玉米大斑病菌支链氨基酸氨基转移酶基因的克隆与原核表达

Gene Cloning and Prokaryotic Expression of Branched-Chain Amino Acid Transaminase in Setosphaeria turcica

支链氨基酸(Branched chain amino acid,BCAA)是生物体内重要的营养物质和信号分子,对蛋白质合成、糖代谢、氧化应激等有重要的调控作用。支链氨基酸氨基转移酶(branched-chain amino acid transaminase,BCAT)是BCAA分解代谢的关键酶,参与许多生理和病理过程。为明确BCAT对玉米大斑病菌(Setosphaeria turcica)生长发育和侵染的影响,克隆玉米大斑病菌BCAT基因(StBCAT1),该基因编码的StBCAT1含有保守结构域BCAT_beta_family,属于PLPDE_IV超家族,与玉米圆斑病菌(Helminthosporium carbonum)产生的寄主选择性毒素HC-toxin ToxF高度同源。为了获得大量高纯度的StBCAT1蛋白,构建该基因的原核表达载体,并转化BL21(DE3)菌株。经IPTG诱导表达,SDS-PAGE和Western Blotting检测,结果表明,StBCAT1基因在大肠杆菌得到高效表达,获得了预期分子量为39.2 KD的StBCAT1蛋白。

The branched chain amino acid(BCAA)is important nutrient and signaling molecule regulating protein synthesis,sugar metabolism and oxidative stress in living cells.Branched chain amino acid transaminase(BCAT)is the key enzyme in the catabolism of BCAA and involves in many physiological and pathological processes.In order to clarify the effects of BCAT on the growth,development and infection of Setosphaeria turcica,the StBCAT1 gene was cloned,whose coding protein contains a conserved domain of BCAT_beta_family belonging to PLPDE_IV superfamily,and is highly homologous to host-selective HC-toxin ToxF of Helminthosporium carbonum.With the aim to obtain highly purified StBCAT1 protein,the prokaryotic expression vector of StBCAT1 was constructed and transformed into bacterial strain BL21(DE3).After induced by IPTG,the StBCAT1 was efficiently expressed in Escherichia coli and the expected protein(about 39 KD)was gotten after detected by SDS-PAGE and Western Blotting.