Preliminary Study On Function Of Dmrt1,a Candidate Gene For Sex Determination Of Large Yellow Croaker(Larimichthys Crocea)

The large yellow croaker,Lrimichthys crocea,is one of the most important marine economic fishes in china.There is a significant sexual dimorphism in growth,but the molecular mechanism of sex determination or sex development is still relatively lacking.Studying on the key gene of L.crocea sex determination mechanism may contribute to future studies of their breeding work.This research starts from the Dmrt1 genes in large yellow croaker testis specific expression,its full-length cDNA cloning and X and Y chromosomes Dmrt1 gene promoter sequences.The expression vector of this gene was constructed and injected into medaka fertilized egg to observe its expression and the influence on medaka gender development.The main results are summarized as follows:1.The full-length cDNA sequence of Dmrt1 gene(LcDmrt1)was obtained by RACE technology.It was 3037 bp including 918 bp ORF,132 bp 5’UTR and 1932 bp 3’UTR.The nucleotide sequence of the promoter region of the Dmrt1 gene was cloned from the X and Y chromosome fragment containing the Dmrt1 gene in the BAC library of the L.crocea.It was found that there were a lot of SNP and insertion deletion mutations between them.The results of bioinformatics software predict that there are several transcription factor binding sites in the starting area of LcDmrt1,including TBP,SMAD2,and the binding sites of Sox family members(Sox5,Sox10,Sox17)and SRY,and TBP binding site in-1375 bp.LcDmrt1~X contains a specific SOX5 binding site and a SRY binding site.The LcDmrt1~Y also have two DMRT1 binding sites at-1813 bp and at-2416 bp.In addition,the LcDmrt1~Y promoter region also contained 3 DMRT1binding sites,located at(-1820 bp,-1816 bp),(-1048 bp,-1040 bp)and(-298 bp,-287 bp)respectively.2.The LcDmrt1 overexpression vector was successfully constructed,and the PCS2-LcDmrt1overexpression vector was introduced into the fertilized eggs of meadaka by microinjection technology.The gene integration and expression and the effect on the sex development of the LcDmrt1 were observed.The results showed that after the injection of 48 h,the LcDmrt1 gene was expressed in a large number of embryos,and the integration rate of LcDmrt1 was 24.3%in the 40d larvae after hatching,and it was observed that the 1 tail genotypes of XX developed into a male,indicating that LcDmrt1 has indeuced medaka female gonadal differentitation development into the testis.3.The LcDmrt1 double luciferase reporter Pro-D2K-X(-1983,+139),Pro-D2K-Y(-1975,+133),Pro-D700-X(-615,+133)and Pro-D700-Y(-607,+133).containing different length promoter sequences were constructed,and were co-transfected into human embryonic kidney 293cells(HEK-293)to detects its expression activity.The results showed that the LcDmrt1 promoter on both X and Y chromosomes is active,and the promoter activity of this gene on the Y chromosome is higher than that on the X chromosome.The constructed Lc Dmrt1 overexpression vectors were co-transfected with LcDmrt1~X and LcDmrt1~Y promoter luciferase reporter vectors of different lengths,respectively,to observe whether LcDmrt1 has a self-feedback regulatory mechanism.The results showed that overexpression of LcDmrt1 reduced the promoter activity,indicating that the Lc DMRT1 has a negative feedback regulation,and the expression activity of the combined LcDmrt1~Y promoter was significantly higher than that of LcDmrt1~X,indicating that the degree of inhibition of LcDmrt1 may be combined with DMRT1 in its promoter region.The number of loci was related to the fact that the promoter activity of LcDmrt1~X containing three DMRT1 binding sites was significantly suppressed compared with LcDmrt1~Y at the two binding sites.However,this self-inhibitory effect of LcDmrt1~Y has a dose-effect,the degree of inhibition of the complete promoter with three DMRT1 binding sites Significantly higher than the promoter fragment with only one DMRT1 binding site.However,the negative feedback regulation of LcDmrt1~X is not related to the number of binding sites.After co-transfection,the decrease of Pro-D700-X expression activity is even slightly greater than that of Pro-D2K-X,suggesting that the binding site in the region of(-615 bp,0)may be the main locus for the negative feedback regulation of the promoter to accept DMRT1.In this study,the full-length cDNA sequence of Dmrt1 gene and the promoter sequences of X and Y chromosomes of L.crocea were obtained.A series of expression plasmids were successfully constructed and the function of Dmrt1 and the promoter of LcDmrt1 on X and Y chromosomes were preliminarily revealed.The expression characteristics and research results provide a good foundation and valuable research data for elucidating the molecular mechanism of sex determination and differentiation of large yellow croaker.