| Paralichthys olivaceus is an important marine economic fish in our country.In the growth process of flounder,the female is larger and grows faster than the male,so it is very important to study on its sex differentiation and gonad development.foxl3(Forkhead box L3),as a paraphylline homologous gene of foxl2(Forkhead box L2),is considered to be two copies unique to fishes,and is highly expressed in the gonads,playing an important role in the development,embryonic development,metabolism and other processes of a variety of fishes.The expression and function of foxl3 in different fishes may not be completely consistent,so the effect of FoxL3 on the gonad differentiation and development of flounder remains to be studied.Therefore,in this study,the correctness of foxl3 gene was verified by PCR cloning and sequencing,and foxl3 gene was analyzed by various bioinformatics methods.Realtime PCR was used to detect the expression of foxl3 in different tissues of P.olivaceus,and the location of Foxl3 fusion protein in cells was analyzed by subcellular localization and the location of foxl3 mRNA in sperms of P.olivaceus was studied by chemical in situ hybridization.Then,the spermatoovarian cells of P.olivaceus were statically passed for several times by primary cell culture method,and si-foxl3 and foxl3 overexpressed plasmid were transfected into spermatoovary cells and ovary cells of P.olivaceus,respectively.The expression changes of foxl3,fbxo47 and sycp3 were detected by RTqPCR.Moreover,miRNAs(miR-200a、miR-200b、miR-200c、miR-141 and miR-429)with foxl3 targeting relationship were screened out by using online target gene prediction website of P.olivaceus gonadal miRNA library constructed in our laboratory,The dual luciferase reporter gene and green fluorescent reporter plasmid were used to verify the targeting relationship in 293 T cells,and the predicted binding sites were further verified after site-specific mutation.The main results of this study are:1.Identification and molecular characteristics of foxl3 gene in P.olivaceusfoxl3 cDNA sequence was obtained by PCR cloning and sequencing in flounder,including 750 bp CDS region and 289 bp 3 ’UTR region.A total of 429 amino acids were encoded,among which the content of proline was the highest.The protein mass was 28.70 kDa,and the theoretical isoelectric point,hydrophilic coefficient and theoretical isoelectric point were 8.77,-0.842 and 63.85.The Foxl3 secondary structure of flounder contains an FH domain.The tertiary structure consisted of 20.48% α helices,15.26%extension chains and 64.26% random curls,which were unevenly distributed in the protein polypeptide chain.Compared with Foxl3 amino acid sequences in various organisms,it was found that the Foxl3 was highly similar to other fish,among which the homology with Turbot was the highest,reaching 91.97%,and the homology with zebrafish was the lowest,only66.27%.Amino acid sequence comparison showed that Foxl3 had more significant differences among different species than Foxl2,indicating that foxl3 evolved faster than foxl2.The phylogenetic tree analysis revealed that the Foxl3 of P.olivaceus was first grouped with Turbot,and then with large yellow croaker,Nile tilapia,half-smooth tongue sole,guasima and zebrafish.The Foxl2 of Paralichthys maximichthys firstly formed a single branch with Turbot,and then formed a single branch with mellifish,Nile tilapia,eel,half-smooth tongue sole and zebrafish,which belong to different clades.2.Expression and function analysis of foxl3 gene in P.olivaceus in male and female gonadsReal-time quantitative PCR results showed that foxl3 was expressed in all tissues of P.olivaceus,but the highest expression level was found in ovary,which was significantly higher than that in testis,muscle,stomach,brain,liver and heart,showing obvious sexual dimorphism.The subcellular location of FOXL3 molecules determines nuclear and intracellular reactions.foxl3 was expressed in the spermatic ovary of flounder by situ hybridization.In the ovary,the positive signal was mainly expressed in the oocte of stageⅡ-Ⅳ,and the expression of FoxL3 was also detected in stromal cells.In the testis,FOXL3 was expressed in primary spermatogonium and sperm,and a small number of positive signals were also detected in somatic cells.These results indicate that foxl3 is widely expressed in the germ cells and somatic cells of the gonads of P.olivaceus,suggesting that foxl3 plays an important role in cell proliferation and meiosis of P.olivaceus.In order to further explore the relationship between foxl3 gene and genes related to gonad development and differentiation,real-time quantitative PCR was used to detect the expressions of foxl3,fbxo47 and sycp3 in the gonad of P.olivaceus.The results showed that foxl3 was highly expressed in the ovary of P.olivaceus,while the expressions of fbxo47 and sycp3 in the testis were higher than those in the ovary.After 36 h of siRNA interference,the expression of foxl3 gene was significantly decreased,and the expressions of meiosis related genes fbxo47 and sycp3 in spermatovaries were significantly inhibited.After overexpression of foxl3,the expressions of fbxo47 and sycp3 in spermatic ovary were significantly increased,indicating that foxl3 gene may indirectly or directly regulate the expressions of fbxo47 and sycp3 genes,and thus affect the gonad differentiation and development of P.olivaceus.3.Prediction and identification of the relationship between miR-200 family and foxl3In order to confirm the interaction between foxl3 and the miR-200 family(miR-200 a,miR-200 b,miR-141 and miR-429),dual luciferase reporter vector and green fluorescent reporter plasmid were constructed.Double luciferase reporter gene test results showed: Compared with the control group,the fluorescence activity of foxl3 wild-type recombinant plasmid and miR-200 a mimics co-transfected group was significantly decreased,,while that of miR-200 b mimics,miR-141 mimics and miR-429 mimics groups had no significant change compared with the control group.After site-specific mutation,the fluorescence activity of foxl3 wild-type recombinant plasmid and miR-200 a mimics co-transfected group did not change significantly.Green fluorescent protein experiment also obtained the same result,proving that foxl3 and miR-200 a have a targeting relationship,and miR-200 a can negatively regulate foxl3,that is,foxl3 is the target gene of miR-200 a.In conclusion,miR-200 a may regulate the expression of foxl3 by targeting,and then regulate the expression of fbxo47 and sycp3,so as to regulate the function of gonads of P.olivaceus. |
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