摘要
该研究在Escherichia coli Transetta（DE3）中表达了C4盐生植物异子蓬的磷酸烯醇式丙酮酸羧化酶基因（PEPC）,并进行了酶学特性及非生物胁迫响应分析,以期初步阐明PEPC基因在非生物胁迫下的生物学功能,为异子蓬PEPC的非生物胁迫的抗性生理研究提供参考依据。基于5′-RACE技术获得异子蓬PEPC全长基因（GenBank登录号为KP985714）,构建了E.coli Transetta：：pGEX-4T-1-PEPC重组菌株。通过分光光度法和酶联免疫吸附技术（ELISA）分别测定了PEPC重组蛋白的酶活和含量,并检测E.coli Transetta：：pGEX-4T-1-PEPC重组菌株在非生物胁迫下的耐受性。生物信息学分析发现,该PEPC基因的cDNA全长为2 901bp,编码966个氨基酸,与甜菜（Beta vulgaris）PEPC的氨基酸序列一致性达90%,具有PEPC的典型保守结构域（PEPcase）以及VlTAHPTQsiRR和VMIGYSDSgKDAG活性位点;PEPC蛋白属PEPC-1型的不含信号肽的非分泌型亲水蛋白。Western blot结果显示,融合GST的PEPC蛋白的分子量约130~140kD;在37℃用0.8 mmol/L IPTG诱导E.coli Transetta：：pGEX-4T-1-PEPC重组菌株显示出更高的PEPC酶活及含量,而且E.coli Transetta：：pGEX-4T-1-PEPC重组菌在200~800mmol/L NaCl、5%~20%聚乙二醇（PEG）6 000、25℃~52℃温度范围、50~400μmol/L甲基紫精和pH 3.0~11.0的非生物胁迫下生长均明显优于对照。研究表明,异子蓬PEPC基因的表达提高了E.coli耐受非生物胁迫的能力。
To elaborate the biological function of PEPC which encodes phosphoenolpyruvate carboxylase from C4 halophyte Suaeda aralocaspica,we investigated the performance of recombinant PEPC in Escherichia coli Transetta（DE3）under various abiotic stresses.The full-length cDNA of PEPC（GenBank accession No.KP985714）was obtained by 5′-RACE technique and its procaryotic expression vector pGEX-4T-1-PEPC was constructed.The content and enzyme activity of PEPC were measured by the spectrophotometric method and enzyme linked immunosorbent assay（ELISA）.Meantime,the tolerance of the recombinant E.coli Transetta：：pGEX-4T-1-PEPCto different abiotic stresses was also investigated.Bioinforma-tics analyses showed that PEPCcontained 2 901 bp nucleotides and encoded 966 amino acids,which possesses the typical conserved domains of PEPC（e.g.active sites VlTAHPTQsiRR and VMIGYSDSgKDAG）,and shares 90% amino acid similarity to PEPC fromBeta vulgaris;our PEPC was a non-secretory PEPC-1type hydrophilic protein without signal peptide sequence,and the molecular weight of the recombinant PEPC was approximately 130-140 kD,which was confirmed by western blot analysis.The recombinant PEPC presented relatively higher enzymatic activity when induced by 0.8 mmol/L IPTG at 37 ℃.Compared with the control strain,the recombinant E.coli Transetta：：pGEX-4T-1-PEPCshowed better growth performance under various abiotic stresses[i.e.200-800mmol/L NaCl,5%-20% PEG 6 000,25 ℃-52℃,50-400μmol/L methyl viologen,and the wide acid and base range（pH 3.0-11.0）].The results showed that overexpression of PEPCgene fromS.aralocaspicain E.coli enhanced the tolerance under various abiotic stresses,which suggests that PEPC may confer stress tolerance to E.coli.Our work can provide more evidence for revealing the biological function of PEPC fromS.aralocaspica.
出处
《西北植物学报》
CAS
CSCD
北大核心
2015年第9期1767-1775,共9页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(31460043)
新疆自治区优秀青年科技人才培养项目(2013721013)
