本文選題：奶山羊 + LXRβ��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：肝臟X受體β(Liver X receptorβ,LXRβ)是核受體LXRs基因的亞型之一,是參與調(diào)控機(jī)體脂質(zhì)代謝的關(guān)鍵轉(zhuǎn)錄因子,它通過(guò)與視黃醛X受體共價(jià)結(jié)合,組成異二聚物后靶定至基因的調(diào)控區(qū)域,如固醇調(diào)節(jié)元件結(jié)合蛋白-1和脂肪酸合酶的啟動(dòng)子上,影響其轉(zhuǎn)錄,進(jìn)而影響到機(jī)體脂質(zhì)的代謝。本研究通過(guò)克隆得到奶山羊LXRβ基因的編碼區(qū),將其重組至腺病毒載體中,進(jìn)一步轉(zhuǎn)染重組質(zhì)粒進(jìn)入293A細(xì)胞,獲取高滴度的腺病毒,同時(shí)在線(xiàn)設(shè)計(jì)并合成siLXRβ,在奶山羊乳腺上皮細(xì)胞中分別超表達(dá)和干擾LXRβ基因后,通過(guò)RT-qPCR和Western Blot等方法檢測(cè)細(xì)胞內(nèi)脂代謝基因表達(dá)量及胞內(nèi)脂質(zhì)含量的變化,為進(jìn)一步明確該基因在乳脂代謝調(diào)控中的功能奠定理論基礎(chǔ)。以下為本研究獲得的主要研究結(jié)果:(1)通過(guò)提取西農(nóng)薩能奶山羊乳腺組織總RNA,根據(jù)GenBank中收錄的羊(XM_018062857)的LXRβ基因序列設(shè)計(jì)引物,利用RT-PCR技術(shù)克隆得到山羊LXRβ基因CDS序列,其長(zhǎng)度為1 368 bp,編碼455個(gè)氨基酸。山羊LXRβ基因CDS區(qū)序列與牛(Bos taurus)、綿羊(Ovisaries)、人(Homo sapiens)和小鼠(Mus musculus)的相似性分別為98%、98%、87%和85%,氨基酸序列相似性分別為99%、99%、90%和91%。(2)成功獲得含有目的基因的重組質(zhì)粒pAdEasy-LXRβ,轉(zhuǎn)染293A細(xì)胞后得到高滴度腺病毒,滴度為109 U/mL。通過(guò)在山羊乳腺上皮細(xì)胞中添加不同體積的病毒液,確定其最佳感染復(fù)數(shù)(MOI)為200。病毒處理細(xì)胞48h后,RT-qPCR結(jié)果顯示LXRβ基因表達(dá)量上升100倍左右,被T0901317激活后,SREBP-1、FASN、SCD、ACSL1、ACSL3、ACCα及ELVOL6基因均顯著上調(diào)(P0.05),FADS1基因的表達(dá)水平無(wú)明顯變化;Western blot結(jié)果表明FASN蛋白表達(dá)量顯著增加;油紅O和甘油三脂檢測(cè)結(jié)果說(shuō)明LXRβ基因可以顯著上調(diào)細(xì)胞內(nèi)的脂滴數(shù)量和胞內(nèi)甘油三酯的含量;脂肪酸組分分析表明細(xì)胞中C18:1的含量顯著上升(P0.05)。(3)根據(jù)LXRβ基因CDS序列,設(shè)計(jì)特異針對(duì)其的siRNA并轉(zhuǎn)染山羊乳腺上皮細(xì)胞,RT-qPCR結(jié)果表明LXRβ基因表達(dá)量顯著下調(diào)了88%,干擾LXRβ表達(dá)后,不添加激動(dòng)劑時(shí)DGAT2基因下調(diào)30%,SREBP1基因上調(diào)50%,其他脂代謝基因變化不顯著,添加激動(dòng)劑之后相對(duì)于對(duì)照組SREBP1和FSAN基因分別下調(diào)15%和35%,ASCL1基因表達(dá)量上調(diào)30%左右,ACSL3、FADS1、ELOVL6以及SCD基因表達(dá)量變化不顯著;甘油三脂檢測(cè)結(jié)果表明當(dāng)LXRβ表達(dá)量下降時(shí),胞內(nèi)甘油三脂含量隨之減少。綜上所述,本試驗(yàn)成功克隆得到了奶山羊LXRβ基因的CDS區(qū),構(gòu)建了超表達(dá)載體,通過(guò)感染山羊乳腺上皮細(xì)胞超表達(dá)LXRβ基因并添加激動(dòng)劑可導(dǎo)致眾多脂代謝基因的mRNA表達(dá)水平顯著上調(diào),胞內(nèi)脂滴含量增加,干擾該基因的表達(dá)對(duì)脂代謝基因的mRNA表達(dá)以及胞內(nèi)甘油三酯合成有一定的抑制作用。為明確LXRβ基因在奶山羊乳腺細(xì)胞中乳脂代謝中的功能提供理論依據(jù)。
[Abstract]:Liver X receptor 尾 -Liver X receptor 尾 -LXR 尾) is one of the subtypes of nuclear receptor LXRs gene and a key transcription factor involved in regulating lipid metabolism. For example, the promoter of steroid regulatory element binding protein 1 and fatty acid synthase affect its transcription, and then affect the metabolism of body lipid. In this study, the coding region of LXR 尾 gene of dairy goat was cloned, and the recombinant plasmid was transfected into 293A cells to obtain high titer adenovirus. At the same time, siLXR 尾 was designed and synthesized online. After overexpression and interference of LXR 尾 gene in dairy goat mammary epithelial cells, the changes of lipid metabolism gene expression and intracellular lipid content were detected by RT-qPCR and Western Blot. In order to further clarify the role of the gene in regulation of milk fat metabolism lay a theoretical foundation. The following is the main result of this study: (1) by extracting the total RNAs from the breast tissue of Sinon Sanen dairy goat, we designed primers according to the LXR 尾 gene sequence of XM018062857 (included in GenBank) and cloned the CDS sequence of LXR 尾 gene from goat by RT-PCR technique. Its length is 1 368 BP, encoding 455 amino acids. The similarity of CDS region of goat LXR 尾 gene with Bos taurus, Ovisariesus, Homo sapiensus and mouse Mus musculus was 98% and 85%, respectively. The amino acid sequence similarity was 990.9999% and 91.1%, respectively. The recombinant plasmid pAdEasy-LXR 尾 containing the target gene was successfully obtained and transfected into 293A cells. Get a high titer adenovirus, The titer was 109 UmL. By adding different volumes of virus to goat mammary epithelial cells, it was determined that the optimal number of infected moi was 200. After 48 hours of virus treatment, RT-qPCR showed that the expression of LXR 尾 gene increased about 100fold, and the expression level of FASN protein increased significantly after activation by T0901317. The expression level of ACSL1ACSL3ACSL3ACC 偽 and ELVOL6 were significantly up-regulated by T0901317. The results of Western blot showed that FASN protein expression was significantly increased. The results of oil red O and triglyceride detection showed that LXR 尾 gene could significantly up-regulate the number of lipid droplets and the content of intracellular triglycerides in cells, and fatty acid component analysis showed that the content of C18: 1 increased significantly (P0.05. 3) according to the CDS sequence of LXR 尾 gene. The results of RT-PCR showed that the expression of LXR 尾 gene was down-regulated by 888.After interfering with the expression of LXR 尾, the DGAT2 gene was down-regulated by 30% SREBP1 gene and no significant change of other lipid metabolism genes was observed without the addition of agonist. Compared with the control group, the expression of SREBP1 and FSAN genes were down-regulated by 15% and 35%, respectively, and the expression of ACSL3, FADS1, ELOVL6 and SCD were up regulated by about 30%, and the results of triglyceride test showed that the expression of LXR 尾 was decreased. The intracellular triglyceride content decreased. In conclusion, the CDS region of LXR 尾 gene of dairy goat was cloned successfully, and the superexpression vector was constructed. The overexpression of LXR 尾 gene in goat mammary epithelial cells and the addition of agonist could significantly upregulate the expression of mRNA and increase the content of lipid droplets in many lipid metabolism genes. Interfering with the expression of this gene can inhibit the mRNA expression and intracellular triglyceride synthesis of lipid metabolism gene. To provide theoretical basis for the function of LXR 尾 gene in milk fat metabolism of milk goat breast cells.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S827

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