中华绒螯蟹ELOVL7基因克隆及功能验证

Cloning and functional verification of ELOVL7 gene in Eriocheir sinensis

  • 摘要: 长链脂肪酸延长酶 (Elongation of long-chain fatty acids, ELOVL) 在长链多不饱和脂肪酸 (Long-chain polyunsaturated fatty acids, LC-PUFA) 的生物合成中起关键作用。为探究中华绒螯蟹 (Eriocheir sinensis) LC-PUFA的合成机制,采用cDNA末端快速扩增 (Rapid amplification of cDNA ends, RACE) 技术克隆获得中华绒螯蟹 ELOVL7基因。该基因cDNA全长序列为1 600 bp,包含1个1 200 bp的开放阅读框 (Open reading frame, ORF),编码399个氨基酸,其5' 非翻译区 (5'-UTR) 和 3'-UTR 长度分别为176和224 bp。ELOVL7具有典型的ELVOL结构,包括8个跨膜结构域、多个保守基序以及特征性的组氨酸盒 (HXXHH)。氨基酸序列相似性比对分析显示,中华绒螯蟹ELOVL7与橄绿青蟹 (Scylla olivacea) 和三疣梭子蟹 (Portunus trituberculatus) ELOVL7的氨基酸序列相似性较高,分别为73.82%和73.76%。系统进化树分析显示,中华绒螯蟹ELOVL7首先与橄绿青蟹和三疣梭子蟹ELOVL7聚为一支,进而与其他甲壳动物ELOVL7共同构成一个独立的甲壳类分支。基因组织表达分析显示,中华绒螯蟹ELOVL7基因在肠道组织中表达量最高,其次为鳃组织。为验证其功能,将中华绒螯蟹ELOVL7基因编码序列重组至pYES2载体,转化酿酒酵母 (Saccharomyces cerevisiae) INVSc1菌株,成功构建了其酿酒酵母异源表达系统。酵母异源表达分析表明,ELOVL7能催化脂肪酸底物C18:1n-9延长为C20:1n-9。

     

    Abstract: Elongation of long-chain fatty acids (ELOVL) plays a critical role in the biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFAs). To investigate the LC-PUFA biosynthesis mechanism in the Chinese mitten crab (Eriocheir sinensis), we cloned the ELOVL7 gene using rapid amplification of cDNA ends (RACE) technology. The full-length cDNA sequence was 1 600 bp, containing a 1 200 bp open reading frame (ORF) that encoded a 399-amino acid protein, with 5'-untranslated region (5'-UTR) and 3'-untranslated region (3'-UTR) lengths of 176 and 224 bp, respectively. The deduced ELOVL7 protein exhibited typical ELOVL structure, including eight transmembrane domains, multiple conserved motifs, and a histidine box (HXXHH). Amino acid sequence similarity analysis reveals high similarity between E. sinensis ELOVL7 and orthologs from the mud crab (Scylla olivacea, 73.82%) and swimming crab (Portunus trituberculatus, 73.76%). Phylogenetic tree analysis shows that E. sinensis ELOVL7 first clustered with S. olivacea and P. trituberculatus ELOVL7 to form a distinct subclade, which subsequently grouped with other crustacean ELOVL7 sequences to comprise an independent monophyletic clade. Tissue expression profiling indicates highest ELOVL7 transcript abundance in the intestine, followed by the gill. To functionally validate the enzyme, we subcloned the coding sequence of E. sinensis ELOVL7 into the pYES2 vector and transformed into Saccharomyces cerevisiae strain INVSc1, successfully establishing a heterologous yeast expression system. Heterologous expression in yeast demonstrates that ELOVL7 can catalyze the elongation of fatty acid substrate C18:1n-9 to C20:1n-9.

     

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