杜长大猪UCP3基因克隆及白藜芦醇对其表达的影响
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摘要
【目的】明确白藜芦醇对杜长大猪UCP3基因表达及脂肪代谢的影响,为揭示猪优良肉质性状形成机制打下基础。【方法】提取2月龄杜长大猪皮下脂肪组织RNA,采用RT-PCR扩增杜长大猪UCP3基因编码区(CDS)序列,运用在线软件对其进行生物信息学分析,并以实时荧光定量PCR检测白藜芦醇(400 mg/kg,饲喂30 d)对杜长大猪脂肪中UCP3基因表达的影响。【结果】从杜长大猪皮下脂肪组织中成功克隆获得UCP3基因CDS序列,全长927 bp,共编码308个氨基酸,其蛋白分子质量33673.09 Da,理论等电点(pI)9.44;与普通猪的UCP3基因序列相比,杜长大猪UCP3基因编码序列共有9处发生碱基突变,其中4处为错义突变,导致UCP3蛋白有4处氨基酸突变,分别为第74位脯氨酸(Pro)→亮氨酸(Leu)、第150位甘氨酸(Gly)→精氨酸(Arg)、第203位Arg→Gly和第259位缬氨酸(Val)→Leu。杜长大猪UCP3基因CDS序列与普通猪UCP3基因CDS序列的同源性为99.0%,其UCP3蛋白二级结构中以α-螺旋占比最高(46.43%)、β-转角占比最低(7.47%),属于亲水性蛋白。饲喂白藜芦醇(400 mg/kg)能极显著上调杜长大猪皮下脂肪UCP3基因表达(P<0.01),其相对表达量是对照组(不饲喂白藜芦醇)的2.06倍。【结论】白藜芦醇是通过上调杜长大猪UCP3基因表达而影响其脂肪代谢。
【Objective】The purpose of the study was to clarify the effects of resveratrol on UCP3 gene expression and fat metabolism in Duroc-Landrace-Yorkshire pigs,and to lay the foundation for revealing the formation mechanism of pig meat quality traits.【Method】The total RNA of subcutaneous adipose tissue of two months old Duroc-Landrace-Yorkshire pig was extracted,and the coding region(CDS)sequence of Duroc-Landrace-Yorkshire pig UCP3 gene was amplified by PCR and analyzed by online bioinformatics software. Real-time quantitative PCR was used to detect the effect of resveratrol(400 mg/kg,fed for 30 d)on UCP3 gene expression in Duroc-Landrace-Yorkshire pig fat.【Result】The UCP3 gene CDS sequence was successfully cloned from the subcutaneous adipose tissue of Duroc-Landrace-Yorkshire pig,which was 927 bp long and encoded a total of 308 amino acids. Its molecular mass was 33673.09 Da,and the theoretical isoelectric point(pI)was 9.44. Compared with the ordinary pig gene sequence,there were nine base mutations in the UCP3 gene coding sequence of Duroc-Landrace-Yorkshire pig,four of which were missense mutations,resulting in four amino acid mutations in UCP3 protein. They were the 74 thproline(Pro)→leucine(Leu),the 150 thglycine(Gly)→arginine(Arg),the203 thArg→Gly and the 259 thproline(Val))→Leu. The homology of the UCP3 gene CDS sequence to the ordinary pig was99.0%,and the UCP3 protein secondary structure had the highest α-helix ratio(46.43%)and low β-turn angle ratio(7.47%). It was a hydrophilic protein. Resveratrol(400 mg/kg)extremely up-regulated UCP3 gene expression in subcutaneous fat of Duroc-Landrace-Yorkshire pigs(P<0.01). The relative expression was 2.06 times as that of the control group(no resveratrol).【Conclusion】Resveratrol affects its fat metabolism by up-regulating the expression of UCP3 gene in Duroc-Landrace-Yorkshire pigs.
【Objective】The purpose of the study was to clarify the effects of resveratrol on UCP3 gene expression and fat metabolism in Duroc-Landrace-Yorkshire pigs,and to lay the foundation for revealing the formation mechanism of pig meat quality traits.【Method】The total RNA of subcutaneous adipose tissue of two months old Duroc-Landrace-Yorkshire pig was extracted,and the coding region(CDS)sequence of Duroc-Landrace-Yorkshire pig UCP3 gene was amplified by PCR and analyzed by online bioinformatics software. Real-time quantitative PCR was used to detect the effect of resveratrol(400 mg/kg,fed for 30 d)on UCP3 gene expression in Duroc-Landrace-Yorkshire pig fat.【Result】The UCP3 gene CDS sequence was successfully cloned from the subcutaneous adipose tissue of Duroc-Landrace-Yorkshire pig,which was 927 bp long and encoded a total of 308 amino acids. Its molecular mass was 33673.09 Da,and the theoretical isoelectric point(pI)was 9.44. Compared with the ordinary pig gene sequence,there were nine base mutations in the UCP3 gene coding sequence of Duroc-Landrace-Yorkshire pig,four of which were missense mutations,resulting in four amino acid mutations in UCP3 protein. They were the 74 thproline(Pro)→leucine(Leu),the 150 thglycine(Gly)→arginine(Arg),the203 thArg→Gly and the 259 thproline(Val))→Leu. The homology of the UCP3 gene CDS sequence to the ordinary pig was99.0%,and the UCP3 protein secondary structure had the highest α-helix ratio(46.43%)and low β-turn angle ratio(7.47%). It was a hydrophilic protein. Resveratrol(400 mg/kg)extremely up-regulated UCP3 gene expression in subcutaneous fat of Duroc-Landrace-Yorkshire pigs(P<0.01). The relative expression was 2.06 times as that of the control group(no resveratrol).【Conclusion】Resveratrol affects its fat metabolism by up-regulating the expression of UCP3 gene in Duroc-Landrace-Yorkshire pigs.
引文
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刘颖,张璐,窦博鑫,关天琪,王秋,石彦国,KALENIK Tatyana K.2013.白藜芦醇对酒精性肝损伤大鼠保护作用的研究[J].哈尔滨商业大学学报(自然科学版),31(1):14-17.[Liu Y,Zhang L,Dou B X,Guan T Q,Wang Q,Shi Y G,Kalenik T K.2013.Study on protective effect of resveratrol on alcoholic liver injury in rats[J].Journal of Harbin University of Commerce(Natural Sciences Edition),31(1):14-17.]
孙延权,陈思凡,朱伟,郑琳,张子丽,凌文华,冯翔.2011.白藜芦醇对KKAy小鼠体质量和脂肪分布的影响及机制[J].食品科学,32(13):289-292.[Sun Y Q,Chen S F,Zhu W,Zheng L,Zhang Z L,Ling W H,Feng X.2011.Effect and mechanism of resveratrol on body weight and adipose tissue distribution of KKAy mice[J].Food Science,32(13):289-292.]
王晓珂,刘天娥,蒋建中,张璇,陈刚,赵健亚.2013.白藜芦醇对肥胖易感和肥胖抵抗大鼠体重的影响[J].营养学报,35(6):577-581.[Wang X K,Liu T E,Jiang J Z,Zhang X,Chen G,Zhao J Y.2013.Effects of resveratrol on body weight in obesity prone and obesity resistant rats fed high-fat diet[J].Acta Nutrimenta Sinica,35(6):577-581.]
王筱婧,林焕冰,祝倩,徐春静.2015.白藜芦醇对大鼠急性酒精性肝损伤的保护作用[J].免疫学杂志,31(11):954-958.[Wang X J,Lin H B,Zhu Q,Xu C J.2015.Protective effects of resveratrol on alcohol-induced acute hepatic injury in rats[J].Immunological Journal,31(11):954-958.]
韦晓,汪奇峰,黄慧,刘超.2018.白藜芦醇治疗糖尿病的分子作用机制研究进展[J].中国药房,29(18):2576-2580.[Wei X,Wang Q F,Huang H,Liu C.2018.Advances in molecular mechanism of resveratrol in the treatment of diabetes[J].Chinese Pharmacy,29(18):2576-2580.]
杨占清,李洋,刘颖,魏利斌,付守鹏,柳巨雄.2013.冷应激对猪解耦联蛋白3 m RNA表达的影响[J].黑龙江畜牧兽医,(10):68-70.[Yang Z Q,Li Y,Liu Y,Wei L F,Fu SP,Liu J X.2013.Effect of cold stress on the expression of decoupling protein 3 mRNA in pigs[J].Heilongjiang Animal Science and Veterinary Medicine,(10):68-70.]
于飞.2015.白藜芦醇对小鼠脂肪代谢的影响研究[D].泰安:山东农业大学.[Yu F.2015.Effect of resveratrol on fat metabolism in mice[D].Tai’an:Shandong Agricultural University.]
赵建国,李辉,孟和,顾志良,王启贵,王宇祥.2002.解偶联蛋白基因(UCP)作为影响鸡脂肪性状候选基因的研究[J].遗传学报,29(6):481-486.[Zhao J G,Li H,Meng H,Gu Z L,Wang Q G,Wang Y X.2002.The study of the uncoulping protein gene as the candidate gene for fatness traits in chicken[J].Acta Genetica Sinica,29(6):481-486.]
郑新杰,刘子惠,廖维瑶,李青蓉,郑琳,罗晓林,冯翔.2017.白藜芦醇对高脂诱导肥胖小鼠脂肪组织分布的影响及其机制[J].中国热带医学,17(12):1176-1180.[Zheng X J,Liu Z H,Liao W Y,Li Q R,Zheng L,Luo X L,Feng X.2017.Effects and mechanism of resveratrol on adipose tissue distribution of mice fed a high-fat diet[J].China Tropical Medicine,17(12):1176-1180.]
周逸亭,万云,黄杉,关蓉媛,张朝云,李益明,张烁,叶红英.2014.白藜芦醇改善高脂饮食小鼠的糖代谢并棕化腹股沟白色脂肪[J].上海医药,35(1):55-58.[Zhou Y T,Wan Y,Huang S,Guan R Y,Zhang C Y,Li Y M,Zhang S,Ye H Y.2014.Resveratrol exerts anti-obesity effects while browning the inguinal white adipose tissue[J].Shanghai Medical&Pharmaceutical Journal,35(1):55-58.]
Fabirs R,Nisoli E,Lombardi A M,Tonello C,Serra R,Granzotto M,Cusin I,Rohner-Jeanrenaud F,Federspil G,Carruba M O,Vettor R.2001.Preferential channeling of energy fuels towards fat rather muscle during high free faty acid availability in rats[J].Diabetes,50(3):601-608.
Gong D W,Monemdjou S,Gavriova O,Leon L R,MarcusSamuels B,Chou C J,Everett C,Kozak L P,Li C,Deng C,Harper M E,Reitman M L.2000.Lack of obesity and normal response to fasting and thyroid hormone in mice lacking uncoupling proteins-3[J].The Journal of Biological Chemistry,275(21):16251-16257.
Seifert E L,Bézaire V,Estey C,Harper M E.2008.Essential role for uncoupling protein-3 in mitochondrial adaptation to fasting but not in fatty acid oxidation to fastty acid anion export[J].The Journal of Biological Chemistry,283(37):25124-25131.
Zhang C,Luo J Q,Yu B,Chen J L,Chen D W.2015.Effects of resveratrol on lipid metabolism in muscle and adipose tissues:A reevaluation in a pig model[J].Journal of Functional Foods,14:590-595.
陈锋,刘志民,赵瑛.2010.白藜芦醇对体外高糖刺激下小鼠脑微血管内皮细胞解偶联蛋白基因表达的影响[J].第二军医大学学报,31(9):952-954.[Chen F,Liu Z M,Zhao Y.2010.Effects of resveratrol on uncoupling protein mRNA expression in brain microvessel endothelial cells under hyperglycemic condition in vitro[J].Academic Journal of Second Military Medical University,31(9):952-954.]
丛军,高弼虎.2012.解偶联蛋白生理功能和调节、周转的研究新进展[J].临床荟萃,27(18):1649-1653.[Cong J,Gao B H.2012.New advances in the study of physiological function,regulation and turnover of uncoupling proteins[J].Clinical Focus,27(18):1649-1653.]
高文荣,张麟,余婷婷,朱万龙,王政昆.2012.解偶联蛋白-1(UCP1)的研究进展及其应用前景[J].生物过程,2(3):128-137.[Gao W R,Zhang L,Yu T T,Zhu W L,Wang Z K.2012.The Research progress and application prospects of uncoupling protein-1(UCP1)[J].Bioprocess,2(3):128-137.]
韩晶晶,刘炜,毕玉平.2008.白藜芦醇的研究进展[J].生物工程学报,24(11):1851-1859.[Han J J,Liu W,Bi Y P.2008.Advances in resveratrol studies[J].Chinese Journal of Biotechnology,24(11):1851-1859.]
孔勇,宋学琴,吴红然.2010.解耦联蛋白3的研究进展[J].医学综述,16(24):3693-3696.[Kong Y,Song X Q,Wu HR.2010.Study of uncoupling protein 3[J].Medical Recapitulate,16(24):3693-3696.]
刘颖,张璐,窦博鑫,关天琪,王秋,石彦国,KALENIK Tatyana K.2013.白藜芦醇对酒精性肝损伤大鼠保护作用的研究[J].哈尔滨商业大学学报(自然科学版),31(1):14-17.[Liu Y,Zhang L,Dou B X,Guan T Q,Wang Q,Shi Y G,Kalenik T K.2013.Study on protective effect of resveratrol on alcoholic liver injury in rats[J].Journal of Harbin University of Commerce(Natural Sciences Edition),31(1):14-17.]
孙延权,陈思凡,朱伟,郑琳,张子丽,凌文华,冯翔.2011.白藜芦醇对KKAy小鼠体质量和脂肪分布的影响及机制[J].食品科学,32(13):289-292.[Sun Y Q,Chen S F,Zhu W,Zheng L,Zhang Z L,Ling W H,Feng X.2011.Effect and mechanism of resveratrol on body weight and adipose tissue distribution of KKAy mice[J].Food Science,32(13):289-292.]
王晓珂,刘天娥,蒋建中,张璇,陈刚,赵健亚.2013.白藜芦醇对肥胖易感和肥胖抵抗大鼠体重的影响[J].营养学报,35(6):577-581.[Wang X K,Liu T E,Jiang J Z,Zhang X,Chen G,Zhao J Y.2013.Effects of resveratrol on body weight in obesity prone and obesity resistant rats fed high-fat diet[J].Acta Nutrimenta Sinica,35(6):577-581.]
王筱婧,林焕冰,祝倩,徐春静.2015.白藜芦醇对大鼠急性酒精性肝损伤的保护作用[J].免疫学杂志,31(11):954-958.[Wang X J,Lin H B,Zhu Q,Xu C J.2015.Protective effects of resveratrol on alcohol-induced acute hepatic injury in rats[J].Immunological Journal,31(11):954-958.]
韦晓,汪奇峰,黄慧,刘超.2018.白藜芦醇治疗糖尿病的分子作用机制研究进展[J].中国药房,29(18):2576-2580.[Wei X,Wang Q F,Huang H,Liu C.2018.Advances in molecular mechanism of resveratrol in the treatment of diabetes[J].Chinese Pharmacy,29(18):2576-2580.]
杨占清,李洋,刘颖,魏利斌,付守鹏,柳巨雄.2013.冷应激对猪解耦联蛋白3 m RNA表达的影响[J].黑龙江畜牧兽医,(10):68-70.[Yang Z Q,Li Y,Liu Y,Wei L F,Fu SP,Liu J X.2013.Effect of cold stress on the expression of decoupling protein 3 mRNA in pigs[J].Heilongjiang Animal Science and Veterinary Medicine,(10):68-70.]
于飞.2015.白藜芦醇对小鼠脂肪代谢的影响研究[D].泰安:山东农业大学.[Yu F.2015.Effect of resveratrol on fat metabolism in mice[D].Tai’an:Shandong Agricultural University.]
赵建国,李辉,孟和,顾志良,王启贵,王宇祥.2002.解偶联蛋白基因(UCP)作为影响鸡脂肪性状候选基因的研究[J].遗传学报,29(6):481-486.[Zhao J G,Li H,Meng H,Gu Z L,Wang Q G,Wang Y X.2002.The study of the uncoulping protein gene as the candidate gene for fatness traits in chicken[J].Acta Genetica Sinica,29(6):481-486.]
郑新杰,刘子惠,廖维瑶,李青蓉,郑琳,罗晓林,冯翔.2017.白藜芦醇对高脂诱导肥胖小鼠脂肪组织分布的影响及其机制[J].中国热带医学,17(12):1176-1180.[Zheng X J,Liu Z H,Liao W Y,Li Q R,Zheng L,Luo X L,Feng X.2017.Effects and mechanism of resveratrol on adipose tissue distribution of mice fed a high-fat diet[J].China Tropical Medicine,17(12):1176-1180.]
周逸亭,万云,黄杉,关蓉媛,张朝云,李益明,张烁,叶红英.2014.白藜芦醇改善高脂饮食小鼠的糖代谢并棕化腹股沟白色脂肪[J].上海医药,35(1):55-58.[Zhou Y T,Wan Y,Huang S,Guan R Y,Zhang C Y,Li Y M,Zhang S,Ye H Y.2014.Resveratrol exerts anti-obesity effects while browning the inguinal white adipose tissue[J].Shanghai Medical&Pharmaceutical Journal,35(1):55-58.]
Fabirs R,Nisoli E,Lombardi A M,Tonello C,Serra R,Granzotto M,Cusin I,Rohner-Jeanrenaud F,Federspil G,Carruba M O,Vettor R.2001.Preferential channeling of energy fuels towards fat rather muscle during high free faty acid availability in rats[J].Diabetes,50(3):601-608.
Gong D W,Monemdjou S,Gavriova O,Leon L R,MarcusSamuels B,Chou C J,Everett C,Kozak L P,Li C,Deng C,Harper M E,Reitman M L.2000.Lack of obesity and normal response to fasting and thyroid hormone in mice lacking uncoupling proteins-3[J].The Journal of Biological Chemistry,275(21):16251-16257.
Seifert E L,Bézaire V,Estey C,Harper M E.2008.Essential role for uncoupling protein-3 in mitochondrial adaptation to fasting but not in fatty acid oxidation to fastty acid anion export[J].The Journal of Biological Chemistry,283(37):25124-25131.
Zhang C,Luo J Q,Yu B,Chen J L,Chen D W.2015.Effects of resveratrol on lipid metabolism in muscle and adipose tissues:A reevaluation in a pig model[J].Journal of Functional Foods,14:590-595.