牦牛肌钙蛋白I基因家族的克隆及组织表达分析

doi:10.7668/hbnxb.20190667

摘要/Abstract

摘要： 为了克隆TNNI基因家族，预测其蛋白结构和功能，并分析其在牦牛不同组织中的表达差异，以0.5岁健康的类乌齐牦牛为试验材料，采用RT-PCR技术克隆牦牛肌钙蛋白I基因家族的CDS区序列并进行生物信息学分析，利用实时荧光定量PCR技术检测TNNI基因家族各成员的mRNA表达水平。结果表明，TNNI1、TNNI2和TNNI3基因CDS区大小分别为564，549，639 bp，分别编码187，182，212个氨基酸残基。预测分析结果显示：TNNI基因家族编码的蛋白质均为偏碱性蛋白，蛋白结构不稳定，均无跨膜结构域，无信号肽，属非分泌型蛋白；二、三级结构均以α-螺旋为主，均含有肌蛋白超家族保守结构域。系统进化树分析表明：类乌齐牦牛TNNI1基因与水牛的亲缘关系最近，其次是绵羊、黄牛，与小鼠亲缘关系最远；TNNI2和TNNI3均与黄牛、水牛的亲缘关系较近，与其他亲缘关系较远。实时荧光定量PCR结果显示，TNNI1和TNNI2基因在臀肌中的相对表达量最高，且TNNI1基因在心脏、肝脏和肺脏中的表达量显著高于TNNI2基因（P<0.05），TNNI3基因在心脏中表达量较高，而在肺脏、臀肌和肝脏中表达量低。

关键词: 牦牛, 肌钙蛋白I基因家族, 克隆, 组织表达

Abstract: In order to clone the TNNI gene family, predict its protein structure and function, and analyze its expression differences in different yak tissues, this study used 0.5-year-old healthy Leiwuqi yak as the experimental material, and the CDS of the yak troponin I gene family was cloned by RT-PCR and then the sequences were analyzed using bioinformatics, the mRNA expression level of TNNI family genes were determined using Real-time PCR. The results showed that the CDS of TNNI1, TNNI2 and TNNI3 were 564, 549 and 639 bp, encoding 187, 182 and 212 amino acid, respectively. The results of predictive analysis showed that the proteins encoded by the TNNI gene family were all alkaline proteins, the protein structure was unstable, no transmembrane domain, no signal peptide, and non-secretory protein; the second and third-order structures were α-helix mainly, both contained the Troponin superfamily conserved domain. Phylogenetic tree analysis showed that the Leiwuqi yak TNNI1 gene had the closest associated with that of Bubalus bubalis, followed by Ovis aries and Bos taurus, and the furthest relationship with Mus musculus; TNNI2 and TNNI3 were closely related to Bos taurus and Bubalus bubalis, and distant with the others. The Real-time quantitative PCR results showed that the relative expression of TNNI1 and TNNI2 genes were the highest in the gluteal muscles, and the expression of TNNI1 in the heart, liver and lung was significantly higher than that of TNNI2 (P<0.05), the TNNI3 gene was expressed in the heart at a higher level, but the expression level was low in the lung, gluteal muscle and liver.

Key words: Yak, Troponin I gene family, Cloning, Tissue expression

中图分类号:

S823.8+5

向娅, 柴志欣, 王吉坤, 信金伟, 王会, 王嘉博, 武志娟, 钟金城, 姬秋梅. 牦牛肌钙蛋白I基因家族的克隆及组织表达分析[J]. 华北农学报, 2020, 35(2): 228-238. doi: 10.7668/hbnxb.20190667.

XIANG Ya, CHAI Zhixin, WANG Jikun, XIN Jinwei, WANG Hui, WANG Jiabo, WU Zhijuan, ZHONG Jincheng, JI Qiumei. Cloning and Tissue Expression of Troponin I Gene Family in Yak[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(2): 228-238. doi: 10.7668/hbnxb.20190667.

http://www.hbnxb.net/CN/Y2020/V35/I2/228

参考文献

[1] Robaszkiewicz K, Ostrowska Z, Cyranka-Czaja A, Moraczewska J. Impaired tropomyosin-troponin interactions reduce activation of the actin thin filament[J]. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2015, 1854(5):381-390. doi:10.1016/j.bbapap.2015.01.004.
[2] Sepúlveda M, Gonano L, Viotti M, Morell M, Blanco P, Alarcón M L, Ramos I P, Carvalho A B, Medei E, Petroff M V. Calcium/calmodulin protein kinase Ⅱ-dependent ryanodine receptor phosphorylation mediates cardiac contractile dysfunction associated with sepsis[J]. Critical Care Medicine, 2017, 45(4):399-408. doi:10.1097/CCM.0000000000002101.
[3] Wong S, Feng H Z, Jin J P. The evolutionarily conserved C-terminal peptide of troponin I is an independently configured regulatory structure to function as a myofilament Ca²⁺-desensitizer[J]. J Mol Cell Cardiol,2019,136:42-52. doi:10.1016/j.yjmcc.2019.09.002.
[4] Sheng J J, Jin J P. TNNI1, TNNI2 and TNNI3:Evolution, regulation, and protein structure function relationships[J]. Gene, 2016, 576(1):385-394. doi:10.1016/j.gene.2015.10.052.
[5] 杨华.猪肌钙蛋白Ⅰ基因家族的基因结构、表达谱和多态性分析[D].武汉:华中农业大学,2009.
Yang H. Porcine troponin I gene family:Genomic structure,expression profiling and polymorphism analysis[D].Wuhan:Huazhong Agricultural University,2009.
[6] Sun Y, Wang G Z, Ji Z B, Chao T L, Liu Z H, Wang X L, Liu G Q, Wu C H, Wang J M. Three slow skeletal muscle troponin genes in small-tailed Han sheep (Ovis aries):molecular cloning, characterization and expression analysis[J]. Molecular Biology Reports, 2016, 43(9):999-1010. doi:10.1007/s11033-016-4027-6.
[7] De Matteis A, dell'Aquila M, Maiese A, Frati P, La Russa R, Bolino G, Fineschi V. The Troponin-I fast skeletal muscle is reliable marker for the determination of vitality in the suicide hanging[J]. Forensic Science International, 2019, 301:284-288. doi:10.1016/j.forsciint.2019.05.055.
[8] Anderson C M, Hu J X, Thomas R, Gainous T B, Celona B, Sinha T, Dickel D E, Heidt A B, Xu S M, Bruneau B G, Pollard K S, Pennacchio L A, Black B L. Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites[J]. Development, 2017, 144(7):1235-1241. doi:10.1242/dev.138487.
[9] Kracklauer M P, Feng H Z, Jiang W R, Lin J L, Lin J J, Jin J P. Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms[J]. FEBS Journal, 2013, 280(3):880-891. doi:10.1111/febs.12076.
[10] Murphy A M, Jones L, Sims H F, Strauss A W. Molecular cloning of rat cardiac troponin I and analysis of troponin I isoform expression in developing rat heart[J]. Biochemistry, 1991, 30(3):707-712. doi:10.1021/bi00217a018.
[11] Bhavsar P K, Brand N J, Yacoub M H, Barton P J. Isolation and characterization of the human cardiac Troponin I gene (TNNI3)[J]. Genomics, 1996, 35(1):11-23. doi:10.1006/geno.1996.0317.
[12] Moses M A, Wiederschain D, Wu I M, Fernandez C A, Ghazizadeh V, Lane W S, Flynn E, Sytkowski A, Tao T, Langer R. Troponin I is present in human cartilage and inhibits angiogenesis[J]. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(6):2645-2650. doi:10.1073/pnas.96.6.2645.
[13] Ahmad F, Seidman J G, Seidman C E. The genetic basis for cardiac remodeling[J]. Annu Rev Genomics Hum Genet,2005, 6(6):185-216. doi:10.1146/annurev.genom.6.080604.162132.
[14] 赵秀丽.不同肢端畸形中 HOXD13、TNNI2 和 TPM2 基因的突变鉴定[D].北京:中国协和医科大学,2006.
Zhao X L. Molecular genetics of limb malformations:mutation identification in HOXD13, TNNI2 and TPM2[D].Beijing:Peking Union Medical College, 2006.
[15] 杨树猛,张海滨,刘汉丽,杨勤,余群力.蕨麻猪血清中激素含量的分析及其与肉质相关性研究[J]. 中国畜牧兽医文摘,2007,1:79-80.
Yang S M, Zhang H B, Liu H L, Yang Q, Yu Q L. Analysis of hormone content in serum of Juerna pig and its correlation with meat quality[J]. Chinese Abstracts of Animal Husbandry and Veterinary Medical,2007,1:79-80.
[16] 朱贵明.秦川牛肉质性状的系统研究[D].杨凌:西北农林科技大学,2003.
Zhu G M. The research on meat quality of Qinchuan cattle[D]. Yangling:Northwest A&F University, 2003.
[17] Li M Z, Li X W, Zhu L, Teng X K, Xiao H H, Shuai S R, Chen L, Li Q, Guo Y J. Differential expression analysis and regulatory network reconstruction for genes associated with muscle growth and adipose deposition in obese and lean pigs[J]. Progress in Natural Science, 2008, 18(4):387-399. doi:10.1016/j.pnsc.2007.10.011.
[18] 孙艳.小尾寒羊三个慢型骨骼肌肌钙蛋白的基因克隆、结构特征及组织表达分析[D].泰安:山东农业大学,2016.
Sun Y. Molecular cloning,characterization and tissue expression analysis of three slow skeletal muscle troponin genes in small-tailed Han sheep[D].Taian:Shandong Agricultural University,2016.
[19] Song Q Q, Chai Z X, Xin J W, Zhao S J, Ji Q M, Zhang C F, Ma Z J, Zhong J C. Genetic diversity and classification of Tibetan yak populations based on the mtDNA COⅢ gene[J]. Genetics and Molecular research, 2015, 14(1):1763-1770. doi:10.4238/2015.
March.13.3.
[20] 吴婷婷. 天府肉羊 TNNI1 和 TNNI3 基因的克隆、表达及其与肌肉组织学性状相关性研究[D]. 雅安:四川农业大学, 2013.
Wu T T. Cloning and expression of Tianfu goat TNNI1 and TNNI3 gene and their relationship with muscle tissue traits[D]. Yaan:Sichuan Agricultural University, 2013.
[21] Karabulut N P, Frishman D. Sequence-and structure-based analysis of tissue-specific phosphorylation sites[J]. PLoS One, 2016, 11(6):e0157896. doi:10.1371/journal.pone.0157896.
[22] Yang H Y, Zhu Q, Zhou N D, Tian Y P. Optimized expression of prolyl aminopeptidase in Pichia pastoris and its characteristics after glycosylation[J]. World Journal of Microbiology and Biotechnology, 2016, 32(11):176. doi:10.1007/s11274-016-2135-z.
[23] 赵伯阳. 山羊 CAPN1 基因和 CAST 基因Ⅱ型转录本克隆及在不同组织中的表达[D]. 雅安:四川农业大学, 2011.
Zhao B Y. cDNA cloning and tissue expression of CAPN1 gene and CAST transcript Ⅱ in goat[D]. Yaan:Sichuan Agricultural University,2011.
[24] 苏强强. 绵羊 MYOZ2 和 MYOZ3 基因的克隆及在凉山半细毛羊组织的时空表达[D]. 雅安:四川农业大学, 2011.
Su Q Q. Cloning of MYOZ2, MYOZ3 gene in Ovine and tissues with time expression in Liangshan semi-fine wool sheep[D]. Yaan:Sichuan Agricultural University,2011.
[25] Wei H G, Jin J P. NH₂-terminal truncations of cardiac troponin I and cardiac troponin T produce distinct effects on contractility and calcium homeostasis in adult cardiomyocytes[J]. Am J Physiol Cell Physiol, 2015, 308(5):397-404. doi:10.1152/ajpcell.00358.2014.
[26] Bollen I A E, Schuldt M, Harakalova M, Vink A, Asselbergs F W, Pinto J R, Krüger M, Kuster D W D, van der Velden J. Genotype-specific pathogenic effects in human dilated cardiomyopathy[J]. The Journal of Physiology, 2017, 595(14):4677-4693. doi:10.1113/JP274145.
[27] 姬改革, 束婧婷, 单艳菊, 章明, 屠云洁, 巨晓军, 刘一帆. 高邮鸭 TNNI1 基因克隆及组织表达分析[J]. 农业生物技术学报, 2018,26(6):970-977. doi:10.3969/j.issn.1674-7968.2018.06.007.
Ji G G, Shu J T, Shan Y J, Zhang M, Tu Y J, Ju X J, Liu Y F. Molecular cloning and tissue expression analysis of TNNI1 gene in Gaoyou duck (Anas platyrhynchos)[J]. Journal of Agricultural Biotechnology, 2018,26(6):970-977.
[28] 张阳. 小尾寒羊 TNNI2 和 TNNI3 基因的克隆、结构分析及组织表达分析[D].泰安:山东农业大学,2016.
Zhang Y. Molecular cloning,characterization and tissue expression analysis of the TNNI2 and TNNI3 genes from small-tailed Han Sheep[D]. Taian:Shandong Agricultural University, 2016.
[29] 李伯江, 李平华, 吴望军, 李齐发, 黄瑞华, 刘红林. 骨骼肌肌纤维形成机制的研究进展[J]. 中国农业科学, 2014,47(6):1200-1207. doi:10.3864/j.issn.0578-1752.2014.06.016.
Li B J, Li P H, Wu W J, Li Q F, Huang R H, Liu H L. Progresses in research of the mechanisms of skeletal muscle fiber formation[J]. Scientia Agricultura Sinica,2014,47(6):1200-1207.
[30] Pang X C, Lin L J, Tang B. Unraveling the role of calcium ions in the mechanical properties of individual collagen fibrils[J]. Scientific Reports, 2017, 7:46042. doi:10.1038/srep46042.
[31] Moreno H M, Jacq C, Montero M P, Gómez-Guillén M C, Borderías A J, Mørkøre T. Effect of selective breeding on collagen properties of Atlantic salmon (Salmo salar L.)[J]. Food Chemistry, 2016, 190:856-863. doi:10.1016/j.foodchem.2015.06.022.
[32] Lesiow T, Rentfrow G K, Xiong Y L. Polyphosphate and myofibrillar protein extract promote transglutaminase-mediated enhancements of rheological and textural properties of PSE pork meat batters[J]. Meat Science, 2017, 128:40-46. doi:10.1016/j.meatsci.2017.02.002.
[33] Luu B E, Tessier S N, Duford D L, Storey K B. The regulation of Troponins I, C and ANP by GATA4 and Nkx2-5 in heart of hibernating thirteen-lined ground squirrels, Ictidomys tridecemlineatus[J]. PLoS One, 2015,10(2):e0117747. doi:10.1371/journal.pone.0117747.
[34] Martin-Garrido A, Biesiadecki B J, Salhi H E, Shaifta Y, Dos Remedios C G, Ayaz-Guner S, Cai W X, Ge Y, Avkiran M, Kentish J C. Monophosphorylation of cardiac troponin-I at Ser23/24 is sufficient to regulate cardiac myofibrillar Ca²⁺, sensitivity and calpain-induced proteolysis[J]. Journal of Biological Chemistry, 2018, 293(22):8588-8599. doi:10.1074/jbc.RA117.001292.
[35] Wang H, Grant J E, Doede C M, Sadayappan S, Robbins J, Walker J W. PKC-betaII sensitizes cardiac myofilaments to Ca²⁺ by phosphorylating troponin I on threonine-144[J]. Journal of Molecular and Cellular Cardiology, 2006, 41(5):823-833. doi:10.1016/j.yjmcc.2006.08.016.
[36] Lu Q W, Hinken A C, Patrick S E, Solaro R J, Kobayashi T. Phosphorylation of cardiac troponin I at protein kinase C site threonine 144 depresses cooperative activation of thin filaments[J]. Journal of Biological Chemistry, 2010, 285(16):11810-11817. doi:10.1074/jbc.M109.055657.
[37] Messer A E, Bayliss C R, El-Mezgueldi M, Redwood C S, Ward D G, Leung M C, Papadaki M, Dos Remedios C, Marston S B. Mutations in troponin T associated with hypertrophic cardiomyopathy increase Ca²⁺-sensitivity and suppress the modulation of Ca²⁺-sensitivity by troponin I phosphorylation[J]. Archives of Biochemistry and Biophysics, 2016, 601:113-120. doi:10.1016/j.abb.2016.03.027.
[38] Akhter S,Zhang Z L, Jin J P. The heart-specific NH2-terminal extension regulates the molecular conformation and function of cardiac troponin I[J]. AJP Heart and Circulatory Physiology, 2012, 302(4):923-933. doi:10.1152/ajpheart.00637.2011.
[39] Zhao Y, Feng Y, Zhang Y M, Ding X X, Song Y Z, Zhang A M, Liu L, Zhang H, Ding J H, Xia S. Targeted next-generation sequencing of candidate genes reveals novel mutations in patients with dilated cardiomyopathy[J]. International Journal of Molecular Medicine, 2015, 36(6):1479-1486. doi:10.3892/ijmm.2015.2361.
[40] 曹晨, 马堃. 蛋白质二级结构指定[J]. 生物信息学, 2016,14(3):181-187. doi:10.3969/j.issn.1672-5565.2016.03.09.
Cao C, Ma K. Protein secondary structure assignment[J]. Chinese Journal of Bioinformatics, 2016,14(3):181-187.
[41] Yang H, Xu Z Y, Ma Z X, Xiong Y Z, Deng C Y, Zuo B. Molecular cloning and comparative characterization of the porcine troponin I family[J]. Animal Biotechnology, 2009, 21(1):64-76. doi:10.1080/10495390903385983.
[42] Sun Y, Wang G Z, Ji Z B, Chao T L, Liu Z H, Wang X L, Liu G Q, Wu C H, Wang J M. Three slow skeletal muscle troponin genes in small-tailed Han sheep (Ovis aries):molecular cloning, characterization and expression analysis[J]. Molecular Biology Reports, 2016,43(9):999-1010. doi:10.1007/s11033-016-4027-6.
[43] Moran C M, Garriock R J, Miller M K, Heimark R L, Gregorio C C, Krieg P A. Expression of the fast twitch troponin complex, fTnT, fTnI and fTnC, in vascular smooth muscle[J]. Cell Motility and the Cytoskeleton, 2008, 65(8):652-661. doi:10.1002/cm.20291.
[44] Ju Y W, Li J, Xie C, Ritchlin C T, Xing L P, Hilton M J, Schwarz E M. Troponin T3 expression in skeletal and smooth muscle is required for growth and postnatal survival:Characterization of Tnnt3^{tm2α(KOMP)Wtsi} mice[J]. Genesis, 2013, 51(9):667-675. doi:10.1002/dvg.22407.
[45] Dhoot G K, Gell P G H, Perry S V. The localization of the different forms of troponin I in skeletal and cardiac muscle cells[J]. Experimental Cell Research, 1978, 117(2):357-370. doi:10.1016/0014-4827(78)90149-0.

选择文件类型/文献管理软件名称

选择包含的内容