本文選題：FGF5　切入點：遼寧絨山羊　出處：《吉林農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：成纖維細胞生長因子5(Fibroblast growth factors 5,FGF5)是影響毛囊周期性生長的重要生長因子。本研究利用CRISPR/Cas9系統(tǒng)對遼寧絨山羊FGF5基因進行敲除,為深入研究FGF5的功能奠定基礎(chǔ)。根據(jù)Genebank中已發(fā)表山羊FGF5(KC236981.1)基因編碼區(qū)的序列信息設(shè)計引物,擴增出遼寧絨山羊FGF5基因的完整CDs區(qū)(Coding sequence)813bp,測序結(jié)果并未發(fā)現(xiàn)其剪接體FGF5s。FGF5基因中CDs區(qū)共編碼270個氨基酸,理論分子質(zhì)量為29.55kDa,理論等電位點為10.59,為親水性蛋白,有信號肽,屬于分泌蛋白,經(jīng)序列對比分析其與牛的同源性為97.2%。采用組織塊貼壁法分離遼寧絨山羊胎兒成纖維細胞,對其進行性別鑒定、生長曲線與核型分析,選出生長旺盛和核型正常的胎兒成纖維細胞用于后續(xù)研究。結(jié)果顯示,成功建立了遼寧絨山羊胎兒成纖維細胞的體外培養(yǎng)體系,細胞系S1為雌性,S2為雄性,其生長旺盛,狀態(tài)良好,核型正常,有30對染色體。利用生物學軟件,針對絨山羊FGF5基因第一外顯子,設(shè)計5個預(yù)測的sgRNA靶位點。構(gòu)建CRISPR/Cas9重組載體,分別為PX459-FGF5-l、PX459-FGF5-2、PX459-FGF5-3、PX459-FGF5-4和PX459-FGF5-5,經(jīng)測序表明載體連接成功。采用脂質(zhì)體3000介導(dǎo)5個PX459-FGF5載體分別轉(zhuǎn)染遼寧絨山羊胎兒成纖維細胞,經(jīng)過T7EI酶酶切檢測靶序列的有效性,選出敲除效率好載體轉(zhuǎn)染的細胞,單克隆細胞擴繁培養(yǎng),再提取基因組,脫靶檢測,PCR及克隆測序驗證。結(jié)果表明,有2個PX459-FGF5載體成功地進行了特異性切割和突變。隨機選取60個單克隆細胞,測序獲得23株遼寧絨山羊成纖維轉(zhuǎn)基因細胞系(包括缺失插入和替換),均在靶位點發(fā)生FGF5突變,總突變率為38.3%。其中有9株發(fā)生插入缺失的單克隆細胞系適于作為體細胞核移植的供體細胞,為高產(chǎn)絨量遼寧絨山羊新品種的培育奠定了基礎(chǔ)。
[Abstract]:Fibroblast growth factors 5 (FGF5) is an important growth factor affecting the periodic growth of hair follicles. In this study, Liaoning Cashmere Goat FGF5 gene was knockout by CRISPR/Cas9 system. In order to further study the function of FGF5, primers were designed according to the sequence information of goat FGF5 (KC236981.1) gene coding region published in Genebank. The complete CDs sequence sequence of Liaoning Cashmere goat FGF5 gene was amplified. The results of sequencing did not show that the CDs region of the splice FGF5s.FGF5 gene encodes 270 amino acids, the theoretical molecular weight is 29.55 kDa, and the theoretical equipotential point is 10.59, which is a hydrophilic protein with a signal peptide. It belongs to secretory protein, and its homology with cattle is 97.2.The fetal fibroblasts of Liaoning cashmere goat were separated by tissue block adhesion method, and their sex identification, growth curve and karyotype analysis were carried out. Fetal fibroblasts with strong growth and normal karyotype were selected for further study. The results showed that the in vitro culture system of Liaoning cashmere goat fetal fibroblasts was successfully established. The cell line S1 was female and S 2 was male and its growth was vigorous. In good condition, normal karyotype and 30 pairs of chromosomes, five predicted sgRNA target sites were designed for the first exon of FGF5 gene in cashmere goats by using biological software. CRISPR/Cas9 recombinant vector was constructed. They were PX459-FGF5-2, PX459-FGF5-3, PX459-FGF5-4 and PX459-FGF5-5, respectively. The ligation of PX459-FGF5-4 and PX459-FGF5-5 showed that the five PX459-FGF5 vectors were transfected into Liaoning cashmere goat fetal fibroblasts by liposome 3000, and the target sequences were detected by T7EI enzyme digestion. The transfected cells with good knockout efficiency vector were selected, and the monoclonal cells were propagated and cultured. Then the genome was extracted, and the PCR and clone sequencing were carried out. The results showed that, Two PX459-FGF5 vectors were successfully dissected and mutated. Twenty three Liaoning Cashmere Goat fibroblast transgenic cell lines (including deletion insertion and substitution) were sequenced from 60 monoclonal cells. All of them had FGF5 mutations at the target sites. The total mutation rate was 38.3. Among them, 9 clones with insertion deletion were suitable for donor cells of somatic nuclear transfer, which laid a foundation for the breeding of new varieties of Liaoning cashmere goat with high yield.
【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S827

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