牛α-actin基因啟動子的改造及其功能驗證
發(fā)布時間：2018-10-11 12:14
【摘要】：培養(yǎng)高產(chǎn)、優(yōu)質(zhì)的肉牛一直是畜牧生產(chǎn)所追求的目標(biāo)。傳統(tǒng)的雜交育種方法存在效率低,周期長等問題。轉(zhuǎn)基因技術(shù)可在較短時間內(nèi)培育出具有優(yōu)質(zhì)性狀的肉牛新品種,應(yīng)用前景廣泛。高效肌肉特異性啟動子能夠啟動外源基因在骨骼肌細(xì)胞中的高效表達(dá),對于提高肌肉產(chǎn)量的轉(zhuǎn)基因肉牛生產(chǎn)具有重要意義。骨骼肌α-肌動蛋白(α-actin)所編碼的蛋白質(zhì)是骨骼肌中肌動蛋白的主要成分。本研究以牛α-actin基因啟動子為對象,通過分子克隆技術(shù)對其啟動子片段進(jìn)行改造,期望篩選出活性較高且具有肌肉組織特異性的啟動子,為今后肉質(zhì)性狀改良相關(guān)的牛轉(zhuǎn)基因研究提供合適的肌肉特異性啟動子。通過PCR定點突變啟動子特定轉(zhuǎn)錄調(diào)控元件;在其上游和下游連接內(nèi)含子;連接Myo G基因的啟動子構(gòu)建雙啟動子;增加正調(diào)控元件拷貝數(shù)等方法改造牛骨骼肌α-actin基因啟動子;構(gòu)建雙熒光素酶報告基因質(zhì)粒;瞬時轉(zhuǎn)染牛骨骼肌衛(wèi)星細(xì)胞和胎兒成纖維細(xì)胞,檢測啟動子的肌肉特異性和表達(dá)效率。最后選擇經(jīng)過改造后活性較高的啟動子來驗證其對牛骨骼肌α-actin基因表達(dá)的影響,具體研究內(nèi)容及結(jié)果為:1.針對牛骨骼肌α-actin基因啟動子,分析確定α-actin啟動子中特定核苷酸序列的功能,應(yīng)用PCR定點突變的方法,定點突變牛α-actin啟動子缺失片段389bp中的Sp1/KLFs元件、430bp中的ZF5F元件和490bp中的Pax3元件;結(jié)果發(fā)現(xiàn),Sp1/KLFs突變后啟動子活性增強(qiáng),為負(fù)調(diào)控元件;ZF5F和Pax3突變后啟動子活性減弱,為牛α-actin啟動子的正調(diào)控元件。2.根據(jù)Gen Bank上公布的序列,將牛骨骼肌α-actin基因的內(nèi)含子I和內(nèi)含子II,分別連在p GL3-α-actinp262的上游和下游,證明內(nèi)含子I和內(nèi)含子II插入啟動子上游時均能提高啟動子轉(zhuǎn)錄活性且具有肌肉特異性,而這兩個內(nèi)含子在啟動子下游時并不提高啟動子活性,證明內(nèi)含子可以影響α-actin基因啟動子活性,且具有位置效應(yīng)。3.將Myo G基因啟動子連接到牛α-actin基因啟動子之前并構(gòu)建了雙啟動子真核表達(dá)載體p GL3-Myo Gp373-α-actinp262。結(jié)果表明雙啟動子的轉(zhuǎn)錄活性較牛Myo G和α-actin基因啟動子活性都顯著提高,且保持較好的肌肉特異性。4.通過PCR擴(kuò)增牛α-actin基因啟動子中含有多個正調(diào)控元件的170bp序列,插入到啟動子上游,構(gòu)建含有兩個和三個這一序列拷貝的重組質(zhì)粒,并構(gòu)建連接Myo G基因啟動子部分調(diào)控元件的牛α-actin基因啟動子,發(fā)現(xiàn)增加調(diào)控元件拷貝數(shù)后牛α-actin基因啟動子活性明顯提高,且具有肌肉特異性。5.根據(jù)Gen Bank上公布的序列,對牛α-actin基因從起始密碼子至終止密碼子進(jìn)行克隆,將改造的含有多拷貝數(shù)調(diào)控元件的牛α-actin基因啟動子連接至其上游,通過Real-time RCR,Western Blot,免疫熒光和微絲染色技術(shù)檢測α-actin基因的表達(dá)量。發(fā)現(xiàn)改造的α-actin基因啟動子可以顯著地提高α-actin基因的表達(dá)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 偽 -actin (偽-actin) is the main component of actin in skeletal muscle. In this study, the promoter of bovine 偽-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 偽-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 偽-actin gene in bovine skeletal muscle. The specific contents and results are as follows: 1. The function of specific nucleotide sequence in 偽-actin promoter was analyzed for bovine skeletal muscle 偽-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 偽-actin promoter. 2. According to the sequence published on Gen Bank, the intron I and intron II, of bovine skeletal muscle 偽-actin gene were linked to the upstream and downstream of p GL3- 偽-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 偽-actin gene and has a position effect of 3. 3%. The eukaryotic expression vector p GL3-Myo Gp373- 偽-actinp262. was constructed by ligating Myo G gene promoter to bovine 偽-actin gene promoter. The results showed that the transcriptional activity of double promoter was significantly higher than that of bovine Myo G and 偽-actin gene promoter, and the activity of double promoter maintained better muscle specificity than that of bovine Myo G and 偽-actin gene promoter. The 170bp sequence containing multiple positive regulatory elements in the promoter of bovine 偽-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 偽-actin gene was constructed with partial regulatory element of Myo G gene promoter. It was found that the promoter activity of bovine 偽-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 偽-actin gene was cloned from the start codon to the termination codon, and the modified bovine 偽-actin gene promoter containing multiple copy number regulatory elements was connected to its upstream. The expression of 偽-actin gene was detected by Real-time RCR,Western Blot, immunofluorescence and microfilament staining. It was found that the modified 偽-actin gene promoter could significantly increase the expression of 偽-actin gene.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S823
本文編號：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 偽 -actin (偽-actin) is the main component of actin in skeletal muscle. In this study, the promoter of bovine 偽-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 偽-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 偽-actin gene in bovine skeletal muscle. The specific contents and results are as follows: 1. The function of specific nucleotide sequence in 偽-actin promoter was analyzed for bovine skeletal muscle 偽-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 偽-actin promoter. 2. According to the sequence published on Gen Bank, the intron I and intron II, of bovine skeletal muscle 偽-actin gene were linked to the upstream and downstream of p GL3- 偽-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 偽-actin gene and has a position effect of 3. 3%. The eukaryotic expression vector p GL3-Myo Gp373- 偽-actinp262. was constructed by ligating Myo G gene promoter to bovine 偽-actin gene promoter. The results showed that the transcriptional activity of double promoter was significantly higher than that of bovine Myo G and 偽-actin gene promoter, and the activity of double promoter maintained better muscle specificity than that of bovine Myo G and 偽-actin gene promoter. The 170bp sequence containing multiple positive regulatory elements in the promoter of bovine 偽-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 偽-actin gene was constructed with partial regulatory element of Myo G gene promoter. It was found that the promoter activity of bovine 偽-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 偽-actin gene was cloned from the start codon to the termination codon, and the modified bovine 偽-actin gene promoter containing multiple copy number regulatory elements was connected to its upstream. The expression of 偽-actin gene was detected by Real-time RCR,Western Blot, immunofluorescence and microfilament staining. It was found that the modified 偽-actin gene promoter could significantly increase the expression of 偽-actin gene.
【學(xué)位授予單位】：東北農(nóng)業(yè)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(xiàn)】
相關(guān)期刊論文 前3條
1 趙曉;莫德林;張悅;龔雯;李安寧;陳瑤生;;豬的骨骼肌生長發(fā)育研究進(jìn)展[J];生命科學(xué);2011年01期
2 吳端欽;賀志雄;喬君毅;冉濤;;牛肉品質(zhì)影響因素及改善技術(shù)的研究進(jìn)展[J];肉類研究;2012年10期
3 李慧敏;石玉;楊志剛;江紹萍;;人管家基因啟動子序列中的組合轉(zhuǎn)錄調(diào)控元件分析[J];生物化學(xué)與生物物理進(jìn)展;2015年01期
相關(guān)碩士學(xué)位論文 前1條
1 張利博;牛Nramp1基因5'端調(diào)控區(qū)的啟動子功能和SNPs分析[D];吉林大學(xué);2009年
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