Cloning, Bioinformatics and Tissue Expression Profile Analysis of PDGFD Gene in Yak (Bos mutus)
Li Xin-Yi1,2, Wang Fu-Bin1,2, Huang Chun2, Ma Xiao-Ming2, La Yong-Fu2, Chu Min2, Guo Xian2, Yan Ping2, Pan He-Ping1, Liang Chun-Nian2,*
1 College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China; 2 Lanzhou Institute of Husbandry and Pharmaceutical Sciences Chinese Academy of Agricultural Sciences, Lanzhou 730050, China
Abstract：Yak (Bos mutus) is an important genetic resource to the plateau region. To study the structure and function of the platelet-derived growth factor D (PDGFD) gene in yaks, and explore the PDGFD gene expression in different tissues at 2 age stages, this study cloned the complete PDGFD gene CDS sequence by using subcutaneous adipose tissue cDNA of 36-month-old Datong yak as the template. Biological software was used for analyzing the PDGFD gene nucleotide sequence and physical and chemical properties of protein. The relative expression of PDGFD in 7 tissues (heart, liver, spleen, lungs, kidney, longissimus muscle and adipose tissue) of embryonic and 36-months old yak was carried out by qPCR. The results showed that the CDS region of PDGFD gene was 1 095bp and encoded 364 amino acids. The most closely genetic relationship of Datong yak was the B. mutus (99.8%), and the most distant was the Sus scrofa (91.2%). The protein analysis of PDGFD showed that PDGFD contains 1 CUB domain of 116 aa, 1 PDGF domain of 93 aa, and no transmembrane structure. PDGFD was predicted to be a stably hydrophilic secretory protein. The PDGFD protein advanced structure was consisted of random coil (48.21%), α-helix (25.62%), extended chain (21.49%), and β-turn (4.68%). In addition, PDGFD mainly interacted with platelet-derived growth factor protein and vascular endothelial growth factor protein. qPCR results showed that PDGFD mRNA was expressed in each tissue at 2 stages. By comparing the 2 periods, PDGFD gene expression was maintained at a high level in the heart. Meanwhile, the expression in adipose tissue changed the most. The mRNA expression level of PDGFD extremely significantly increased in adipose tissue with the growth and development of yaks (P<0.001). The result showed that PDGFD may be closely related to yak's heart, and affected adipose growth and development. This study offers a theoretical foundation for the regulation mechanism of yak fat deposition and provides references for the genetic breeding of yaks.
[1] 柴顺仓,康延珍,王雪祯,等. 2020. 大通牦牛改良青海环湖型牦牛效果分析[J]. 畜牧兽医科学: 电子版, (04): 9-10. (Chai S C, Kang Y Z, Wang X Z, et al. 2020. Effect analysis on improving Qinghai Lake type yak with Datong Yak[J]. Animal Science and Veterinary Science with Electronic Form, (04): 9-10.) [2] 陈美, 柴志欣, 武志娟, 等. 2021. 牦牛ACAA2基因克隆及组织表达分析[J]. 东北农业大学学报, 51(12): 51-57. (Chen M, Chai Z X, Wu Z J, et al.2021. Cloning and tissue expression analysis of ACAA2 gene in yak[J]. Journal of Northeast Agricultural University, 51(12): 51-57.) [3] 郭勇, 张雄, 史开志等. 2021. 猪脂肪沉积相关酶基因表达与胴体及肉质性状的相关性研究[J]. 中国畜牧杂志, 57(3): 91-96. ( Guo Y, Zhang X, Shi K Z, et al.2021. Correlation analysis between the expression of porcine fat deposition-related enzyme gene and carcass and mest quality traits[J]. China Animal Husbandary& Veterinary Medicine, 57(3): 91-96.) [4] 韩建刚. 2020. 绵羊胚胎期尾脂沉积关键功能基因挖掘[D]. 硕士学位论文, 中国农业科学院, 导师: 马月辉. pp. 1-69. (Han J G.2020. The detection of key genes for embryonic tail development of fat-tailed sheep[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences Thesis, Supervisor: Ma Y H. pp. 1-69.) [5] 郝力壮, 刘书杰, 胡令浩等. 2020. 牦牛营养需要量与饲草料营养价值评价研究进展[J]. 动物营养学报, 32(10): 4725-4732. (Hao L Z, Liu S J, Hu L H, et al.2020. Research progress on nutrient requirements of yak and nutritional value evaluation of forage/feed[J]. Chinese Journal of Animal Nutrition, 32(10): 4725-4732.) [6] 胡金丽, 庄蕾, 吴森. 2021. 牦牛肉质性状相关候选基因研究进展[J]. 中国畜牧杂志, 57(8): 6. (Hu J L, Zhuang L, Wu S, 2021. Research progress on candidate genes influencing meat quality traits in yaks[J]. Chinese Journal of Animal Science, 57(8): 6 ) [7] 李青. 2019. 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]. 硕士学位论文, 中国农业科学院, 导师: 魏彩虹. pp. 1-52. ( Li Q.2020. Association of BMP2 and PDGF-D polymorphisms with tail type in sheep and functional verification at the cellular level[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences Thesis, Supervisor: Wei C H. pp. 1-52.) [8] 刘伟伟, 赵塔娜, 李丽. 2019. PDGF家族在肾纤维化中的研究进展[J]. 微量元素与健康研究, 36(3): 65-67. (Liu W W, Zhao T N, Li L.2019. Research progress of PDGF family in renal fibrosis[J]. Weiliang Yuansu Yu Jiankang Yanjiu, 36(3): 65-67.) [9] 刘真. 2015. 肥尾和瘦尾羊全基因组选择信号检测[D]. 硕士学位论文, 中国农业科学院, 导师: 魏彩虹. pp. 1-44. ( Li Q.2020. Genome-wide detection of selection signatures between fat-tailed and thin-tailed sheep populations[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences Thesis, Supervisor: Wei C H. pp. 1-44.) [10] 石红梅, 丁考仁青, 杨勤等. 2013. 甘南牦牛一年一产试验[J]. 中国牛业科学, 39(6): 35-37. (Shi H M, Ding K R Q, Yang Q.2013. Experiment of annual litter size of Gannan yark[J]. China Cattle Science, 39(6): 35-37) [11] 王晓龙, 王秀然, 卢天成. 2017. 蛋白质糖基化修饰的研究进展[J]. 基因组学与应用生物学, 36(10): 4380-4384. (Wang X L, Wang X R, Lu T C.2017. The research progress of protein glycosylation madification[J]. Genomics and Appplied Biology, 36(10): 4380-4384.) [12] 王钰安. 2017. 自体脂肪纯化均质仪器开发[D]. 硕士学位论文, 杭州电子科技大学, 导师: 李宏. pp. 1-65. (Wang J A.2017. Development of purification and homogenization instrument of autologous fat[D]. Thesis for M.S., Hangzhou Dianzi University, Supervisor: Li H. pp. 1-65.) [13] 席斌, 王莉蓉, 郭天芬等. 2018. 大通牦牛肉与高原牦牛肉营养品质比较分析[J]. 黑龙江畜牧兽医, (23): 198-201. (X B, W L R, G T F, et al. 2018. Comparative analysis on nutritional quality of muscle between Datong yak and Mountain yak[J]. Heilongjiang Animal Science and Veterinary Medicine, (23): 198-201.) [14] 许晓波, 陈杰. 2004. 脂肪细胞的分化及其调控[J]. 畜牧与兽医, 36(11): 41-43. (Xu X B, Chen J.2004. Differentiation and regulation of adipocytes[J]. Animal Husbandry and Veterinary Medicine, 36(11): 41-43.) [15] 阎萍, 陆仲璘, 何晓林. 2006. "大通牦牛"新品种简介[J]. 中国畜禽种业, 2(5): 49-51. (Yan P, Lu Z L, He X L.2006. Brief introduction of new variety "Datong Yak"[J]. The Chinese Livestock and Poultry Breeding, 2(5): 49-51.) [16] 杨烨城, 黄秋艳, 辛海云, 等. 2021. 藏猪和杜藏猪生长、肉质性状及脂肪沉积相关基因的表达差异分析[J]. 南方农业学报, 53(04): 926-934. (Yang Y C, Huang Q Y, Xin H Y, et al.2021. Difference analysis of growth, meat quality and expression of genes related with adipose deposition between Duzang pig and Tibetan pig[J]. Journal of Southern Agriculture, 53(04): 926-934.) [17] 张宏山, 许明, 张阳, 等. 2010. 小鼠心肌组织3种总RNA提取方法比较[J]. 临床心血管病杂志, (2): 149-150. (Zhang H S, Xu M, Zhang Y, et al. 2010. Comparison of three different methods for total RNA isolation from mouse heart tissue[J]. Jounal of Clinical Cardiology, (2): 149-150.) [18] 张紫菡. 2019. 王不留行黄酮苷对小鼠脂质沉积的调控作用[D]. 硕士学位论文,西北农林科技大学, 导师: 吴江维. pp. 1-60. (Zhang Z H.2019. The effect of vaccarin on lipid deposition in mice[D]. Thesis for M.S., Thesis Submitted to Northwest A＆F University, Supervisor: Wu W J. pp, 1-60.) [19] 朱国兴,张云玲. 2005. 浅谈我国牦牛业生产现状及发展思路[J]. 中国畜牧杂志, 41(1): 63-65. (Zhu G X, Zhang Y L.2005. Discussion on the present situation and development of Yak industry in China[J]. China Animal Husbandary& Veterinary Medicine, 41(1): 63-65.) [20] 朱良瑞, 王家圣, 杨军等. 2013. 清平猪杂交组合胴体品质和肉质性状相关分析[J]. 畜牧与兽医, 45(11): 39-41. (Zhu L R, Wang J S, Yang J, et al.2013. Correlation analysis of carcass quality and meat quality traits in Qingping pig crosses[J]. Animal Husbandry and Veterinary Medicine, 45(11): 39-41.) [21] Acosta J R, Joost S, Karlsson K, et al.2017. Single cell transcriptomics suggest that human adipocyte progenitor cells constitute a homogeneous cell population[J]. Stem Cell Research Therapy, 8(1): 250. [22] Aguilar A.2016. Renal fibrosis: PDGF-D in renal fibrosis[J]. Nature Reviews. Nephrology, 12(5): 257. [23] Artemenko Y, Gagnon A, Aubin D, et al.2010. Anti‐adipogenic effect of PDGF is reversed by PKC inhibition[J]. Journal of Cellular Physiology, 204(2): 646-653. [24] Borkham-Kamphorst E, Meurer S, Leur K E Van, et al.2015. PDGF-D signaling in portal myofibroblasts and hepatic stellate cells proves identical to PDGF-B via both PDGF receptor type alpha and beta[J]. Cellular Signaling, 27(7): 1305-1314. [25] Dennis E, Bradshaw R.2009. Handbook of Cell Signaling[M]. Beijing: Science Press. pp. 308. [26] Fredriksson, Linda, Ehnman, et al.2005. Structural requirements for activation of latent platelet-derived growth factor cc by tissue plasminogen activator[J]. Journal of Biological Chemistry, 280(29): 26856-26862. [27] Fredriksson L, Li H, Fieber C, Li, X, et al.2014. Tissue plasminogen activator is a potent activator of PDGF‐CC[J]. Embo Journal, 23(19): 3793-3802. [28] Hausman G J, Dodson M V, Ajuwon K, et al.2009. Board-invited review: The biology and regulation of preadipocytes and adipocytes in meat animals[J]. Journal of Animal Science, 87(4): 1218. [29] Heldin C H, Ostman A, Rnnstrand L.1998. Signal transduction via platelet-derived growth factor receptors[J]. Biochim Biophys Acta, 1378(1): F79-113. [30] Hilgendorf K I, Johnson C T, Mezger A, et al.2019. Omega-3 fatty acids activate ciliary FFAR4 to control adipogenesis[J]. Cell, 179(6): 1289-1305. [31] Holmstrm T E, Mattsson C L, FaLting J M, et al.2008. Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes[J]. Experimental Cell Research, 314(19): 3581-3592. [32] Ji H K, Sang G P, Kim W, et al.2015. Functional regulation of adipose‐derived stem cells by PDGF-D[J]. Stem Cells, 33(2): 542-556. [33] Kohler N, Lipton, A.1974. Platelets as a source of fibroblast growth-promoting activity[J]. Experimental Cell Research, 87(2): 297-301. [34] Koza R A, Nikonova L, Hogan J, et al.2006. Changes in gene expression foreshadow diet-induced obesity in genetically identical mice[J]. PLOS Genetics, 2(5): 769-780. [35] Kumar J, Yumnam S, Basu T, et al.2011. Association of polymorphisms in 9p21 region with CAD in North Indian population: Replication of SNPs identified through GWAS[J]. Clinical Genetics, 79(6): 588-593. [36] Mori R, Wang Q, Danenberg K D, et al.2010. Both β-actin and GAPDH are useful reference genes for normalization of quantitative RT-PCR in human FFPE tissue samples of prostate cancer[J]. Prostate, 68(14): 1555-1560. [37] Qin Z, Feng J, Liu Y, et al.2015. PDGF-D promotes dermal fibroblast invasion in 3-dimensional extracellular matrix via snail-mediated MT1-MMP upregulation[J]. Tumor Biology, 37(1): 591-599. [38] Ross R.1999. Atherosclerosis-an in flammatory disease[J]. The New England Journal of Medicine, 340(2): 115-126. [39] Lee Y H, Nair S, Rousseau E, et al.2005. Microarray profiling of isolated abdominal subcutaneous adipocytes from obese vs non-obese Pima Indians: Increased expression of inflammation-related genes[J]. Diabetologia, 48(9): 1776-1783. [40] Zhao T Q, Zhao W Y, Chen Y J, et al.2013. Platelet-derived growth factor-D promotes fibrogenesis of cardiac fibroblasts[J]. American Journal of Physiology-Heart and Circulatory Physiology, 304(12): H1719-H1726. [41] Zhao Y X, Yang J, Lv F H, et al.2017. Genomic reconstruction of the history of native sheep reveals the peopling patterns of nomads and the expansion of early pastoralism in east asia[J]. Molecular Biology and Evolution, 34(9): 2380-2395. [42] Zhu C Y, Fan H Y, Yuan Z H, et al.2016. Genome-wide detection of CNVs in Chinese indigenous sheep with different types of tails using ovine high-density 600K SNP arrays[J]. Scientific Reports, 6: 27822.
