牛α-actin基因启动子的改造及其功能验证
发布时间：2018-10-11 12:14
【摘要】：培养高产、优质的肉牛一直是畜牧生产所追求的目标。传统的杂交育种方法存在效率低,周期长等问题。转基因技术可在较短时间内培育出具有优质性状的肉牛新品种,应用前景广泛。高效肌肉特异性启动子能够启动外源基因在骨骼肌细胞中的高效表达,对于提高肌肉产量的转基因肉牛生产具有重要意义。骨骼肌α-肌动蛋白(α-actin)所编码的蛋白质是骨骼肌中肌动蛋白的主要成分。本研究以牛α-actin基因启动子为对象,通过分子克隆技术对其启动子片段进行改造,期望筛选出活性较高且具有肌肉组织特异性的启动子,为今后肉质性状改良相关的牛转基因研究提供合适的肌肉特异性启动子。通过PCR定点突变启动子特定转录调控元件;在其上游和下游连接内含子;连接Myo G基因的启动子构建双启动子;增加正调控元件拷贝数等方法改造牛骨骼肌α-actin基因启动子;构建双荧光素酶报告基因质粒;瞬时转染牛骨骼肌卫星细胞和胎儿成纤维细胞,检测启动子的肌肉特异性和表达效率。最后选择经过改造后活性较高的启动子来验证其对牛骨骼肌α-actin基因表达的影响,具体研究内容及结果为:1.针对牛骨骼肌α-actin基因启动子,分析确定α-actin启动子中特定核苷酸序列的功能,应用PCR定点突变的方法,定点突变牛α-actin启动子缺失片段389bp中的Sp1/KLFs元件、430bp中的ZF5F元件和490bp中的Pax3元件;结果发现,Sp1/KLFs突变后启动子活性增强,为负调控元件;ZF5F和Pax3突变后启动子活性减弱,为牛α-actin启动子的正调控元件。2.根据Gen Bank上公布的序列,将牛骨骼肌α-actin基因的内含子I和内含子II,分别连在p GL3-α-actinp262的上游和下游,证明内含子I和内含子II插入启动子上游时均能提高启动子转录活性且具有肌肉特异性,而这两个内含子在启动子下游时并不提高启动子活性,证明内含子可以影响α-actin基因启动子活性,且具有位置效应。3.将Myo G基因启动子连接到牛α-actin基因启动子之前并构建了双启动子真核表达载体p GL3-Myo Gp373-α-actinp262。结果表明双启动子的转录活性较牛Myo G和α-actin基因启动子活性都显著提高,且保持较好的肌肉特异性。4.通过PCR扩增牛α-actin基因启动子中含有多个正调控元件的170bp序列,插入到启动子上游,构建含有两个和三个这一序列拷贝的重组质粒,并构建连接Myo G基因启动子部分调控元件的牛α-actin基因启动子,发现增加调控元件拷贝数后牛α-actin基因启动子活性明显提高,且具有肌肉特异性。5.根据Gen Bank上公布的序列,对牛α-actin基因从起始密码子至终止密码子进行克隆,将改造的含有多拷贝数调控元件的牛α-actin基因启动子连接至其上游,通过Real-time RCR,Western Blot,免疫荧光和微丝染色技术检测α-actin基因的表达量。发现改造的α-actin基因启动子可以显著地提高α-actin基因的表达量。
[Abstract]:Cultivating high-yield and high-quality beef cattle has always been the goal of livestock production. The traditional hybrid breeding methods have some problems, such as low efficiency and long period. Transgenic technology can produce new beef cattle varieties with high quality traits in a short time, and it has a wide application prospect. High efficient muscle specific promoters can activate the high expression of exogenous genes in skeletal muscle cells, which is of great significance for the production of transgenic beef cattle with increased muscle yield. The protein encoded by 伪 -actin (伪-actin) is the main component of actin in skeletal muscle. In this study, the promoter of bovine 伪-actin gene was modified by molecular cloning technique, and the promoter with high activity and specific muscle tissue was expected to be screened. To provide appropriate muscle-specific promoters for cattle transgenic research related to meat quality improvement in the future. The specific transcriptional regulatory elements of PCR site-directed promoter were used; the intron was connected upstream and downstream; the promoter linked with Myo G gene was constructed; and the 伪-actin gene promoter of bovine skeletal muscle was reconstructed by increasing the copy number of positive regulatory elements. Double luciferase reporter gene plasmids were constructed and transient transfected into bovine skeletal muscle satellite cells and fetal fibroblasts to detect the muscle specificity and expression efficiency of the promoter. Finally, the modified promoter with high activity was selected to verify the effect of the promoter on the expression of 伪-actin gene in bovine skeletal muscle. The specific contents and results are as follows: 1. The function of specific nucleotide sequence in 伪-actin promoter was analyzed for bovine skeletal muscle 伪-actin gene promoter. PCR site-directed mutation method was used to detect Sp1/KLFs element in 389bp, ZF5F element in 430bp and Pax3 element in 490bp by site-directed mutation. The results showed that the activity of promoter was increased after Sp1/KLFs mutation, and the activity of promoter was decreased after ZF5F and Pax3 mutation, which was the positive regulatory element of bovine 伪-actin promoter. 2. According to the sequence published on Gen Bank, the intron I and intron II, of bovine skeletal muscle 伪-actin gene were linked to the upstream and downstream of p GL3- 伪-actinp262, respectively. It was proved that both intron I and intron II could increase the promoter's transcription activity and have muscle specificity when inserted upstream, but the two introns did not improve the promoter activity at the downstream of the promoter. It is proved that intron can affect the promoter activity of 伪-actin gene and has a position effect of 3. 3%. The eukaryotic expression vector p GL3-Myo Gp373- 伪-actinp262. was constructed by ligating Myo G gene promoter to bovine 伪-actin gene promoter. The results showed that the transcriptional activity of double promoter was significantly higher than that of bovine Myo G and 伪-actin gene promoter, and the activity of double promoter maintained better muscle specificity than that of bovine Myo G and 伪-actin gene promoter. The 170bp sequence containing multiple positive regulatory elements in the promoter of bovine 伪-actin gene was amplified by PCR and inserted into the upstream of the promoter to construct a recombinant plasmid containing two and three copies of the sequence. The promoter of bovine 伪-actin gene was constructed with partial regulatory element of Myo G gene promoter. It was found that the promoter activity of bovine 伪-actin gene was significantly increased after increasing the copy number of regulatory element, and the promoter was muscle-specific. According to the sequence published on Gen Bank, bovine 伪-actin gene was cloned from the start codon to the termination codon, and the modified bovine 伪-actin gene promoter containing multiple copy number regulatory elements was connected to its upstream. The expression of 伪-actin gene was detected by Real-time RCR,Western Blot, immunofluorescence and microfilament staining. It was found that the modified 伪-actin gene promoter could significantly increase the expression of 伪-actin gene.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
本文编号：2264151
[Abstract]:Cultivating high-yield and high-quality beef cattle has always been the goal of livestock production. The traditional hybrid breeding methods have some problems, such as low efficiency and long period. Transgenic technology can produce new beef cattle varieties with high quality traits in a short time, and it has a wide application prospect. High efficient muscle specific promoters can activate the high expression of exogenous genes in skeletal muscle cells, which is of great significance for the production of transgenic beef cattle with increased muscle yield. The protein encoded by 伪 -actin (伪-actin) is the main component of actin in skeletal muscle. In this study, the promoter of bovine 伪-actin gene was modified by molecular cloning technique, and the promoter with high activity and specific muscle tissue was expected to be screened. To provide appropriate muscle-specific promoters for cattle transgenic research related to meat quality improvement in the future. The specific transcriptional regulatory elements of PCR site-directed promoter were used; the intron was connected upstream and downstream; the promoter linked with Myo G gene was constructed; and the 伪-actin gene promoter of bovine skeletal muscle was reconstructed by increasing the copy number of positive regulatory elements. Double luciferase reporter gene plasmids were constructed and transient transfected into bovine skeletal muscle satellite cells and fetal fibroblasts to detect the muscle specificity and expression efficiency of the promoter. Finally, the modified promoter with high activity was selected to verify the effect of the promoter on the expression of 伪-actin gene in bovine skeletal muscle. The specific contents and results are as follows: 1. The function of specific nucleotide sequence in 伪-actin promoter was analyzed for bovine skeletal muscle 伪-actin gene promoter. PCR site-directed mutation method was used to detect Sp1/KLFs element in 389bp, ZF5F element in 430bp and Pax3 element in 490bp by site-directed mutation. The results showed that the activity of promoter was increased after Sp1/KLFs mutation, and the activity of promoter was decreased after ZF5F and Pax3 mutation, which was the positive regulatory element of bovine 伪-actin promoter. 2. According to the sequence published on Gen Bank, the intron I and intron II, of bovine skeletal muscle 伪-actin gene were linked to the upstream and downstream of p GL3- 伪-actinp262, respectively. It was proved that both intron I and intron II could increase the promoter's transcription activity and have muscle specificity when inserted upstream, but the two introns did not improve the promoter activity at the downstream of the promoter. It is proved that intron can affect the promoter activity of 伪-actin gene and has a position effect of 3. 3%. The eukaryotic expression vector p GL3-Myo Gp373- 伪-actinp262. was constructed by ligating Myo G gene promoter to bovine 伪-actin gene promoter. The results showed that the transcriptional activity of double promoter was significantly higher than that of bovine Myo G and 伪-actin gene promoter, and the activity of double promoter maintained better muscle specificity than that of bovine Myo G and 伪-actin gene promoter. The 170bp sequence containing multiple positive regulatory elements in the promoter of bovine 伪-actin gene was amplified by PCR and inserted into the upstream of the promoter to construct a recombinant plasmid containing two and three copies of the sequence. The promoter of bovine 伪-actin gene was constructed with partial regulatory element of Myo G gene promoter. It was found that the promoter activity of bovine 伪-actin gene was significantly increased after increasing the copy number of regulatory element, and the promoter was muscle-specific. According to the sequence published on Gen Bank, bovine 伪-actin gene was cloned from the start codon to the termination codon, and the modified bovine 伪-actin gene promoter containing multiple copy number regulatory elements was connected to its upstream. The expression of 伪-actin gene was detected by Real-time RCR,Western Blot, immunofluorescence and microfilament staining. It was found that the modified 伪-actin gene promoter could significantly increase the expression of 伪-actin gene.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823
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