海带PEPC功能区的原核重组表达及功能鉴定  

Expression and identification of phosphoenolpyruvate carboxylase(PEPC)of Saccharina japonica

作　　者：秦天明 金迷娜 方佩佩 毕燕会 周志刚 QIN Tianming;JIN Mina;FANG Peipei;BI Yanhui;ZHOU Zhigang(Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China;International Research Center for Marine Biosciences,Ministry of Science and Technology,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学,水产种质资源发掘与利用教育部重点实验室,上海201306 [2]上海海洋大学,国家海洋生物科学国际联合研究中心,上海201306

出　　处：《水产学报》2024年第10期40-50,共11页Journal of Fisheries of China

基　　金：国家重点研发计划“蓝色粮仓科技创新”专项(2018YFD0901500);国家“双一流”水产学科。

摘　　要：与其他大型海藻一样,海带同样具备无机碳浓缩机制(CCM),以实现高光合作用效率和生产力。胞质磷酸烯醇式丙酮酸羧化酶(phosphoenolpyruvate carboxylase,PEPC)被认为是海带CCM的重要组件,但目前尚没有关于海带胞质PEPC相关研究报道。本研究自海带配子体全长转录组数据库中筛选到一条可能位于细胞质的PEPC编码基因的cDNA全长序列。通过RT-PCR方法验证,获得SjPEPC基因的cDNA全长,为3503 bp,包含2850 bp的完整开放阅读框、50 bp的5'-UTR和603 bp的3'-UTR。SjPEPC编码一个含949个氨基酸残基的蛋白,相对分子质量为104.91 ku,等电点为7.31。多序列比对显示PEPC的功能位点在不同物种中高度保守,系统演化及序列结构分析表明SjPEPC属于细菌型的PEPC。选取编码SjPEPC功能区的核苷酸序列,利用同源重组技术构建了pET32a-SjPEPC原核表达载体,经过诱导和纯化,得到了分子质量大小为86.3 ku的重组蛋白(rSjPEPC),酶活性测定结果显示,rSjPEPC具有催化PEP发生羧化的活性,比活力为(9.37±0.46)U/mg prot。研究表明,PEPC在海带无机碳储存中可能具有重要功能。本研究可为进一步解析海带CCM提供了分子和生物化学证据。Like other macroalgae,Saccharina japonica also has CO2-concentrating mechanism(CCM)to realize high photosynthesis efficiency as well as elevated biomass.Phosphoenolpyruvate carboxylase(PEPC)in the cytoplasm was suggested to be an essential component of CCM.However,there was no report about cytosolic PEPC of S.japonica.In this study,the full length cDNA sequence(SjPEPC)of a PEPC gene,which might be located in cytoplasm,was screened from the full-length transcriptome of this kelp and was identified by RT-PCR.The fulllength cDNA sequence of SjPEPC was 3503 bp in length,consisting of a ORF of 2850 bp,a 5′-UTR of 50 bp,and a 3′-UTR of 603 bp.It encoded a protein of 949 amino acids with a molecular weight of 104.91 ku,and a pI of 7.31.Multi-sequence alignment showed functional sites of PEPC were highly conserved in the selected species.Both phylogenetic analysis and sequence characterization showed that SjPEPC was a bacterial-type PEPC.pET32a-SjPEPC was constructed for expressing the recombinant SjPEPC harbouring all the enzyme active sites.After being induced by IPTG and purified,rSjPEPC with molecular weight of 86.3 ku was obtained.Enzyme activity assay results showed that rSjPEPC could catalyze the carboxylation of PEP,and the specific activity was(9.37±0.46)U/mg prot.These findings provide molecular and biochemical data for analysis the role of cytosolic PEPC in the storage of inorganic carbon in S.japonica,and furtherly for analysis CCM of this kelp.

关 键 词：海带 配子体 磷酸烯醇式丙酮酸羧化酶 原核表达 酶活性测定 

分 类 号：Q786[生物学—分子生物学] S917.3[农业科学—水产科学]