牛MYOZ1、MYOZ2基因启动子活性分析

发布时间：2018-05-19 00:44

本文选题：牛MYOZ1基因 + 牛MYOZ2基因　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：MYOZ1基因和MYOZ2基因编码的蛋白都参与肌纤维Z线形成,肌纤维Z线在肌小节组装和不同类型肌纤维形成等过程中起广泛调控作用。其中,MYOZ1基因在成年哺乳动物的快肌纤维中表达,MYOZ2基因在成年哺乳动物的慢肌纤维和心肌中表达。目前对于MYOZ1基因启动子的研究主要集中在猪和小鼠上,对于MYOZ2基因启动子的研究主要集中在小鼠上,在牛上相关研究未见报道。本研究旨在寻找牛MYOZ1、MYOZ2基因核心启动子区域,为研究牛MYOZ1基因和MYOZ2基因的转录调控机制奠定基础。首先以秦川肉牛肌肉cDNA为模板,设计5'RACE试验,确定目的基因转录起始位点。再以秦川肉牛基因组DNA为模板,通过PCR的方法,克隆获得约2kb大小的基因5'转录调控区片段。通过预测可能包含的转录因子结合位点,设计逐段缺失引物,进行PCR试验,获得7个亚克隆片段,分别与pGL3-Basic载体连接,得到目的基因5'转录调控区片段荧光素酶报告基因重组子。通过脂质体法转染C2C12细胞系,检测7个重组质粒的启动子活性。本研究有如下结果:1.不同物种间MYOZ1蛋白氨基酸序列比对,结果显示牛与山羊、绵羊的相似性都高达96%,与猪,狗和鼠的相似性较低;不同物种间MYOZ2蛋白氨基酸序列比对,结果显示牛与山羊、绵羊的相似性都高达98.9%,与猪,狗和鼠的相似性较低。我们推测,MYOZ1基因与MYOZ2基因可能在反刍动物中相对保守,与单胃动物差异较大。2.试验确定了牛MYOZ1基因与牛MYOZ2基因转录起始位点,结果表明牛MYOZ1基因的转录起始位点为碱基C,牛MYOZ2基因的转录起始位点为碱基A。3.构建了牛MYOZ1、MYOZ2基因启动子荧光素酶报告基因重组子,测序证实牛MYOZ1、MYOZ2基因启动子双荧光素酶报告基因重组载体构建成功。4.将牛MYOZ1、MYOZ2基因启动子重组子,分别通过脂质体法转染C2C12细胞系,检测重组子的启动子活性。确定了牛MYOZ1核心启动子位于片段-116/+61,牛MYOZ2基因启动子核心区域位于-84/+125。5.牛MYOZ1基因核心启动子区片段-116/+61可能包含SP1,GC Box,CAAT等多个重要转录因子结合位点。牛MYOZ2基因核心启动子区片段-84/+125可能包含SRF,MyoD,MEF2等多个重要转录因子结合位点。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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