绵羊Fsp27基因组织表达、多态性及其与不同绵羊品种尾脂沉积能力的关联性分析

doi:10.11843/j.issn.0366-6964.2020.05.007

摘要/Abstract

摘要： 旨在研究绵羊Fsp27基因的组织表达/多态性及其与不同绵羊品种尾脂沉积能力的关联性。本研究采用qRT-PCR检测了营养充足期阿勒泰羊不同组织中Fsp27基因的表达情况，同时对营养充足期和营养匮乏期阿勒泰羊尾脂组织中Fsp27基因的表达情况进行了定量分析，采用PCR-SSCP结合测序技术检测了5个不同尾脂沉积能力绵羊品种Fsp27基因的突变情况，并分析了相关突变与绵羊尾脂沉积能力的关联性。结果表明，Fsp27基因在营养充足期阿勒泰羊尾脂中高表达，其表达量极显著高于其他组织（P<0.01），在肾周脂肪和皮下脂肪组织中的表达量也较高，极显著高于心、肝、脾、肺、肾、胃、肠、皮肤和骨骼肌组织（P<0.01）。在心、肝、脾、肺、肾、胃、肠、皮肤和骨骼肌组织中呈微量表达，且各组织间差异不显著（P>0.05）。另外，营养充足期阿勒泰羊尾脂中Fsp27基因的表达量极显著高于营养匮乏期（P<0.01）。绵羊Fsp27基因在不同尾脂沉积能力品种群体中共检测到25个突变位点，其中位于第3外显子g.16771741的G/A突变以及第5外显子g.16774969的C/T突变均为错义突变，且与绵羊尾脂沉积能力高度相关。g.16771741的G/A突变在尾脂沉积能力较强的阿勒泰羊、小尾寒羊和湖羊群体中以G等位基因为主，分别占到86.8%、83.7%和85.7%，而尾脂沉积能力较差的中国美利奴细毛羊和萨福克羊群体中G等位基因分别仅占30.3%和11.1%。g.16774969的C/T突变在阿勒泰羊群体中以T等位基因为主，TT基因型占84.7%，CT基因型占15.3%，没有检测到CC基因型；在短脂尾型的小尾寒羊和湖羊群体中，TT和CT基因型也分别占到65.9%、50.4%和22.7%、41.1%，而在长瘦尾的中国美利奴细毛羊和萨福克羊群体中则以CC基因型为主，分别占到97.4%和69.4%，没有检测到TT基因型。以上研究结果表明，Fsp27基因在阿勒泰羊尾脂中高表达，且其在尾脂中的表达量与阿勒泰羊的营养状态密切相关，Fsp27基因第3外显子g.16771741的G/A突变以及第5外显子g.16774969的C/T突变与绵羊尾脂沉积能力高度相关，可以作为理想的分子标记用于低脂肪绵羊品种的选育。

关键词: 绵羊, Fsp27, 尾脂, SNP

Abstract: The aim of this study was to investigate tissue expression profiles and polymorphism of Fsp27 gene in sheep (Ovis aries) and their association with tail fat deposition of different sheep breeds. The qRT-PCR was applied to detect the expression level of Fsp27 gene in different tissues of Altay sheep in adequate nutrition stage, and its expression levels in tail fat of Altay sheep in adequate and deficient nutrition stages, then the PCR-SSCP combined with sequencing were used to detect polymorphism of Fsp27 gene in 5 sheep breeds with different tail fat deposition ability and their association with tail fat deposition in sheep were analyzed. The results showed that Fsp27 gene was expressed at a high level in tail fat of Altay sheep in adequate nutrition stage and extremely higher than those in the other tissues (P<0.01), and expressed at a relatively high level in perirenal fat and subcutaneous fat which was extremely higher than those in heart, liver, spleen, lung, kidney, stomach, intestine, skin and skeletal muscle tissues (P<0.01), but expressed at a very low level in heart, liver, spleen, lung, kidney, stomach, intestine, skin and skeletal muscle tissues, and there were no significant difference among them (P>0.05). In addition, the expression level of Fsp27 gene in tail fat tissue of Altay sheep in adequate nutrition stage was significantly higher than that in deficient nutrition stage (P<0.01). Twenty-five SNPs were detected in Fsp27 gene sequence of sheep with different tail fat deposition ability, G/A mutation at g. 16771741 site in exon3 and C/T at g. 16774969 site in exon5 were missense mutations, and they were significantly associated with tail fat deposition ability of sheep. G was dominant allele in Altay sheep, Small Tail Han sheep and Hu sheep at g. 16771741 site, the allele frequency was 86.8%, 83.7% individuals and 85.7%, respectively, but only 30.3% and 11.1% individuals of Chinese Merino and Suffolk sheep were G allele at this site. T was dominant allele in Altay sheep at g. 16774969 site, 84.7% were TT genotype, 15.3% individuals were CT genotype, and no CC genotype was detected, and the genotype frequency of TT and CT in Small Tail Han sheep and Hu sheep were 65.9%, 50.4% and 22.7%, 41.1%, respectively, but CC was dominant genotype in Chinese Merino and Suffolk sheep, their frequency were 97.4% and 69.4%, and no TT genotype was detected. These results indicated that Fsp27 gene expressed at a high level in tail fat tissue of Altay sheep, and its expression level was closely related with nutrition state of Altay sheep. The G/A mutation at g. 16771741 site in exon3 and C/T mutation at g. 16774969 site in exon5 of Fsp27 gene were highly correlated with tail fat deposition ability of sheep, so could be used to help selecting low fat sheep breed.

Key words: sheep, Fsp27, tail fat, SNP

中图分类号:

S826.2

张伟, 王世银, 高莉, 徐梦思, 王新华, 甘尚权. 绵羊Fsp27基因组织表达、多态性及其与不同绵羊品种尾脂沉积能力的关联性分析[J]. 畜牧兽医学报, 2020, 51(5): 952-964.

ZHANG Wei, WANG Shiyin, GAO Li, XU Mengsi, WANG Xinhua, GAN Shangquan. Tissue Expression Profiles and Polymorphism of Fsp27 Gene in Sheep (Ovis aries) and Their Association with Deposition of Tail Fat[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(5): 952-964.

参考文献 40

[1]	RYDER M L.Sheep and Man[M].London:Duckworth Press,1983.
[2]	DAVIDSON A.The Oxford companion to food[M].2nd ed.London:Oxford University Press,2006.
[3]	马桢,郝耿,杨会国,等.阿勒泰羊品种资源现状及发展思路[J].草食家畜,2012(2):10-12,15.MA Z,HAO G,YANG H G,et al.Altay sheep status and development[J].Grass-feeding Livestock,2012(2):10-12,15.(in Chinese)
[4]	许瑞霞,高磊,赵伟利,等.FABP4基因在阿勒泰羊尾脂沉积与代谢模型中的表达变化规律[J].遗传,2015,37(2):174-182.XU R X,GAO L,ZHAO W L,et al.Analysis of FABP4 expression pattern in rump fat deposition and metabolism of Altay sheep[J]. Hereditas (Beijing),2015,37(2):174-182.(in Chinese)
[5]	刘丹,何鑫,李涛,等.不同品种脂臀羊尾脂品质的比较分析[J].现代食品科技,2019,35(4):244-249,300.LIU D,HE X,LI T,et al.Comparative analysis of the quality of different varieties of fat buttocks[J].Modern Food Science and Technology,2019,35(4):244-249,300.(in Chinese)
[6]	NEDERGAARD J,GOLOZOUBOVA V,MATTHIAS A,et al.UCP1:the only protein able to mediate adaptive non-shivering thermogenesis and metabolic inefficiency[J].Biochim Biophys Acta,2001,1504(1):82-106.
[7]	WALTHER T C,FARESE R V JR.The life of lipid droplets[J].Biochim Biophys Acta,2009,1791(6):459-466.
[8]	KATO Y,ARAKAWA S,TERASAWA K,et al.The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl) decanamide induces large lipid droplet accumulation and highlights the effect of LAMP-2 deficiency on lipid droplet degradation[J]. Bioorg Med Chem Lett,2020,30(3):126891,doi:10.1016/j.bmcl.2019.126891.
[9]	WALTHER T C,CHUNG J,FARESE R V J R.Lipid droplet biogenesis[J].Annu Rev Cell Dev Biol,2017,33:491-510.
[10]	CHAPMAN K D,AZIZ M,DYER J M,et al.Mechanisms of lipid droplet biogenesis[J].Biochem J,2019,476(13):1929-1942.
[11]	GAO M M,HUANG X,SONG B L,et al.The biogenesis of lipid droplets:lipids take center stage[J].Prog Lipid Res,2019, 75:100989.
[12]	CHEN F J,YIN Y S,CHUA B T,et al.CIDE family proteins control lipid homeostasis and the development of metabolic diseases[J].Traffic,2020,21(1):94-105.
[13]	许祥,董维鹏,张少华,等.Fsp27基因沉默载体的构建及其对细胞脂解的影响研究[J].生物技术通报,2020,36(1):88-94.XU X,DONF W P,ZHANG S H,et al.Construction of Fsp27 gene silencing vector and its effect on cell lipolysis[J].Biotechnology Bulletin,2020,36(1):88-94.(in Chinese)
[14]	潘洪彬,赵素梅,孙泽威,等.脂肪特异蛋白27(Fsp27)与脂肪代谢[J].畜牧兽医学报,2014,45(3):347-353.PAN H B,ZHAO S M,SUN Z W,et al.Fat-specific protein 27(Fsp27) and lipid metabolism[J].Acta Veterinaria et Zootechnica Sinica,2014,45(3):347-353.(in Chinese)
[15]	PRICE A M,DONER N M,GIDDA S K,et al.Mouse Fat-Specific Protein 27(FSP27) expressed in plant cells localizes to lipid droplets and promotes lipid droplet accumulation and fusion[J].Biochimie,2020,169:41-53.
[16]	李红强,李海玉,李婧实,等.CIDEC的亚细胞定位及其功能初步研究[J].核农学报,2017,31(8):1494-1499.LI H Q,LI H Y,LI J S,et al.Localization and preliminarily functional analysis of CIDEC[J].Journal of Nuclear Agricultural Sciences,2017,31(8):1494-1499.(in Chinese)
[17]	NISHINO N,TAMORI Y,TATEYA S,et al.Fsp27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets[J].J Clin Invest,2008,118(8):2808-2821.
[18]	董维鹏,张少华,许祥,等.下调Fsp27基因表达联合杨梅素干预对3T3-L1细胞脂解的影响[J].中国生物工程杂志, 2018, 38(12):7-13.DONG W P,ZHANG S H,XU X,et al.The effect and mechanism of down-regulated Fsp27 gene combined with myricetin on lipolysis of 3T3-L1 adipocytes[J].China Biotechnology,2018,38(12):7-13.(in Chinese)
[19]	GONG J Y,SUN Z Q,WU L Z,et al.Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites[J].J Cell Biol,2011,195(6):953-963.
[20]	李聪,周林康,李蓬.Fsp27通过抑制HSL的脂滴定位调控脂肪水解[J].中国科学:生命科学,2014,44(10):1073-1081.LI C,ZHOU L K,LI P.Fsp27 inhibits lipolysis by excluding HSL from lipid droplet surface[J].Scientia Sinica Vitae,2014, 44(10):1073-1081.(in Chinese)
[21]	甘尚权,张伟,沈敏,等.绵羊X染色体59383635位点多态性与脂尾性状的相关性分析[J].遗传,2013,35(10):1209-1216.GAN S Q,ZHANG W,SHEN M,et al.Correlation analysis between polymorphism of the 59383635th locus on X chromosome and fat-tail trait in sheep[J].Hereditas (Beijing),2013,35(10):1209-1216.(in Chinese)
[22]	MATSUSUE K.A physiological role for fat specific protein 27/cell death-inducing DFF45-Like effector C in adipose and liver[J].Biol Pharm Bull,2010,33(3):346-350.
[23]	LI Y,YU S,CHEN L L,et al.Involvement of PPARγ/FSP27 in the pathogenic mechanism underlying insulin resistance:tipping the balance between lipogenesis and fat storage in adult catch-up growth rats[J].Nutr Metab,2019,16(1):11,doi:10.1186/s12986-019-0336-9.
[24]	LI Y H,LEI T,CHEN X D,et al.Molecular cloning,chromosomal location and expression pattern of porcine CIDEa and CIDEc[J]. Mol Biol Rep,2009,36(3):575-582.
[25]	WANG J,CAO X K,PAN H,et al.Cell death-inducing DFFA-like effector c (CIDEC/Fsp27) gene:molecular cloning,sequence characterization,tissue distribution and polymorphisms in Chinese cattles[J].Mol Biol Rep,2013,40(12):6765-6774.
[26]	HU J Y,WANG M F,ZHOU Y,et al.Bariatric surgery in rats upregulates FSP27 expression in fat tissue to affect fat hydrolysis and metabolism[J].BioMed Res Int,2019,2019:6415732,doi:10.1155/2019/6415732.
[27]	MATSUSUE K,KUSAKABE T,NOGUCHI T,et al.Hepatic steatosis in leptin-deficient mice is promoted by the PPARγ target gene Fsp27[J]. Cell Metab,2008,7(4):302-311.
[28]	LEE S W,RHO J H,LEE S Y,et al.Dietary fat-associated osteoarthritic chondrocytes gain resistance to lipotoxicity through PKCK2/STAMP2/FSP27[J].Bone Res,2018,6(1):20,doi:10.1038/s41413-018-0020-0.
[29]	KELLER P,PETRIE J T,DE ROSE P,et al.Fat-specific protein 27 regulates storage of triacylglycerol[J].J Biol Chem,2008, 283(21):14355-14365.
[30]	KARKI S.FSP27 and links to obesity and diabetes mellitus[J].Curr Obes Rep,2019,8(3):255-261.
[31]	巩建飞,岳静伟,赵玲玲,等.猪HMGA1基因的组织表达及其多态性与背膘厚的关联分析[J].畜牧兽医学报, 2019,50(7):1340-1346.GONG J F,YUE J W,ZHAO L L,et al.Tissue expression profiles of HMGA1 gene in pigs and its association with backfat thickness[J].Acta Veterinaria et Zootechnica Sinica,2019,50(7):1340-1346.(in Chinese)
[32]	安清明,周辉通,吴震洋,等.绵羊脂联素基因(ADIPOQ)多态性及其与生长及胴体性状关联性分析[J].中国农业科学,2019,52(10):1807-1817.AN Q M,ZHOU H T,WU Z Y,et al.Polymorphisms of ADIPOQ gene and their association with growth and carcass traits in sheep[J].Scientia Agricultura Sinica,2019,52(10):1807-1817.(in Chinese)
[33]	惠嫣婷,刘若余,张依裕,等.贵州地方猪脂肪特异蛋白27基因SNPs筛查与生物信息学分析[J].中国畜牧兽医,2012,39(7):83-86.HUI Y T,LIU R Y,ZHANG Y Y,et al.SNPs screening and bioinformatics analysis of Fsp27 gene in the breeds of Guizhou local pigs[J].China Animal Husbandry & Veterinary Medicine,2012,39(7):83-86.(in Chinese)
[34]	RUBIO-CABEZAS O,PURI V,MURANO I,et al.Partial lipodystrophy and insulin resistant diabetes in a patient with a homozygous nonsense mutation in CIDEC[J].EMBO Mol Med,2009,1:280-287.
[35]	WANG J,HUA L S,PAN H,et al.Haplotypes in the promoter region of the CIDEC gene associated with growth traits in Nanyang cattle[J]. Sci Rep,2015,5(1):12075,doi:10.1038/srep12075.
[36]	张伟,沈敏,李欢,等.绵羊X染色体59571364与59912586位点在脂尾、瘦尾绵羊群体中的多态性检测及分析[J].遗传,2013,35(12):1384-1390.ZHANG W,SHEN M,LI H,et al.Detection and analysis of polymorphisms of 59571364 and 59912586 loci on X chromosome in fat-tail and thin-tail sheep flocks[J].Hereditas (Beijing),2013,35(12):1384-1390.(in Chinese)
[37]	SEO S W,YANG J S,KIM I,et al.Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency[J].Metab Eng,2013,15:67-74.
[38]	LEPPEK K,DAS R,BARNA M.Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them[J]. Nat Rev Mol Cell Biol,2018,19(3):158-174.
[39]	MIYAMOTO Y,MABUCHI A,SHI D Q,et al.A functional polymorphism in the 5'UTR of GDF5 is associated with susceptibility to osteoarthritis[J].Nat Genet,2007,39(4):529-533.
[40]	CHANG C K,WU S R,CHEN Y C,et al.Mutations in VP1 and 5'-UTR affect enterovirus 71 virulence[J].Sci Rep,2018, 8(1):6688.