Screening Of Gonadal Marker Genes Of Pristella Maxillaris And Cloning Male Marker Gene DMRT1

The transparent Pristella maxillaris whose visceral and skeletal can be observed for its transparent body color,is rather suitable for the study of fish gonadal sex determination development,but also for non-invasive in-vivo observation and elucidation of target gene function.Moreover,the feeding and reproduction of Pristella maxillaris is relatively simple.It is a promising way to developed it as a model animal of fish,which can be used to expand the study of the development of fish in the organs and bones,and also provide an efficient way for the elucidation of the development of adult tissues and organs of vertebrates and the pathogenic mechanism.In this study,Pristella maxillaris was choosen as the research object.The maker genes of gonad were screened by transcriptome sequencing technology,and one male maker named DMRT1 was cloned and verified.The following results were obtained in this study.1.8 samples from 4 tissues which including brain,muscle,testis and ovary of Pristella maxillaris were independently sequenced to construct sequencing library.The sequencing data were obtained after filtering.The lowest Q30 was 89.78% higher than 85%,indicating the high quality of sequencing and a reliable result.114 227 Unigenes were spliced from the transcriptome sequencing results,with an average length of 1 658 bp.2.The spliced Unigenes was compared with KEGG,GO,NR,NT,Swiss Prot,Pfam and KOG databases for annotation.A Venn map of Unigenes distribution was made according to the annotation results by NR database,finding that 1 688 Unigenes expressed only in the testis tissue of Pristella maxillaris and 281 Unigenes expressed only in ovarian tissue.The top 10 average FPKM Unigenes for testis and ovary were selected as candidate genes for gonadal marker genes.3.The full sequence of DMRT1 cDNA of Pristella maxillaris was cloned.It has a length of 1 160 bp,of which the length of 5’UTR is 185 bp,the length of 3’UTR is108 bp.The coding region is 867 bp corresponding to 289 aa-translation.Amino acid comparison results of DMRT1 in Pristella maxillaris showed a high homology of Pristella maxillari with other fishes and is not very similar to mammals and birds.However,they shared rather similarity DM domain.4.Tissue semi-quantitative analysis showed that DMRT1 gene was only expressed in the testis tissue of Pristella maxillaris.It indicated DMRT1 gene could be used as a marker gene for male gonad of Pristella maxillaris.This transcriptome sequencing provides a data basis for the molecular biology research of Pristella maxillaris.10 male gonadal marker genes and 10 female gonadal marker candidate genes have been screened out.A further study is needed to verify the candidated gonadal maker genes.Additionanlly,DMRT1 was screened out from the analysis of the transcriptome.The cDNA cloning and analysis results confirmed it was specific for the testis of Pristella maxillaris,which made a base for gonadal research of Pristella maxillaris.