海带配子体γ-碳酸酐酶的基因克隆与功能鉴定

本研究采用cDNA末端快速扩增(RACE)等技术获得海带配子体细胞一个γ-亚型碳酸酐酶(CA)基因的cDNA及DNA全长序列。其cDNA长为1618 bp,编码由305个氨基酸组成的蛋白;DNA长为11812 bp,具6个内含子,将开放阅读框(ORF)分割成7个外显子;其具有一个gamma-CA的结构域,并具有一个独特的左手平行β-螺旋结构域;由36个CA的氨基酸序列所构建的聚类图显示,该CA与其他物种的γ-CA聚为一支。因而,将该克隆的基因命名为Sjγ-CA。为了解其编码蛋白的功能,将Sjγ-CA的ORF亚克隆至表达载体pET28a上,导入大肠杆菌表达菌株BL21中,培养并诱导目的基因表达,亲和层析以纯化重组蛋白。SDS聚丙烯酰胺凝胶电泳(SDS-PAGE电泳)、免疫印迹和质谱技术鉴定结果表明,纯化所获得的重组蛋白是Sjγ-CA。利用电极法和分光光度计法分别检测重组的Sjγ-CA在CO2与示,重组Sjγ-CA的水合酶比活力为0.82 U/mg,但没有酯酶活性,从而从功能上鉴定了Sjγ-CA。该研究为后续探讨Sjγ-CA在海带配子体乃至孢子体细胞或组织中的亚细胞定位等研究奠定了基础。

Cloning and functional characterization of aγ-carbonic anhydrase(CA)gene from the gametophytes of Saccharina japonica

In this study,the full-length cDNA and DNA of aγ-type carbonic anhydrase(CA)gene was obtained from the gametophyte of Saccharina japonica by the rapid amplification of cDNA ends(RACE)technique.The results showed that the cDNA of this gene was 1618 bp in length,encoding a protein consisting of 305 amino acids,and the genomic DNA was 11812 bp long,with 6 introns,so that the open reading frame(ORF)was divided into 7 exons.It had a gamma-CA domain and a unique LβH domain.The Neighbor-joining and the Maximum Likelihood phylogenetic tree constructed from the deduced amino acid sequences of 36 CAs showed that the cloned CA was clustered with otherγ-CAs.Therefore,the gene was designated Sjγ-CA.In order to understand the function of the encoded protein,the ORF of Sjγ-CA was subcloned and ligated into the expression vector pET-28 a to generate pET28 a-SiγCA.Subsequently,this construct was introduced into Escherichia coli BL21 for the heterologous expression of target protein.The recombinant Sjγ-CA was purified by affinity chromatography.After SDS-PAGE electrophoresis,Western blotting analysis and mass spectrometry,the purified was detected by electrode method and its specific activity was 0.82 U/mg.The hydrolysis of p-nitrophenyl acetate was detected by spectrophotometer,but no esterase activity was detected.Sjγ-CA was thus identified functionally.This study provides a basis for the subcellular localization of Sjγ-CA in gametophyte and sporophyte cells or tissues of S.japonica.