本文關(guān)鍵詞： MC1R 羊駝 綿羊黑色素細(xì)胞 熒光定量PCR Western blot　出處：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：經(jīng)過多項研究和實驗得出,黑色素皮質(zhì)素受體-1 (Melanocortin-1 Receptor, MC1R)在控制哺乳動物毛色基因中是關(guān)鍵性的主要控制基因。本試驗將羊駝MC1R基因和慢病毒載體連接,構(gòu)建一個轉(zhuǎn)基因表達(dá)載體。將載體轉(zhuǎn)入綿羊黑色素細(xì)胞中,使其在綿羊黑色素細(xì)胞中特異性表達(dá),從基因和蛋白水平鑒定轉(zhuǎn)基因表達(dá)載體的活性和效果,為生產(chǎn)表達(dá)羊駝MC1R基因的轉(zhuǎn)基因綿羊奠定基礎(chǔ)。(1)根據(jù)NCBI上羊駝MC1R (GenBank:FJ517582.2)的基因序列,設(shè)計帶有酶切位點的特異性引物,擴(kuò)增到大小為1069bp的完整的羊駝MC1R CDS區(qū),該序列與其他物種MC1R的相似度達(dá)99%,可用于下一步連接載體。(2) MC1R基因經(jīng)限制性內(nèi)切酶Sal I和Xba Ⅰ酶切后,與同樣經(jīng)Sal I和Xba Ⅰ酶切后的慢病毒載體通過T4 DNA連接酶連接,測序鑒定正確。(3)重組質(zhì)粒轉(zhuǎn)染綿羊黑色素細(xì)胞,36h后熒光顯微鏡下觀察到綠色熒光,48h提取細(xì)胞RNA和總蛋白質(zhì)。(4)運用熒光定量PCR分析MC1R、MITF、TYR和YRP2在實驗組(轉(zhuǎn)染重組質(zhì)粒)、Negtive Control組(轉(zhuǎn)染慢病毒空載體質(zhì)粒)和空白細(xì)胞組(不進(jìn)行轉(zhuǎn)染)的mRNA表達(dá)量。結(jié)果顯示實驗組MC1R, MITF, TYR和YRP2基因的mRNA表達(dá)量分別為空白對照組的3.193倍,2.021倍,3.218倍,3.140倍,并呈差異顯著。(5)運用蛋白免疫印跡(Western blot)技術(shù)檢測MITF、TYR和YYRP2在實驗組,轉(zhuǎn)染Negtive Control組和空白細(xì)胞組的平均蛋白含量。結(jié)果為：轉(zhuǎn)染表達(dá)載體的實驗組的MITF、TYR、TYRP2蛋白表達(dá)量是KB組的1.852倍、3.537倍,2.045倍,且差異均顯著。綜上所述,綿羊黑色素細(xì)胞轉(zhuǎn)入羊駝MC1R轉(zhuǎn)基因表達(dá)載體后,調(diào)控黑色素細(xì)胞的活性和黑素生成的主效基因MC1R,MITF,TYR和TYRP2的基因和蛋白表達(dá)量均提高。羊駝MC1R轉(zhuǎn)基因表達(dá)載體構(gòu)建成功,效果良好。
[Abstract]:After many studies and experiments, it was found that Melanocortin-1 Receptor-1 (MC1R) is the key gene in the control of mammalian coat color. In this study, the MC1R gene of alpaca was linked with lentivirus vector. A transgenic expression vector was constructed. The vector was transferred into sheep melanocytes and specifically expressed in sheep melanocytes. The activity and effect of the transgenic expression vector were evaluated at the gene and protein levels. To lay the foundation for the production of transgenic sheep expressing the MC1R gene of alpaca. According to the gene sequence of MC1R GenBank: FJ517582.2) on NCBI, a specific primer with enzyme cutting site was designed, and the complete MC1R CDS region of alpaca was amplified. The sequence is similar to MC1R of other species and can be used to ligate the MC1R gene by restriction endonuclease Sal I and Xba 鈪，

本文編號：1550964