中华绒螯蟹内源性纤维素酶EsGH9-1基因的克隆、表达及其对不同饵料的响应

Molecular cloning and expression analysis of endogenous cellulase EsGH9-1 from Eriocheir sinensis and its response to different diets

中文关键词: 中华绒螯蟹  纤维素酶  β-1,4内切葡聚糖酶  基因克隆  表达分析  不同饵料

英文关键词: Eriocheir sinensis  Cellulase  β-1,4-endoglucanase  Gene cloning  Expression analysis  Different diet

中文摘要:

      本研究从中华绒螯蟹(Eriocheir sinensis)转录组数据库中比对筛选到内源性纤维素酶(β-1,4-内切葡聚糖酶)基因序列信息，通过克隆获得该纤维素酶(EsGH9-1)基因组DNA和cDNA序列。EsGH9-1基因组DNA序列长度为11 679 bp，包含15个外显子和14个内含子；cDNA序列全长为2044 bp，编码580个氨基酸，具有糖基水解酶第9家族的典型催化功能域。RT-PCR结果显示，EsGH9-1主要分布在肝胰腺组织，在大眼幼体期和仔蟹早期高表达。不同饵料条件下，EsGH9-1相对表达量在植物性饵料组显著上调，与β-1,4内切葡聚糖酶活性趋势相一致，推测EsGH9-1参与纤维素的消化与分解。本研究证明中华绒螯蟹自身具有纤维素酶基因，为今后深入研究内源性纤维素酶在水生甲壳类的摄食与消化机制中的作用奠定了基础。

英文摘要:

      Cellulases are a class of modular enzymes with a catalytic glycoside hydrolase (GH) module that hydrolyzes the β-1,4-glucosidic bond of the cellulose chain. Cellulases had been broadly divided into three types: endo-β-1,4-glucanases, exo-β-1,4-glucanases and β-glucosidases, which of synergistic effect lead to the complete degradation of cellulose to glucose. The endo-β-1,4-glucanase is one of the key cellulases in cellulose digestion, which are likely to be the product of a glycosyl hydrolase family 9 (GHF9) gene. In this study, an endogenous cellulase gene, EsGH9-1, was identified from the transcriptome database of the Chinese mitten crab, Eriocheir sinensis, and the sequence of EsGH9-1 was cloned by PCR. The expression levels of EsGH9-1 in different tissues and different developmental stages were studied. In addition, EsGH9-1 mRNA expression and corresponding enzyme activity under different diet conditions were analysed. The result showed that EsGH9-1 genomic DNA (11 679 bp) consists of 15 exons interrupted by 14 introns; EsGH9-1 cDNA (2044 bp) contains an open reading frame of 1740 bp, corresponding to a polypeptide of 580 amino acid residues, with a typical catalytic domain of the glycosyl hydrolase family 9 (GHF9). EsGH9-1 was expressed in various tissues of E. sinensis, particularly high in the hepatopancreas. A significant increase in expression of EsGH9-1 was also observed in megalopa and early-stage larvae. Under different dietary conditions, the relative expression of EsGH9-1 in the plant diet group was significantly up-regulated, which was consistent with the trend of β-1,4-endoglucanase activity. The finding of the endogenous cellulase gene (EsGH9-1) in E. sinensis implies that EsGH9-1 may be involved in the decomposition of plant diet. Furthermore, this work is a fundamental step toward understanding the role of endogenous cellulase in the digestion mechanism of aquatic crustaceans.