西农萨能奶山羊脂肪酸合酶基因启动子的克隆、测序及活性区域分析
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摘要
脂肪酸合酶(Fatty acid synthase, FAS;EC 2.3.1.85)是哺乳动物的脂肪酸合成过程中具有重要作用的关键酶。它利用乙酰-CoA、丙二酸单酰-CoA和NADPH催化饱和脂肪酸的合成。山羊乳腺短、中链脂肪酸合成受到FAS中的乙酰/丙二酸单酰基转移酶(Acetyl-CoA and malonyl-CoA transacylases, AT/MT)功能域的转酰基作用调控。可以设想对脂肪酸合酶基因进行调控或改造,从而达到生产出高品质乳品的目标。通过调控FAS基因表达可以达到改变山羊乳脂肪酸组成、提高羊奶中对人体有益的脂肪酸含量、解决山羊乳膻味问题等目的。所以对其启动子的结构和功能的研究,可进一步揭示脂肪酸合酶基因表达所依赖的调控信息以及这些信息相互作用的分子机制。因此,本研究以脂肪酸合酶基因的启动子活性功能探索为切入点,对FAS基因启动子的全长进行克隆、测序和生物信息学分析,在对其结构了解比较清楚的基础上进行启动子缺失片段载体的构建,找出其最小活性中心。用不同浓度雌激素作用于此最小活性中心,对其启动活性和功能调控进行进一步的探索,为FAS基因的功能调控等深入研究打下基础。该研究旨在为西农萨能奶山羊乳腺FAS基因的功能研究奠定基础,通过掌握FAS基因启动子的调控规律,为山羊乳腺脂肪酸代谢、FAS基因功能以及山羊奶膻味的遗传调控机理研究提供试验依据。
本研究以西北农林科技大学萨能羊原种场健康的纯种西农萨能奶山羊为试验材料,扩增获得了西农萨能奶山羊乳腺FAS基因启动子序列全长,并对其进行了生物信息学分析;采用荧光素酶活性分析的方法检测了不同启动子缺失片段(pGL3—E、pGL3—3、pGL3—4、pGL3—5、pGL3—6、pGL3—8、pGL3—10、pGL3—14、pGL3—19、pGL3—21)的启动活性。用不同浓度的雌激素作用于启动子的最小活性中心,研究雌激素对启动子活性的影响。试验结果如下:
1.克隆获得了西农萨能奶山羊FAS基因启动子全长2640bp,登录GenBank收录号为EF556550(数据已公布)。
2.对克隆得出的启动子序列进行生物信息学分析,表明羊FAS基因启动子和牛、人FAS启动子存在90%以上同源性,启动子还存在数个潜在SP1、Ets、LSF等转录因子结合位点。没有典型的TATA框,存在CCAAT框和GC框,在-1040bp~-340bp处可能存在启动子活性中心。
3.启动子缺失片段的荧光素酶活性分析表明,启动子前段区域可能存在负调控元件,-721~-540bp中间存在着FAS启动子的核心区域,-540bp后无启动子活性序列。
4.雌激素对FAS启动子活性影响试验表明,-721~-540bp序列存在雌激素的结合区域。雌激素浓度为4-100μmol/L时,雌激素对FAS启动子活性有显著的提高作用。其中20μmol/L时作用最为显著。
Fatty acid synthase(FAS) plays an important role in de novo fatty acids synthesis in mammals. FAS helps to catalyze all the reaction steps in the conversion of acety1-CoA, malonyl-CoA and NADPH to saturated fatty acids. Short- and medium- chain fatty acids in goat mammary synthesis are functioned by a transferase reaction in the region of Acetyl-CoA and malonyl-CoA transacylases(AT/MT). It’s one of the important means to solve goat milk odor from genetics and breeding aspect by regulating FAS gene expression in order to change fatty acids composition in goat mammary gland. So the study of FAS gene promotor’s struture and function can further reveal the regulated informaton and its interacted mechanism of which FAS gene expression depending on. Therefore, this study take FAS gene promoter active and function as a point, cloned the FAS gene promoter, sequenced and bioinformation analyzed it. Construct PGL-3 expression vector based on structure foundation, found out core promoter sequences. Use estrogen of concentration gradient on the core promoter sequence, further explore its starting activity and function regulation. Laid a foundation for FAS gene founctional regulation research. This study aims at consummating fundamental research of Xinong Saanen Goat FAS gene promoter, Provided experimental basis on Goat Mammary Gland fatty acid metabolism,FAS gene’s function and genetic regulation mechnism of goat milk flavor by mastering the FAS promoter’s regulation rules.
The study uses purebred and healthy Xinong saanen goat in Xinong Saanen Goat Breeding Farm of Northwest A&F University as experimental materials, cloned and analyzed the sequence of Xinong Saanen Dairy Goat mammary FAS gene promoter full length region; Detected activity of different promoter deletion fragments(pGL3—E, pGL3—3, pGL3—4, pGL3—5, pGL3—6, pGL3—8, pGL3—10, pGL3—14, pGL3—19, pGL3—21)by luciferase assays. Selected estrogen of different concentrations act on the core promoter sequences, study the estrogen effects on promoter’s activity. The experimental results are as follows:
1. Cloned sequence of FAS gene promoter is 2640bp, The Genbank number of the sequence is EF556550 (published).
2. Sequence analysis shows that the homology of Xinong Saanen Goat FAS gene promoter with bovine and homo is over 90%, there are several potential transcription factor binding sites such as SP1、Ets、LSF. There is no typical TATA box, and similar CCAAT box and GC box exist. The core promoter sequences potentially located in -1040bp~-340bp.
3. Luciferase assays of different FAS gene promoter deletion fragments shows that the potential negative control element may located in forepart of FAS gene promoter, The core promoter sequences potentially located in-721~-540bp, and there is no promoter activity sequencese after-540bp.
4. Use estrogen of concentration gradient on the core promoter sequence, we draw a conclusion that the estrogen binding domain exsit in-721~-540bp. When the consentration of estrogen is 4-100μmol/L, estrogen has a significant effect on the improvement of promoter activity, the improvement was most remarkable when the estrogen concentration is 20μmol/L.
本研究以西北农林科技大学萨能羊原种场健康的纯种西农萨能奶山羊为试验材料,扩增获得了西农萨能奶山羊乳腺FAS基因启动子序列全长,并对其进行了生物信息学分析;采用荧光素酶活性分析的方法检测了不同启动子缺失片段(pGL3—E、pGL3—3、pGL3—4、pGL3—5、pGL3—6、pGL3—8、pGL3—10、pGL3—14、pGL3—19、pGL3—21)的启动活性。用不同浓度的雌激素作用于启动子的最小活性中心,研究雌激素对启动子活性的影响。试验结果如下:
1.克隆获得了西农萨能奶山羊FAS基因启动子全长2640bp,登录GenBank收录号为EF556550(数据已公布)。
2.对克隆得出的启动子序列进行生物信息学分析,表明羊FAS基因启动子和牛、人FAS启动子存在90%以上同源性,启动子还存在数个潜在SP1、Ets、LSF等转录因子结合位点。没有典型的TATA框,存在CCAAT框和GC框,在-1040bp~-340bp处可能存在启动子活性中心。
3.启动子缺失片段的荧光素酶活性分析表明,启动子前段区域可能存在负调控元件,-721~-540bp中间存在着FAS启动子的核心区域,-540bp后无启动子活性序列。
4.雌激素对FAS启动子活性影响试验表明,-721~-540bp序列存在雌激素的结合区域。雌激素浓度为4-100μmol/L时,雌激素对FAS启动子活性有显著的提高作用。其中20μmol/L时作用最为显著。
Fatty acid synthase(FAS) plays an important role in de novo fatty acids synthesis in mammals. FAS helps to catalyze all the reaction steps in the conversion of acety1-CoA, malonyl-CoA and NADPH to saturated fatty acids. Short- and medium- chain fatty acids in goat mammary synthesis are functioned by a transferase reaction in the region of Acetyl-CoA and malonyl-CoA transacylases(AT/MT). It’s one of the important means to solve goat milk odor from genetics and breeding aspect by regulating FAS gene expression in order to change fatty acids composition in goat mammary gland. So the study of FAS gene promotor’s struture and function can further reveal the regulated informaton and its interacted mechanism of which FAS gene expression depending on. Therefore, this study take FAS gene promoter active and function as a point, cloned the FAS gene promoter, sequenced and bioinformation analyzed it. Construct PGL-3 expression vector based on structure foundation, found out core promoter sequences. Use estrogen of concentration gradient on the core promoter sequence, further explore its starting activity and function regulation. Laid a foundation for FAS gene founctional regulation research. This study aims at consummating fundamental research of Xinong Saanen Goat FAS gene promoter, Provided experimental basis on Goat Mammary Gland fatty acid metabolism,FAS gene’s function and genetic regulation mechnism of goat milk flavor by mastering the FAS promoter’s regulation rules.
The study uses purebred and healthy Xinong saanen goat in Xinong Saanen Goat Breeding Farm of Northwest A&F University as experimental materials, cloned and analyzed the sequence of Xinong Saanen Dairy Goat mammary FAS gene promoter full length region; Detected activity of different promoter deletion fragments(pGL3—E, pGL3—3, pGL3—4, pGL3—5, pGL3—6, pGL3—8, pGL3—10, pGL3—14, pGL3—19, pGL3—21)by luciferase assays. Selected estrogen of different concentrations act on the core promoter sequences, study the estrogen effects on promoter’s activity. The experimental results are as follows:
1. Cloned sequence of FAS gene promoter is 2640bp, The Genbank number of the sequence is EF556550 (published).
2. Sequence analysis shows that the homology of Xinong Saanen Goat FAS gene promoter with bovine and homo is over 90%, there are several potential transcription factor binding sites such as SP1、Ets、LSF. There is no typical TATA box, and similar CCAAT box and GC box exist. The core promoter sequences potentially located in -1040bp~-340bp.
3. Luciferase assays of different FAS gene promoter deletion fragments shows that the potential negative control element may located in forepart of FAS gene promoter, The core promoter sequences potentially located in-721~-540bp, and there is no promoter activity sequencese after-540bp.
4. Use estrogen of concentration gradient on the core promoter sequence, we draw a conclusion that the estrogen binding domain exsit in-721~-540bp. When the consentration of estrogen is 4-100μmol/L, estrogen has a significant effect on the improvement of promoter activity, the improvement was most remarkable when the estrogen concentration is 20μmol/L.
引文
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