【摘要】：Wnt10b基因作為"第一表皮信號"候選基因,對毛囊生長發(fā)育和周期性循環(huán)生長具有重要意義。研究發(fā)現(xiàn),Wnt10b基因在小鼠毛囊形成和毛囊生長期初期有表達量顯著上調(diào)的趨勢,Wnt10b蛋白在體外培養(yǎng)的胚胎表皮中通過經(jīng)典的Wnt通路,誘導未成熟的表皮細胞向毛干及內(nèi)根鞘細胞分化。Wnt10b基因作為改善羊毛品質(zhì)的重要候選基因,具有巨大研究價值。本研究主要進行以下方面工作:1)本研究首次延長綿羊KAP6.1啟動子至1523bp,對綿羊KAP6.1啟動子轉(zhuǎn)錄因子結(jié)合位點進行預測分析,并通過EMSA實驗驗證,表明本研究延長的KAP6.1啟動子區(qū)域存在一個NF-kappa-B轉(zhuǎn)錄因子結(jié)合位點,有助于增強啟動子轉(zhuǎn)錄活性。2)本研究首次克隆了綿羊完整的Wnt10b基因cDNA序列并首次發(fā)現(xiàn)一個可變剪接體Wnt10b-AS。利用熒光定量PCR檢測Wnt10b基因和可變剪接體在綿羊心、肝、脾、肺、腎、皮膚、肌肉和脂肪組織中的表達。結(jié)果表明Wnt10b和Wnt10b-AS在檢測組織中普遍表達。本研究構(gòu)建Wnt10b-EGFP融合表達載體,利用激光共聚焦顯微鏡觀察Wnt10b蛋白在細胞內(nèi)的表達。結(jié)果表明,綿羊Wnt10b蛋白屬于分泌型蛋白,在細胞質(zhì)中合成后分泌到細胞外,通過與細胞膜上受體結(jié)合發(fā)揮生物學作用。本研究發(fā)現(xiàn)的可變剪接體Wnt10b-AS可能以mRNA形式存在,并通過與主轉(zhuǎn)錄本不同機制高效調(diào)節(jié)Wnt通路活性。3)本研究構(gòu)建KAP6.1-Wnt10b真核表達載體,通過原核注射方法得到5只原代Wnt10b轉(zhuǎn)基因小鼠(F0代)。通過對F0代小鼠基因表達水平進行分析,66號家系小鼠皮膚組織中Wnt10b、frizzled和β-catenin基因高表達,差異顯著(P0.05)。利用皮膚組織冰凍切片分析毛囊形態(tài)和密度,結(jié)果表明66號家系F1代個體中,陽性個體毛囊由生長期進入靜止期的時間相對陰性個體滯后;29和66號家系F1代個體中,同性別個體相比,陽性個體毛囊密度高于陰性個體,雌性個體高于雄性個體。4)本研究雙酶切KAP6.1-Wnt10b表達載體,保留啟動子和外源基因片段,通過原核注射方法制備轉(zhuǎn)基因羊。對18只美利奴供體進行超數(shù)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只陽性羔羊。W002個體在羊毛長度、細度和毛囊密度上優(yōu)于半同胞陰性個體;W003個體在羊毛長度、強度和伸長率上具有明顯優(yōu)勢。Wnt10b和β-catenin基因可作為改良羊毛長度、細度的重要候選基因。
[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.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S826

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