Cloning, Spatiotemporal Expression And Association Analysis Of 4 Genes With Growth Traits In Hard Clam Meretrix Meretrix

The Meretrix meretrix is an important commercial species which is widely cultured in the coast of China, which has high nutritional and medicinal value. It’s very popular in coastal residents. In recent years, with the technological breakthroughs of artificial breeding and large-scale farming, the aquaculture industry of M. meretrix presented the trend of rapid development, resulting in a huge economic and social benefits. Despite the economic value and research importance, knowledge of genetics and genome in M. meretrix has been largely limited, and breeding work has just begun up to date. In order to develop molecular resources and expedite gene discovery, EST of 4 genes, GRB2、HDAC1、MSTN and α-amylase, were identificated on the basis of 454 c DNA library of M. meretrix. c DNA was cloned and spatiotemporal expression was analysed of 4 genes, SNPs were screened out in GRB2 and HDAC1, and the differences between α-amylase m RNA expression and enzyme activity in four vibrant shell color(thin checkered, white shell, dark fringe and red shell) were analysed, to study the relationship between α-amylase and growth traits. Our results are as follows:1. GRB2 c DNA was cloned by SMART RACE techniques, then the bioinformatics and expression profiles in different tissues and developmental stages were analyzed and SNPs were screened out in exon. The results indicated that the full length c DNA of GRB2 gene was 1791 bp, containing a complete 669 bp ORF encoding 223 amino acids. There were three functional domains of GRB2 protein(SH3-SH2-SH3). Comparisons of animal acid sequence, the GRB2 of M. meretrix has highly homologous with Tegillarca granosa and shares 65.6% similarity. It shares more than 60% similarity with vertebrates and proves that GRB2 was highly conservative. The result of q RT-PCR showed that GRB2 expressed in all six tissues and ten developmental stages, but the expression of tissues did not have significantly difference. The relative expression in different stages revealed that the expression of GRB2 gradually increased with the process of the development, and showed the highest in umbo larvae stage. 16 SNPs in the exon of GRB2 were identified.2. Mm-HDAC1 c DNA was cloned by SMART RACE techniques, then the bioinformatics and expression profiles in different tissues and developmental stages were analyzed, and SNPs were screened out in exon. The results indicated that the full length c DNA of Mm-HDAC1 gene was 3065 bp, containing a complete 1599 bp ORF encoding 532 amino acids. Comparison of animal acid sequence, M. meretrix shares 74.3%-78.5% similarity with others and proves that Mm-HDAC1 was highly conservative. The result of q RT-PCR showed that Mm-HDAC1 expressed in all six tissues, and the expression of mantle was significantly higher than those of other tissues(P<0.01). The relative expression in different stages revealed that the expression of Mm-HDAC1 gradually increased with the process of the development, and showed the highest in umbo larvae stage(P<0.01). 19 SNPs in the exon of Mm-HDAC1 were identified and 3 SNPs potentially associated with M. meretrix growth(627A>T 、924T>C and 1266T>C).3. MSTN c DNA was cloned by SMART RACE techniques, then the bioinformatics and expression profiles in different tissues and developmental stages were analyzed and SNPs were screened out in exon. The results indicated that the full length c DNA of MSTN gene was 2265 bp, containing a complete 1671 bp ORF encoding 557 amino acids, including a 19-amino acid signal peptide. Comparisons of animal acid sequence, the MSTN of M. meretrix has highly homologous with Azumapecten farreri and shares 72% similarity. The result of q RT-PCR showed that MSTN expressed in all six tissues, and the expression of adductor muscle was significantly higher than those of other tissues(P<0.05). The relative expression in different stages revealed that the expression of MSTN gradually increased with the process of the development, and showed the highest in trochophore stage(P<0.05).4. α-Amylase(Mm Amy) c DNA nucleotide sequence was investigated, by expressed sequence tag and rapid amplification of c DNA ends(RACE) approaches. The full length c DNA of Mm Amy was 1783 bp, with a 1566 bp of open reading frame(ORF) encoding 522 amino acids, including a 19-amino acid signal peptide. The molecular mass and the estimated isoelectric point(p I) of the deduced mature Mm Amy were 57.58 k Da and 6.72, respectively. Sequence alignment revealed that Mm Amy shared the highest identity(73.6%) with that of C. gigas, Consistent with this, the phylogenetic tree showed that it was closed related to Haliotis discus hannai. The genomic DNA sequence was 6689 bp, which contains 7 exons and 6 introns. Real-time q RT-PCR shows that(1) Mm Amy only expressed in viscera and without expression in other five tissues;(2) Mm Amy was not expressed in the stage of unfertilized mature eggs, fertilized eggs, 4 cells, blastula, gastrulae, trochophore, and was expressed from the stage of D-shaped larva, the highest expression observed in the umbo larvae;(3) the highest expression observed in the dark fringe and the lowest expression in the red shell. Assay for α-amylase activity of four shell colors results shows the highest activity was observed in dark fringe and lowest in red shell.