海带碳酸酐酶基因Sjα-CA3的分离与鉴定  

Isolation and identification of the carbonic anhydrase gene Sjα-CA3 in Saccharina japonica

作　　者：翁佳建 许玲 王雯 毕燕会 周志刚[1,3] WENG Jiajian;XU Ling;WANG Wen;BI Yanhui;ZHOU Zhigang(Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China;National Demonstration Center for the Experimental Teaching of Fisheries Science,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]上海海洋大学水产科学国家级实验教学示范中心,上海201306 [3]上海海洋大学海洋生物科学国际联合研究中心,上海201306

出　　处：《上海海洋大学学报》2025年第1期44-55,共12页Journal of Shanghai Ocean University

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

摘　　要：自海带配子体转录组数据库中筛选到一个属于α亚型碳酸酐酶(CA)的表达序列标签(EST)。该序列与已报道的海带的α-CA家族成员Sjα-CA1和Sjα-CA2的相似度分别为68.02%和77.32%,表明其可能是该家族的一个新成员,命名为Sjα-CA3。通过RACE技术,获得了Sjα-CA3的全长cDNA序列,为1 469 bp,其中包含840 bp的完整开放阅读框(ORF)、332 bp的5′-非编码区(UTR)和297 bp的3′-UTR。Sjα-CA3基因编码1个由279个氨基酸残基组成的蛋白质,理论相对分子质量为31.19 ku,等电点为4.85。多序列比对表明Sjα-CA3的功能位点具有高度保守性。系统发育分析结果显示,Sjα-CA3与其他藻类的α-CA以高的置信度(99/81,NJ/ML)聚类在一起,进一步证实了其属于α-CA家族。通过异源重组技术构建了pET32a-SjαCA3原核表达载体,将其转入E. coli BL21(DE3)感受态细胞。在诱导表达和纯化后,获得了分子量约为45 ku的重组蛋白(rSjα-CA3)。酶活性测定结果显示,rSjα-CA3具有水合酶和酯酶活性,其比活力分别为0.82 U/mg和2.157 U/g。Sjα-CA3的成功分离与鉴定为进一步解析其在海带无机碳储存机制中的作用,以及海带无机碳浓缩机制CCM的研究提供了重要的数据支持。In this study,an expressed sequence tag(EST)belonging to theα-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 reportedα-CA family members in S.japonica,namely Sjα-CA1 and Sjα-CA2,suggesting that it might be a new member of theα-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 withα-CA proteins from other algae with high confidence(99/81,NJ/ML),further supporting its classification within theα-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.

关 键 词：海带 配子体 碳酸酐酶 原核表达 酶活测定 

分 类 号：S917.3[农业科学—水产科学]