陆川猪FTO基因的克隆及生物信息学分析
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摘要
为了获得广西陆川猪肥胖相关基因(FTO)编码区序列(CDS),试验利用基因克隆技术,从陆川猪背最长肌组织中提取总RNA,扩增出FTO基因CDS,并运用Lasergene 7.0、ExPASy ProtParam、ExPASy ProtScale、NPS@在线蛋白分析系统和SWISS-MODEL在线分析预测系统等分析软件对扩增出的序列进行生物信息学分析。结果表明:通过克隆测序得到的陆川猪FTO基因CDS全长为1 518 bp,与GenBank中公布的乌金猪FTO基因CDS的同源性为99.9%,与苏钟猪FTO基因CDS的同源性为99.6%;陆川猪存在第1 050位点的C→G,碱基G为陆川猪所特有,导致天冬氨酸突变为谷氨酸;通过建立系统进化树发现,陆川猪与乌金猪的遗传距离最近,它们聚为一支;陆川猪FTO氨基酸组成中亮氨酸含量最高,占总氨基酸的比例为11.7%,FTO蛋白具有较强的亲水性;陆川猪FTO蛋白的二级、三级结构预测显示包含α-螺旋、无规则卷曲和延伸链。说明陆川猪FTO基因具有高度的保守性,今后在陆川猪脂肪沉积研究中, FTO基因可作为主要候选基因之一。
To obtain the coding sequence(CDS) of obesity related gene(FTO) in Luchuan pig, total RNA was extracted from the longissimus dorsi muscle of Luchuan pig, and the CDS of FTO was amplified by gene cloning. Then the amplified sequences were analyzed by using Lasergene 7.0, ExPASy ProtParam, ExPASy ProtScale, NPS@ online protein analysis system and SWISS-MODEL online analysis prediction system etc. The results showed that the length of the FTO gene CDS in Luchuan pig was 1 518 bp. Compared with the FTO gene CDS of Wujin pig and Suzhong pig in GenBank, the homology was 99.9% and 99.6%, respectively. The mutation(C→G) at the 1 050 site of FTO gene in Luchuan pig resulted in the mutation of aspartic acid to glutamic acid. The mutated base G was unique to Luchuan pig. Through the establishment of phylogenetic tree, it was found that Luchuan pig and Wujin pig had the closest genetic distance, and they were clustered into the same branch. The content of leucine in the Luchuan pig FTO amino acid composition was the highest, accounting for 11.7% of the total amino acids. At the same time, it was found that the FTO protein had a strong hydrophilic property. The prediction of secondary and tertiary structure of FTO protein in Luchuan pig showed that there were α-helix, random coil and extended chain. The results indicated that the porcine FTO gene was highly conserved, and FTO gene could be considered as one of the major candidate genes in the future studies of fat deposition in Luchuan pigs.
To obtain the coding sequence(CDS) of obesity related gene(FTO) in Luchuan pig, total RNA was extracted from the longissimus dorsi muscle of Luchuan pig, and the CDS of FTO was amplified by gene cloning. Then the amplified sequences were analyzed by using Lasergene 7.0, ExPASy ProtParam, ExPASy ProtScale, NPS@ online protein analysis system and SWISS-MODEL online analysis prediction system etc. The results showed that the length of the FTO gene CDS in Luchuan pig was 1 518 bp. Compared with the FTO gene CDS of Wujin pig and Suzhong pig in GenBank, the homology was 99.9% and 99.6%, respectively. The mutation(C→G) at the 1 050 site of FTO gene in Luchuan pig resulted in the mutation of aspartic acid to glutamic acid. The mutated base G was unique to Luchuan pig. Through the establishment of phylogenetic tree, it was found that Luchuan pig and Wujin pig had the closest genetic distance, and they were clustered into the same branch. The content of leucine in the Luchuan pig FTO amino acid composition was the highest, accounting for 11.7% of the total amino acids. At the same time, it was found that the FTO protein had a strong hydrophilic property. The prediction of secondary and tertiary structure of FTO protein in Luchuan pig showed that there were α-helix, random coil and extended chain. The results indicated that the porcine FTO gene was highly conserved, and FTO gene could be considered as one of the major candidate genes in the future studies of fat deposition in Luchuan pigs.
引文
[1] 王辉, 孟雁. 肥胖基因FTO的研究进展 [J]. 国际内分泌代谢杂志,2010,30(2):116-118.
[2] FISCHER J, KOCH L, EMMERLING C, et al. Inactivation of the Fto gene protects from obesity[J]. Nature, 2009, 458(7240): 894-898.
[3] GERKEN T, GIRARD C A, TUNG Y C, et al. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase [J]. Science, 2007, 318(5855): 1469-1472.
[4] HAUPT A, THAMER C, STAIGER H, et al. Variation in the FTO gene influences food intake but not energy expenditure [J]. Exp Clin Endocr Diab, 2009, 117(4):194-197.
[5] 陈静, 孙琳. FTO基因rs9939609研究进展 [J]. 中国医药导刊, 2011, 13(4): 682-683.
[6] DU Z Q, FAN B, ZHAO X, et al. Association analyses between type 2 diabetes genes and obesity traits in pigs [J]. Obesity, 2009, 17(2): 323-329.
[7] 黄英, 杨明华, 毕保良, 等. 乌金猪FTO基因的克隆和表达分析[J]. 生物技术通讯, 2014,25(3):316-322.
[8] 付言峰, 李兰, 王学敏, 等. 苏钟猪FTO基因的mRNA表达、克隆测序及其生物信息学分析 [J]. 华北农学报, 2013, 28(1): 128-134.
[9] FREDRIKSSON R, HAGGLUND M, OLSZEWSKI P K, et al. The obesity gene, FTO, is of ancient origin, up regulated during food deprivation and expressed in neurons of feeding-related nuclei of the brain[J]. Endocrinology, 2008, 149 (5): 2062-2071.
[10]林亚秋, 廖红海, 贺庆华, 等. 山羊FTO基因克隆及其表达谱 [J]. 畜牧兽医学报, 2016, 47(5): 888-898.
[11]李倩, 林亚秋, 廖红海, 等. 成都麻羊FTO基因组织表达及其与IMF含量的相关性研究 [J]. 黑龙江畜牧兽医, 2016(04上): 97-99.
[12]林森, 林亚秋, 朱江江, 等. 藏鸡FTO基因表达的时空特性及与肌内脂肪含量的相关分析[J]. 畜牧兽医学报, 2017,48(7): 1191-1201.
[13]MADSEN M B, BIRCK M M, CIRERA S, et al. Expression studies of the obesity candidate gene FTO in pig [J]. Anim Biotechnol, 2010,21(1):51-63.
[14]陈友贵, 冯文武, 龙威海, 等. 白洗猪不同组织FTO基因的表达研究[J]. 基因组学与应用生物学, 2015, 34(5): 926-930.
[15]朱琳娜.FTO、METTL3基因表达对猪脂肪细胞mRNA N6-甲基腺苷水平及脂肪沉积的影响研究[D]. 杭州: 浙江大学, 2014.
[16]HU Z L, REECY J M. Animal QTLdb: beyond a repository[J]. Mamm Genome, 2007, 18:1-4.
[2] FISCHER J, KOCH L, EMMERLING C, et al. Inactivation of the Fto gene protects from obesity[J]. Nature, 2009, 458(7240): 894-898.
[3] GERKEN T, GIRARD C A, TUNG Y C, et al. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase [J]. Science, 2007, 318(5855): 1469-1472.
[4] HAUPT A, THAMER C, STAIGER H, et al. Variation in the FTO gene influences food intake but not energy expenditure [J]. Exp Clin Endocr Diab, 2009, 117(4):194-197.
[5] 陈静, 孙琳. FTO基因rs9939609研究进展 [J]. 中国医药导刊, 2011, 13(4): 682-683.
[6] DU Z Q, FAN B, ZHAO X, et al. Association analyses between type 2 diabetes genes and obesity traits in pigs [J]. Obesity, 2009, 17(2): 323-329.
[7] 黄英, 杨明华, 毕保良, 等. 乌金猪FTO基因的克隆和表达分析[J]. 生物技术通讯, 2014,25(3):316-322.
[8] 付言峰, 李兰, 王学敏, 等. 苏钟猪FTO基因的mRNA表达、克隆测序及其生物信息学分析 [J]. 华北农学报, 2013, 28(1): 128-134.
[9] FREDRIKSSON R, HAGGLUND M, OLSZEWSKI P K, et al. The obesity gene, FTO, is of ancient origin, up regulated during food deprivation and expressed in neurons of feeding-related nuclei of the brain[J]. Endocrinology, 2008, 149 (5): 2062-2071.
[10]林亚秋, 廖红海, 贺庆华, 等. 山羊FTO基因克隆及其表达谱 [J]. 畜牧兽医学报, 2016, 47(5): 888-898.
[11]李倩, 林亚秋, 廖红海, 等. 成都麻羊FTO基因组织表达及其与IMF含量的相关性研究 [J]. 黑龙江畜牧兽医, 2016(04上): 97-99.
[12]林森, 林亚秋, 朱江江, 等. 藏鸡FTO基因表达的时空特性及与肌内脂肪含量的相关分析[J]. 畜牧兽医学报, 2017,48(7): 1191-1201.
[13]MADSEN M B, BIRCK M M, CIRERA S, et al. Expression studies of the obesity candidate gene FTO in pig [J]. Anim Biotechnol, 2010,21(1):51-63.
[14]陈友贵, 冯文武, 龙威海, 等. 白洗猪不同组织FTO基因的表达研究[J]. 基因组学与应用生物学, 2015, 34(5): 926-930.
[15]朱琳娜.FTO、METTL3基因表达对猪脂肪细胞mRNA N6-甲基腺苷水平及脂肪沉积的影响研究[D]. 杭州: 浙江大学, 2014.
[16]HU Z L, REECY J M. Animal QTLdb: beyond a repository[J]. Mamm Genome, 2007, 18:1-4.