本文選題：雞　切入點：性腺分化　出處：《廣西大學》2017年碩士論文　論文類型：學位論文

【摘要】：哺乳動物 FOXL2(forkhead transcription factor gene 2)在卵巢分化和卵巢功能維持上具有重要的作用。FOXL2是雞卵巢分化的重要候選基因,但目前其功能還不清楚。為了探討FOXL2在雞性腺分化過程中的功能,本研究通過構(gòu)建FOXL2慢病毒過表達載體并在DF1細胞系驗證其功能;通過胚盤下腔注射雞胚,探討FOXL2對雞胚性腺分化的影響;通過睪丸注射,探討FOXL2對成年雞性腺功能維持的影響。本研究得出如下結(jié)果:(1)成功克隆了廣西麻雞FOXL2基因CDS全長序列,共918bp,將測序結(jié)果與GenBank上雞參考序列(NM_001012612.1)相比,同源性99.7%,存在三處堿基突變,分別是T226C,C501T和T690C。物種間同源性比較結(jié)果顯示,廣西麻雞FOXL2基因CDS區(qū)序列與豬,鼠和人的同源性分別為80.5%,80.3%和80.1%。進化樹分析顯示,廣西麻雞與人的親緣關(guān)系最遠,與鼠的親緣關(guān)系最近。(2)用BamHI和EcoRI同時雙酶切pMD-FOXL2克隆質(zhì)粒和pLV慢病毒空質(zhì)粒,然后連接轉(zhuǎn)化到Trans 5α感受態(tài)細胞中,經(jīng)菌液PCR、雙酶切和測序鑒定,成功獲得了 pLV-FOXL2慢病毒過表達質(zhì)粒。利用脂質(zhì)體轉(zhuǎn)染法,將pLV-FOXL2重組質(zhì)粒轉(zhuǎn)入雞DF1細胞系,在倒置熒光顯微鏡下,觀察到轉(zhuǎn)染pLV-FOXL2重組質(zhì)粒后,DF1能檢測到綠色熒光蛋白的表達,說明構(gòu)建的pLV-FOXL2慢病毒過表達質(zhì)粒具有表達活性。QRT-PCR檢測其FOXL2的過表達效率,結(jié)果表明FOXL2的過表達效率與對照差異極顯著。(3)將pLV-FOXL2質(zhì)粒和pLV空質(zhì)粒與脂質(zhì)體混合,制作質(zhì)粒-脂質(zhì)體復合物,通過胚盤下腔注射法將復合物注入孵化到第2天的雞胚,孵化到16.5天,在體視顯微鏡下觀察性腺的表型變化,收集性腺組織用作后續(xù)的實時熒光定量(qPCR),組織切片和免疫組化分析,采集肌肉組織分別用于遺傳性別鑒定和陽性個體鑒定。結(jié)果顯示,pLV-FOXL2組共注射260枚,存活41枚,存活率為15.8%,其中遺傳性別為公的23只,遺傳性別為母的18只,表型性別為公的21只,表型性別為母的18只,其中有2只的表型性別不典型,左側(cè)性腺膨大變黃類似卵巢結(jié)構(gòu)。pLV組共注射100枚,存活21枚,存活率為21.0%,遺傳性別為公的9只,遺傳性別為母的12只,表型性別與遺傳性別一致。陽性個體鑒定結(jié)果示顯,pLV-FOXL2組有10只可檢測到GFP,陽性率為24.4%;pLV組有8只可檢測到GFP,陽性率為38.1%。(4)雞胚性腺HE染色的結(jié)果顯示,pLV-FOXL2組和pLV組公雞睪丸的組織結(jié)構(gòu)相似,與正常母雞卵巢結(jié)構(gòu)不同。免疫組化的結(jié)果顯示CYP19A1蛋白在pLV-FOXL2組左右側(cè)睪丸中的表達量與pLV組左右側(cè)睪丸和正常母雞卵巢相當;FOXL2蛋白在pLV-FOXL2組左右側(cè)睪丸中的表達量高于pLV組左右睪丸,與正常母雞卵巢中的表達相似。性別相關(guān)基因的qPCR結(jié)果顯示,在pLV-FOXL2組中,AMH和CYP19A1的表達量顯著下調(diào)(P0.05),而SOX9的表達量顯著上調(diào)(P0.05)。本試驗說明,胚胎期FOXL2可能通過下調(diào)AMH抑制睪丸發(fā)育,使曲精細管壁變厚,結(jié)締組織增生。(5)將pLV-FOXL2和pLV質(zhì)粒-脂質(zhì)體復合物,通過睪丸直接注射法從左側(cè)睪丸注入13周齡公雞睪丸中,20天后,觀察公雞雞冠顏色,檢測兩側(cè)睪丸的解剖學結(jié)構(gòu),組織學結(jié)構(gòu),免疫組化和性別相關(guān)基因的相對表達情況。結(jié)果顯示,與pLV組相比,pLV-FOXL2組公雞雞冠的顏色明顯變白;pLV-FOXL2組左右測睪丸曲精細管變小,曲精管上皮變厚,精子生成出現(xiàn)障礙;免疫組化的結(jié)果顯示,與pLV組比,pLV-FOXL2組睪丸FOXL2和CYP19A1的表達量增加;性別相關(guān)基因的表達結(jié)果示顯,在pLV-FOXL2組中,FOXL2和CYP19A1基因的表達量均顯著高于pLV組(P0.05)。本試驗說明,FOXL2可通過調(diào)控CYP19A1的表達來維持睪丸的功能。本研究的結(jié)果提示,FOXL2可通調(diào)節(jié)AMH和CYP19A1基因的表達調(diào)節(jié)雞胚性腺的發(fā)育,與CYP19A1協(xié)同作用維持成年性腺的功能。
[Abstract]:Mammalian FOXL2 (forkhead transcription factor gene 2) in the differentiation of ovarian and ovarian function in maintaining an important role for.FOXL2 is an important candidate gene of chicken ovarian differentiation, but its function is not clear. In order to explore the role of FOXL2 in chicken gonadal differentiation in the process of function, this paper constructed the lentivirus expression vector of FOXL2 and its verification the function in the DF1 cell line; the subgerminal cavity injection of chicken embryo, effects of FOXL2 on gonadal differentiation in the chick embryo; by testicular injection, the effect of FOXL2 on adult chicken gonadal function maintenance. This study draws the following results: (1) successfully cloned the full-length sequence of FOXL2 gene of Guangxi chicken, CDS 918bp, and the sequencing results GenBank chicken (NM_001012612.1) compared with the reference sequence, 99.7% homology, three mutations were T226C, C501T and T690C. species homology comparison results show, Guangxi Ma And the pig CDS gene sequence of chicken FOXL2, mouse and human homology were 80.5%, 80.3% and 80.1%. phylogenetic analysis showed that the genetic relationship between Guangxi chicken and the most far genetic relationship with rats recently. (2) and double enzyme digestion of plasmid pMD-FOXL2 and pLV lentivirus empty plasmid with BamHI and EcoRI then, the connection is transformed into Trans 5 alpha competent cells, the bacteria PCR, double enzyme digestion and sequencing, successfully obtained pLV-FOXL2 lentiviral expression plasmid by liposome transfection, pLV-FOXL2 recombinant plasmid was transfected into chicken DF1 cells under an inverted fluorescence microscope to observe transfection of pLV-FOXL2 recombinant plasmid. DF1, can detect the expression of green fluorescent protein, build pLV-FOXL2 lentiviral expression plasmid with the expression efficiency of.QRT-PCR activity detection of FOXL2. The results showed that overexpression of FOXL2 efficiency and extremely significant difference (3 pLV-). FOXL2 plasmid and empty plasmid pLV mixed with liposome, making plasmid liposome complex, the subgerminal cavity was injected into the complex into the incubation to second day chick embryos, hatching to 16.5 days, to observe the phenotypic changes in the gonad under stereomicroscope, collected for gonadal tissue real-time follow-up (qPCR), organization biopsy and immunohistochemical analysis were used for genetic sex identification and individual identification of positive acquisition muscle tissue. The results showed that pLV-FOXL2 group were injected 260 pieces, 41 survived, the survival rate was 15.8%, of which the genetic sex is 23 male, 18 female sex genetic and phenotypic sex for 21 male. 18 female sex phenotype, including 2 atypical phenotypic sex, left gonadal swellings yellow similar ovarian structure.PLV group were injected 100 pieces, 21 survived, the survival rate was 21%, the genetic sex is 9 male, 12 female sex genetic, Phenotypic sex is consistent with the genetic sex. Positive individual identification results show that the pLV-FOXL2 group of 10 GFP can be detected, the positive rate was 24.4%; pLV group of 8 GFP can be detected, the positive rate was 38.1%. (4) chicken gonadal HE staining indicated that the tissue structure of pLV-FOXL2 group and pLV group of cock testis similar, different from the normal hen ovary structure. Immunohistochemical results showed that CYP19A1 protein in the pLV-FOXL2 group about testicular expression in pLV group with the left and right side of testis and normal hens ovarian FOXL2 expression; in group pLV-FOXL2, left and right side in the testes in high pLV group about similar expression in normal testis, hen ovary the sex related gene qPCR results showed that in the pLV-FOXL2 group, the expression of AMH and CYP19A1 significantly decreased (P0.05), and the expression of SOX9 was significantly increased (P0.05). The experiment shows that the embryonic FOXL2 can downregulate AMH suppression For testicular development, make seminiferous tubule wall thickening, hyperplasia of connective tissue. (5) the pLV-FOXL2 and pLV plasmid liposome complex, injected into the 13 week old Rooster testes, after 20 days from the left testis testis by direct injection method, observe the cock comb color testing on both sides of the anatomy structure of testicular tissue. The chemical structure, relative expression of immunohistochemistry and sex related genes. The results showed that, compared with the pLV group, pLV-FOXL2 group of rooster combs the color became white; the pLV-FOXL2 group measured around seminiferous tubules became small, seminiferous duct epithelial thickening, spermatogenesis failure; immunohistochemistry results showed that, with the the pLV group, pLV-FOXL2 group expression levels of FOXL2 and CYP19A1 increased; the expression of sex related gene results show that in the pLV-FOXL2 group, the expression of FOXL2 and CYP19A1 genes were significantly higher than pLV group (P0.05). The experiment that FOXL2 can be regulated by CYP19A1 The results indicate that FOXL2 can regulate the expression of AMH and CYP19A1 genes, regulate the development of chick embryo gonads, and maintain the function of adult gonads with CYP19A1.

【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S831

【參考文獻】

相關(guān)期刊論文 前7條

1 楊秀榮;蔣和生;楊寧;;鳥類性別決定與性別分化機制[J];遺傳;2012年04期

2 何川;孟和;;雞性別決定與分化分子機制研究進展[J];農(nóng)業(yè)生物技術(shù)學報;2011年06期

3 ;Molecular cloning,characterization and expression analysis of Sox9a and Foxl2 genes in half-smooth tongue sole(Cynoglossussemilaevis)[J];Acta Oceanologica Sinica;2011年01期

4 淮亞紅;許尚忠;;牛Dmrt7基因的cDNA克隆及遺傳變異[J];安徽農(nóng)業(yè)科學;2009年30期

5 肖調(diào)義;吳寶林;葛熹凱;蘇建明;陳開健;許寶紅;崔樹良;章懷云;;中國大鯢Dmrt基因DM結(jié)構(gòu)域的克隆及序列分析[J];水生生物學報;2009年01期

6 ;Evolutionary conservation of Dmrt gene family in amphibi-ans, reptiles and birds[J];Chinese Science Bulletin;2001年23期

7 內(nèi)藤充,江益民;雞的胚胎控制及其在育種中的應(yīng)用[J];洛陽農(nóng)專學報;1995年03期

，

本文編號：1581785