Sreening Of Sex Differentiation Genes Of Chlamys Farreri And Expression Analysis Of Two Sex-related Genes

Sex differentiation is one of the most important research fields in development biology. The study about sex differentiation genes in vertebrates, especially in mammal, is deeper than in other species. Up to now, many genes related to sex determination and differentiation, such as Sry, Sox9, Wnt4, have been identified. But the research in invertebrates especially in marine bivalves is limited. In the present study, we selected the important commercial species-Chlamys farreri as the research object. Based on solexa sequencing, we analyzed the differentially expressed transcripts between femal and male gonads at proliferative stage. We totally obtained 295262 tags which are differentially expressed between sexes, and 1354 of which could be annotated with informations in GenBank. 5 of theses genes were selected and analyzed with RT-PCR. We find the concordance rate of Solexa sequencing results and RT-PCR results is about 40%.We amplified the full-length cDNA sequence of CL3256 gene using RACE technology. Sequence aligiment indicated it as the ribosomal protein L24 gene (Cf-rlp24). The full-length cDNA of Cf-rlp24 gene is 965 nucleotides which could encodes 175 amino acid residues. Alignment analysis indicated that the RLP24 protein shares a high homology among different species. Real-time PCR analysis showed that the expression of Cf-rlp24 gene is different between testis and ovary, and between different stages of testis. The expressions of Cf-rlp24 gene in the testis of growing and mature stage are significant higher than that of ovary, 2.200±0.446 fold and 6.036±1.283 fold, respectively. In the development of testis, the expression of Cf-rlp24 increased with the maturation of testis and reached the peak value at mature stage, then declined rapidly. However, the expression level maintained a low and steady state in ovary. We conclude that Cf-rlp24 gene may play a positive role in gonad development and is more effective in testis.We also performed real-time PCR to analyse the expression regulation in gonads cycle and female hepatopancreas. The result showed that Cf-vtg gene mainly expresses in ovary during the gonad cycle and the level increases as the development of ovary. The expression level is 1.917±0.035 (mean±S.E.) and 2.570±0.252 times higher during the growing and mature stage, respectively, than during the proliferative stage. There was negligible expression during the resting stage. Meanwhile, we observe weak expression in the female hepatopancreas. The level was 0.045±0.009 (Mean±S.E.) of that in the ovary during the proliferative stage. We did not detect any expression in the testis or other tissues. Further researches about the inducing effects of estradiol-17βon Cf-vtg expression with injuction method were performed. Real-time PCR showed that the expression level of Cf-vtg gene significantly increased in the ovary. It was concluded that estradiol-17βcould promote the synthesis of Cf-VTG.