| There are many reports on gene identification and functional analysis of gonadal development and differentiation in Paralichthys olivaceus, but such research are more concentrated in the expression of genes encoding and functions analysis of these genes, micro RNA(mi RNA) studies rarely reported. We has already established the flounder gonad mi RNA library, and identified 141 mature mi RNA by a second-generation sequencing(NGS) and technology and bioinformations analysis, selected a group mi RNA or specific expression differences in male and female gonads of flounder. On this basis, we selected a higher expression levels and significantly differentially expressed in fine ovary mi RNA-200 b, then we use real-time quantitative PCR(RT-q PCR) and in situ hybridization to analysis the pol-mi R-200 b expression in flounder gonads, and to predict the effect of target genes, and we cloned and identified dazl、wt1a genes which are related to reproductive. In addition, we use real-time quantitative PCR(RT-q PCR) analysis of temperature, exogenous sex hormones on pol-mi R-200b、dazl and wt1 a genes expression. The study contribute to researching mi RNA-target gene flounder gonadal differentiation and development.First, we extracted the flounder gonad total RNA, and tested mi RNA-200 b expression in the flounder gonad using RT-q PCR method, the results showed that: there are differences expression in ovary and testis, and the expression in the ovary was significantly higher than testis. The gonads of flounder were put into 4% paraformaldehyde before paraffin sections, followed by in situ hybridization, the positioning results showed: pol-mi R-200 b had positive signal with the blue-violet patchy distribution and more concentrated in the egg follicular oocyte membrane, however, there was a rare positive signals patchy distribution in a mature sperm cells edge, mainly in lobular cavity of testis. It suggest that pol-mi R-200 b may play an important role in the development of the gonads in flounder.DAZL(Deleted in AZoospermia-like) gene is an important regulator of a variety of species of spermatogenesis. To explore the role of dazl gene in gonad differentiation and development of Paralichthys olivaceus, the full-length c DNA of dazl gene was cloned using homology cloning and RACE technology, its sequence structure was analyzed by bioinformatics method, and its gene expression in different adult tissues and during early development of P. olivaceus using real-time PCR technology. The results showed that the full-length c DNA sequence of P. olivaceus dazl is 2031 bp, including 105 bp of 5’ untranslated region, 1275 bp of 3’ untranslated region and 651 bp of open reading frame encoding 217 amino acid residues. The dazl had a highly conserved RNA recognition motif, which shared 100% sequence similarities among fish species by amino acid sequence homology comparison. RT-q PCR results showed that the dazl gene is mainly expressed in gonad, and it has a higher expression level in testis than in ovary. During the early development, the dazl gene had higher transcriptions in the unfertilized egg, fertilized egg and blastocysts, and its expression began to reduce at gastrula stage, and remained a low level until to 3 days after hatching. These results suggest that the abundant maternal dazl m RNA may be involved in the formation of primitive germ cells in the embryonic development. This study provides a bias to further clarify the role of dazl gene in P. olivaceus reproductive development.WT1 gene was originated adults various tumor, including pancreatic, lung, colon, brain tumors, desmoid tumor and breast cancer, the gene is also associated with testis development. In this study, the full-length c DNA of wt1 a gene was cloned using homology cloning and RACE technology, its sequence structure was analyzed by bioinformatics method, and its gene expression in different adult tissues and during early development of P. olivaceus using real-time PCR technology. The results showed that the full-length c DNA sequence of P. olivaceus wt1 a is 2051 bp, including 416 bp of 5 ’untranslated region, 390 bp of the 3’-UTR and coding 415 amino acid residues 1245 bp open reading frame; The wt1 a had four highly conserved zinc finger motif, which shared 100% sequence similarities among fish species by amino acid sequence homology comparison; RT-q PCR results showed that the wt1 a gene is mainly expressed in gonad, and it has a higher expression level in testis than in ovary. During the early development, the wt1 a gene had lower transcriptions after fertilization, embryo nerve sudden rise followed by a slow decrease until hatching period, but slowly increased after incubation at 14 d after reaching the maximum incubation subsequently reduce and maintain stable expression levels. These results suggest that the abundant maternal wt1 a m RNA may be involved in the formation of primitive germ cells in the embryonic development. This study provides a bias to further clarify the role of wt1 a gene in P. olivaceus reproductive development.To investigate the expression of exogenous hormones and high temperature flounder gonads related mi R-200 b, dazl and Wt1 a, the study set a high temperature(28 ℃) treatment group, 17β- estradiol(17β-E2) treated group, 17α- methyl testosterone(17α-MT) treatment group and the control group, to analysis the flounder gonadal differentiation in the expression of mi R-200 b, dazl and wt1 a use RT-q PCR method. It was found that pol-mi R-200 b in 45 d, 55 d and 85 d juveniles, the expression of mi R-200 b is gradually increased, and the estrogen group than other groups; at 65 d the ovaries begin to differentiate, and the high temperature group mi R-200 b expression higher than other groups; but in the 105 d, its expression didn’t continue to rise. Flounder dazl gene throughout the gonad differentiation process remained generally rising level of expression, and the highest expression level is high temperature group; until the 65 d, the expression levels of high-temperature treatment group and androgen treatment group decreased slightly, while the expression level of estrogen treatment higher; wt1 a gene expression levels in the 35 d, 45 d, 55 d and 65 d flounder each treatment group was very low levels, tended to increase very slowly, and in the 85 d and 105 d, its expression increased rapidly. 55 d and 65 d after hatching time are flounder ovary and testis began to differentiate, and 85 d and 105 d are fine flounder ovary early development period, the above results show wt1 a gene may be involved in the early formation of the testes and ovaries. |
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