黔北麻羊FTO基因SNPs筛选及其生物信息学分析
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摘要
筛选150只黔北麻羊(0.5岁、1岁、1.5岁各50只)FTO基因的SNPs,为进一步开展FTO基因多态性与山羊肉质性状相关性研究提供参考。通过构建DNA混合池结合PCR产物直接测序技术,对黔北麻羊FTO基因外显子区域进行多态性分析,采用生物信息学软件对PCR扩增所获序列进行m RNA二级结构分析及对SNPs突变位点峰高进行等位基因频率估算,并应用相应软件对FTO基因编码的蛋白质结构、理化性质、信号肽、跨膜结构、亚细胞定位等结构进行生物信息学分析。在FTO基因外显子区域共筛选出8个SNPs,分别为Exon2-C139T、Exon3-A242G、Exon3-T282A、Exon3-C339T、Exon3-G387T、Exon3-T399C、Exon9-C1615G、Exon9-G1718A。分析表明,Exon3-A242G、Exon3-T282A、Exon3-C339T、Exon3-G387T、Exon3-T399C位点的突变在CDS区内,其中Exon3-A242G(Gln-His)、Exon3-G387T引起m RNA二级结构的改变,Exon3-C339T、Exon3-G387T突变导致其m RNA二级结构最小自由能增大,即二级结构稳定性降低。对FTO基因序列进行生物信息学分析,得出FTO是一种非分泌型的亲水性蛋白,不存在跨膜区域,发挥生物学作用的主要场所是细胞质,有7个Ser位点,5个Thr位点,5个Tyr位点,二级结构中ɑ螺旋结构数量最多。
A total of 150 SNPs from FTO gene were screened from 150 Qianbei Ma goat to provide reference for further studies on the association between FTO gene polymorphisms and meat quality traits in goats. By constructing a mixed DNA pool and direct PCR product sequencing technique, the polymorphism of the FTO gene exon of Qianbei Ma goat was analyzed, and the secondary structure of m RNA and SNPs were analyzed by using bioinformatics software for the PCR amplification sequence. The allele frequency was estimated at the peak height of the mutation site. Bioinformatics analysis of the protein structure, physicochemical properties, signal peptide, transmembrane structure, and subcellular localization of theFTO gene was performed using the corresponding software. Eight SNPs were selected in the exon region of FTO gene, Exon2-C139 T, Exon3-A242 G, Exon3-T282 A, Exon3-C339 T, Exon3-G387 T, Exon3-T399 C, Exon9-C1615 G, and Exon9-G1718 A. The analysis showed that mutations in Exon3-A242 G, Exon3-T282 A, Exon3-C339 T, Exon3-G387 T, and Exon3-T399 C sites were within the CDS region, in which Exon3-A242 G(Gln-His) and Exon3-G387 T caused changes in m RNA secondary structure. Among them, the Exon3-C339 T and Exon3-G387 T mutations resulted in the increase of the minimum free energy of the m RNA secondary structure, ie, the decrease of the secondary structure stability. The bioinformatics analysis of the FTO gene sequence revealed that FTO is a non-secreting hydrophilic protein and that there is no transmembrane region. The main site of biological function is cytoplasm, there are 7 Ser sites, 5 at the Thr site, 5 Tyr sites, the number of helium helix structures in the secondary structure was the most.
A total of 150 SNPs from FTO gene were screened from 150 Qianbei Ma goat to provide reference for further studies on the association between FTO gene polymorphisms and meat quality traits in goats. By constructing a mixed DNA pool and direct PCR product sequencing technique, the polymorphism of the FTO gene exon of Qianbei Ma goat was analyzed, and the secondary structure of m RNA and SNPs were analyzed by using bioinformatics software for the PCR amplification sequence. The allele frequency was estimated at the peak height of the mutation site. Bioinformatics analysis of the protein structure, physicochemical properties, signal peptide, transmembrane structure, and subcellular localization of theFTO gene was performed using the corresponding software. Eight SNPs were selected in the exon region of FTO gene, Exon2-C139 T, Exon3-A242 G, Exon3-T282 A, Exon3-C339 T, Exon3-G387 T, Exon3-T399 C, Exon9-C1615 G, and Exon9-G1718 A. The analysis showed that mutations in Exon3-A242 G, Exon3-T282 A, Exon3-C339 T, Exon3-G387 T, and Exon3-T399 C sites were within the CDS region, in which Exon3-A242 G(Gln-His) and Exon3-G387 T caused changes in m RNA secondary structure. Among them, the Exon3-C339 T and Exon3-G387 T mutations resulted in the increase of the minimum free energy of the m RNA secondary structure, ie, the decrease of the secondary structure stability. The bioinformatics analysis of the FTO gene sequence revealed that FTO is a non-secreting hydrophilic protein and that there is no transmembrane region. The main site of biological function is cytoplasm, there are 7 Ser sites, 5 at the Thr site, 5 Tyr sites, the number of helium helix structures in the secondary structure was the most.
引文
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[11] Szydlowski M, Salamon S, Grzes M, et al. SNP in the 5′flanking region of the pig FTO gene is associated with fatness in Polish Landrace[J]. Livest Sci, 2012, 150(1-3):397-400.
[12] Zhang L F, Miao X T, Hua X C, et al. Polymorphism in 5'regulatory region of the porcine fat mass and obesity associated(FTO)gene is associated with intramuscular fat content in a jinhua x pietrain freference population[J]. J Anim Vet Adv,2012(11):2329-2334.
[13] Gan W, Song Q, Zhang N N, et al. Association between FTO polymorphism in exon 3 with carcass and meat quality traits in crossbred ducks[J]. Gen Mol Res, 2015, 14(2):6699-6714.
[14]康晓龙,张莉莉,王昊升,等. FTO基因多态性及其与2个绵羊群体生长性状的相关性[J].西南农业学报, 2017,30(11):2603-2608.
[2] Frayling T M, Timpson N J, Weedon M N, et al. A common variant in the FTO gene is associated with Body Mass Index and predisposes to childhood and adult obesity[J]. Science,2007, 316(5826):889-894.
[3] Zhang G W, Gao L, Chen S Y, et al. Single nucleotide polymorphisms in the FTO gene and their association with growth and meat quality traits in rabbits[J]. Gene, 2013, 527(2):553-557.
[4] Bin F, Zhi Q D, Max F. Rothschild. The Fat mass and obesityassociated(FTO)gene is associated with intramuscular fat content and growth rate in the pig[J]. Anim Biotechnol, 2009,20(2):58-70.
[5]罗卫星,张启林,穆林,等.黔北麻羊的肉用性能和肉质特性研究[J].西南农业学报, 2010, 23(5):1706-1710.
[6]于欣,杨震,楚元奎,等. IL-6基因结构和功能生物信息学预测[J].国际检验医学杂志, 2016, 37(21):2959-2960.
[7] Dai H J, Wu C Y, Tsai T H, et al. T-HOD:a literature-based candidate gene database for hypertension, obesity and diabetes[J]. Database, 2013, 2013(4):1093-1105.
[8] Loos R J, Bouchard C. FTO:the first gene contributing to common forms of human obesity[J]. Obes Rev, 2008, 9(3):246-250.
[9]尹靖东.动物肌肉生物学与肉品科学[M].北京:中国农业大学出版社, 2011.
[10] Jevsinek S D, Kunej T, Kovac M, et al. FTO gene variants are associated with growth and carcass traits in cattle[J]. Anim Gen,2015, 47(2):219-222.
[11] Szydlowski M, Salamon S, Grzes M, et al. SNP in the 5′flanking region of the pig FTO gene is associated with fatness in Polish Landrace[J]. Livest Sci, 2012, 150(1-3):397-400.
[12] Zhang L F, Miao X T, Hua X C, et al. Polymorphism in 5'regulatory region of the porcine fat mass and obesity associated(FTO)gene is associated with intramuscular fat content in a jinhua x pietrain freference population[J]. J Anim Vet Adv,2012(11):2329-2334.
[13] Gan W, Song Q, Zhang N N, et al. Association between FTO polymorphism in exon 3 with carcass and meat quality traits in crossbred ducks[J]. Gen Mol Res, 2015, 14(2):6699-6714.
[14]康晓龙,张莉莉,王昊升,等. FTO基因多态性及其与2个绵羊群体生长性状的相关性[J].西南农业学报, 2017,30(11):2603-2608.
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