Cloning And Expression Analysis Of Sex-related Genes Of Spotted Halibut(Verasper Variegatus)

The spotted halibut(Verasper variegatus)is a large benthic cold-blooded fish,it is mainly distributed in the north of China's yellow,bohai seas and the east China sea.It has a wide range of food,fast growth,strong disease resistance,high nutritional value,delicious taste and other characteristics,is an excellent breed.The Mature females are much larger than males because of their faster growth rate.It will create substantial economic benefits to establish an all-female breeding technique for V.variegatus.A better understanding of sex-related genes will contribute to the improvement of a single-sex breeding technique.1.Cloning and expression of the piwil2 gene in V.variegatusIn this study,we successfully isolated the piwil2 gene of V.variegatus,which was named as Vvpiwil2.The total length was 3872 bp,including a 3192 bp ORF,encoding1063 amino acids;the 5?UTR was 140 bp and the 3?UTR was 540 bp.Based on Ex PASy ? SMART ? Signal4.1 and NCBI Conservative Domain Database(CDD)biological analysis,the ORF encoded a putative protein,with a predicted molecular weight of 118.6 k Da and an isoelectric point of 9.02,no transmembrane structure and signal peptide site were detected.There were three domains: Argo L1 domain,PAZ domain and PIWI domain.Real-time fluorescence quantitative PCR technique was used to analyze the expression patterns of Vvpiwil2 gene at different stages of embryo and larvae.The results showed that: the Vvpiwil2 gene was abundantly expressed from the early development to the high blastocyst stage,and then declined until the hatching stage.The developmental stage of the embryo from cleavage to blastocyst stage is mainly guided by the cytoplasmic component.The mRNAs begin to been transcribed and synthesized in a large amount at the early gastrula,then the transition from maternal to zygote occur.Therefore,the results indicated that the Vvpiwil2 gene was a maternal gene.After hatching,the expression of Vvpiwil2 gene at 68 days post hatching was significantly higher than other stages,which demonstrated that Vvpiwil2 gene was associated with gonadal differentiation.The expression level of Vvpiwil2 gene in gonads was significantly higher than other tissues,and the expression level in ovary was significantly higher than testis,revealing that Vvpiwil2 gene might play an important role in the maintenance of ovarian functions.The results of this study provide a potential sex determinant gene for V.variegatus and lay a solid theoretical foundation for the establishment of all-female breeding technique.2.Cloning and expression of the wt1 gene in V.variegatusIn this study,we successfully isolated the wt1 gene of V.variegatus,which is named Vvwt1 a and Vvwt1b.The total length of Vvwt1 a was 3263 bp,including a 1245 bp open reading frame(ORF),encoding 415 amino acids,the 5?UTR was 372 bp and the 3?UTR was 1640 bp.The total length of Vvwt1b was 2312 bp,including a 1281 bp open reading frame(ORF),encoding 427 amino acids,the 5?UTR was 369 bp and the 3?UTR was 659 bp.Through biological analysis,the Vvwt1a gene encoded amino acids,with a predicted molecular weight of 46.2 k Da and an isoelectric point of 9.24,no transmembrane structure or signal peptide site was detected,there were four zinc finger structures at the end of ORF and encode KTS.The Vvwt1b gene encoded amino acids,with a predicted molecular weight of 46.95 k Da and an isoelectric point of 8.99,no transmembrane structure or signal peptide site was detected,there were four zinc finger structures at the end of ORF and encode KTS.Real-time fluorescence quantitative PCR technique was used to analyze the expression patterns of the wt1 gene at different stages of embryo and larvae.The results showed that the expression level of Vvwt1 a and Vvwt1b genes in gonads was significantly higher than in other tissues,and the expression level in the testis was significantly higher than in the ovary.Therefore,we speculated that Vvwt1 a and Vvwt1b genes play important roles in gonad and kidney development and function;In the early stage of development,Vvwt1 a gene weakly expressed before the early gastrula,then the expression level gradually rose to the highest level in the neural embryo stage,followed by decline until the hatching stage.It is speculated that Vvwt1 a gene might played a role in the differentiation process of primitive germ cells and gonad development.3.Cloning and expression of the cyp19a1 b gene in V.variegatusIn this study,we successfully isolated the cyp19a1 b gene of V.variegatus,which is named Vvcyp19a1 b.The total length of Vvcyp19a1 b was 1748 bp,including a 738 bp open reading frame(ORF),encoding 245 amino acids,the 5?UTR was 394 bp and the 3?UTR was 615 bp.Through biological analysis,the Vvcyp19a1 b gene encoded amino acids,with a predicted molecular weight of 27.55 k Da and an isoelectric point of 6.31,there were I-helix region,aromatase specific region,Ozol' s petide region and Heme-binding region.Real-time fluorescence quantitative PCR technique was used to analyze the expression patterns of Vvcyp19a1 b gene at different stages of embryo and larvae.The results showed that: the expression of Vvcyp19a1 b gene increased from the blastocyst stage to the neuroembryonic stage,reached the peak,and then began to decline until the hatching stage,indicating that Vvcyp19a1 b gene may be involved in the development of the nervous system.After hatching,the expression of Vvcyp19a1 b gene at 68 days post hatching was significantly higher than other stages,which demonstrated that Vvcyp19a1 b gene plays a role in the sexual differentiation of V.variegatus.Vvcyp19a1 b gene is expressed to different degrees in both male and female adult fish tissues.The expression level in brain tissues is significantly higher than that in other tissues,and the expression level in female brain tissues is higher than that in male brain tissues.We speculate that Vvcyp19a1 b gene,on the one hand,can regulate the sexual behavior of fish through the central nervous system,and may also be related to gonadal development and reproduction of V.variegatus.4.Cloning and expression of the amh gene in V.variegatusIn this study,we successfully isolated the amh gene of V.variegatus,which is named Vvamh.The total length of Vvamh was 1434 bp,including a 478 bp open reading frame(ORF),encoding 245 amino acids,the 1-20 amino acids are signal peptide sequences,The 36?387 amino acids were the AMH-N region,and the 404?469amino acids were the TGF-? region.The expression of Vvamh gene at 68 days post hatching was significantly higher than other stages,it is speculated that its function is to inhibit the development of male gonads into ovaries.Through real-time fluorescent quantitative PCR research Vvamh gene expression in male and female fish and organizations,The results showed that the expression levels in Vvamh gonad tissues were significantly higher than other tissues,and the expression levels in the testis tissues were significantly higher than that in the ovarian tissues.We speculated that Vvamh gene also plays an important role in the development of testis.In addition,theVvamh gene is also highly expressed in the ovary,suggesting that this gene also has a role in the ovary.