Genomic Structure, Growth Association SNP Screening And Ecological Response Of A-amylase Gene In The Abalone Haliotis Diversicolor Supertexta

Marine organisms have the ability of producting enzymes with unique properties compared to those of the same enzymes from terrestrial organisms.a-amylases are among the most important extracellular enzymes found in various group of organisms such as plants, animals and microorganisms. They play important roles in their carbohydrates metabolism and growth of each organism. In this study, the alpha-amylase gene c DNA full-length was cloned in Haliotis diversicolor supertexta supertexta, and analyzing physicochemical properties of alpha-amylase, exploring the expression of amylase gene in different tissues. Study the single nucleotide polymorphisms(SNPs) loci was based on the research of alphamylase gene structure, the author screened of SNPs loci in alpha-amylase gene as well as association analysis of SNPs with growth-related traits were carried on. Then effects of salinity and treatment time on the expression of alpha-amylase were analysed using real-time quantitative polymerase chain reaction(Real-time q RT-PCR). The detailed results were as follows:1. Cloning and characterization of alpha-amylase c DNA full-length in Haliotis diversicolor supertexta supertexta and analyzing the expression of alpha-amylase in different tissues.Experiments using rapid amplification of cDNA ends(RACE) and reverse transcription polymerase chain reaction(RT-PCR) technique on alpha-amylase gene in Haliotis diversicolor supertexta. The results showed that alpha- amylase gene Cdna full-length is 2260 bp, the open reading frame is 2088 bp, 5’UTR length is 68 bp, 3’UTR length is 104 bp, which encoded 695 amino acids. By Singal P analyzing the structure of signal peptide in amylase gene, the signal peptide contains 18 amino acids(M1WAQYGIVSALLVLSASA18), suggested that the protein is a secreted protein. The analysis of alpha-amylase domain, results showed that the protein contains two domains(domain A, domain C), there is a catalytic center in domain A, which located at 28 ~ 396 amino acids. The results of homology analysis showed that there is the highest homology between Haliotis diversicolor supertexta supertexta and Haliotis discus hannai(87.9%). In contrast, there is lowest homology among goats, humans and other vertebrates amylase sequence(<50%), indicating that the Haliotis diversicolor supertexta alpha-amylase Phylogenetic tree shows that Haliotis diversicolor supertexta and invertebrates gather in the same cluster. Using real-time PCR technology analysis the expression of alpha-amylase gene in Haliotis diversicolor supertexta different tissues, the results showed that, there is alphaamylase gene expression in adductor muscle, intestine, digestive gland, stomach, mantle, rostral, gastropods seven kinds tissues in Haliotis diversicolor supertexta. The α- amylase gene expression in stomach tissues is highest among others tissues, which up to 326.803, and the expression of amylase gene in the the mantle is the lowest(0.035). After ANOVA analysis, there is significant different between the expression of seven tissues, and the expression of amylase in stomach reach significant level(P<0.01).2. Indentification of SNPs in the alpha-amylase gene of Haliotis diversicolor supertexta supertexta and their association with growth traits.In this study, polymorphisms of the α-amylase gene were evaluated for association with growth traits in 72 individuals. Using direct sequencing, two single nucleotide polymorphisms(SNPs) were identified in the α-amylase gene, with a mutation from cytidine to guanosine at 18 bp in a partial α-amylase segment. The other mutation is at 299 bp in the same α-amylase gene fragment, with a change from cytidine to thymidine. Notably, these two polymorphisms were highly correlated with all three growth traits examined. At the g.18C>G position, individuals with the GG genotype had significantly greater values for body weight and shell length than those with the GC or CC genotypes(P<0.05). At the g.299C>T position, the TT genotype had significantly greater values for all analyzed growth traits than those with the CC or CT genotype(P<0.05). In conclusion, our results demonstrated that these SNPs in the α-amylase gene could influence growth performance in H. d. supertexta and could be used for marker-assisted selection in this species.3. Synergistic effects of salinity and threaten time on the alpha-amylase expression of H.d supertexta.In this study, the synergistic effects of salinity and threaten time on the expression of alpha-amylase gene by using the central composite design(CCD) and response surface methodology(RSM). The results showed that the linear effects of salinity on the alpha-amylase gene expression was not significant(P>0.05), but the linear effects of stressing time was significant(P<0.05), which suggested that the expression of amylase gene was easily be impacted by the stressing time. And quadratic effects of salinity on the amylase gene expression was significant(P<0.05), the quadratic effects of stressing time on the amylase gene expression was not significant(P>0.05). There is no synergistic effects between salinity and threaten time.Through response surface methodology, a model equation about the relationship of the gene expression to the two factors was established, with the R2 as high as 80.05%, Adj as high as 65.80%, the Pre.R2 reached to 50.18%, suggesting that the fitting capability of the model was practicably applied for prediction.