| Tegillarca granosa is one of the four traditional cultured shellfish in China,which has high nutritional and medicinal value. It’s very popular in coastal residents. But at present, the growth mechanism of invertebrates are not clear including the clam, which restricts the development of breeding the clam.TGF-β superfamily is a multifunctional cytokine group, they have correlated structure. It involved in cell multiplication, differentiation, growth and other biological processes. Many growth related genes of the super family have been studied. From the family, we have a research about the mechanism of Smad1/5 gene and BMP2/4 gene in the development of the clam, to provide a theoretical basis for the MAS, be more targeted, shorten the cycle of breeding the clam, reduce the breeding costs, improve the traditional breeding technology, vigorously develop the healthy aquaculture of Tegillarca granosa.The results are as follows:1.The full-length c DNA sequence, genomic structure and the spatiotemporal expression of Tg-Smad1/5. In order to explore the molecular structure and biological function of Smad1/5 in molluscs, Smad1/5 gene of Tegillarcagranosa(Tg-Smad1/5) was cloned by SMART RACE techniques. The full length c DNA of Tg-Smad1/5 was 2424 bp, and its complete ORF(Open Reading Frame) was 1386 bp encoding 462 amino acids. The homologous similarity between the clam and Pinctada fucata, Crassostrea gigas was92.3%, 91.2%, respectively. On the basis of c DNA, the 3 introns of Tg-Smad1/5 were cloned by common PCR method, the size was 504 bp,553bp, 874 bp, respectively. And they were all GT-AG- introns. Tg-Smad1/5promoter was cloned by genome walking,the fragment length from the end of5’ to the start codon ATG was 934 bp,contained GATA frame, Oct-1 and so on, conformed to the promoter sequence features. The results of 6tissue-specific expression by real time PCR showed that Tg-Smad1/5 gene expressed in all tissues, and the expression of foot showed significantly higher than other tissues. The results of relative expression in 9 development stages revealed that the expression of Tg-Smad1/5 gene showed the highest expression in the eyebot larvae stage, which was significantly higher than other stages. And the expression decreased significantly from eyebot larvae stage to juvenile clams. The results showed that Tg-Smad1/5 had the similar molecular structure as the vertebrata, and the relative expression revealed differences in different tissues and development stages, which laid the theoretical foundation for further study on function and mechanisms of Smad1/5 in molluscs.2.The c DNA sequence of Tg-BMP2/4 gene and its spatiotemporal expression.BMP2/4 gene of Tegillarca granosa(Tg- BMP2/4) was cloned by SMART RACE techniques. The c DNA fragment of Tg-BMP2/4 was 1583 bp, and its ORF(Open Reading Frame) was 896 bp encoding 298 amino acids. The homology between Tg-BMP2/4 and other species is not very high, the homologous similarity between the clam and Crassostrea gigas, Chlamys farreri was 53%, 52%, respectively only. The results of 6 tissue-specific expression by real time PCR showed that Tg-BMP2/4 gene expressed in all tissues, and the expression of gill, mantle and foot showed significantly higher than blood, adductor muscle and visceral mass. The results of relative expression in 9 development stages revealed that the expression of Tg-BMP2/4 gene showed the highest expression in the D-shaped larvae stage and juvenile clams stage, which were significantly higher than other stages,and the juvenile clams stage was the highest. The expression results in this study showed that, there was a significant difference between Tg-BMP2/4 in different tissues and developmental stage of the clam. It might be involved in the growth of the organs, tissues and shell. This has laid an important foundation for further study on the function and mechanism of BMP 2/4 gene in shellfish.3.Screening the growth traits related SNP of Tg-Smad1/5. Sequencing directly about 10 randomly selected individuals, candidated 78 SNPs. The average density was 1SNP/43 bp, the density of exon region was 1SNP/198 bp, the density of intron region was 1SNP/28 bp. The mutation types respectively:conversion 34.62%, transversion 28.21% and insertion / deletion 37.18%. 7SNPs were in the coding region, included 5 synonymous mutations and 2 non synonymous mutations. Using HRM technology to verify the 82 clams in a group, obtained a growth traits related SNP c.74G>A in the first intron. The 5growth indexes, shell length, shell width, shell height, shell weight and soft tissue weight of GG genotype were significantly higher than that of AA genotype(P<0.05), also higher than the GA genotype. The results of this study can be applied to the breeding, but there are a lot of work must to be started, and also needed more in-depth researches. |
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