皱纹盘鲍肝胰腺和肾脏维生素C缺乏诱导的差异表达cDNA文库的构建

Construction of the hepatopancreas and kidney cDNA subtractive library of abalone Haliotis discus hannai Ino. fed with vitamin C-deficiency diet

采用抑制消减杂交(suppression subtractive hybridization,SSH)技术研究皱纹盘鲍(Haliotis discus hannai Ino.)Vc缺乏诱导的差异表达基因。实验用皱纹盘鲍成鲍初始体质量为(74.32±0.43)g,初始壳长为(84.36±0.24)mm。配制了Vc缺乏(0.0mg/kg)和高剂量添加(4967.5mg/kg)2个水平的人工饲料进行饲喂。经过170d的养殖后,分别构建肝胰腺和肾脏Vc缺乏消减cDNA文库。消减杂交效率分析显示,差异表达的基因分别被富集了大约25和25～210倍。从两个文库中随机挑选阳性克隆进行测序。在肝胰腺消减cDNA文库中获得63个片段,其中已知功能基因占54.0%,Gene ontology(GO)按照功能将其分为4类:代谢相关基因占15.9%,细胞代谢相关基因占30.2%,生物调节相关基因占4.8%,其他功能基因占3.2%。未知功能基因占46.0%。在肾脏消减cDNA文库中获得39个片段,其中已知功能基因占53.8%,GO将其分为3类:代谢相关基因占5.1%,细胞代谢相关基因占28.2%,生物调节相关基因占20.5%。未知功能基因占46.2%。其中,获得了包括细胞色素c氧化酶、葡萄糖-1脱氢酶、α-葡萄糖苷酶、β-1,3-D-葡聚糖酶、纤维素酶、藻酸盐裂解酶以及铁蛋白等在其他动物中被证明与Vc合成相关的基因,为进一步研究皱纹盘鲍Vc的合成和代谢奠定了基础。

The study was conducted to clone the differentially expressed genes in hepatopancreas and kidney of abalone（ Haliotis discus hannai Ino.） under vitamin C deficiency. Abalone,initial body weight（ 74.32±0.43） g, initial shell length（84.36±0.24） mm,were fed 170 d with purified diets containing 2 levels of vitamin C at 0 mg/kg and 4 967.5 mg/kg. cDNA subtractive libraries of hepatopancreas and kidney of abalone vitamin C deficiency-treated were constructed using the method of suppression subtractive hybridization（ SSH）. Subtraction efficiencies were about 25 and 25-210-fold enrichment in hepatopancreas and kidney cDNA subtractive library respectively. Positive clones were randomly selecte and sequenced. Sixty three clones from hepatopancreas cDNA subtractive library were sequenced. The sequences were subjected to GO annotation. According to their physiological function,they could be subdivided into 4 categories：15.9% of metabolic processes genes,30.2% of cellular metabolic processes genes, 4.8% of biological regulation genes,3.2% of other functions genes,and unknown genes. Thirty nine cDNA fragments from kidney cDNA subtractive library were sequenced,and they could be subdivided into 3 categories：5.1% of metabolic processes genes,28.2% of cellular metabolic processes genes,20.5% of biological regulation genes,and 46.2% unknown genes. A series of genes related to vitamin C biosynthesis have been obtained in this study,which includes cytochrome c oxidase,glucose 1-dehydrogenase,α-glucosidase,β-1,3-D-glucanase,cellulase,alginate lyase and ferritin. This research provides the basic data to further study the biosynthesis and metabolism of vitamin C in abalone.