海带PEPC功能区的原核重组表达及功能鉴定OA北大核心CSTPCD

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.