Study Of L-gulono-1, 4-lactone Oxidase (GLO) Of Abalone (Haliotis Discus Hannai Ino.)

A detailed review of studying status on L-gulono-1,4-lactone oxidase (GLO), a key enzyme required for biosynthesis of ascorbic acid in vertebrate. A series of experiments were conducted to elucidate whether or not the abalone(Haliotis discus hannai Ino.) has GLO activity and the ability to synthesize VC by using molecular biology techniques. The current studies include the followings:(1) Isolation, cDNA cloning and gene expression of GLO in different tissue and development phase of abalone (Haliotis discus hannai Ino.). (2) The effects of dietary vitamin C on growth, tissue concentration of ascorbic acid and GLO mRNA relative expression level in different tissue of Haliotis discus hannai Ino..(3) Construction of the hepatopancreas and kidney cDNA subtractive library of Haliotis discus hannai Ino. fed with vitamin C-deficiency diet. The results are summarized as follows.(1) The study was conducted to isolate and clone full-length cDNA sequence of GLO from Haliotis discus hannai Ino., and to investigate the relative expression level of GLO mRNA in different tissue and different development phase. Abalone, (initial weight:74.66±1.0 g; initial shell length:84.36±1.07 mm) were fed 170 days with purified vitamin C deficiency diet (VC 0.0 mg/kg). The full-length sequence (1606 bp) was determined from kidney of abalone fed with vitamin C deficiency diet. The open reading frames contained 454 amino acid which exhibited high amino acid identity (>48%) with vertebrate GLOs. The GLO mRNA expression was demonstrated in the abalone hepatopancreas, kidney, muscle, mantle, gill, blood cells and different development phase from trochosphere to 140d-old juvenile abalone. The GLO mRNA relative expression level of different development phase was also conducted by real-time quantitative PCR (RT-PCR). The result shows that the GLO mRNA relative expression level reaches the highest in the larva with pallial tentacle. W ith the development of abalone, relative expression level increased first and then decreased. The result suggested Haliotis discus hannai Ino. has the ability to synthesize VC.(2) The study was conducted to investigate the effects of dietary vitamin C on growth, tissue ascorbic acid concentration and GLO mRNA relative expression level in different tissue of Haliotis discus hannai Ino.. Abalone, (initial weight:74.66±1.0 g; initial shell length:84.36±1.07 mm) were fed 170 days with purified diets containing 4 levels of vitamin C:0.0,70.3,829.8 and 4967.5 mg/kg. The result shows that survival and growth were not significantly affected by dietary treatments (p>0.05). Ascorbic acid concentration had significantly difference in different tissue (p<0.01). It was significantly higher (p<0.01) in gill and lower (p<0.05) in serum than the other tissues. Ascorbic acid concentration in different tissue ranked as follows:gill>kidney and hepatopancreas> muscle and mantle>serum. Ascorbic acid concentration was not significantly affected in kidney, gill, mantle and serum by dietary vitamin C (p>0.05), but significantly increased in hepatopancreas and muscle (p<0.05) with the increase dietary vitamin C. The mRNA relative expression level of two species of housekeeping geneβ-actin and ribosome protein S9 had significantly difference in different tissue(p<0.01). The mRNA relative expression level ofβ-actin in different tissue ranked as follows:blood cells> gill, muscle, mantle, and kidney> hepatopancreas. The mRNA relative expression level of ribosome protein S9 in different tissue ranked as follows:gill> blood cells, mantle, kidney and hepatopancreas> muscle. The mRNA relative expression level ofβ-actin was not significantly affected in hepatopancreas, kidney, mantle and muscle (p>0.05), but significantly affected in gill and blood cell (p<0.05) by dietary vitamin C. The mRNA relative expression level of ribosome protein S9 was not significantly affected in every tissue by dietary vitamin C (p>0.05). The mRNA relative expression level of GLO was not significantly affected in every tissue by dietary vitamin C when ribosome protein S9 was selected as reference gene (p>0.05). The mRNA relative expression level of GLO was not significantly affected in other tissues except gill by dietary vitamin C whenβ-actin was selected as reference gene (p>0.05). The mRNA relative expression level of GLO had significantly difference in different tissue(p<0.01).It was ranked as follows:kidney> gill, blood cells, mantle, and hepatopancreas> muscle.(3) The study was conducted to clone the diffierentially expressed genes in the hepatopancreas and kidney of abalone (Haliotis discus hannai Ino.) under vitamin C deficiency. Abalone, (initial weight:74.66±1.0 g; initial shell length:84.36±1.07 mm) were fed 170 days with purified diets containing 2 levels of vitamin C:0.0 mg/kg and 4967.5 mg/kg. The cDNA subtractive library of the hepatopancreas·and kidney of abalone vitamin C deficiency-treated was constructed using the method of suppression subtractive hybridization Analysis of subtraction efficiency shows that diffierentially expressed genes had at least 25 and 25-10 - fold enrichment in two cDNA subtractive librarys respectively. Sixty three and thirty nine cDNA fragments which might be differentially expressed genes in the vitamin C deficiency group were obtained from the hepatopancreas and kidney cDNA subtractive library respectively by randomly picking and sequencing positive clones. This study plays an important role in researching the vitamin C metabolic mechanism of abalone at the molecular level.