Molecular Cloning And Expression Analysis Of Piwil2and Pik3r1Gene In The Half-smooth Tongue-sole, Cynoglossus Semilaevis

The half-smooth tongue sole, Cynoglossus semilaevis (Pleuronectiformes, Cynoglossidae)is an important economic marine fish species in the north of China, and is highly valued becauseof its agreeable taste. But in nature, this species is sexually dimorphic and the development ofmales is lagging far behind the females. Furthermore the reproductive performance of males isoften weak which has a negative impact on female spawning and leads to a low quantity andquality of fertilized eggs, and low survival rate. Further research on spermatogenesis in this fishneeds to be done to improve sperm quality and promote the development of the aquacultureindustry.According to part information of genomic sequencing, in this study, we cloned the completecDNA of piwil2from C. semilaevis and assessed its relative expression in different tissues ofsexually mature fish and early larval stages. To analyze the expression differences amongdifferent sexes, promoter methylation detection and fluorescence in situ hybridization (FISH)were conducted. After RT-PCR and RACE, a3314bp piwil2cDNA was obtained. The completecDNA sequence contains a60bp5’UTR, a3162bp open reading frame (ORF), along with a92bp3’UTR. The ORF encodes a putative protein with1053amino acid residues, with a predictedmolecular weight of117kDa and an isoelectric point (pI) of8.81. Based on bioinformaticsanalysis, two principal domains of the Piwi-subfamily were found in this sequence without asignal peptide site. One was the RNA-binding PAZ domain and the other was the RNase-likePiwi domain. As shown in RT-PCT results, piwil2transcripts were highly abundant in ovary, andeven higher in testis, while weakly in other tissues. The expression of piwil2at earlydevelopmental stages of C. semilaevis gonads revealed that the piwil2gene didn’t come intoeffect until day95after hatching. Starting from day95after hatching, a marked increaseappeared in ovary and testis, and the expression level was higher in testis than in ovary. ByFluorescence In Situ Hybridization (FISH), we concluded that the piwil2was a Z-linked geneand located on the Z sex chromosome. Although not a candidate gene in C. semilaevis sexdetermination, piwil2played an essential role in spermatogenesis. In this study, we analyzed thedegree of methylation in the5’ flanking region of the piwil2DNA sequence utilizing the bisulfitesodium sequencing method (bisulfite-sequencing PCR, BSP). As shown in the result, a relativelylower level of methylation was observed in testis tissue of males and neo-males than in ovary.These results indicated that the high level of promoter methylation of piwil2in females may leadto low expression, while in males and neo-males, the piwil2gene was expressed actively as aresult of a low degree of promoter DNA methylation. Here, we obtained the full length of pik3r1gene from C. semilaevis by RT-PCR and RACE.The full-length cDNA of pik3r1was2443bp containing2154bp ORF,116bp3’UTR and173bp5’UTR. This sequence encoded717amino acids, with a predicted molecular weight of81.7kDa and an isoelectric point (pI) of5.88. Physiological histology slice of different gonadalstages and RT-PCR results showed that the pik3r1transcripts began to increase from95day afterhatching the time when spermatogonial stem cells (SSC) started to divide rapidly. In sexualmature C. semilaevis, the relative expression of pik3r1was higher in testis than in ovary andother tissues. All that implied that pik3r1gene may involve in testis mature and spermatogenesisin C. semilaevis.