Cloning, Identification And Expression Analysis Of Differentially Expressed Genes In Gonad Of Cynoglossus Semilaevis

Half-smooth tongue sole (Cynoglossus semilaevis) is a rare marine flatfish and one of the most valuable fishes in Chinese coastal. In nature, the size difference between females and males of half-smooth tongue sole is great. It is very important to search method for sex control and improving the efficiency of fertilization because of weak reproduction of male. Recent years, the research of reproduction in half-smooth tongue sole has carried out in histology and embryology, but no report about gene profile. Therefore, identification of differentially expressed genes in gonad of half-smooth tongue is not only help to understand the different development mechanism but also help to look for new method for sex control and improving the efficiency of fertilization. In this paper, a subtracted cDNA library was constructed between ovary and testis by SSH. A total of 41 nonredundant clones were identified, from which 5 genes were cloned by RACE method, followed by analysis of characterization and spatio-temporal expression. In addition, the DMRT1 gene was cloned using a homologous cloning strategy and its tissues expression was analysed.Using suppression subtractive hybridization (SSH), a subtractive cDNA library was constructed with the ovary as tester and testis as driver. The total RNA was isolated from tester and driver respectively and double-strand cDNA molecules were synthesized. After two times of subtractive hybridization and then two times of PCR amplification, the forward subtracted PCR products were ligated with T vector (pMD18T) and the subtractive cDNA library construction was finished. 1000 clones of the total 2000 clones were detected by nest PCR, and about 600 clones contained the inserted fragments with the length between 250bp and 2000bp. Dot blot was performed on the nest PCR products of the 600 clones hybridized with the Dig-High Prime labeled probes and 196 clones were obtained as positive, which were analyzed by sequencing, and 166 generated adequate sequencing results. Comparison of the obtained cDNA sequences against GenBank databases resulted in the identification of 41 nonredundant cDNAs, of which 20 corresponded to known genes and 21 to unkown genes.Five reproduction-related genes including zp3a, Aquaporin1o, survivin, PABP and RacGAP1 were colned using RACE method. All these genes were first identified in half-smooth tongue sole. ZP3 protein is one of Zona pellucida (ZP) proteins and it play important role in oogenesis and fertilization. Two zp3 gene were cloned from a subtractive cDNA library and a full-length cDNA library, named zp3a and zp3b. zp3a and zp3b cDNAs were 1638bp and 1135bp. The open reading frames(ORFs) encode proteins of 518 and 312 amino acids. The two genes belong to ZP3 sub-family by characterization analysis and phylogenetic analysis. They were all highly expressed in ovary and low expressed in some other tissues of female by RT-PCR analysis. Interestingly the mRNA of zp3b also was identified in testis and kidney of male. Aquaporin1o was mainly expressed in ovary and related to egg hydration. From subtractive cDNA library, aquaporin1o was cloned and aquaporin1 was cloned by homologous cloning strategy. Aquaporin1o and aquaporin1 cDNAs were 993bp and 1335bp. The open reading frames(ORFs) encode proteins of 262 and 261 amino acids. Aquaporin1o was highly expressed in ovary and slightly in kidney of female.Survivin of one kind IAPs (inhibitor of apoptosis proteins) gene was cloned in Cynoglossus semilaevis. Sequence analysis revealed that the full-length cDNA was 704bp containing 444bp open reading frame (ORF). This sequence encoded 147 amino acids. RT-PCR analysis showed that the gene was highly expressed in ovary and slightly in kidney of female. In different embryo developmental stages, its expression was decline gradually . This result indicated that the survivin may play important role in oogenesis and early embryogenesis. One PABP gene was cloned from the subtractive cDNA library. It was considered that the PABP gene might be homologous to ePABP2 gene of other species by sequence analysis and expression analysis. RacGAP1 was one of the main regulator of Rac, one small G protein. One RacGAP1 was cloned and was mainly expressed in ovary. Until now, it is the first time to find one RacGAP1 that is mainly expressed in ovary. The expression pattern of different tissue and embryo development suggests that the RacGAP1 might play important role in oogenesis and embryogenesis.The DMRT1 gene was obtained by homologous cloning in Cynoglossus semilaevis. Sequence analysis revealed that the full-length cDNA was 1232bp containing 774bp open reading frame (ORF). This sequence encoded 258 amino acids. RT-PCR analysis showed that the gene expressed in all the analyzed developmental stages, from early embryo to 35-day juveniles, but the expression reached the highest level in juveniles at 22-days after hatching. In adult fish, however, its expression was detected only in male testis. This result indicated that the DMRT1 may play important role in the gonad differentiation and maturation of male C. semilaevis.