绵羊PDGFD基因克隆及其在不同部位脂肪组织中的表达

doi:10.16431/j.cnki.1671-7236.2021.09.004

摘要/Abstract

摘要： 试验旨在获得绵羊血小板源性生长因子D （PDGFD）基因的编码区（coding sequence，CDS）全长序列，并了解其在体内不同部位脂肪组织中的表达规律。以阿勒泰母羊和中国美利奴母羊为研究对象，克隆获得了PDGFD基因CDS区全长序列并对其进行了生物信息学分析，利用实时荧光定量PCR方法对PDGFD基因在2个品种羊沉积在尾部、肠系膜、背部和肾脏周围4个不同部位脂肪组织中的表达水平进行定量分析。结果显示，绵羊PDGFD基因CDS区序列长度为1 113 bp，编码370个氨基酸；PDGFD基因CDS区序列及其编码序列与山羊的相似性最高，其次是牛、猪、马、人，与鼠的相似性最低，系统进化树分析结果与其一致；预测PDGFD蛋白属于稳定的亲水性蛋白，且无跨膜区，存在信号肽序列，属于分泌型蛋白；PDGFD蛋白含有1个糖基化位点、1个CUB结构域和1个PDGF结构域，PDGFD蛋白二级结构由α-螺旋、β-转角、延伸链、无规则卷曲构成，三级结构与结构域和二级结构分析结果基本吻合。实时荧光定量PCR结果显示，PDGFD基因在阿勒泰羊4个脂肪组织的表达量均高于中国美利奴羊，其中阿勒泰羊尾脂和背部脂肪的表达量显著高于中国美利奴羊（P<0.05）。本研究结果为进一步探究PDGFD基因在绵羊脂肪沉积中的作用机制提供参考。

关键词: 绵羊; PDGFD基因; 基因克隆; 脂肪

Abstract: The aim of this experiment was to obtain the full-length coding sequence (CDS) of PDGFD gene in sheep, and understand its expression in adipose tissue deposited in different parts of the body. The full-length CDS sequence of PDGFD gene was cloned from Altay ewes and Chinese Merino ewes, and the bioinformatics analysis of the sequence was carried out. The expression of PDGFD gene in adipose tissues of the tail, mesentery, back and kidney of the two breeds of sheep was quantitatively analyzed by Real-time quantitative PCR. The results showed that the total CDS region sequence of PDGFD gene was 1 113 bp, which encoded 370 amino acids. The sequence of CDS region and its coding sequence of PDGFD gene had the highest similarity with Capra hircus, followed by Bos taurus, Sus scrofa, Equus caballus and Homo sapiens, and the lowest similarity with Mus musculus, which was consistent with the result of phylogenetic tree analysis. It was predicted that PDGFD protein was a stable hydrophilic protein, no transmembrane structure, had signal peptide sequence. PDGFD protein contained a glycosylation site, a CUB domain and a PDGF domain, and the secondary structure of PDGFD protein were composed of alpha helix, beta turn, extended chain and random coil, and tertiary structure was basically consistent with the analysis results of domain and secondary structure. Real-time quantitative PCR results showed that the expression of PDGFD gene in four adipose tissues of Altay sheep was higher than that of Chinese Merino sheep, especially the expression in tail fat and back fat of Altay sheep was significantly higher than of Chinese Merino sheep (P<0.05). The results of this study provided reference for further research on the mechanism of PDGFD gene in adipose deposition in sheep.

Key words: sheep; PDGFD gene; gene cloning; adipose

中图分类号:

S826.9⁺2

刘金瑞, 李忠慧, 蒋芳芳, 玛依拉, 李文蓉, 李海英. 绵羊PDGFD基因克隆及其在不同部位脂肪组织中的表达[J]. 中国畜牧兽医, 2021, 48(9): 3138-3146.

LIU Jinrui, LI Zhonghui, JIANG Fangfang, MA Yila, LI Wenrong, LI Haiying. Cloning of PDGFD Gene and Its Expression in Different Adipose Tissue in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3138-3146.

参考文献

[1] KOHLER N, LIPTON A.Platelets as a source of fibroblast growth-promoting activity[J]. Experimental Cell Research, 1974, 87(2):297-301.
[2] SHELTON M.Studies on tail length of Rambouillet and Mouflon sheep[J]. Narnia, 1977, 68(2):128-130.
[3] BERGSTEN E, UUTELA M, LI X, et al. PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor[J]. Nature Cell Biology, 2001, 3(5):512-516.
[4] LORIN E O, PHILIPPE S.PDGFRβ signaling regulates mural cell plasticity and inhibits fat development[J]. Developmental Cell, 2011, 20(6):815-826.
[5] KIM J H, PARK S G, KIM W K, et al. Functional regulation of adipose-derived stem cells by PDGFD[J]. Stem Cells, 2015, 33(2):542-556.
[6] ACOSTA J R, JOOST S, KARLSSON K, et al. Single cell transcriptomics suggest that human adipocyte progenitor cells constitute a homogeneous cell population[J]. Stem Cell Research & Therapy, 2017, 8(1):250.
[7] HILGENDORF K I, JOHNSON C T, MEZGER A, et al. Omega-3 fatty acids activate ciliary FFAR4 to control adipogenesis[J]. Yearbook of Paediatric Endocrinology, 2020, 179(6):1289-1305.
[8] ROBERT A K, LARISSA N, JESSICA H, et al. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice[J]. PLoS Genetics, 2006, 2(5):769-780.
[9] LEE Y H, NAIR S, ROUSSEAU E, et al. Microarray profiling of isolated abdominal subcutaneous adipocytes from obese vs non-obese Pima Indians:Increased expression of inflammation-related genes[J]. Diabetologia, 2005, 48(9):1776-1783.
[10] PETRUS P, MEJHERT N, CORRALES P, et al. Transforming growth factor-β3 regulates adipocyte number in subcutaneous white adipose tissue[J]. Cell Reports, 2018, 25(3):551-560.
[11] 朱才业.不同尾型绵羊全基因组关联分析、拷贝数变异及选择信号检测[D].北京:中国农业大学, 2017. ZHU C Y.Genome-wide assiociation study, copy number variations and selection signatures detect for sheep with different types of tails[D].Beijing:China Agricultural University, 2017.(in Chinese)
[12] ZHAO F P, DENG T Y, SHI L Y, et al. Genomic scan for selection signature reveals fat deposition in Chinese indigenous sheep with extreme tail types[J]. Animals, 2020, 10(5):773.
[13] LI X, YANG J, SHEN M, et al. Whole-genome resequencing of wild and domestic sheep identifies genes associated with morphological and agronomic traits[J]. Nature Communications, 2020, 11(1):2815.
[14] DONG K Z, YANG M, HAN J G, et al. Genomic analysis of worldwide sheep breeds reveals PDGFD as a major target of fat-tail selection in sheep[J]. BMC Genomics, 2020, 21(1):800.
[15] 袁泽湖.利用选择信号分析方法筛选与绵羊尾型相关的基因[D].北京:中国农业科学院, 2016. YUAN Z H.Analysis of selection signatures identify gene associated with tail type in sheep[D].Beijing:Chinese Academy of Agricultural Sciences, 2016.(in Chinese)
[16] 刘真, 王慧华, 刘瑞凿, 等.肥尾和瘦尾羊全基因组选择信号检测[J]. 畜牧兽医学报, 2015, 46(10):1721-1732. LIU Z, WANG H H, LIU R Z, et al. Genome-wide delection of selection signatures of distinct tail types in sheep populations[J]. Chinese Journal of Animal and Veterinary Sciences, 2015, 46(10):1721-1732.(in Chinese)
[17] 李青.绵羊BMP2和PDGF-D基因多态性与尾型的关联及细胞水平功能验证[D].北京:中国农业科学院, 2019. LI Q.Association analysis of BMP2 and PDGF-D polymorphisms with tail type in sheep and functional verification at the cellular level[D].Beijing:Chinese Academy of Agricultural Sciences, 2019.(in Chinese)
[18] 徐红伟.三种尾脂型绵羊脂肪沉积性状表型测定及关联基因筛选研究[D].兰州:甘肃农业大学, 2019. XU H W.Research on phenotypic determination of fat deposition traits and screening of associated genes in three kinds of tail fat sheep[D].Lanzhou:Gansu Agricultural University, 2019.(in Chinese)
[19] 王晓龙, 王秀然, 卢天成.蛋白质糖基化修饰的研究进展[J]. 基因组学与应用生物学, 2017, 10(63):4380-4384. WANG X L, WANG X R, LU T C.Research progress in glycosylation modification of proteins[J]. Genomics and Applied Biology, 2017, 10(63):4380-4384.(in Chinese)
[20] LAROCHELLE W J, JEFFERS M, MCDONALD W F, et al. PDGFD, a new protease-activated growth factor[J]. Nature Cell Biology, 2001, 3(5):517-521.
[21] PERRY S E, ROBINSON P, MELCHER A, et al. Expression of the CUB domain containing protein 1(CDCP1) gene in colorectal tumour cells[J]. FEBS Letters, 2007, 581(6):1137-1142.
[22] FREDRIKSSON L, LI H, ERIKSSON U.The PDGF family:Four gene products form five dimeric isoforms[J]. Cytokine & Growth Factor Reviews, 2004, 15(4):197-204.
[23] UUTELA M, LAUREN J, BERGSTEN E, et al. Chromosomal location, exon structure, and vascular expression patterns of the human PDGFC and PDGFD genes[J]. Circulation, 2001, 103(18):2242-2247.
[24] 吴丹.血小板衍生生长因子D及其在癌症中的作用[J]. 生命的化学, 2020, 40(5):750-761. WU D.Platelet-derived growth factor D and its role in carcer[J]. The Chemistry of Life, 2020, 40(5):750-761.(in Chinese)
[25] 普琰, 刘明军, 李文蓉, 等.绵羊不同部位脂肪沉积表型性状的相关分析[J]. 中国畜牧杂志, 2019, 55(7):86-89. PU Y, LIU M J, LI W R, et al. Correlation analysis of phenotypic traits of fat deposition in different parts of sheep[J]. Chinese Journal of Animal Science, 2019, 55(7):86-89.(in Chinese)