Construction Of The Mantle CDNA Subtractive Library Of Haliotis Discus Hannai Ino Fed With Vitamin E-deficiency Diet And Cloning Of The Full Length Of The Related Genes

This article summarized the technologies polymerase chain reaction (PCR) and nucleic acid hybridization in the field of nutrition and gene expression of aquatic animals) and its productions. In addition, we made the foreground on the development of nutrition and gene expression of aquatic animals.We suggest to use suppression subtractive hybridization (SSH) to cloning the differentially expressed genes in the mantle of abalone Haliotis discus hannai during vitamin E deficiency. Juvenile abalone (initial weight: 0.71±0.00 g; initial shell length: 15.49±0.04 mm) were fed 240 days with semi-purified diets containing 2 levels of vitamin E: 3.5 mg/kg and 52.8 mg/kg. The cDNA subtractive library of the mantle of abalone vitamin E deficiency-treated was constructed using the method of SSH.The results showed that the constructed cDNA subtractive library was a highly efficient one: the cDNAs were significantly enriched for differentially expressed sequences. One hundred positive clones were randomly picked and identified by PCR method, that 95 % clones contained 100～900 bp inserts, these inserts might be the cDNA fragments of differentially expressed genes in the vitamin E deficiency group. Forty eight clones were sequenced, including 16 new genes, 10 glutathione-s-transferase genes, 5 glutathione peroxidase genes, 2 catalase genes, 3 hydroxysteroid dehydrogenase genes, 4 tyrosine kinase genes, 1 thyroid peroxidase-like protein gene, 3 genes for cellulose, 1 cAMP responsive element binding protein gene and 3 shell biomineralization related genes.In addition,we successful cloned the full length of two peroxidase related gene and calmodulin bindingt protein like gene, also abtained 3'product of cellulose-like gene and 5'product of two matrix related protein gene,using RACE technology.