本文關(guān)鍵詞： 牦牛 毛色 熒光定量PCR 表達(dá)量　出處：《甘肅農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：牦牛毛色影響毛的利用經(jīng)濟(jì)價(jià)值。本研究以天祝白牦牛和甘南牦牛為研究對(duì)象,通過(guò)基因克隆、DNA混合池測(cè)序、熒光定量PCR等方法。克隆牦牛MC1R、MITF及Agouti基因CDS區(qū)序列,進(jìn)行生物信息學(xué)分析。對(duì)調(diào)控毛色候選MITF、Agouti基因的編碼區(qū)與牦牛毛色性狀的相關(guān)性進(jìn)行分析。通過(guò)實(shí)時(shí)熒光定量PCR技術(shù),分析MC1R、MITF及Agouti基因在不同毛色牦牛的皮膚組織相對(duì)表達(dá)量。研究結(jié)果如下:(1)克隆獲得了牦牛MC1R基因、MITF基因及Agouti基因完整編碼區(qū)序列(CDS)。MC1R基因編碼區(qū)(CDS)序列長(zhǎng)度為1021 bp,其編碼317個(gè)氨基酸,該蛋白無(wú)信號(hào)肽,但有七個(gè)跨膜區(qū)。MITF基因編碼區(qū)(CDS)序列長(zhǎng)度為1460 bp。其編碼413個(gè)氨基酸,MITF-M蛋白存在10個(gè)氨基酸構(gòu)成的M域,即MLEMLEYNHY。該氨基酸序列無(wú)信號(hào)肽,且不具有跨膜結(jié)構(gòu)域。MITF-M有25個(gè)磷酸化位點(diǎn)。Agouti基因是一條長(zhǎng)593 bp的DNA序列。該序列編碼133個(gè)氨基酸,ASIP有一個(gè)22個(gè)氨基酸的信號(hào)肽,且沒(méi)有跨膜結(jié)構(gòu)域。ASIP是一種膜外蛋白。該基因的序列在第6-129位氨基酸之間具有完整的DNA binding ASIP功能域。ASIP有5個(gè)磷酸化位點(diǎn)。MC1R、MITF-M和ASIP均是親水性蛋白。(2)利用DNA混合池、直接測(cè)序法對(duì)牦牛MITF基因和Agouti基因序列的編碼區(qū)進(jìn)行檢測(cè)分析。所得結(jié)果顯示,在牦牛MITF基因和Agouti基因序列編碼區(qū)并無(wú)突變位點(diǎn)。表明牦牛MITF基因和Agouti基因編碼區(qū)序列不存在與毛色相關(guān)聯(lián)的功能突變。(3)實(shí)時(shí)熒光定量PCR檢測(cè)分析得到MC1R基因、MITF基因的僅在皮膚組織中特異性表達(dá)。Agouti基因在卵巢、肌肉組織及皮膚組織中均有表達(dá),且其皮膚組織中表達(dá)最高。Agouti基因m RNA表達(dá)量在黑色毛皮膚組織和白色毛皮組織中差異不顯著。MC1R基因m RNA相對(duì)表達(dá)量在純黑色毛皮組織中顯著高于純白色毛皮組織。說(shuō)明皮膚組織中MC1R基因m RNA的高表達(dá)可能利于黑色素的沉著。MITF基因m RNA相對(duì)表達(dá)量在純黑色毛皮組織中顯著高于白色毛皮組織。說(shuō)明牦牛白色被毛與黑色被毛性狀可能與MITF基因在皮膚組織中m RNA的表達(dá)量高低有關(guān)聯(lián)。
[Abstract]:In this study, Tianzhu White Yak and Gannan Yak were selected as the research objects, and the DNA mixed pool was sequenced by gene cloning. Fluorescence quantitative PCR and other methods were used to clone the CDS region of MC1RN MITF and Agouti gene of Yak, and to analyze the bioinformatics. The candidate MITF for controlling coat color was analyzed. The correlation between the coding region of Agouti gene and the hair color traits of yak was analyzed, and MC1R was analyzed by real-time fluorescence quantitative PCR. The relative expression of MITF and Agouti genes in the skin of different hairy yaks was studied. The results were as follows: 1) the yak MC1R gene was obtained by cloning. The length of the complete coding region of MITF gene and Agouti gene was 1021 BP, encoding 317 amino acids. The protein has no signal peptide, but has seven transmembrane regions. The length of CDSs of MITF gene coding region is 1460 BP, which encodes 413 amino acids. There are 10 amino acids in the M domain of MITF-M protein, that is, MLEMLEYNHY. the amino acid sequence has no signal peptide. Moreover, there are 25 phosphorylation sites. Agouti gene is a 593bp DNA sequence with no transmembrane domain .MITF-M, which encodes 133 amino acids. ASIP has a signal peptide of 22 amino acids. There is no transmembrane domain. ASIP is an extracellular protein. The sequence of the gene has complete DNA binding between the 6-129 amino acids. ASIP functional domain. ASIP has 5 phosphorylation sites. MC1R. MITF-M and ASIP are both hydrophilic proteins. The coding regions of MITF gene and Agouti gene of yak were detected by direct sequencing. There was no mutation in the coding region of yak MITF gene and Agouti gene, which indicated that there was no functional mutation associated with coat color in the coding region of MITF gene and Agouti gene of yak. 3) MC1R gene was obtained by real-time fluorescence quantitative PCR analysis. The specific expression of MITF gene was only in the skin tissue. The MITF gene was expressed in the ovary, muscle tissue and skin tissue. There was no significant difference in the expression of m RNA between black fur skin and white fur tissue. MC1R gene m. The relative expression of RNA in pure black fur tissue was significantly higher than that in pure white fur tissue, indicating that the high expression of MC1R gene m RNA might be beneficial to melanin deposition. MITF gene m. The relative expression of RNA in pure black fur tissue was significantly higher than that in white fur tissue, indicating that the characters of white coat and black coat of yak might be associated with MITF gene in skin tissue m. The level of RNA expression is related.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823.85

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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本文編號(hào)：1442176