Molecular Cloning And Expression Patterns Of Sex-rclatcd Genes In Paralichthys Olivaceus

The olive flounder, Paralichthys olivaceus, is one of the most important marineeconomic fish in China. The study on flounder sex control has been attractingresearchers’interests because the females grow faster and larger than the males. Inthis paper, wnt4, ar, Lhx1and rspo1were cloned from flounder using the RACEmethod. RT-PCR and real time PCR were used to detect the distribution of thesegenes in different tissues of male and female flounder, and also detect the expressionof wnt4and Lhx1in different gonads stages. Expression of wnt4in gonads of juvenilecompared with cyp19a1a and dmrt1was also analyzed by real-time PCR. Studyresults showed that all these genes were sex-related genes, and they may playimportant roles in flounder gonad.The full-length cDNA of wnt4contains1584bp, including129bp5’UTR,396bp3’UTR and a1059bp ORF encoding a protein with a signal peptide domain and a wntfamily domain. Homology comparison result showed that it could be close to wnt4a.Flounder wnt4was widely expressed in all male and female tissues, and had sexualdimorphism in gonads, the expression in ovary is1.5times than that in testis; we alsofound a descending expression of wnt4in gonads stages III and IV. Comparison ofwnt4and cyp19a1a, dmrt1expression during differentiation developmental stage ofgonads of gynogenetic and its pseudo-male juveniles showed that wnt4kept a slowlyincreasing at first, and there was a sudden increasing before the differentiation oftestis. It is implied that wnt4could play an important role during the flounder gonaddifferentiation period.The full-length cDNA of ar contains3215bp, including97bp5’UTR,1051bp3’UTR and a2067bp ORF. The binding sites of sex-related transcription factors suchas er, Pax4and Pax6on DBD domain were showed by TRANSFAC6.0analysis. Andan840bp CpG island acrossing5’UTR and ORF was predicted. The flounder ar geneencodes a protein with688amino acids, which is more close to arα by homologicalcomparison analysis. Flounder ar was widely expressed in all male and femaleflounder tissues, and had sexual dimorphism in gonads, the expression in testis is1.8 times than that in ovary.The flounder erα cDNA, erβ cDNA and upstream promoter region of erα wereanalyzed by TRANSFAC6.0. The binding sites of sex-related transcription factorssuch as Pax4, gata4, oct1and foxl2in erα DBD domain were expected, but no CpGislands in cDNA or promoter region were detected. In erβ DBD domain, binding sitesof sex-related transcription factors such as Pax4and oct1of were found, and4CpGislands were detected in its total cDNA area. Flounder erα was widely expressed in allmale and female tissues except muscle and kidney, and had sexual dimorphism ingonads, the expression in ovary is1.14times than that in testis.The ORF of Lhx1a and Lhx1b lengths are1224bp and1206bp, respectively.LHX1a and LHX1b proteins all have two LIM and one HOX high conserved domains.The RT-PCR different expression patterns of Lhx1a and Lhx1b genes in male andfemale tissues showed that Lhx1a was expressed in testis but not in ovary. Theexpression of Lhx1b in gonads was totally opposite to that of Lhx1a. It was higherexpressed in ovary and lower expressed in testis. Moreover, the expression patterns ofthese two genes at different gonad developmental stages showed that there werealmost no signals of these two genes at stage I，II and V in gonads. But, at stage IIIand IV, the expression signals could be detected. Lhx1a was lower expressed at stageIII and IV of testis tissues, and Lhx1b was higher expressed in ovary and lowerexpressed in testis.The ORF of rspo1was also firstly cloned from olive flounder, encoding a260amino acids protein with two FU domain and one TSP domain. RT-PCR resultsshowed a wide expression in male and female issues, and with a sexual dimorphism ingonads, the expression in ovary is stronger than testis.In summary, the flounder wnt4, ar, er, Lhx1and rspo1are all sex-related genes,they have sexually dimorphic expressions in gonads, and wnt4plays an important roleduring the flounder gonad differentiation and development periods, Lhx1alsoinvolved in the flounder gonad development.