带,分子量分别是78kD和80kD。

Expression of PEPCase cDNA in E. coli with T7 RNA Polymerase/Promoter System

Phosphoenolpyruvate carboxylase(PEPCase) is one of the key enzymesin C_4-dicarboxylic assimilation, andan important turget for genetic mani-pulation for increasing photosyntheticefficiency in higher plants. For intro-ducing PEPCase gene into C_3 a plant,we preliminarily selected the T7 RNApolymerase/promoter system, a veryusefully expression plasmid, to expressPEPCase cDNA in E. coli, and triedto find out the optimum conditions forits expression in higher plants. pPEP3055 plasmid was digestedwith EcoR I, and 2. 0 kb fragmentof PEPCase cDNA was recovered byelectroelution method, then it wassubcloned into a T7 plasmid with T7RNA polymerase promoter and intro-duced into E. coli K38 harboring aresident plasmid of PGP1-2. A recom-binant clone of PEPCase cDNA(pGTE1) was screened by Southern(1975) hybridization. As there was aheat-sensitive repressor geneC1857 inappropriate concentration of ri-fampicin was added to inhibit the ex-pression of RNA polymerase of E. col-i, the PEPCase cDNA in recombinantplasmid was effectively expressed. Anadditional band of protein was ob-served on the SDS-PAGE, which washeavily contaminated by backgroundproteins. Then Maxcell method wasused to identify the expression pro-duct. When the sample was pulsedwith ~(35)S-Met, two small more clear-cut bands appeared on the autoradio-graphic film, but other newly synthe-sized proteins also appeared. It showsthat there is not high enough expres-sion specificity. In order to increasethe speicificity, a concentrations gra-dient of rifampicin was used, andgood results were obtained. Only twoadditonal protein bands were selective-ly expressed at a concentration of 200ug/ml rifampicin. In that case, pro-tein product was more than 90% ofthe total protein. Western blot test re-vealed that the two additional bandswere subunits of PEPCase with amolecular weight of 78 and 80 kD re-spectively.