Research On Variation Of The Content Of Ascorbic Acid In Different Developmental Stages And The Homologous Genes Of Its Key Synthetase In Bombyx Mori

Ascorbic acid, also known as vitamin C, has important physiological functions in vivo. From the low single-celled organisms to the higher plants and animals, many organisms are able to de novo synthesis of reduced ascorbic acid(AsA). But it is not clear whether insect itself has the ability to synthesize As A at present. Although AsA is an essential nutrient for silkworm larva, Bombyx mori, but in the non-feeding stage, such as the egg and pupa stages, the content of AsA have remained relatively stable in the specific developmental stages.There is no research to determine that silkworm only obtain AsA from food and can not be synthesized AsA so far. For this the sake, our study determined variation of the content of ascorbic acid of silkworm in different developmental stages and discussed the homologous gene of the As A anabolic key enzyme and variation of its expression.In the first instance, our test measured variation of the content of total ascorbic acid(TAA) and AsA of silkworm in different developmental stages and conditions. The results show that, in the feeding stages, the growth and development of the silkworm larva using artificial fodder lack of VC is relatively reared compared to the normal feeding silkworm larva, but it still growth to 4th instar. In the diapause graine, the content of TAA within spawning 72 h and the content of As A within spawning 24 h are increasing and then both of them have different degrees reduced, meanwhile they maintain relatively steady between them. During the normal embryonic developmental stage of granie, the content of As A within spawning 24 h is increasing significantly and in spawning 48 h~144 h keeping stable in a high level, until later stage of the embryonic development significantly decreasing, and the content of As A of the non-diapause eggs higher than that of the diapause eggs all the time. The content of TAA and AsA of silkworm larvae feeding on mulberry leaves or the artificial feed of adding VC are increased, while that of molting, feeding on the artificial feed of lacking VC or under fasting conditions are significantly declined, of which the content of AsA of larvae under fasting conditions decreased to very low levels. In the pupal stage, the content of TAA of silkworm have no significant change, but the content of AsA of female pupae have increased significantly in pupating 0 h~24 h and that of male pupae in pupating 0 h~144 h overall remained relatively stable in a high level and until the pupal later stage have a more significant reduction.Secondly, by searching the AsA synthetase homologous genes in the silkworm database with the amino acid sequence of the known AsA synthase gene and carrying out analysis and alignment for the amino acid sequence of these genes using bioinformatics software such as Mega, we have obtained the homologous genes in silkworm of the AsA anabolic key enzyme in the last step(L-gulono-1,4-lactone oxidase, GULO) and the penultimate step(aldonolactonase, ALase): BmGULO_like1 ? BmGULO_like2 ? BmALase_like1 and BmALase_like2. Due to the low single-celled organisms to higher primates, almost the contain complete or partial AsA synthase gene, and AsA has a wide physiological functions, suggesting that there should be this gene in silkworm.Finally, we design primers of 4 homologous genes of AsA synthase in silkworm using Primer software and detect variaty in the expression of homologous genes of the relative AsA synthase of silkworm in different insect state and organ using real time fluorescence quantitative PCR(RT-PCR). The detection result of the expression of 4 homologous genes, BmGULO_like1?BmGULO_like2?BmALase_like1 and BmALase_like2, indicate that, in midgut and fat body, the expression of 4 homologous genes in the egg stage and the pupal stage as a whole significantly higher than that of larval stage, and with the development rising trends.Ours results indicate that there may be the pathway of the source of AsA in the embryonic developmental stage and the pupal stage, including the way of de novo synthesis of AsA and oxidized ascorbic acid(DHA) reduced to AsA, while the larvae mainly absorb VC from diet and lack other sources. According to the research results, although the similarity of homologous genes of GULO in silkworm is lower than other known organisms, but silkworm still might have the ability of synthesis of AsA; the structure of ALase contained multiple functions is relative conservative and its distribution of species is extremely extensive and the homologous genes in silkworm are also highly conservative.