文章摘要
海带配子体γ-碳酸酐酶(CA)的基因克隆与功能鉴定
Cloning and functional characterization of a ?-carbonic anhydrase (?-CA) gene in the gametophytes of Saccharina japonica
投稿时间:2019-05-27  修订日期:2019-10-23
DOI:
中文关键词: 海带,碳酸酐酶,亚型,基因克隆,功能鉴定
英文关键词: Saccharina japonica, Carbonic anhydrase, Isoforms, Gene cloning, Functional characterization
基金项目:国家重点研发计划“蓝色粮仓科技创新”重点专项(2018YFD0901500);国家自然科学(41376136);国家“双一流”水产学科
作者单位E-mail
许玲 上海海洋大学 1272220168@qq.com 
毕燕会 上海海洋大学  
周志刚 上海海洋大学 zgzhou@shou.edu.cn 
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中文摘要:
      本研究采用cDNA末端快速扩增(RACE)等技术获得海带(Saccharina japonica)配子体细胞中一个γ亚型碳酸酐酶(CA)基因的cDNA及DNA全长序列,该基因的cDNA全长为1618 bp,编码由305个氨基酸组成的蛋白,基因组DNA全长为11812 bp,具6个内含子,将开放阅读框(ORF)分割成7个外显子;它具有一个gamma-CA的结构域,并具有一个独特的左手平行β-螺旋结构域,自36个CA的氨基酸序列所构建的邻接和最大似然聚类图显示,该CA与其它物种的 γ-CA聚为一支。因此,将该基因命名为Sjγ-CA。为了解它编码蛋白的功能,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,进行诱导表达和亲和层析纯化,获得重组蛋白rSjγ-CA。经过SDS-PAGE电泳、免疫印迹和质谱技术鉴定后,最后利用电极法和分光光度计法分别检测rSjγ-CA在CO2与HCO 的水合反应及酯酶反应中的活性。结果显示,rSjγ-CA大小约38 kD,水合酶比活力为0.82 U/mg,但未检测到酯酶活性,从而从功能上鉴定了Sjγ-CA。该研究为后续进行Sjγ-CA在海带配子体细胞乃至孢子体细胞和组织中的定位研究奠定了基础。
英文摘要:
      In the present study, the full-length cDNA and DNA of the first gamma carbonic anhydrase (CA) gene was obtained in the gametophyte of Saccharina japonica by RACE. The results showed that the cDNA of this gene is 1618 bp, encoding a protein consisting of 305 amino acids, and the genomic DNA is 11812 bp, with 6 introns, the open reading frame (ORF) is divided into 7 exons; It has a gamma-CA domain and a unique LβH domain; The Neighbor-Joining and the Maximum Likelihood clustering map constructed from the amino acid sequences of 36 CAs shows that the CA is clustered with γ-CA of other species. Therefore, the gene was designated Sjγ-CA. In order to understand the function of the encoded protein, the gene was inserted into the expression vector pET-28a, then introduced into E. coli BL21 for heterologous expression and affinity chromatography to obtain a pure recombinant protein rSjγ-CA. After SDS-PAGE, western blot and mass spectrometry identification, the activity of it in the hydration reaction of CO2 and HCO and esterase reaction was detected by electrode method and spectrophotometer, respectively. The results reavealed molecular mass of the recombinant protein is about 38 kD, the activity for hydration is 0.82 U/mg, but no esterase activity was detected. Then Sjγ-CA was identified functionally. This study provides a basis for the localization of Sjγ-CA in gametophyte cells, sporophyte cells and tissues of Saccharina japonica.
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