Molecular Mechanism Of Sex Differentiation Of Ruditapes Philippinarum Under The Action Of Estradiol

Ruditapes philippinarum(Mollusk phylum,Bivalves,Veneroida,Veneridae,Ruditapes),It is distributed in both the north and south coasts of China.It is one of the traditional "four kinds of shellfish" in China.It has fast growth,short breeding cycle,strong adaptability(wide temperature,wide salt and wide distribution)and broad market prospect.In this study,the blank group and the experimental group were sliced by tissue sectioning method.The ratio of male to female in each group was observed by microscopic observation.qPCR was used to analyze the Dmrt gene of clams under different developmental stages,short-term estradiol treatment,and long-term estradiol stimulation.Transcriptome analysis was performed on the estradiol-stimulated experimental group and blank group by high-throughput sequencing.The results provide a theoretical basis for the mechanism of early gonadal development,sex differentiation and sex regulation.1 Observation on the gonad tissue of clams under the stimulation of estradiolIn the preliminary experiment,the estradiol content of the estradiol group with 10 g/L was significantly higher than that of the blank group when the estradiol content of each group was determined.n the formal experiment,the clams were cultured in estradiol sea water at 10 g/L and ordinary control sea water respectively.After the gonads were mature for two months,the gonad tissue sections of the clams were observed.The results showed that 85 females and 61 males were treated with estradiol in the experimental group,and the ratio of female to male was 1.39:1.There were 68 females and 73 males in the blank group,and the ratio of female to male was 0.93:1.At the same time,there were 3 androgynous clams in the experimental group,accounting for 2%.This study showed that estradiol had an effect on sex differentiation of clams and could induce sex differentiation of some clams into females.2 Transcriptome analysis of different sex clam under the stimulation of estradiolThe female and male clams with mature gonads in the experimental group(10?g/L estradiol)and the normal blank group were selected,and the transcriptome analysis of the hermaphroditic clams in the experimental group was performed.Experimental group was stimulated by estradiol for two months(E),blank group D was cultured normally for two months(D),and ET was hermaphrodite in experimental.The results showed that there were 697 differentially expressed genes in group E and group D,among which 288 genes were up-regulated and 409 genes were down-regulated.Compared with group E and group ET,there were 208 differentially expressed genes,among which 73 genes were up-regulated and 135 genes were down-regulated.This study provided candidate genes for sex differentiation of clam and provided theoretical basis for sex differentiation mechanism of clam.From the perspective of GO function clustering(ETvE),the differentially expressed genes were mainly concentrated in GO term,which was related to membrane transport and glucolipid metabolism.In the KEGG pathway(EvD),the pathways related to signal transduction,nucleotide metabolism and infectious diseases are the main enrichment items.In the KEGG pathway(ETvE),pathways related to signal transduction,neurological diseases and carbohydrate metabolism are the main enrichment terms.Therefore,it was speculated that estradiol had a certain effect on the communication and material transport of clam.By selecting sex-related differentially expressed genes for transcriptome verification,it was found that some differentially expressed genes related to male gonads were down-regulated.As a whole,it can be speculated that hormones regulate the process of sex differentiation by affecting the transmembrane transport capacity,signal transduction process and regulating gender-related genes of a.chinensis.3 Analysis of Dmrt gene characteristics and qPCRDmrt(double-sex and mab-3 relatated transcription factor)associated with gametogenesis,gonad development,gender differentiation.In the unpublished data of clam genome,BLASTe comparison was used to identify three Dmrt genes of Ruditapesphilippinarum,and genetic identification was carried out using MEGA,expasy,SMART and other analysis software to identify the evolutionary relationship.Three Dmrt genes were named into Dmrt3-like,Dmrt4-like-1,Dmrt4-like-2 according to the clustering of genetic evolution tree.qT-PCR was used to study the spatiotemporal expression of these three Dmrt genes in clams and their response to estradiol treatment.The results showed that there was an explosive increase in the expression levels of the three genes from the stage of trochophore to the stage of umbo larvae.Dmrt4-like-2 was expressed in every developmental stages of clam larva.In normal clams,Dmrt4-like-1 and Dmrt4-like-2 were the highest expression levels in gills and mantle,while Dmrt3-like was the highest expression levels in gills and viscera,but not in mantle and aqua.In a short period of time,estradiol treatment increased the expression levels of these three genes in the gonads of the clams.In the long period of time,the expression levels of the three genes in the male gonads of the blank group were higher than that in the female gonads.In the experimental group,the expression levels of Dmrt4-like-1 and Dmrt4-like-2 in the androgynous gonads were higher than that in female gonads and lower than that in male gonads.