Cloning, Expression And Functional Analysis Of Cf-dmrt4-like Genes In Chlamys Farreri

Study on animal sex determination-and gonad development-related genes, an important part in developmental biology, is a theoretical basis for artificial sex control in aquaculture. Dmrt gene, originally found in drosophila and nematode, was isolated in succession in many vertebrates and invertebrates. These dmrt genes in different animals share a DM conserved domain and show a functional conservation to a certain extent. They are involved in some biological processes such as sex determination, gonadal differentiation and development, ontogeny and maintaining tissue function in vertebrate.In the present study, full-length cDNA of Cf-dmrt4-like gene and two alternative splice isoforms (Cf-dmrt4a-like and Cf-dmrt4b-like) were isolated from Chlamys farreri testis using a homologous cloning strategy, followed by analysis of sequence characteristic and spatio-temporal expression with methods of RT-PCR, qRT-PCR, in situ hybridization (ISH). To detect the function of this gene, RNA interference (RNAi) was conducted by injecting dsRNA of Cf-dmrt4-like gene into testes at the growing stage, and then mRNA levels were determined by qRT-PCR.cDNA of Cf-dmrt4-like gene is 2312 bp long containing an open reading frame (ORF) of 1110 bp that encodes the protein of 369 amino acids. The amino acid sequence shares the DM domain conserved in DMRT proteins, and contains both the DMA conserved domain and the characteristic short conserved domain (short motif 1), sharing with DMRT4 and DMRT5 proteins at C end. Sequence comparation and phylogenetic analysis showed that Cf-DMRT4-LIKE protein was most similar to other DMRT4 and appeared more closely related to DMRT4 protein, indicating that Cf-dmrt4-like gene is highly conserved through evolution. The Cf-dmrt4a-like isoform includes 1638bp encoding 143 amino acids, and Cf-dmrt4b-like isoform includes 1641 bp encoding 104 amino acids, which were preliminary speculated as the alternative splice isoforms.Cf-dmrt4-like gene was expressed in the gill, mantle, kidney, adductor muscle, and gonad of adult female, and the gill and gonad of adult male detected by RT-PCR. The real time qRT-PCR also showed a widely expression of Cf-dmrt4-like gene during the gonadal development cycle both in male and female. The expression level in mature testis was 20-time higher significantly than those in other stages, but no significant difference was observed between testes at other development stages, which showed similar express level with all ovary stages. These results reveal that this gene is involved in the gonadal development both of male and female and especially play an important role in the mature of testis. The ISH assay showed that Cf-dmrt4-like mRNA was distributed in the cytoplasm and nucleus of oogonia and oocytes during ovary development, and the positive signal appeared stronger in mature ovary than in proliferative and growing stages. During testis development, Cf-dmrt4-like mRNA was located in spermatogonia, spermatocytes and spermatids, and the signal was stronger in germ cells at the mature stage. No signal was found in somatic cells of the gonad. Above results imply that Cf-dmrt4-like gene is involved in differentiation and functional maintenance of germ cells.The semi-quantitative RT-PCR assay showed that Cf-dmrt4-like mRNA was distributed in unfertilized eggs, fertilized eggs, cleavage embryos and all larva stages. The expression level in unfertilized eggs was higher than those in other embryo stages, and the level in D-shaped larva higher than those in umbo larva and creeping larva (p <0.05). Results of ISH showed that the blue positive signal of Cf-dmrt4-like mRNA mainly appeared in the center of unfertilized eggs and early embryos, and migrated to the gill and mantle of larvae with the embryogenesis, which suggests this gene is involved in early establishment of gill and mantle. It is supposed that this gene also play a role in the functional maintenance of some tissues including gill and mantle due to the expression in adult tissues.The RNAi and qRT-PCR showed that injection of dsRNA can instantly silence the expression of Cf-dmrt4-like gene. The expression level of target gene in dsRNA treatment was same as the control at 48 h post injection, but was significantly lower than the control at 72 h post injection. In this study, a RNAi method for scallop was preliminary established. Further functional analysis including morphological observation of germ cell development in gonads and the expression variety of Cf-vtg are being performed.