Researches On MRNA Expression Of Four Polysaccharide Lyase Genes In Pacific Abalone, Haliotis Discus Hannai Ino

Polysaccharides are major components of algae which adult abalone is naturally feed on. Diatoms includs cellulose and starch, kelp contains cellulose, alginate and laminarin, for example.The mRNA expression of alginate lyase, cellulase,α-amylase and laminarinase gene in Pacific abalone, Haliotis discus hannai Ino, on the process of growth and development has been researched with the Real-time RT-PCR technology in this article. We also analyzed the effect of temperature on mRNA expression of these four genes. We investigated mRNA expression of these four polysaccharidase genes in order to reveal utilization of polysaccharide in abalone at different developmental stages and physiological state.1. The sequence and tissue-specific expression of four polysaccharide lyase genes in Pacific abalone, Haliotis discus hannai InoReal-time PCR primers of polysaccharidase genes andβ-actin gene were designed based on the cDNA sequences of Haliotis discus hannai Ino or Haliotis discus discus Reeve. Sequnences of cDNA PCR products of alginate lyase, cellulase,α-amylase, laminarinase gene andβ-actin gene were 116bp, 162bp, 188bp, 177bp and 175bp, and the DNA PCR products of these genes were 116bp, 829bp, 626bp, 177bp and 175bp respectively. The results of bioinformatics analysis indicated that, (1) Sequences of cDNA PCR products of alginate lyase, cellulase,α-amylase, laminarinase gene andβ-actin gene were almost the same as compared with the published sequences of certain corresponding gene, while two SNPs were found in intron of cellulase gene. (2) The DNA PCR products of the cellulase andα-amylase gene contain introns of 667bp and 438bp respectively.The mRNA expression of these genes has been researched in gill, muscle, antenna, pallium and digestive gland of Pacific abalone, Haliotis discus hannai Ino. The results show that these four genes have the highest mRNA expression in digestive gland there are low or no expression in other organizations. The distribution of these four polysaccharide lyase genes has the significant tissue-specific.2. The mRNA expression of four polysaccharide lyase genes on embryonic development period and food conversion period of Pacific abaloneThe expression of these four genes on the embryonic development period were decreased first and then increased. The results are as follows. 0h-6d in the early stages of embryonic development, the expression level of these four genes are at a low volume, and there was the lowest level at 24h. During the period of 7d-22d and 30d-45d, the expression was increased slowly. However the expression of these four genes on period of 7d-22d ware significantly higher than the expression of 0h-6d, and the expression of 30d-45d ware significantly higher than the expression of 7d-22d.The expression of these four genes was decreased sharply during 24 hours after spin-off on the food conversion period, and then increased after 24 hours.3. The effect of different temperature on mRNA expression of four polysaccharide lyase genes in Pacific abaloneWe set up seven treated groups. They were 8℃, 12℃, 16℃, 20℃, 22℃, 24℃and 30℃group. There were three repeats in every one group. And each repeat, there were seventy ones in the shell length of 2.3cm. We began to up/down (1℃/day) operation after a week of natural water temperature, until reached the target. The expression of these four genes from digestive gland of Haliotis discus hannai was researched with Real-time RT-PCR technology. The results showed that there was significant effect on the expression of these four genes. The expression of the 24℃group was significant higher than that of the others.