The Transcriptome Analysis Of The Giant Salamander Gonadal And The Screening Of Sex Determination And Differentiation Genes

Chinese giant salamander(Andrias davidianus)is our country unique Urodele Amphibia,belonging to the class Ⅱ protected wild animal.With Chinese giant salamander breeding technology breakthrough,Chinese giant salamander breeding industry as a new special aquaculture is booming.But farmers raised young Chinese giant salamander with well water,which led to a serious imbalance in the proportion of sex differentiation,and most of the individual were male Chinese giant salamander.Thus,our laboratory was always using exogenous estradiol to treat with young Chinese giant salamanders in the sensitive period of gonadal differentiation for four years since 2011.As a result,all treated young Chinese giant salamanders differentiated into famale Chinese giant salamander.In conclusion,the Chinese giant salamander in the sensitive period of gonadal differentiation is very sensitive to the external environment change.However,a series of major theoretical issues,such as the mechanism of different sex differentiation caused by the changes of the external environment and the molecular mechanism of sex determination,still remain to be solved at present.In view of the transcriptome study’ great potential in the aspect of sex determination and sex gland differentiation,Illumina Hiseq2000 technology was used to sequence undifferentiated gonad(UD),famale gonad(F)and male gonad(M)from Chinese giant salamander,to select with the differentially expressed genes related to sex determination and differentiation,to obtain significant enrichment of metabolic pathways in the gonads.We hope our research could accumulate basic experiment data for building gene regulatory network related to sex determination and differentiation and discussing molecular mechanism of sex determination and gonadal differentiation,also to provid theoretical basis for the sustainable development of the Chinese giant salamander breeding industry and continuation of this ancient species.The results and conclusion included the following aspects:1.Sequenced on the platform of Illumina Hiseq 2000,24,623,084,880 nt bases were generated totally.In the results of mixed assembly,146,292 unigenes were obtained,total length of unigenes was 146,321,850 nt,By blast comparison,unigenes were annotated with the databases of NR,NT,Swiss-Prot,KEG,COG and GO.Then counted the number of Unigenes annotated with each database.We respectively obtained 57,862\36,773\45,054\42,365\16,960\29,268 unigenes.The total annotation unigenes were 62,102,accounted for 42.5%of total unigene 146,292.2.By Unigene Expression Difference analysis,from Chinese giant salamander UD-vs-M,22,544 significiant differrentially expressed genes(DEGs)were selected which involved 7,907 up-regulated genes and 14,637 down-regulated genes in M.From UD-vs-F,34,800 significiant DEGs were selected which involved 16,751 up-regulated genes and 18,049 down-regulated gene’s in F.From M-vs-F,29,530 significiant DEGs were selected which involved 17,240 up-regulated genes and 12,290 down-regulated genes in F.3.By GO function and Pathway enrichment,we preliminary understand DEGs main function,main biochemical pathways and signal transduction pathways.We screened with some pathways related to gonadal differentiation,such as Progesterone-mediated oocyte maturation,Steroid hormone biosynthesis.Further we preliminary analysised suspected Hsdl7b3 and Cyp19a genes in steroid hormone biosynthesis pathway.4.From GO function enrichment of DEGs,the DEGs related to sex determination were screened,mainly including Dmrt1,Fgf9,Wnt-4,Foxl2 etc.,and gene function was described.Among them,the common genes associated with male sex determination were Dmrtl,Fgf9,and gene related to female sex determination was Wnt-4,analysised their differences expression among these gonad samples further.In addition,we also screened with other possible genes related to sex determination,such as Dmrt2,Tcf21,Foxl2 etc..5.From GO function enrichment of DEGs,the DEGs related to sex differentiation were screened,mainly including Dmrtl,Dmrt2,Fgf8,Wnt-4,Fst,Dmcl etc.,and gene function was described.Among them,the common genes associated with male sex differentiation was Dmrt1,and genes related to female sex differentiation were Wnt-4 and Fsh-R,analysised Fsh-R differences expression among these gonad samples further.In addition,we also screened with other possible genes related to sex differentiation,such as Dmrt2,Fgf8,Fst,Dmc1 etc..