Cloning And Expression Analysis Of Dmrt1 Gene And The Role In TSD Of Gekko Japonicus

Gekko japonicus is a small nocturnal reptile, which belongs to Reptilia, Squamata , Gekkonidae, Gekko. G. japonicus as a temperature-dependent sex determination (TSD) species. This paper collected the gravid female to launch egg hatching experiment and exogenous hormone manipulation experiment, aimed to detect the offspring sex ratio under different incubation temperatures and verified the temperature dependent sex determination model of G. japonicus;Through gene cloning technology to clone and analysis Dmrtl gene;Through qPCR to detect the Dmrtl expression level in different tissues and the expression pattern of Dmrtl in AKG at different developing stages during TSP to forecast the potential ability of Dmrt1 gene in Temperature-dependent sex determination of G. japonicus, provide basic information for further studies on Dmrtl gene in temperature dependent sex determination.1. Effect of Letrozole manipulation on sex of G. JaponicusIncubation temperature significantly affected the offsprings' sex ratio of G. japonicus , and conformed G. japonicus belongs to the TSD species. But the gender is not completely decided by temperature, and exogenous hormone manipulation experiment showed that at three temperatures, female gender was significantly decreased by Letrozole,but also failed to completely reverse the gender. This article speculated that G. japonicus may have sex chromosomes, might also have two kinds of mode of sex, GSD and TSD. This result supports the hypothesis of a continuous spectrum of TSD-GSD.2. Cloning and sequence analysis of Dmrtl geneWe used 3 'and 5' RACE technology to cloned Dmrt1 gene from G.japonicus. The length of Dmrt1 was 1884 bp, including 3'/5' non encoding region and open reading frame (ORF). ORF encodes 285 amino acids with a molecular weight of 30.94 kDa and an isoelectric point is about 4.95. Open reading frame from eighty-fifth to 939th nucleotides.And the predicted Dmrt1 protein sequence was used to multiple analysis,found three conserved segment of Dmrtl gene (DM binding domain, a P/S-rich region, male-specific motif) are highly conserved in vertebrates.Phylogenetic analysis also showed that Dmrtl amino acid sequence in vertebrates is also highly conserved.3. Expression of Dmrtl gene in different tissues and its expression pattern in sex determination of G. japonicusThe experimental results showed that the Dmrt1 gene expression in testis of G. japonicus was significantly higher than that in ovary. At the same time, we also examined other male related genes in testis, Sox9, Sfl,AMH, Lhx9 and WT1 gene, results was significantly higher in testis than that in ovary. We detected the expression of Dmrtl gene in the testis during breeding season, the results showed that Dmrt1 gene expression reached significant level in April the recovery phase of testis, indicating that the Dmrtl gene may be involved in the Spermatogenesis of G.japonicus; We detected the expression of Dmrt1 in AKG though different developing stage of embryos during TSP. The results showed that under 28 degree, the level of gene expression of Dmrtl during the thirty-fifth stage was significantly higher than 32 degree, we hypothesized that Dmrtl gene may be involved in male sex determination process of G.japonicus.