Study On The Expression And Localization Of Primordial Germ Cell Marker Genes In Different Ploidy Of Rainbow Trout

Rainbow trout(Oncorhynchus mykiss)is a typical cold-water fish.Because of its delicious meat and rich nutrition,it has become the main cultured species of salmon in China.Triploid female rainbow trout has abnormal gonad development and cannot normally produce gametes for reproduction.This advantage effectively reduces the energy loss required during the ovarian development stage,but also avoids the occurrence of stagnation growth,fish quality degradation and high mortality caused by ovarian development and spawning season.Therefore,it is favored by breeders,and all female triploid rainbow trout has gradually become the focus of breeding.At present,the mechanism of sterility of triploid female rainbow trout has been studied from many aspects in the world,but it has not been explored from the perspective of germ cells.Therefore,this research studies the critical period of embryo and gonad differentiation of diploid and triploid female rainbow trout through the primordial germ cells(PGCs)marker genes Vasa and Dead end(Dnd).By comparison the expression of two genes in diploid and triploid and the changes in their expression positions,we explored the cause of infertility from the perspective of germ cells.The results provided a theoretical basis for further study on the mechanism of gonad abortion in triploid female rainbow trout.The main results were as follows:1.The Dnd gene sequence of rainbow trout was 1,231 bp by cloning and amplification,encoding a total of 364 amino acids.Multiple amino acid alignments showed that the highest homology between rainbow trout Dnd and Oncorhynchus tshawytscha was 98.36 %,and the similarity with other teleost fish was 98.36-44.4 %.The phylogenetic tree showeds that the Dnd of rainbow trout was very conservative in evolution,and it was clustered with salmon such as Oncorhynchus tshawytscha,Oncorhynchus kisutch and so on in the branch of teleost,indicating that it hads high homology.2.Studies on different tissues of female rainbow trout have shown that Vasa and Dnd genes are only highly expressed in rainbow trout gonads,and are almost not expressed in other tissues.They can be used as germ cell markers and play an important role in the differentiation and development of rainbow trout.3.The Vasa and Dnd genes were continuously expressed throughout the embryonic development of diploid and triploid rainbow trout,confirmed that both genes were maternal genes and play an important role in PGCs migration and the whole process of embryo development.4.During the critical period of gonad differentiation of 240-330 dpf rainbow trout,the analysis of the expression of the two genes in the gonads of female rainbow trout under different ploidy showed that at 240 dpf stage,the triploid female rainbow trout could not further differentiate and develop due to the abnormal structure of the oocyte,So that no obvious expression can be detected in both genes.At 270 dpf,the triploid gonads began to reconstruct,and the digested maternal mRNA gradually synthesized new RNA needed by itself.At this time,the expression reached the maximum,but the difference was still significant compared with the diploid expression at the same period(P<0.05).5.The expression of Vasa and Dnd genes at the mRNA level and the protein level were not synchronized,and there was a difference in the expression time.In diploid,Vasa mRNA was continuously consumed for translation and synthesis of Vasa protein.Since 240 dpf in triploid,the ovary has stopped developing,the number of oocytes gradually decreased,and the synthesis of proteins associated with germ cells have been inhibited,so the content of Vasa and Dnd proteins is extremely low.6.During the embryonic period,the expression patterns of Vasa and Dnd genes were the same between the two ploidy,and the difference in expression was small,but in the later studies of gonads,the expression of the two genes in triploid was significantly lower than that in diploid(P<0.05),which proves that the triploid female rainbow trout did not have a large difference from the diploid due to chromosome doubling during embryonic development.Germ cells can develop normally in the early stage,but as the gonad develops in the later stage,the special regulatory mechanism in the triploid makes the oocytes unable to differentiate normally,resulting in infertility.