Study On Lentiviral Vector Construction And Functional Of FOXL2 Gene In Chicken

FOXL2(forkhead transcription factor gene 2)plays an important role in ovarian differentiation and maintenance of ovarian function.FOXL2 is an important candidate gene during ovarian differentiation in chicken,but its function is not clear.In order to explore the role of FOXL2 in chicken gonadal differentiation,the current study constructed the lentivirus expression vector of FOXL2 and verified its function in chicken cell line,analyized the function of FOXL2 on gonadal differentiation in chick embryo and maintance of gonadal function in adult chicken by subgermial cavity injection and testis injection,respectively.The results of current study are as follows:(1)Successfully cloned the CDS sequence of FOXL2 gene in Guangxi Ma chicken,the full-length sequence is 918bp.Comparing with the chicken sequence of FOXL2 released in GenBank(NM 001012612.1),the homology is 99.7%.Three mutations were founded,T226C,C501T,and T690C,respectively.Homologous analysis among different species showed the homology were 80.5%,80.3%,and 80.1%in pig,rat,and human,respectively.The phylogenetic tree analysis showed that chicken had a far genetic distance compared with pig,a short distance comparied with human.(2)pMD-FOXL2 cloning plasmid and pLV lentiviral plasmid digesteed with BamHI and EcoRI double enzyme,ligated under ligase,and transfect into Trans 5a competent cells.After identification of bacterial PCR,double enzyme digestion,and sequencing,respectively.We successfully obtained pLV-FOXL2 lentivirus expression plasmid.LTX lipofection was used to transfect pLV-FOXL2 recombinant plasmid into chicken DF1 cells.The result showed that the expression of green fluorescent protein was tested in DF1 treated with pLV-FOXL2 plasmid under the inverted fluorescence microscope.The expression efficiency of QRT-PCR was detected by FOXL2,and the results showed that the overexpression of FOXL2 was very significant(P<0.01).(3)The pLV-FOXL2 and pLV empty plasmid-lipofection mixture were injected into day2 chicken embryo from subgerminal cavity.At day 16.5,phenotypes of the gonad were observed under microscope and the goand samples for analysis of histology and immunohistochemy,real-time fluorescence quantitative PCR(qPCR)and muscle tissue for genetic identification and positive identification were collected.The results showed that 260 fertilized eggs were treated and 41 chick embryos were survival in pLV-FOXL2,the survival rate was 15.8%,of which the genetic sex of male and female were 23 and 18,respectively,while the phenotypic sex of male and female were 21 and 18,respectively.Phenotypic sexes of two chick embryos were not typical,left gonadal swelling large yellow ovary like structure.In pLV,100 fertilized eggs were treated and 21 chick embryos were survival in pLV-FOXL2,the survival rate was 21%,of which the genetic sexes of male and female were 9 and 12,respectively.Phenotypic sex was consistent with genetic sex.The results of positive individuals with GFP showed that 10 chick embryos were detected the GFP in pLV-FOXL2 group.The positive rate was 24.4%.8 chick embryos were detected the GFP in pLV group.The positive rate was 38.1%.(4)The results of HE staining showed that testis in pLV-FOXL2 group and the pLV group were a similar stuctue,which was different with ovary of normal female chicken.The result of immunohistochemistry showed that the expression of CYP19A1 protein in the left and right testis of the pLV-FOXL2 Group was equal to that of the pLV group and the normal hen ovary.The expression levels of FOXL2 protein in left and right testis in pLV-FOXL2 group were similar with the ovary of normal chicken.The expression levels of sex-related genes showed that the expression levels of AMH and CYP19A1 were significantly reduced(P<0.05)in pLV-FOXL2 group compared with pLV group.Th expression levels of SO3X9 were significantly up-regulated(P<0.05).This study suggests that embryonic phase FOXL2 may inhibit testicular development by lowering the AMH,making the curved fine tube wall thicker and connective tissue hyperplasia.(5)The pLV-FOXL2 and pLV plasmid-liposome complex were injected directly from the left testicular at 13 weeks old rooster,after 20 days of injection,the color of comb,anatomical structure and histological structure of testis,immunohistochemy,expression of sex-related genes.The results show that the color of combs became pale in pLV-FOXL2 group compared with pLV group.In pLV-FOXL2 group,seminiferous tubules of testis became small,epithelial of seminiferous tubule became thicken,spermatogenesis was abnormal.Immunohistochemistry results showed that expression of FOXL2 and CYP19A1 were increased in pLV-FOXL2 group compared with pLV.The expression levels of sex-related genes showed that the expression levels of FOXL2 and CYP19A1 were significantly up-regulated(P<0.05)in pLV-FOXL2 group compared with pLV group.The current study suggest that FOXL2 affects the goandal differentiation in chicken embryo and maintances the gonadal function in adult by regulating the expression of CYP19A1.The results of this study suggest that FOXL2 can regulate the expression of AMH and CYP19A1 genes to regulate the development of chicken embryo gonad,and to maintain the function of adult gonad with CYP19A1 Synergy.