本文選題：前黑素小體蛋白基因(PMEL)　切入點(diǎn)：啟動子　出處：《農(nóng)業(yè)生物技術(shù)學(xué)報(bào)》2017年06期

【摘要】：貂皮顏色是水貂皮重要的質(zhì)量性狀,也是影響其價(jià)格的重要因素,前黑素小體蛋白基因(premelanosome protein gene,PMEL)作為影響動物被毛顏色變異的重要候選基因,近年來得到廣泛重視。本研究旨在篩選調(diào)控水貂(Mustela vison)毛色基因PMEL啟動子活性區(qū)域及轉(zhuǎn)錄因子結(jié)合位點(diǎn),為探究該基因的表達(dá)調(diào)控機(jī)制提供理論依據(jù),并為彩色水貂的育種和改良提供思路。以與水貂同源性較高的雪貂(M.putorius furo)PMEL基因序列(GenBank:NW_004569320.1)為參考進(jìn)行引物設(shè)計(jì),以黑色水貂毛皮基因組DNA為模板擴(kuò)增PMEL基因啟動子片段并克隆到pMD19載體,通過PCR和測序鑒定為陽性克隆,利用限制性內(nèi)切酶KpnⅠ和HindⅢ對陽性質(zhì)粒和pGL3-Basic載體同時(shí)進(jìn)行雙酶切,膠回收酶切產(chǎn)物,將二者的酶切產(chǎn)物通過T4連接酶連接成環(huán)狀質(zhì)粒,再利用PCR、雙酶切和測序鑒定為陽性克隆,然后提取無內(nèi)毒素質(zhì)粒,利用lip2000脂質(zhì)體轉(zhuǎn)染到A375細(xì)胞和293T細(xì)胞,通過雙熒光素酶檢測系統(tǒng)測定相對熒光素酶活性值,并對核心啟動子區(qū)的轉(zhuǎn)錄因子結(jié)合位點(diǎn)進(jìn)行預(yù)測及活性驗(yàn)證。成功構(gòu)建了6個不同長度的啟動子片段,水貂PMEL基因-671/+87為核心啟動子區(qū)域,從-671缺失到-477時(shí)啟動子活性由最高降低到最低,表明在-671/-477可能存在正調(diào)控元件增強(qiáng)其啟動活性。利用生物信息學(xué)軟件分析該區(qū)域的轉(zhuǎn)錄因子結(jié)合位點(diǎn),提示有十幾種轉(zhuǎn)錄因子結(jié)合位點(diǎn)的存在,綜合至少2個軟件的預(yù)測結(jié)果,選擇出Sp1(-516/-506)、Sp1(-505/-495)和Sp1(-499/-489)結(jié)合位點(diǎn),對其分別構(gòu)建了3個Sp1突變體,檢測結(jié)果表明,-516/-506、-505/-495和-499/-489為轉(zhuǎn)錄的正調(diào)控區(qū)域。確定了水貂PMEL基因啟動子核心區(qū)域-671/+87,預(yù)測的3個Sp1結(jié)合位點(diǎn)為水貂PMEL基因轉(zhuǎn)錄的正調(diào)控區(qū)域。本研究結(jié)果為了解水貂PMEL基因的生物學(xué)功能提供了重要信息,為進(jìn)一步研究其調(diào)控水貂被毛顏色分子遺傳機(jī)制提供新的理論依據(jù)。
[Abstract]:Mink color is an important quality trait of mink skin and an important factor affecting its price. Premelanosome protein gene PMEL is an important candidate gene to influence the color variation of animal hair. In recent years, the aim of this study was to screen the active region of the PMEL promoter and transcription factor binding sites of the coat color gene of mink Mustela visonon, so as to provide a theoretical basis for exploring the regulation mechanism of the expression of this gene. The primer design was based on the sequence of M. putorius furo)PMEL gene (GenBank: NW004569320.1), which had high homology with mink. The promoter fragment of PMEL gene was amplified from the genomic DNA of black mink fur and cloned into pMD19 vector. The positive plasmid and pGL3-Basic vector were digested by restriction endonuclease Kpn 鈪，

本文編號：1674836