鸡FOXL2基因慢病毒过表达载体的构建和功能研究

发布时间：2018-03-08 01:18

本文选题：鸡　切入点：性腺分化　出处：《广西大学》2017年硕士论文　论文类型：学位论文

【摘要】：哺乳动物 FOXL2(forkhead transcription factor gene 2)在卵巢分化和卵巢功能维持上具有重要的作用。FOXL2是鸡卵巢分化的重要候选基因,但目前其功能还不清楚。为了探讨FOXL2在鸡性腺分化过程中的功能,本研究通过构建FOXL2慢病毒过表达载体并在DF1细胞系验证其功能;通过胚盘下腔注射鸡胚,探讨FOXL2对鸡胚性腺分化的影响;通过睾丸注射,探讨FOXL2对成年鸡性腺功能维持的影响。本研究得出如下结果:(1)成功克隆了广西麻鸡FOXL2基因CDS全长序列,共918bp,将测序结果与GenBank上鸡参考序列(NM_001012612.1)相比,同源性99.7%,存在三处碱基突变,分别是T226C,C501T和T690C。物种间同源性比较结果显示,广西麻鸡FOXL2基因CDS区序列与猪,鼠和人的同源性分别为80.5%,80.3%和80.1%。进化树分析显示,广西麻鸡与人的亲缘关系最远,与鼠的亲缘关系最近。(2)用BamHI和EcoRI同时双酶切pMD-FOXL2克隆质粒和pLV慢病毒空质粒,然后连接转化到Trans 5α感受态细胞中,经菌液PCR、双酶切和测序鉴定,成功获得了 pLV-FOXL2慢病毒过表达质粒。利用脂质体转染法,将pLV-FOXL2重组质粒转入鸡DF1细胞系,在倒置荧光显微镜下,观察到转染pLV-FOXL2重组质粒后,DF1能检测到绿色荧光蛋白的表达,说明构建的pLV-FOXL2慢病毒过表达质粒具有表达活性。QRT-PCR检测其FOXL2的过表达效率,结果表明FOXL2的过表达效率与对照差异极显著。(3)将pLV-FOXL2质粒和pLV空质粒与脂质体混合,制作质粒-脂质体复合物,通过胚盘下腔注射法将复合物注入孵化到第2天的鸡胚,孵化到16.5天,在体视显微镜下观察性腺的表型变化,收集性腺组织用作后续的实时荧光定量(qPCR),组织切片和免疫组化分析,采集肌肉组织分别用于遗传性别鉴定和阳性个体鉴定。结果显示,pLV-FOXL2组共注射260枚,存活41枚,存活率为15.8%,其中遗传性别为公的23只,遗传性别为母的18只,表型性别为公的21只,表型性别为母的18只,其中有2只的表型性别不典型,左侧性腺膨大变黄类似卵巢结构。pLV组共注射100枚,存活21枚,存活率为21.0%,遗传性别为公的9只,遗传性别为母的12只,表型性别与遗传性别一致。阳性个体鉴定结果示显,pLV-FOXL2组有10只可检测到GFP,阳性率为24.4%;pLV组有8只可检测到GFP,阳性率为38.1%。(4)鸡胚性腺HE染色的结果显示,pLV-FOXL2组和pLV组公鸡睾丸的组织结构相似,与正常母鸡卵巢结构不同。免疫组化的结果显示CYP19A1蛋白在pLV-FOXL2组左右侧睾丸中的表达量与pLV组左右侧睾丸和正常母鸡卵巢相当;FOXL2蛋白在pLV-FOXL2组左右侧睾丸中的表达量高于pLV组左右睾丸,与正常母鸡卵巢中的表达相似。性别相关基因的qPCR结果显示,在pLV-FOXL2组中,AMH和CYP19A1的表达量显著下调(P0.05),而SOX9的表达量显著上调(P0.05)。本试验说明,胚胎期FOXL2可能通过下调AMH抑制睾丸发育,使曲精细管壁变厚,结缔组织增生。(5)将pLV-FOXL2和pLV质粒-脂质体复合物,通过睾丸直接注射法从左侧睾丸注入13周龄公鸡睾丸中,20天后,观察公鸡鸡冠颜色,检测两侧睾丸的解剖学结构,组织学结构,免疫组化和性别相关基因的相对表达情况。结果显示,与pLV组相比,pLV-FOXL2组公鸡鸡冠的颜色明显变白;pLV-FOXL2组左右测睾丸曲精细管变小,曲精管上皮变厚,精子生成出现障碍;免疫组化的结果显示,与pLV组比,pLV-FOXL2组睾丸FOXL2和CYP19A1的表达量增加;性别相关基因的表达结果示显,在pLV-FOXL2组中,FOXL2和CYP19A1基因的表达量均显著高于pLV组(P0.05)。本试验说明,FOXL2可通过调控CYP19A1的表达来维持睾丸的功能。本研究的结果提示,FOXL2可通调节AMH和CYP19A1基因的表达调节鸡胚性腺的发育,与CYP19A1协同作用维持成年性腺的功能。
[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.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S831

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