水产学报2024，Vol.48Issue(10)：40-50,11.DOI:10.11964/jfc.20220413468
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
摘要
Abstract
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 cyto-plasm 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 full-length cDNA sequence of SjPEPC was 3 503 bp in length,consisting of a ORF of 2 850 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 ana-lysis 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Key words
Saccharina japonica/gametophytes/phosphoenolpyruvate carboxylase(PEPC)/prokaryotic expres-sion/enzyme activity assay分类
生物科学引用本文复制引用
秦天明,金迷娜,方佩佩,毕燕会,周志刚..海带PEPC功能区的原核重组表达及功能鉴定[J].水产学报,2024,48(10):40-50,11.基金项目
国家重点研发计划"蓝色粮仓科技创新"专项(2018YFD0901500) （2018YFD0901500）
国家"双一流"水产学科 National Key R & D Program of China(2018YFD0901500) （2018YFD0901500）
National"Double First-class"Aquatic Science （）
