The Development Of SNP And The Clone And Expression Anlysis Of Tyrosinase Gene In Hard Clam Meretrix Meretrix

The hard clam, Meretrix meretrix Linnaeus, is one of the major edible farming shellfish. It is one of the main sea outlet products known as “The most delicious dish in the world” because of its delicate meat and nutrition nourishing and is loved by the people at home and abroad. In this paper, we developed 166 EST-SNP loci based on M. meretrix transcriptome sequencing, which provides available molecular markers resources for genetic diversity research and genetic breeding of M. meretrix; We also cloned tyrosinase gene which is related to the synthesis of melanin genes and analysised its expression, which provide a reference for the study of M. Meretrix shell colors.1. Development of166 SNP markers based on the transcriptome in M.meretrixM.meretrix transcriptome sequencing get a total of 164,789 transcripts, obtained 94,525 unigene by splicing the sequences and won 416 989 EST-SNP candidate site by SNP Calling variation detection software. A total of 445 pairs of primers were designed from randomly selected candidate SNP loci based on primer design principles. Among them, 203 pairs of primers obtain a single purpose amplified bands in M.meretrix genome, 191 pairs of primers had accurate genotyping in M.meretrix populations and 166 loci were polymorphic loci. The observed heterozygosity and expected heterozygosity were ranged from 0.0000 to 0.7667 and 0.1831 to 0.5085, respectively. The minor allele frequency changed from 0.1000 to 0.5000. 76 SNP loci deviate significantly from the Hardy-Weinberg equilibrium(P<0.05). This study provides available molecular markers resources for genetic diversity research and molecular marker-assisted breeding of M. meretrix.2. Genge cloning and genetic analysis of tyrosinase gene from M.meretrixTyrosinase(TYR) is an important enzyme in the biosynthesis of animal melanin and plays an important role in the formation of shell color in shellfish. We amplified the cDNA fragment of TYR gene from transcriptome datebase by RACE technology. Then we stitched the sequencing results and obtained the full-length sequence of TYR cDNA gene which is 2406 bp. ORF analysis indicates that TYR includes 42 bp 5' non- coding region(5'-UTR), 2067 bp open reading frame(ORF) and 297 bp 3' untranslated region(3'-UTR), as well as we find the polyA tail at the 140 bp of AATAAA polyadenylation signal in 3' sequence. The analysis of the biological information software showed that, TYR gene encoded a protein of 689 amino acids with the molecular mass of 78.25 kDa and the isoelectric point of 6.69, there are 12 transmembrane domains but no significant signal peptide sequence. Three introns with the sizes of 807 bp, 754 bp and 328 bp were cloned by conventional PCR methodsand, and they all belonged to GT-AG introns. Both semi-quantitative and real-time RT-PCR analyses showed that this gene expressed highly in the black shell, while expressed at very low levels in other color.