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  本文選題：牛MYOZ1基因 + 牛MYOZ2基因　； 參考：《西北農(nóng)林科技大學(xué)》2015年碩士論文

【摘要】：MYOZ1基因和MYOZ2基因編碼的蛋白都參與肌纖維Z線形成,肌纖維Z線在肌小節(jié)組裝和不同類型肌纖維形成等過程中起廣泛調(diào)控作用。其中,MYOZ1基因在成年哺乳動物的快肌纖維中表達,MYOZ2基因在成年哺乳動物的慢肌纖維和心肌中表達。目前對于MYOZ1基因啟動子的研究主要集中在豬和小鼠上,對于MYOZ2基因啟動子的研究主要集中在小鼠上,在牛上相關(guān)研究未見報道。本研究旨在尋找牛MYOZ1、MYOZ2基因核心啟動子區(qū)域,為研究牛MYOZ1基因和MYOZ2基因的轉(zhuǎn)錄調(diào)控機制奠定基礎(chǔ)。首先以秦川肉牛肌肉cDNA為模板,設(shè)計5'RACE試驗,確定目的基因轉(zhuǎn)錄起始位點。再以秦川肉�；蚪MDNA為模板,通過PCR的方法,克隆獲得約2kb大小的基因5'轉(zhuǎn)錄調(diào)控區(qū)片段。通過預(yù)測可能包含的轉(zhuǎn)錄因子結(jié)合位點,設(shè)計逐段缺失引物,進行PCR試驗,獲得7個亞克隆片段,分別與pGL3-Basic載體連接,得到目的基因5'轉(zhuǎn)錄調(diào)控區(qū)片段熒光素酶報告基因重組子。通過脂質(zhì)體法轉(zhuǎn)染C2C12細胞系,檢測7個重組質(zhì)粒的啟動子活性。本研究有如下結(jié)果:1.不同物種間MYOZ1蛋白氨基酸序列比對,結(jié)果顯示牛與山羊、綿羊的相似性都高達96%,與豬,狗和鼠的相似性較低;不同物種間MYOZ2蛋白氨基酸序列比對,結(jié)果顯示牛與山羊、綿羊的相似性都高達98.9%,與豬,狗和鼠的相似性較低。我們推測,MYOZ1基因與MYOZ2基因可能在反芻動物中相對保守,與單胃動物差異較大。2.試驗確定了牛MYOZ1基因與牛MYOZ2基因轉(zhuǎn)錄起始位點,結(jié)果表明牛MYOZ1基因的轉(zhuǎn)錄起始位點為堿基C,牛MYOZ2基因的轉(zhuǎn)錄起始位點為堿基A。3.構(gòu)建了牛MYOZ1、MYOZ2基因啟動子熒光素酶報告基因重組子,測序證實牛MYOZ1、MYOZ2基因啟動子雙熒光素酶報告基因重組載體構(gòu)建成功。4.將牛MYOZ1、MYOZ2基因啟動子重組子,分別通過脂質(zhì)體法轉(zhuǎn)染C2C12細胞系,檢測重組子的啟動子活性。確定了牛MYOZ1核心啟動子位于片段-116/+61,牛MYOZ2基因啟動子核心區(qū)域位于-84/+125。5.牛MYOZ1基因核心啟動子區(qū)片段-116/+61可能包含SP1,GC Box,CAAT等多個重要轉(zhuǎn)錄因子結(jié)合位點。牛MYOZ2基因核心啟動子區(qū)片段-84/+125可能包含SRF,MyoD,MEF2等多個重要轉(zhuǎn)錄因子結(jié)合位點。
[Abstract]:The protein encoded by MYOZ1 gene and MYOZ2 gene is involved in the formation of Z line of muscle fiber, and the Z line of muscle fiber plays an important role in the assembly of muscle section and the formation of different types of muscle fiber. MYOZ1 gene was expressed in fast muscle fibers of adult mammals and MYOZ2 gene was expressed in slow muscle fibers and myocardium of adult mammals. At present, the research on MYOZ1 gene promoter is mainly focused on pigs and mice, while the research on MYOZ2 gene promoter is mainly focused on mice. The purpose of this study was to search for the core promoter region of bovine MYOZ1 and MYOZ2 gene, and to lay a foundation for studying the transcriptional regulation mechanism of bovine MYOZ1 gene and MYOZ2 gene. Firstly, using Qinchuan beef muscle cDNA as template, 5'RACE test was designed to determine the target gene transcription initiation site. Using the genomic DNA of Qinchuan beef cattle as template, the 5 'transcriptional regulatory region of 2kb gene was cloned by PCR. By predicting the possible transcription factor binding sites, we designed one fragment by segment deletion primer, and carried out PCR test. Seven subcloned fragments were obtained, respectively, which were linked to pGL3-Basic vector. Recombinant of luciferase reporter gene of target gene 5 'transcriptional regulatory region was obtained. The promoter activity of 7 recombinant plasmids was detected by transfection of C2C12 cell lines by liposome method. The results of this study are as follows: 1. The results of amino acid sequence alignment of MYOZ1 protein among different species showed that the similarity between cattle and goat and sheep was as high as 96%, but the similarity with pig, dog and mouse was lower, and the amino acid sequence alignment of MYOZ2 protein between different species showed that cattle and goat were similar to each other. The similarity of sheep was as high as 98.9, and the similarity with pigs, dogs and mice was lower. We speculate that the MYOZ1 gene and MYOZ2 gene may be relatively conserved in ruminants, and are different from monogastric animals. The transcriptional initiation sites of bovine MYOZ1 gene and bovine MYOZ2 gene were determined. The results showed that the transcription initiation site of bovine MYOZ1 gene was base C, and that of bovine MYOZ2 gene was base A.3. The recombinant luciferase reporter gene promoter of bovine MYOZ1 and MYOZ2 gene was constructed. Sequencing confirmed that the recombinant vector of double luciferase reporter gene of bovine MYOZ1 and MYOZ2 gene promoter was constructed successfully. The promoter of bovine MYOZ1 and MYOZ2 gene was transfected into C2C12 cell line by liposome method to detect the promoter activity. Bovine MYOZ1 core promoter was located in fragment -116 / 61 and bovine MYOZ2 gene promoter was located at -84 / 125.5. The core promoter fragment -116 / 61 of bovine MYOZ1 gene may contain several important transcription factor binding sites such as SP1GCBoxCAAT. The core promoter fragment -84 / 125 of bovine MYOZ2 gene may contain several important transcription factor binding sites, such as SRF MYOZ2 Myo DX MEF2 and so on.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823

【參考文獻】

相關(guān)期刊論文 前1條

1 陳代文,張克英,胡祖禹;豬肉品質(zhì)特征的形成原理[J];四川農(nóng)業(yè)大學(xué)學(xué)報;2002年01期

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本文編號：1907977