皮肤组织特异性表达绵羊Wnt10b基因转基因小鼠和转基因绵羊研究

发布时间：2018-11-04 08:24

【摘要】：Wnt10b基因作为"第一表皮信号"候选基因,对毛囊生长发育和周期性循环生长具有重要意义。研究发现,Wnt10b基因在小鼠毛囊形成和毛囊生长期初期有表达量显著上调的趋势,Wnt10b蛋白在体外培养的胚胎表皮中通过经典的Wnt通路,诱导未成熟的表皮细胞向毛干及内根鞘细胞分化。Wnt10b基因作为改善羊毛品质的重要候选基因,具有巨大研究价值。本研究主要进行以下方面工作:1)本研究首次延长绵羊KAP6.1启动子至1523bp,对绵羊KAP6.1启动子转录因子结合位点进行预测分析,并通过EMSA实验验证,表明本研究延长的KAP6.1启动子区域存在一个NF-kappa-B转录因子结合位点,有助于增强启动子转录活性。2)本研究首次克隆了绵羊完整的Wnt10b基因cDNA序列并首次发现一个可变剪接体Wnt10b-AS。利用荧光定量PCR检测Wnt10b基因和可变剪接体在绵羊心、肝、脾、肺、肾、皮肤、肌肉和脂肪组织中的表达。结果表明Wnt10b和Wnt10b-AS在检测组织中普遍表达。本研究构建Wnt10b-EGFP融合表达载体,利用激光共聚焦显微镜观察Wnt10b蛋白在细胞内的表达。结果表明,绵羊Wnt10b蛋白属于分泌型蛋白,在细胞质中合成后分泌到细胞外,通过与细胞膜上受体结合发挥生物学作用。本研究发现的可变剪接体Wnt10b-AS可能以mRNA形式存在,并通过与主转录本不同机制高效调节Wnt通路活性。3)本研究构建KAP6.1-Wnt10b真核表达载体,通过原核注射方法得到5只原代Wnt10b转基因小鼠(F0代)。通过对F0代小鼠基因表达水平进行分析,66号家系小鼠皮肤组织中Wnt10b、frizzled和β-catenin基因高表达,差异显著(P0.05)。利用皮肤组织冰冻切片分析毛囊形态和密度,结果表明66号家系F1代个体中,阳性个体毛囊由生长期进入静止期的时间相对阴性个体滞后;29和66号家系F1代个体中,同性别个体相比,阳性个体毛囊密度高于阴性个体,雌性个体高于雄性个体。4)本研究双酶切KAP6.1-Wnt10b表达载体,保留启动子和外源基因片段,通过原核注射方法制备转基因羊。对18只美利奴供体进行超数排卵,共得到胚胎177枚,其中可用胚胎166枚;共得到活羔21只,2只阳性羔羊。W002个体在羊毛长度、细度和毛囊密度上优于半同胞阴性个体;W003个体在羊毛长度、强度和伸长率上具有明显优势。Wnt10b和β-catenin基因可作为改良羊毛长度、细度的重要候选基因。
[Abstract]:As a candidate gene for the first epidermal signal, Wnt10b gene plays an important role in hair follicle growth and cyclic growth. It was found that the expression of Wnt10b gene was significantly up-regulated in mouse hair follicle formation and early hair follicle growth, and that Wnt10b protein was expressed through the classical Wnt pathway in the embryonic epidermis cultured in vitro. Immature epidermal cells were induced to differentiate into hair stem and inner root sheath cells. As an important candidate gene for improving wool quality, Wnt10b gene has great research value. The main work of this study was as follows: 1) the KAP6.1 promoter of sheep was extended to 1523 BP for the first time. The transcription factor binding sites of sheep KAP6.1 promoter were predicted and verified by EMSA experiment. It is suggested that there is a NF-kappa-B transcription factor binding site in the extended KAP6.1 promoter region in this study. In this study, the complete cDNA sequence of sheep Wnt10b gene was cloned for the first time and a variable splice Wnt10b-AS. was first found. The expression of Wnt10b gene and mutable splice in heart, liver, spleen, lung, kidney, skin, muscle and adipose tissue of sheep were detected by fluorescence quantitative PCR. The results showed that Wnt10b and Wnt10b-AS were widely expressed in tissues. In this study, Wnt10b-EGFP fusion expression vector was constructed and the expression of Wnt10b protein in cells was observed by confocal laser microscopy. The results showed that sheep Wnt10b protein belongs to secretory type. It is synthesized in the cytoplasm and secreted out of the cell, which plays a biological role by binding to the receptor on the cell membrane. The variable splicing Wnt10b-AS found in this study may exist in the form of mRNA and regulate the activity of Wnt pathway efficiently through different mechanisms compared with the principal transcripts. 3) the eukaryotic expression vector of KAP6.1-Wnt10b was constructed in this study. Five primary Wnt10b transgenic mice (F 0 generation) were obtained by prokaryotic injection. Through the analysis of gene expression level of F 0 generation mice, the expression of Wnt10b,frizzled and 尾-catenin genes in skin tissues of F 0 generation mice was higher than that of F 0 generation mice (P0.05). The morphology and density of hair follicles were analyzed by frozen sections of skin tissue. The results showed that the hair follicles of positive individuals in F _ 1 generation of family 66 lag behind those of negative individuals. The hair follicle density of positive individuals was higher than that of negative individuals, and that of female individuals was higher than that of male individuals. 4) in this study, KAP6.1-Wnt10b expression vectors were digested by double enzyme to retain promoter and foreign gene fragments. Transgenic sheep were prepared by prokaryotic injection. A total of 177 embryos were obtained from 18 Merino donors, including 166 embryos, 21 live lambs, 2 positive lambs. W002 individuals were superior to half sibling negative individuals in wool length, fineness and hair follicle density. W003 individuals have obvious advantages in wool length, strength and elongation. Wnt10b and 尾-catenin genes can be used as candidate genes for improved wool length and fineness.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S826

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