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Spatio-temporal Expression And Functional Analysis Of Sex-biased-related MiRNA-26a,26b In Paralichthys Olivaceus

Posted on:2016-05-09Degree:MasterType:Thesis
Country:ChinaCandidate:C YinFull Text:PDF
GTID:2283330479487426Subject:Biology
Abstract/Summary:PDF Full Text Request
Due to the high protein content, fat individual, full of delicious taste and easy to be digested, Paralichthys olivaceus has become a kind of precious temperate species. It has a high nutritional and economic value and has been classified as an important marine fish aquaculture species. In the process of atificial breeding, the female fish matures sexually around 2 years, the first year has reached sexual maturity in male fish. But in view of the male testis development consumed more energy than mature females, and after sexual maturity the male will breed nearly 5 months, the intake of nutrition are mostly used for reproduction, so the artificial cultivation process can be easily seen the Paralichthys olivaceus female were larger, the increasing aquaculture production through sex control has become an important research field. Research of sex determination and related molecular mechanism has been paid more attention.Recently, it has been a lot of gonadal development and differentiation related gene expression function analysis of the genes have been reports in Paralichthys olivaceus. But more focused on the gene regulation of post transcription level, especially like micro RNA(mi RNA) has not been studie. In our previous study, mi RNA libraries derived from adult gonads of Paralichthys olivaceus were generated by using NGS method. Bioinformatics analysis was fulfild to distinguish several mature mi RNA sequences expressed differentially in the gonads from two classes in the sequencing data.On this basis, this paper selected 2 high expression and the difference between the expression of mi RNA in the ovary(pol-mi R-26 a, 26b) by using RT-q PCR method and in situ hybridization for further experimental verification. The results show that pol-mi R-26 a, 26 b showed the highest relative expression in the testis of adult flounder, and there are higher level in the brain tissues, but the expression of the two mi RNAs in the ovary showed relatively lower in Paralichthys olivaceus. The situ hybridization experiments showed that the localization and expression of pol-mi R-26 a, 26 b, positive signal was showed blue purple flake distribution and more concentrated in the lobular cavity in the mature sperm cells. In the adult ovary, the positive signals of pol-mi R-26 a, 26 b gene were showed flake sparse distribution. And the expression were mainly concentrated in the V follicular membrane of oocytes and the cell edges. Through the construction the spatial and temporal expression of pol-mi R-26 a, 26 b in ovary of spectrum, the success of this experiment verified that the sequence of about 2 mi RNA is highly expressed in the gonad in theoretical results in lower level of expression.Detection of mi RNA targets has become an pivotal part of the research of mi RNA function. This experiment using Target Scan, Pic Tar and mi RBase target genes prediction software,the result shows that pol-mi R-26 a, 26 b has more than one target m RNA. Among these, we picked out the fish sex determination and sex differentiation gene, and the gene empty spiracles homeobox 2(emx2) was proposed to be a candidate target by bioinformatics prediction. In this study, the identification and characterization of P. olivaceus emx2 gene has been reported. A 1665 bp c DNA of P. olivaceus emx2 was identified, including the CDS of 741 bp, the 3’-UTR of 912 bp and the 5’-UTR of 12 bp(Gen Bank accession number: KP260631). The predicted protein had 246 bp amino acids. It has a molecular mass(MM) of 27.9 k Da and an isoelectric point(PI) of 9.83. Its deduced amino acid sequence is 71-98% homologous to the Emx2 of other species from teleosts to mammals. The result showed the emx2 protein is highly conserved and especially its homeodomain region is 100% identical from teleosts to mammals. A phylogenetic tree of P. olivaceus and other vertebrates Emx2 was constructed by Neighbor-Joining method. P. olivaceus Emx2 is located in the same group with that of C. semilaevis. Real-time PCR results showed that the emx2 is not only highly expressed in embryonic neurula stage and adult brain but also has abundant expression in adult gonad, moreover, it exhibits higher expression in ovary than testis. Compared with the expression patterns of emx2 and pol-mi R-26 a and pol-mi R-26 b in the P. olivaceus testis and ovary, it also supported that the emx2 may be targeted regulation by pol-mi R-26 a and pol-mi R-26 b in the P. olivaceus gonad development.The Paralichthys olivaceus emx2 3’UTR was successfully cloned, the achieved mutation of the sequence of the target sites GACTTGA was found to AGTCCAG. The wild-type and mutant luciferase reporter plasmids used in mi RNA target detection were constructed successfully. To determine the relationship between emx2 and mi RNAs, a luciferase reporter assay was performed and verified that the emx2 is a common target gene of pol-mi R-26 a and pol-mi R-26 b. It was also supported by the inverse expression patterns of the emx2, pol-mi R-26 a and pol-mi R-26 b in bisexual gonad. After cotransfecting pol-mi R-26 a, 26 b and psi CHECK-emx2-3’UTR, Renilla luciferase(RL) / firefly luciferase(FL) activity decreased significantly compared with the blank control group and negative control, which indicated "seed sequence" of pol-mi R-26 a, 26 b combined with psi CHECK-emx2-3’ UTR to cut with degradation of target gene. But the mutant plasmid psi CHECK-mutated emx2-3’UTR and pol-mi R-26 a,26b cotransfected Renilla luciferase,(RL) /(FL) activity had no significant difference with the blank, NC and inhibitor. Due to the mutations, pol-mi R-26 a, 26 b and emx2 binding ability of the lack of 3 ’UTR to play on the degradation of target gene cutting. Therefore, dual luciferase reporter assay indicated that emx2 gene is a target gene of pol-mi R-26 a, 26 b, which provides molecular evidence for pol-mi R-26 a, 26 b in regulation of gonadal development.
Keywords/Search Tags:Paralichthys olivaceus, gonad development, pol-miR-26a, polmiR-26b, emx2
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