Expression Characteristics And Functional Studies Of Piwi And Dmrt In Sea Cucumber,Apostichopus Japonicus

Sea cucumber(Apostichopus japonicus)belonged to the Echinopermata,Eicutherozoa,Holothuroidea,Aspidochirotidae,Stichopodidae,and Apostichopus.Sea cucumber ranked first among the eight treasures of seafood,and was mainly produced in the Dalian sea area of our country.The market supply and demand relationship had promoted the large-scale production of the sea cucumber industry,and the demand for seed had also increased year by year.Although the genetic sex of sea cucumber can be identified by a male-specific DNA marker,the mechanisms of sex determination and sex differentiation of the sea cucumber remain a mystery.In addition,the gonads of sea cucumbers were also called "sea cucumber flowers",which were a high-quality source of peptides and had rich nutritional value.Therefore,more and more researchers had focused their attention on the developmental mechanism of the gonads of sea cucumbers.The first step to study the gonadal development of sea cucumbers was to obtain candidate genes related to gonadal development.In this study,multiple genes related to gonadal development of sea cucumbers were screened from the whole genome level through homologous sequence alignment,such as: piwi,dmrt gene family,nodal and foxl2.Subsequently,we used piwi and dmrt gene family as the entry points to conduct preliminary investigations on their molecular characteristics,expression patterns and functions in germ cells.The piwi1 gene of sea cucumber was a maternal factor(Ajpiwi1),and its expression had been detected throughout the embryonic development.In adult tissues,it was mainly expressed in the gonads and respiratory tree,and the expression level in the ovary was 21 times that of the testis.In the ovary,as the process of oogenesis progresses,the expression of Ajpiwi1 gradually increaseed.A large number of Ajpiwi1 transcripts were detected in primary oocytes and previtellogenic oocytes,reaching the peak during the growing stage,and the expression of Ajpiwi1 gradually decreased significantly in the mature and post-spawning stage,and only a small amount of Ajpiwi1 was detected in mature oocytes.During the early growing stage and growing stage of the testis,the expression of Ajpiwi1 had no significant change.As the process of sperm cells mature,the expression of Ajpiwi1 gradually increased,and reached the highest during the post spawning stage,and the transcript of Ajpiwi1 was detected in spermatogonia and spermatocytes,and no positive Ajpiwi1 cells were observed in the somatic cells and spermatids.Members of the dmrt gene family play significant regulatory roles in sex determination/differentiation and gonad development;however,their genome-wide identification has not yet been performed in the sea cucumber Apostichopus japonicus.In this study,a total of five members of the Dmrt gene family were identified from the genome of sea cucumber,consisting of Aj Dmrt1,Aj Dmrt2,Aj Dmrt3 a,Aj Dmrt3 b and Aj Dmrt5;Dmrt4 could not be found,suggesting that it was lost in the sea cucumber genome.These Dmrt genes were predicted to encode the 282 aa,342 aa,216 aa,314 aa,and 168 aa proteins,respectively.The analysis of gene structure showed that the number of introns in sea cucumber Dmrt genes varied from 0 to 2.Further analysis revealed that Aj Dmrt1,Aj Dmrt2,Aj Dmrt3 a,and Aj Dmrt3 b genes may be linearly arranged,and Aj Dmrt5 had no collinearity with the others.The expression pattern results revealed that the expression patterns also differed among the Dmrt genes.Aj Dmrt1,Aj Dmrt3 a,and Aj Dmrt3 b were maternal factors and were detected continuously during embryogenesis.In contrast,the transcripts of Aj Dmrt2 and Aj Dmrt5 were not detected during embryogenesis.Tissue expression specificity analysis revealed that Aj Dmrt1 was expressed predominantly in the testis,with slight expression in the ovaries.Aj Dmrt2 and Aj Dmrt5 were predominantly expressed in the tube feet.Aj Dmrt3 a and Aj Dmrt3 b were predominantly expressed in the coelomocytes and tube feet.At present,the method for studying the gene function of sea cucumbers at the adult level had not been established.Considering the small eggs and low survival rate of sea cucumbers,it is difficult to carry out gene function research directly through embryo microinjection.We had established a gene function research method in sea cucumbers by direct injection of ds RNA into the body cavity.After injection of the specific double-stranded RNA(ds RNA)of Ajpiwi1,it successfully caused target gene expression in ovary and testis to be down-regulation by 24% and62%,respectively,and led to the expression of genes such as dmrt1 and foxl2 changing in the sex differentiation pathway.The establishment of this technology laid a foundation for studying the functions of genes related to gonadal development in sea cucumbers at the adult level.