The Analysis Of Molecular Mechanisms Of Shell Color Formation For Meretrix Meretrix

The Meretrix meretrix is an important commercial species which is widely cultured in thecoast of China. The colorful shell-color and varied patterns, which can be inherited stably and issignificantly correlated with growth, resistance and other economic traits, has high economic valueand the value of academic research, and the commercial value of some brightly-colored clams wassignificantly increased.The color polymorphism of body and shell for aquatic animals is due to themutation of pigment synthesis genes, and the color of yellow and red are closely related to levels ofcarotenoid in vivo, a new line of brightly-color has an important significance for M. meretrix.aquaculture industry. In the study of epigenetics, DNA cytosine methylation affects the change inthe phenotype, is one of the ways that eukaryotic genomic DNA modification processing methods.In this study four clam strain (red shell, black spot, white shell and thin checkered) as the material,microscopic observation for pigment band from the mantle of white black spot and red clam wastested, and the content of carotenoids were examined by spectrophotometer method. ESTsequences of GGPS and crtO, which were two key enzymes in the process of arotenoid synthesishas screened. The full length cDNA of GGPS and crtO from red-shell clam were cloned andcharactered by the method of rapid amplification of cDNA ends using polymerase chain reaction(RACE-PCR). qRT-PCR was employed to investigate the protein of GGPS and crtO expression inmantle of different shell color, different tissues and different developmental stages of M. merereix. Expected toprovide theoretical basis and technical support for exploring the laws of carotenoids deposited in mantle of thedifferent shell-color clam and coloring mechanism of the clam. What’s more, methylation-sensitive amplifiedpolymorphism(MSAP) analysis was used to screed the different sites from the four kinds of clam and explorethe relationship between DNAmethylation and shell color. The main results are as follows:1. Microscopic observed that shading of the black spot individuals mantle edge near shell surface has abelt of dark pigment, and in the same shell no-shading area of pigment on the belt is clear. Red shell mantleedge of individuals is a belt of the pigment of red, and white shell color individuals with no pigmentband.SPASS software analysis found that there were significant differences between the two clam shells,andthe total content of carotenoid in red and white shell mantle were1.703μg/100g and0.50μg/100g,respectively.2. The full-length cDNA of GGPS was1130bp, an open reading frame consisted of293aminoacids.GGPS shared a remarkable high degree of structural conservation with the Isoprenoid-Biosyn-C1super-family, such as Pfam-A. Comparative analysis showed that GGPS had a high degree of similarity toother species’ GGPS proteins, and there has a higher sequence identity with Xenopus tropicalis (74.1%) and Aplysia californica (73.7%). Its structure prediction shows that it has two conserved domains characteristic(LLIDDIEDNSNLRRG and LGLYFQIRDDYAN) of the polypreny l synthetase family.The full-lengthcDNA of crtO comprises2967bp with5’UTR of120bp, a3’UTR of1020bp and putative ORF of1827encoding608amino acid residues. Deduced amino acid of crtO shared higher identities with other reportedcrtO proteins and its3D model was successfully built as single chain. Comparative analysis showed that crtOhas a high degree of similarity to Gallus gallus(50.1%) and Chrysemys picta bellii(45.5%).3. qRT-PCR was employed to investigate the protein of GGPS and crtO expression in mantle ofdifferent shell color, different tissues and different developmental stages of M. merereix. Both of the GGPSand crtO mRNA transcripts were expressed in all examined mantles of different shell-color with the highestexpressions in mantle of red-shell, and followed by pink, white-shell was the lowest. Compared to othertissues, a relative higher expression level in mantle was detected, and the transcriptions of GGPS and crtOwere also expressed in all of the developmental stages, which were increasing with the growth of organism,and to the point of maximum in eyespot, then, with the further growth the content of GGPS and crtO began todecline.4. Methylation patterns、 extent and heterosexual sites shell color were detected using the method ofMSAP. The results shows that for the red shell, black spot, fine lines and white shell, the semi-methylationrates is5%,9%,0%and4.5%, the degree of methylation is20.39%,18.18%,19.23%and10%, respectively.Black spot clams over the other three strains of hard clam has a specific missing under the primer ofE70HM1、E35HM8�'�E42HM3.