金华猪PPARGC1A基因克隆、生物信息学分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2020.03.006

摘要/Abstract

摘要： 试验旨在研究过氧化物酶体增殖物激活受体γ辅助激活因子1α（PPARGC1A）在金华猪和大白猪中的遗传特征和表达情况，探究PPARGC1A基因在mRNA水平和蛋白质水平的表达模式。以金华猪和大白猪为试验动物，分别提取背脂组织的总RNA，根据GenBank中公布的猪PPARGC1A基因序列（登录号：NM_213963.2）设计编码区和实时定量PCR引物，以猪GAPDH基因和β-actin蛋白作为内参，应用多种生物信息学方法对PPARGC1A基因编码蛋白进行功能分析，并通过实时荧光定量PCR及Western blotting检测其在金华猪背脂中的表达水平。结果表明，金华猪PPARGC1A基因CDS区全长2 361 bp，编码786个氨基酸，该蛋白分子大小90 336.01 u，其中丝氨酸（ser）所占比例最高（13.7%），色氨酸（Trp）所占比例最低（0.8%）。同源性比对结果显示，金华猪PPARGC1A基因与山羊、牛和绵羊的同源性较高，分别为95.0%、94.9%和94.9%，在物种进化中具有较强的保守性；金华猪PPARGC1A蛋白不稳定指数为74.88，属于不稳定亲水蛋白，无跨膜结构，其二级结构由α-螺旋、延伸链、β-转角及无规则卷曲4种结构组成，所占比例分别为26.59%、5.73%、5.73%和61.96%，PPARGC1A蛋白同源建模经折叠、弯曲等一系列复杂的过程获得三级结构模型；实时荧光定量PCR试验和Western blotting试验结果一致，显示PPARGC1A基因在背脂较厚的金华猪中表达量显著低于瘦肉型的大白猪（P<0.05）。本研究为探明PPARGC1A基因对猪脂肪沉积的分子生物学功能提供了理论基础。

关键词: 金华猪; PPARGC1A基因; 生物信息学分析; 组织表达; 脂肪沉积

Abstract: The aim of this study was to investigate the genetic characteristics and expression patterns of PPARGC1A gene in Jinhua pigs and Yorkshire pigs.The Jinhua pigs and Yorkshire pigs were used as samples to extract total RNA from the dorsal lipid tissue,and the coding region and real-time quantitative PCR primers were designed according to the sequence of porcine PPARGC1A gene in GenBank (accession No.:NM_213963.2).The porcine GAPDH gene and β-actin protein were used as internal parameters,and the PPARGC1A gene-encoded protein was applied by various bioinformatics methods.Functional analysis was performed and its expression level in dorsal lipid tissue of Jinhua pigs was detected by Real-time quantitative PCR and Western blotting assay.The results showed that the full length of PPARGC1A gene CDS region was 2 361 bp and encoded 786 amino acids.The protein had a molecular size of 90 336.01 u,with serine (Ser) accounting for the highest proportion (13.7%) and tryptophan (Trp) accounting for the lowest proportion (0.8%).Homology alignment results showed that PPARGC1A gene in Jinhua pigs had high homology with goats,cattle and sheep,which were 95.0%,94.9% and 94.9%,respectively,and was highly conserved in species evolution.The PPARGC1A protein of the instability index in Jinhua pigs was 74.88,belonging to unstable hydrophilic protein,and no transmembrane structure.Its secondary structure consists of alpha helix,extended chain,beta turn and random coil,and the proportion of them were 26.59%,5.73%,5.73% and 61.96%,respectively.The PPARGC1A protein homology modeling obtained a series of complex processes such as folding and bending to obtain a three-level structural model.The results of the Real-time quantitative PCR and Western-blotting were consistent,indicating that the expression of PPARGC1A gene in Jinhua pigs with dorsal lipid tissue was significantly lower than that of lean pigs (P<0.05).This study provides a theoretical basis for the discovery of the molecular biological function of PPARGC1A gene in pig fat deposition.

Key words: Jinhua pigs; PPARGC1A gene; bioinformatics analysis; tissue expression; fat deposition

中图分类号:

S828.9

刘玉芳, 郭思武, 张清阳, 张鑫, 杨俊琦, 白莹. 金华猪PPARGC1A基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2020, 47(3): 696-705.

LIU Yufang, GUO Siwu, ZHANG Qingyang, ZHANG Xin, YANG Junqi, BAI Ying. Cloning,Bioinformatics Prediction and Tissue Expression Analysis of PPARGC1A Gene in Jinhua Pigs[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(3): 696-705.

参考文献

[1] PUIGSERVER P,WU Z,PARK C W,et al.A Cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis[J].Cell,1998,92(6):829-839.
[2] 苏燕,彭姝彬,李智琼,等.PPARGC1A基因Thr394Thr/Gly482Ser多态性与2型糖尿病的关联研究[J].遗传,2008,30(3):304-308. SU Y,PENG S B,LI Z Q,et al.Association study between PPARGC1A Thr394Thr/Gly482Ser polymorphisms and type 2 diabetes[J].Hereditas,2008,30(3):304-308.(in Chinese)
[3] LI J D,FENG Q C,QI Y,et al.PPARGC1A is upregulated and facilitates lung cancer metastasis[J].Experimental Cell Research,2017,359(2):356-360.
[4] ZHANG S,JIANG J,CHEN Z,et al.Relationship of PPARG,PPARGC1A,and PPARGC1B polymorphisms with susceptibility to hepatocellular carcinoma in an eastern Chinese Han population[J].OncoTargets and Therapy,2018,11:4651-4660.
[5] SOYAL S M,ZARA G,FERGER B,et al.The PPARGC1A locus and CNS-specific PGC-1α isoforms are associated with Parkinson's disease[J].Neurobiology of Disease,2019,121:34-46.
[6] 王杰,原清会,张明,等.中国荷斯坦奶牛PPARGC1A基因内含子9的SNP与产奶性状的相关分析[J].四川农业大学学报,2010,28(2):219-222. WANG J,YUAN Q H,ZHANG M,et al.Association analysis between SNP of intron 9 in PPARGC1A gene and milking traits in Chinese Holstein breed[J].Journal of Sichuan Agricultural University,2010,28(2):219-222.(in Chinese)
[7] 刘婷婷,孟军,王建文,等.三个品种马PPARGC1A基因PCR-SSCP多态性分析[J].新疆农业科学,2015,52(10):1926-1931. LIU T T,MENG J,WANG J W,et al.Polymorphism analysis of three different horse types PPARGC1A gene by PCR-SSCP[J].Xinjiang Agricultural Sciences,2015,52(10):1926-1931.(in Chinese)
[8] 安世钰,张国敏,尤佩华,等.PPARGC1A/NRF1/TFAM通路核心基因在湖羊睾丸发育过程中的表达变化[J].南京农业大学学报,2019,42(2):345-351. AN S J,ZHANG G M,YOU P H,et al.Research on the change of PPARGC1A/NRF1/TFAM pathway genes expression in the development of Hu sheep testis[J].Journal of Nanjing Agricultural University,2019,42(2):345-351.(in Chinese)
[9] 韩雪蕾,刘榜,彭勇波,等.猪PPARGC1A基因的遗传变异检测及与胴体性状的关联分析[J].华中农业大学学报,2009,28(5):587-590. HAN X L,LIU B,PENG Y B,et al.Hereditary variation of pig PPARGC1A gene and its association with carcass traits of porcine[J].Journal of Huazhong Agricultural University,2009,28(5):587-590.(in Chinese)
[10] GANDOLFI G,CINAR M U,PONSUKSILI S,et al.Association of PPARGC1A and CAPNS1 gene polymorphisms and expression with meat quality traits in pigs[J].Meat Science,2011,89(4):478-485.
[11] 徐芹芹,刘玉芳,康晓龙,等.滩羊EphA3基因克隆表达分析及生物信息学初步研究[J].中国畜牧杂志,2019,3:33-38. XU Q Q,LIU Y F,KANG X L,et al.Cloning,expression and bioinformatics analysis of EphA3 gene in Chinese Tan sheep[J].Chinese Journal of Animal Science,2019,3:33-38.(in Chinese)
[12] 瞿秋红,黄叶,蒋钦杨,等.影响猪脂肪沉积主要候选基因的研究进展[J].动物营养学报,2019,7:2981-2987. QU Q H,HUANG Y,JIANG Q Y,et al.Research progress of major candidate genes affecting pig fat deposition[J].Chinese Journal of Animal Nutrition,2019,7:2981-2987.(in Chinese)
[13] 吕亚宁,叶文文,兰旅涛.牛肌内脂肪沉积影响因素及相关基因研究进展[J].中国草食动物科学,2019,4:55-57. LYU Y N,YE W W,LAN L T.Research progress on influencing factors and related genes of fat deposition in bovine muscle[J].China Herbivore Science,2019,4:55-57.(in Chinese)
[14] 农秋雲,刘嘉琪,单体中.猪肉中脂肪酸组成的品种差异及脂肪酸沉积的调控机制[J].动物营养学报,2019,6:2507-2514. NONG Q Y,LIU J Q,SHAN T Z.Breed difference of fatty acid composition and regulation mechanism of fatty acid deposition in pork[J].Chinese Journal of Animal Nutrition,2019,6:2507-2514.(in Chinese)
[15] 张雨,施力光,荀文娟,等.能量水平和激素对脂肪代谢酶基因的调控研究进展[J].动物营养学报,2019,8:3505-3510. ZHANG Y,SHI L G,XUN W J,et al.Research progress of regulation of fat metabolism enzyme gene by energy level and hormone[J].Chinese Journal of Animal Nutrition,2019,8:3505-3510.(in Chinese)
[16] RAMOSLOPEZ O,RIEZUBOJ J I,MILAGRO F I.Association of the Gly482Ser PPARGC1A gene variant with different cholesterol outcomes in response to two energy-restricted diets in subjects with excessive weight[J].Nutrition,2018,47:83-89.
[17] PARDO R,ENGUIX N,LASHERAS J,et al.Rosiglitazone-induced mitochondrial biogenesis in white adipose tissue is independent of peroxisome proliferator-activated receptor γ coactivator-1α[J].PLoS ONE,2011,6(11):e26989.
[18] PEI X,LIU L,CAI J,et al.Haplotype-based interaction of the PPARGC1A and UCP1 genes is associated with impaired fasting glucose or type 2 diabetes mellitus[J].Medicine,2017,96(23):e6941.
[19] 乌日古木拉.蒙古马运动候选基因PPARGC1A多态性和表达量研究[D].呼和浩特:内蒙古农业大学,2012. WURI G.The investigation of polymorphism and expression's quantity on athletics candidate gene PPARGC1A in Mongolian horses[D].Hohhot:Inner Mongolia Agricultural University,2012.(in Chinese)
[20] 梁爽,王伟伟,孙力,等.PPARGC1启动子甲基化与Ⅱ型糖尿病发病相关性分析[J].分子诊断与治疗杂志,2019,2:91-95. LIANG S,WANG W W,SUN L,et al.Correlation analysis between PPARGC1 promoter methylation and type Ⅱ diabetes[J].Journal of Molecular Diagnosis and Therapy,2019,2:91-95.(in Chinese)
[21] WANG B,NIE W,FU X.Neonatal vitamin A injection promotes cattle muscle growth and increases oxidative muscle fibers[J].Journal of Animal Science and Biotechnology,2019,1:140-149.
[22] 李密杰.秦川牛PPARGC1A基因功能验证及遗传变异研究[D].杨凌:西北农林科技大学,2013. LI M J.Function analysis and genetic variations of PPARGC1A gene in Qinchuan cattle[D].Yangling:Northwest A&F University,2013.(in Chinese)
[23] POPOV D V,LYSENKO E A,MAKHNOVSKII P A,et al.Regulation of PPARGC1A gene expression in trained and untrained human skeletal muscle[J].Physiological Reports,2017,5(23):doi:10.14814/phy2.13543.
[24] AUDREY C,AUDREN F,ALESSIA B,et al.Hepatocyte nuclear factor-1β controls mitochondrial respiration in renal tubular cells[J].Journal of the American Society of Nephrology,2017,28(11):3205-3217.
[25] VOILLET V,CRISTOBAL M S,PERE M C,et al.Integrated analysis of proteomic and transcriptomic data highlights late fetal muscle maturation process[J].Molecular & Cellular Proteomics,2018,17(4):672-693.