Transcriptome Analysis During Sex Differentiation And Expression And Regulation Of Dmrt1 In The Large Yellow Croaker (Larimichthys Crocea)

In order to understand the molecular mechanism of sex differentiation and determination of the large yellow croaker,Larimichthys crocea,we generated and analyzed the transcriptome sequencing(RNA-Seq)data of males and females in the early stage of sex differentiation.We further studied the activity of LcDmrt1(LcDmrt1X and LcDmrt1Y)promoter and the transcriptional regulation of LcDmrt1 product on the LcDmrt1 promoter.We also determined the function of different Dmrt1 transcription binding sites in LcDmrt1X and LcDmrt1Y promoter.The main results are as follows:(1)Comparison of gonad transcriptome between males and females in the early stage of sex differentiation of the large yellow croaker.RNA-Seq of the female and male gonadal tissues were carried out at five different time points(37dph,39dph,41dph,44dph and 50dph),when gonad differentiation initiates in the large yellow croaker.After quality control and data trimming,a total of 153.98 GB clean data were obtained.The reads map rate to the reference genome was 95.09%-96.27%,and 27,392 genes and 51,761 transcripts were finally annotated.According to the analysis of differential expression genes(DEGs),a total of 1,115 differentially expressed genes(|log2fc|>2,Padj<0.05)were detected at five stages.The maximum quantity of differentially expressed genes was 823 at 44 dph,followed by 529 at 37 dph,and relatively few at 39 dph,41 dph and 50 dph.The KEGG analysis clustered on the steroid biosynthesis and steroid hormone biosynthesis,related to gender differentiation.In total,108 and 1,005 genes mainly expressed in male and female gonads were obtained in all five time points,respectively.This genes included several sex related genes,such as Hsd17B1,Cyp19a,Dmrt1,Amh,Cyp17Ⅱ.In the differential expression gene analyses between different time points among the same sex,904 female differentially expressed genes(F-DEGs)and 1,545 male differentially expressed genes(M-DEGs)were found;Over time,the number of differential expression genes decreased and increased in turn in female and male.(2)Analysis of the active region of the LcDmrt1 promoter.Fourteen luciferase reporter vectors with seven different length fragment in LcDmrt1X and LcDmrt1Y promoter region were constructed-Pro-1X(2130bp),Pro-3X(1531bp),Pro-4X(1138bp),Pro-5X(767bp),Pro-6X(658bp),Pro-7X(449bp),Pro-8X(236bp),Pro-1Y(2120bp),Pro-3Y(1518bp),Pro-4Y(1022bp),Pro-5Y(761bp),Pro-6Y(653bp),Pro-7Y(450bp)and Pro-8Y(234bp).Through double Luciferase Report System transfection experiments,the basic regulatory regions of LcDmrt1X and LcDmrt1Y promoter were located at(-304,-91)and(-305,-89),and positive and negative regulatory elements in different regions were also found to affect promoter activity.(3)Studying on the self-expression regulation and key action sites of LcDmrt1.Through co-transfection experiment of LcDmrt1 expression vector with LcDmrt1X or LcDmrt1Y promoter vector,we found that LcDMRT1 could enhance LcDmrt1X promoter activity in low concentration and inhibit its activity in high concentration;LcDMRT1 can inhibit the activity of LcDmrt1Y promoter activity,regardless of the concentration of LcDMRT1.Four LcDmrt1 promoters vectors with different DMRT1 binding sites mutated were constructed.Using co-transfection with LcDmrt1 expression vector,we found that DMRT1 transcription binding site 2 in the promoter region of LcDmrt1X was the key site to affect DMRT1’s positive regulation.On the contrary,this site in the promoter region of LcDmrt1Y was the key site to affect DMRT1’s negative regulation.