Isolation and identification of the carbonic anhydrase gene Sja-CA3 in Saccharina japonica
In this study,an expressed sequence tag(EST)belonging to the α-type carbonic anhydrase(CA)was identified from the transcriptome database of Saccharina japonica gametophytes.This sequence shares 68.02％and 77.32％similarity with previously reported α-CA family members in S.japonica,namely Sjα-CA1 and Sjα-CA2,suggesting that it might be a new member of the α-CA family,designated as Sjα-CA3.Using RACE technology,the full-length cDNA sequence of Sjα-CA3 was obtained,measuring 1469 bp in total and comprising an 840 bp open reading frame(ORF),a 332 bp 5'-untranslated region(UTR),and a 297 bp 3'-UTR.The Sjα-CA3 gene encodes a protein of 279 amino acid residues with a theoretical molecular weight of 31.19 kDa and an isoelectric point of 4.85.Multiple sequence alignments indicate that the functional sites of Sjα-CA3 are highly conserved.Phylogenetic analysis shows that Sjα-CA3 clusters with α-CA proteins from other algae with high confidence(99/81,NJ/ML),further supporting its classification within the α-CA family.A pET32a-SjαCA3 prokaryotic expression vector was constructed through heterologous recombination technology and introduced into E.coli BL21(DE3)competent cells.Following induction and purification,a recombinant protein(rSjα-CA3)with an approximate molecular weight of 45 ku was obtained.Enzyme activity assays revealed that rSjα-CA3 exhibits both hydration and esterase activities,with specific activities of 0.82 U/mg protein and 2.157 U/g protein,respectively.The successful isolation and identification of Sjα-CA3 provide crucial data for further analysis of its role in the inorganic carbon storage mechanism in S.japonica,as well as for advancing studies on the carbon concentrating mechanism(CCM)in this kelp.
国家科技期刊平台
NSTL
万方数据
