沐川乌骨黑鸡LIPE基因第1外显子多态性及其与体重、肌内脂肪含量的关联分析

doi:10.16431/j.cnki.1671-7236.2023.12.019

摘要/Abstract

摘要： 【目的】探究沐川乌骨黑鸡激素敏感脂酶(hormone-sensitive lipase,LIPE)基因第1外显子单核苷酸多态性(single nucleotide polymorphism,SNP)位点的遗传变异,并分析其与体重、肌内脂肪(intramuscular fat,IMF)含量的相关性。【方法】采集106只沐川乌骨黑鸡血液,利用PCR扩增和直接测序技术检测LIPE基因第1外显子SNP位点,采用SPSS 21.0软件分析LIPE基因第1外显子SNP位点多态性及其与沐川乌骨黑鸡体重、胸肌IMF含量的相关性;通过生物信息学在线工具分析LIPE基因第1外显子SNP位点对其编码氨基酸的影响;利用实时荧光定量PCR检测LIPE基因在沐川乌骨黑鸡不同组织和不同基因型个体胸肌中的相对表达量。【结果】沐川乌骨黑鸡LIPE基因第1外显子共检测到3个SNP位点,66 bp处SNP位点(SNP1:c.66 A＞C)上发现AA、AC和CC 3种基因型;440 bp处SNP位点(SNP2:c.440 A＞G)上发现AA和AG 2种基因型;462 bp处SNP位点(SNP3:c.462 G＞A)上发现GG和GA 2种基因型。卡方适合性检验表明,沐川乌骨黑鸡LIPE基因第1外显子的SNP2、SNP3位点符号哈迪-温伯格平衡状态(P＞0.05),SNP1位点偏离哈迪-温伯格平衡状态(P＜0.05)。关联分析表明,SNP2位点的AG基因型个体胸肌IMF含量显著高于AA基因型(P＜0.05),各位点不同基因型个体体重均无显著差异(P＞0.05)。氨基酸序列比对分析显示,SNP2为非同义突变,其导致赖氨酸变为精氨酸。实时荧光定量PCR结果显示,LIPE基因在脂肪组织中表达量较高,SNP2位点AA基因型个体胸肌中LIPE基因mRNA表达水平极显著高于AG基因型(P＜0.01)。【结论】沐川乌骨黑鸡LIPE基因第1外显子存在3个SNP位点,其中SNP2位点对沐川乌骨鸡IMF含量具有显著影响,该位点可作为沐川乌骨黑鸡IMF含量选育的候选分子遗传标记。

关键词: 沐川乌骨黑鸡; LIPE基因; 第1外显子; 肌内脂肪; 遗传变异

Abstract: 【Objective】 The aim of this study was to investigate the genetic variation of single nucleotide polymorphism (SNP) of hormone-sensitive lipase (LIPE) gene exon 1 in Muchuan Black-bone chickens,and analyzed the correlation with body weight and intramuscular fat (IMF) content.【Method】 The blood of 106 Muchuan Black-bone chickens was collected,and the SNP site of LIPE gene exon 1 was detected by PCR amplification and direct sequencing.SPSS 21.0 was used to analyze the SNP polymorphism of LIPE gene exon 1 and its correlation with body weight and IMF content of chest muscle in Muchuan Black-bone chickens.The effect of SNP of LIPE gene exon 1 on its amino acid was analyzed by bioinformatics online tool.Real-time quantitative PCR was used to detect the relative expression of LIPE gene in different tissues and chest muscles of different genotypes of Muchuan Black-bone chickens.【Result】 A total of 3 SNP sites were detected of LIPE gene exon 1 in Muchuan Black-bone chickens.Genotypes of AA,AC and CC were found in SNP1 (c.66 A＞C).Genotypes of AA and AG were found in SNP2 (c.440 A＞G).Genotypes of GG and GA were found in SNP3 (c.462 G＞A).Chi-square fitness test showed that SNP2 and SNP3 of LIPE gene exon 1 in Muchuan Black-bone chickens were in Hardy-Weinberg equilibrium (P＞0.05),and SNP1 deviated from Hardy-Weinberg equilibrium (P＜0.05).Association analysis showed that the IMF content in chest muscle of AG genotype with SNP2 locus was significantly higher than that of AA genotype (P＜0.05),and there was no significant difference in body weight among different genotypes with each locus (P＞0.05).Amino acid sequence alignment analysis showed that SNP2 was a non-synonymous mutation,which caused lysine to become arginine.Real-time quantitative PCR results showed that the expression level of LIPE gene was higher in adipose tissue,and the mRNA expression level of LIPE gene in chest muscle of AA genotype with SNP2 locus was extremely significantly higher than that of AG genotype (P＜0.01).【Conclusion】 There are three SNP of LIPE gene exon 1 in Muchuan Black-bone chickens,among which SNP2 locus has a significant effect on IMF content in Muchuan Black-bone chickens,and it could be used as a candidate molecular genetic marker for IMF content selection in Muchuan Black-bone chickens.

Key words: Muchuan Black-bone chickens; LIPE gene; exon 1; intramuscular fat; genetic variation

中图分类号:

S831.99

苟丹妮, 王钢, 沈雪梅, 聂亮, 惠文静, 古丽帕日·艾克拜, 杨雅玲, 廖娟, 喻世刚. 沐川乌骨黑鸡LIPE基因第1外显子多态性及其与体重、肌内脂肪含量的关联分析[J]. 中国畜牧兽医, 2023, 50(12): 4973-4982.

GOU Danni, WANG Gang, SHEN Xuemei, NIE Liang, HUI Wenjing, GULIPARI Aikebai, YANG Yaling, LIAO Juan, YU Shigang. Polymorphism of LIPE Gene Exon 1 and Its Association with Body Weight and Intramuscular Fat Content in Muchuan Black-bone Chickens[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 4973-4982.

参考文献

[1] 瞿丞,贺稚非,李少博,等.我国肉鸡生产加工现状与发展趋势[J].食品与发酵工业,2019,45(8):262-270. QU C,HE Z F,LI S B,et al.Current status and development trend of processing and producing broilers in China[J].Food and Fermentation Industries,2019,45(8):262-270.(in Chinese)
[2] 沙尔山别克·阿不地力大,李海英,努尔江·买地亚尔,等.不同饲养方式对拜城油鸡生长、屠宰性能及肉品质的影响[J].新疆农业科学,2011,48(11):2121-2128. SARSENBEK A,LI H Y,NURJAN M,et al.Effects of different feeding patterns on growth and slaughter performances and meat quality of Baicheng-You chickens[J].Xinjiang Agricultural Sciences,2011,48(11):2121-2128.(in Chinese)
[3] 陈继兰,文杰,王述柏,等.鸡肉肌苷酸和肌内脂肪沉积规律研究[J].畜牧兽医学报,2005,36(8):843-845. CHEN J L,WEN J,WANG S B,et al.Studies on the characteristics of deposition of chicken IMP and IMF[J].Acta Veterinaria et Zootechnica Sinica,2005,36(8):843-845.(in Chinese)
[4] CHARTRIN P,MÉTEAU K,JUIN H,et al.Effects of intramuscular fat levels on sensory characteristics of duck breast meat[J].Poultry Science,2006,85(5):914-922.
[5] 姜琳琳.不同品种鸡的肌肉化学成分及其与风味关系的比较研究[D].武汉:华中农业大学,2006. JIANG L L.Comparation of the chemical composition in different kinds of broilers meat and the relationship with the flavour[D].Wuhan:Huazhong Agricultural University,2006.(in Chinese)
[6] CHEN J L,ZHAO G P,ZHENG M Q,et al.Estimation of genetic parameters for contents of intramuscular fat and inosine-5'-monophosphate and carcass traits in Chinese Beijing-You chickens[J].Poultry Science,2008,87(6):1098-1104.
[7] JIANG M,FAN W L,XING S Y,et al.Effects of balanced selection for intramuscular fat and abdominal fat percentage and estimates of genetic parameters[J].Poultry Science,2017,96(2):282-287.
[8] LIU R,ZHENG M,WANG J,et al.Effects of genomic selection for intramuscular fat content in breast muscle in Chinese local chickens[J].Animal Genetics,2019,50(1):87-91.
[9] JENNEN D G,VEREIJKEN A L,BOVENHUIS H,et al.Confirmation of quantitative trait loci affecting fatness in chickens[J].Genetics Selection Evolution,2005,37(2):215-228.
[10] NASSAR M K,GORAGA Z S,BROCKMANN G A.Quantitative trait loci segregating in crosses between New Hampshire and White Leghorn chicken lines:Ⅲ.Fat deposition and intramuscular fat content[J].Animal Genetics,2013,44(1):62-68.
[11] 王小亚,陈大海,饶永超,等.基于RNA-Seq技术挖掘影响贵州黄鸡肌内脂肪沉积的关键基因[J].农业生物技术学报,2022,30(1):85-95. WANG X Y,CHEN D H,RAO Y C,et al.Exploration of key genes affecting intramuscular fat deposition in Guizhou Yellow chicken (Gallus gallus domesticus) based on RNA-Seq technology[J].Journal of Agricultural Biotechnology,2022,30(1):85-95.(in Chinese)
[12] 王明发,王浩宇,王浩,等.固始鸡和AA肉鸡肝脏中脂肪酸合成酶基因表达及肌肉脂肪酸的比较研究[J].南京农业大学学报,2021,44(6):1162-1168. WANG M F,WANG H Y,WANG H,et al.Comparative study on livers fatty acid synthase gene expression and muscle fatty acids of Gushi chicken and AA broiler[J].Journal of Nanjing Agricultural University,2021,44(6):1162-1168.(in Chinese)
[13] 李京徽,邢思远,王希彩,等.鸡肌内脂肪沉积相关候选基因筛选[J].中国畜牧兽医,2020,47(6):1828-1836. LI J H,XING S Y,WANG X C,et al.Screening of candidate genes related to intramuscular fat deposition in chickens[J].China Animal Husbandry&Veterinary Medicine,2020,47(6):1828-1836.(in Chinese)
[14] 李冬立,文杰,赵桂苹,等.鸡肌内脂肪双向选择对脂肪性状及相关基因mRNA表达的影响[J].畜牧兽医学报,2008,39(1):24-28. LI D L,WEN J,ZHAO G P,et al.Studies on the differences of phenotypic values and mRNA expression of genes associated with fat deposition in the divergent-selected chicken lines[J].Acta Veterinaria et Zootechnica Sinica,2008,39(1):24-28.(in Chinese)
[15] CUI H X,LIU R R,ZHAO G P,et al.Identification of differentially expressed genes and pathways for intramuscular fat deposition in pectoralis major tissues of fast-and slow-growing chickens[J].BMC Genomics,2012,13:213.
[16] ANTHONSEN M W,DEGERMAN E,HOLM C.Partial purification and identification of hormone-sensitive lipase from chicken adipose tissue[J]. Biochemical and Biophysical Research Communications,1997,236(1):94-99.
[17] 王美玲,陈仲建,吕林,等.不同形态锰对肉仔鸡脂肪代谢关键酶活性及其基因表达的影响[J].中国农业科学,2011,44(18):3850-3858. WANG M L,CHEN Z J,LYU L,et al.Effect of different manganese sources on activities and gene expression of key enzymes in fat metabolism of broilers[J]. Scientia Agricultura Sinica,2011,44(18):3850-3858.(in Chinese)
[18] 丁能水,黄路生,任军,等.猪激素敏感脂肪酶(HSL)基因的研究概况[J].遗传,2000,22(5):331-333. DING N S,HUANG L S,REN J,et al.The review on the HSL gene research[J].Hereditas,2000,22(5):331-333.(in Chinese)
[19] 聂靖茹,马黎,严达伟,等.大型迪庆藏猪不同生长阶段肌内脂肪沉积差异表达基因及其调控通路分析[J].中国畜牧兽医,2022,49(8):2855-2868. NIE J R,MA L,YAN D W,et al.Analysis of differentially expressed genes and regulation pathways of intramuscular fat deposition in large Diqing Tibetan pigs at different growth stages[J].China Animal Husbandry&Veterinary Medicine,2022,49(8):2855-2868.(in Chinese)
[20] XUE W,WANG W,JIN B,et al.Association of the ADRB3,FABP3,LIPE,and LPL gene polymorphisms with pig intramuscular fat content and fatty acid composition[J].Czech Journal of Animal Science,2015,60(2):60-66.
[21] GUI L S,RAZA S H A,MEMON S,et al.Association of hormone-sensitive lipase (HSL) gene polymorphisms with the intramuscular fat content in two Chinese beef cattle breeds[J].Genomics,2020,112(6):3883-3889.
[22] KONG Y,YUAN Z,LIU X,et al.A novel SNP within LIPE gene is highly associated with sheep intramuscular fat content[J].Small Ruminant Research,2022,209:106658-106666.
[23] LUO N,SHU J,YUAN X,et al.Differential regulation of intramuscular fat and abdominal fat deposition in chickens[J].BMC Genomics,2022,23(1):308.
[24] GE K,CHEN X,KUANG J,et al.Comparison of liver transcriptome from high-and low-intramuscular fat Chaohu ducks provided additional candidate genes for lipid selection[J].3 Biotech,2019,9(7):251.
[25] ZHU Y,ZHANG X,DU P,et al.Dietary herbaceous mixture supplementation reduced hepatic lipid deposition and improved hepatic health status in post-peak laying hens[J].Poultry Science,2022,101(6):101870.
[26] ZHAN X A,WANG M,REN H,et al.Effect of early feed restriction on metabolic programming and compensatory growth in broiler chicken[J].Poultry Science,2007,86(4):654-660.
[27] 喻世刚,王钢,廖娟,等.一种禽类血液基因组DNA高效提取通用方法的建立[J].黑龙江畜牧兽医,2018,13:195-198. YU S G,WANG G,LIAO J,et al.A general method for extraction of DNA from avian blood[J]. Heilongjiang Animal Science and Veterinary Medicine,2018,13:195-198.(in Chinese)
[28] HOCQUETTE J F,GONDRET F,BAÉZA E,et al.Intramuscular fat content in meat-producing animals:Development,genetic and nutritional control,and identification of putative markers[J].Animal,2010,4(2):303-319.
[29] 陈继兰,文杰,赵桂苹,等.鸡肉肌苷酸和肌内脂肪等肉品风味性状遗传参数的估计[J].遗传,2005,27(6):898-902. CHEN J L,WEN J,ZHAO G P,et al.Genetic parameter estimation for inosine-5-monophosphate and intramuscular fat contents and other meat quality traits in chicken muscle[J]. Hereditas,2005,27(6):898-902.(in Chinese)
[30] BEUZEN N D,STEAR M J,CHANG K C.Molecular markers and their use in animal breeding[J].Veterinary Journal,2000,160(1):42-52.
[31] 王安娜,冉雪琴,王嘉福,等.贵州地方猪品种HSL基因多态性与屠宰性状的关联性研究[J].畜牧兽医学报,2009,40(5):627-632. WANG A N,RAN X Q,WANG J F,et al.Polymorphism of exon 1 in hormone-sensitive lipase gene and its association with carcass traits in two Guizhou native pig breeds[J].Acta Veterinaria et Zootechnica Sinica,2009,40(5):627-632.(in Chinese)
[32] 强巴央宗,商鹏,杨涛,等.藏猪激素敏感脂肪酶(HSL)基因遗传多样性分析[J].西南农业学报,2012,25(5):1904-1906. CHAMBA Y Z,SHANG P,YANG T,et al.Polymorphisms of hormone-sensitive lipase gene in Tibet pigs[J]. Southwest China Journal of Agricultural Sciences,2012,25(5):1904-1906.(in Chinese)
[33] 马志杰,钟金城,字向东,等.三个牦牛群体激素敏感脂肪酶(HSL)基因外显子Ⅰ的多态性[J].中国农业科学,2009,42(1):370-376. MA Z J,ZHONG J C,ZI X D,et al.Polymorphism of exon Ⅰ of hormone-sensitive lipase (HSL) gene in three Chinese yak populations[J].Scientia Agricultura Sinica,2009,42(1):370-376.(in Chinese)
[34] KRAEMER F B,SHEN W J.Hormone-sensitive lipase[J].Journal of Lipid Research,2002,43(10):1585-1594.
[35] SZTALRYD C,KOMAROMY M C,KRAEMER F B.Overexpression of hormone-sensitive lipase prevents triglyceride accumulation in adipocytes[J].Journal of Clinical Investigation,1995,95(6):2652-2661.
[36] FANG X,ZHAO Z,JIANG P,et al.Identification of the bovine HSL gene expression profiles and its association with fatty acid composition and fat deposition traits[J].Meat Science,2017,131:107-118.
[37] 王怡平,徐晨晨,罗海玲.动物肌内脂肪沉积相关候选基因的研究进展[J].动物营养学报,2017,29(5):1475-1480. WANG Y P,XU C C,LUO H L.Research progress in relevant candidate genes of animal intramuscular fat deposition[J]. Chinese Journal of Animal Nutrition,2017,29(5):1475-1480.(in Chinese)
[38] ZHANG H,GUAN W.The response of gene expression associated with intramuscular fat deposition in the longissimus dorsi muscle of Simmental×Yellow breed cattle to different energy levels of diets[J].Animal Science Journal,2019,90(4):493-503.
[39] 栾兆进,刘开东,贺建宁,等.绵羊FAM134B、PPARγ、HSL和FAS基因表达量及与肌内脂肪含量的关系[J].畜牧兽医学报,2016,47(12):2379-2389. LUAN Z J,LIU K D,HE J N,et al.The FAM134B,PPARγ,HSL and FAS gene expression patterns and their association with intramuscular fat content in sheep[J].Acta Veterinaria et Zootechnica Sinica,2016,47(12):2379-2389.(in Chinese)
[40] 乔永,黄治国,李齐发,等.绵羊肌肉中FAS基因和HSL基因的发育性变化及其对肌内脂肪含量的影响[J].遗传学报,2007,10:909-917. QIAO Y,HUANG Z G,LI Q F,et al.Developmental changes of the FAS and HSL mRNA expression and their effects on the content of intramuscular fat in Kazak and Xinjiang sheep[J].Journal of Genetics and Genomics,2007,10:909-917.(in Chinese)