西藏牦牛源牛支原体T10株全基因组测序及其序列分析

doi:10.11843/j.issn.0366-6964.2024.05.033

摘要/Abstract

摘要： 旨在进一步完善牛支原体全基因组数据库，阐明其生物学功能和遗传进化关系，对西藏牦牛源牛支原体T10株进行了全基因组测序及其序列分析。使用Nanopore和Illumina PE150测序平台对T10株进行全基因组序列测定，利用生物信息学对其进行基因组特征分析和利用基因系统进化树与国内外分离株进行遗传进化关系比对。基因测序结果显示，T10株全基因组大小为987 812 bp，GC含量为29.27%，预测到822个编码基因，编码基因总长度为709 129 bp；通过功能数据库注释结果显示，注释到与膜转运蛋白相关基因22个，碳水化合物酶相关基因7个、糖基转移酶类相关基因4个，分泌蛋白基因相关43个、T3SS效应蛋白相关基因51个，病原与宿主互作相关基因28个，细菌毒力相关基因14个，抗性相关基因10个；通过比较基因组学分析结果显示，T10与08M、CQ-W70、Hubei-1、Ningxia-1和PG45均存在氨基酸突变和基因片段的插入或缺失，以及基因家族数量的差异，其中基因家族数量差异在8~32个。通过遗传进化关系分析结果显示：T10与HB0801、16M、Hubei-1、CQ-W70、NM2012、Ningxia-1、08M、JF4278、KG4397和PG45均在同一分支，但与PG1、PG2处于不同分支，其中与HB0801亲缘关系最近，与PG45亲缘关系较远。本研究不仅完善了牛支原体全基因组数据库，详细阐述了与致病性相关基因，还充分阐明了T10株以及与国内外其他牛支原体之间的遗传进化关系，明确了T10株基本基因组信息以及与国内外菌株亲缘关系，为后续进行致病机制以及疫苗研究提供理论依据。

关键词: 牦牛, 牛支原体, 全基因组测序, 基因功能注释, 比较基因组

Abstract: This study aims to further improve the complete genome database of Mycoplasma bovis, elucidate its biological characteristics and genetic evolution relationship, the whole genome sequencing and sequence analysis of Mycoplasma bovis T10 strain derived from Xizang yaks were conducted. The entire genome of T10 strain was sequenced using the Nanopore and Illumina PE150 sequencing platforms, and the genomic characteristics were analyzed using bioinformatics. Genetic phylogenetic trees were used to compare genetic evolution relationships with domestic and foreign isolates. The gene sequencing results showed that the total genome size of T10 strain was 987 812 bp, with a GC content of 29.27%. It was predicted that 822 coding genes were present, with a total length of 709 129 bp; According to the annotation results of the functional database, 22 genes related to membrane transport proteins, 7 genes related to carbohydrate enzymes, 4 genes related to glycosyltransferases, 43 genes related to secretion proteins, 51 genes related to T3SS effector proteins, 28 genes related to pathogen host interaction, 14 genes related to bacterial virulence, and 10 genes related to resistance were annotated; Through comparative genomic analysis, it was found that T10 and 08M, CQ-W70, Hubei-1, Ningxia-1, and PG45 all exhibit amino acid mutations and gene fragment insertions or deletions, as well as differences in the number of gene families, with a difference in the number of gene families ranging from 8 to 32. The results of genetic evolution analysis show that T10 is in the same branch as HB0801, 16M, Hubei-1, CQ-W70, NM2012, Ningxia-1, 08M, JF4278, KG4397, and PG45, but in different branches from PG1 and PG2. Among them, T10 has the closest genetic relationship with HB0801 and the farther genetic relationship with PG45. This study not only improved the complete genome database of Mycoplasma bovis and elaborated on the genes related to pathogenicity, but also fully elucidated the genetic evolution relationship between T10 strain and other Mycoplasma bovis strains at home and abroad. It clarified the basic genome information of T10 strain and its genetic relationship with domestic and foreign strains, providing a theoretical basis for future research on pathogenic mechanism vaccines.

Key words: yak, Mycoplasma bovis, whole-genome sequencing, gene function annotation, comparative genomics

中图分类号:

S852.62

罗婷, 韩著, 徐业芬, 蔡林, 索朗斯珠, 徐晋花, 牛家强. 西藏牦牛源牛支原体T10株全基因组测序及其序列分析[J]. 畜牧兽医学报, 2024, 55(5): 2154-2167.

LUO Ting, HAN Zhu, XU Yefen, CAI Lin, SUOLANG Sizhu, XU Jinhua, NIU Jiaqiang. Whole Genome Sequencing and Sequence Analysis on T10 of Mycoplasma bovis Strain from Yaks in Xizang[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2154-2167.

参考文献

[1] MAUNSELL F P, WOOLUMS A R, FRANCOZ D, et al. Mycoplasma bovis infections in cattle[J]. J Vet Intern Med, 2011, 25(4):772-783.
[2] 武琪, 张玉娟, 刘桐, 等. 牛支原体黏附素及黏附素结合蛋白研究进展[J]. 畜牧兽医学报, 2023, 54(8):3206-3216.
WU Q, ZHANG Y J, LIU T, et al. Research progress of Mycoplasma bovis adhesins and adhesin-binding proteins[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8):3206-3216. (in Chinese)
[3] HALE H H, HELMBOLDT C F, PLASTRIDGE W N, et al. Bovine mastitis caused by a Mycoplasma species[J]. Cornell Vet, 1962, 52:582-591.
[4] KIRK J H, GLENN K, RUIZ L, et al. Epidemiologic analysis of Mycoplasma spp isolated from bulk-tank milk samples obtained from dairy herds that were members of a milk cooperative[J]. J Am Vet Med Assoc, 1997, 211(8):1036-1038.
[5] FEENSTRA A, BISGAARD MADSEN E, FRIIS N F, et al. A field study of Mycoplasma bovis infection in cattle[J]. J Vet Med, 1991, 38(1-10):195-202.
[6] TER LAAK E A, NOORDERGRAAF J H, DIELTJES R P J W. Prevalence of mycoplasmas in the respiratory tracts of pneumonic calves[J]. J Vet Med Ser B, 1992, 39(1-10):553-562.
[7] POUMARAT F, LE GRAND D, PHILIPPE S, et al. Efficacy of spectinomycin against Mycoplasma bovis induced pneumonia in conventionally reared calves[J]. Vet Microbiol, 2001, 80(1):23-35.
[8] 石磊, 龚瑞, 尹争艳, 等. 肉牛传染性牛支原体肺炎流行的初步诊断[J]. 华中农业大学学报, 2008, 27(4):572.
SHI L, GONG R, YIN Z Y, et al. Preliminary diagnosis of cattle infectious Mycoplasma bovis pneumonia[J]. Journal of Huazhong Agricultural University, 2008, 27(4):572. (in Chinese)
[9] 辛九庆, 李媛, 郭丹, 等. 国内首次从患肺炎的犊牛肺脏中分离到牛支原体[J]. 中国预防兽医学报, 2008, 30(9):661-664.
XIN J Q, LI Y, GUO D, et al. First isolation of Mycoplasma bovis from calf lung with pneumoniae in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2008, 30(9):661-664. (in Chinese)
[10] 冉智光, 谢建华, 骆璐, 等. 我国部分地区牛支原体肺炎和关节炎的病原体诊断[J]. 中国预防兽医学报, 2010, 32(1):40-43.
RAN Z G, XIE J H, LUO L, et al. Etiological diagnosis of Mycoplasma bovis pneumonia and arthritis in some provinces in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2010, 32(1):40-43. (in Chinese)
[11] 郭澍强, 蒋万, 罗海峰, 等. 银川地区某奶牛场牛支原体病的诊断[J]. 中国预防兽医学报, 2015, 37(3):203-206.
GUO S Q, JIANG W, LUO H F, et al. Diagnosis of Mycoplasma bovis in a cattle farm of Yinchuan[J]. Chinese Journal of Preventive Veterinary Medicine, 2015, 37(3):203-206. (in Chinese)
[12] 高航飞. 牛支原体NM2012株生物学特性研究[D]. 呼和浩特:内蒙古农业大学, 2017.
GAO H F. Studies on the biological characteristics of Mycoplasma bovis NM2012 strain[D]. Hohhot:Inner Mongolia Agricultural University, 2017. (in Chinese)
[13] 姚永进, 剡根强, 王静梅, 等. 致犊牛肺炎和关节炎牛支原体新疆株的分离与鉴定[J]. 中国畜牧兽医, 2011, 38(12):76-79.
YAO Y J, YAN G Q, WANG J M, et al. Isolation and identification of Mycoplasma bovis Xinjiang strain inducing pneumonia and arthritis of calf[J]. China Animal Husbandry & Veterinary Medicine, 2011, 38(12):76-79. (in Chinese)
[14] 李媛, 闫磊, 刘桐, 等. 中国西部和北部边境4省区牛支原体血清流行病学调查及分析[J]. 中国预防兽医学报, 2021, 43(7):717-721.
LI Y, YAN L, LIU T, et al. Seroprevalence and analysis of Mycoplasma bovis infection in four provinces of western and northern China[J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(7):717-721. (in Chinese)
[15] NIU J Q, LI K W, PAN H C, et al. Epidemiological survey of Mycoplasma bovis in yaks on the Qinghai Tibetan Plateau, China[J]. Biomed Res Int, 2021, 2021:6646664.
[16] NIU J Q, WANG D J, YAN M S, et al. Isolation, identification and biological characteristics of Mycoplasma bovis in yaks[J]. Microb Pathog, 2021, 150:104691.
[17] 王冬经. 牦牛牛支原体病流行病学调查及其病原生物学特性研究[D]. 林芝:西藏农牧学院, 2020.
WANG D J. Epidemiological investigation of yak mycoplasma disease and study on the biological characteristics of its pathogen[D]. Linzhi:Xizang Institute of Agriculture and Animal Husbandry, 2020. (in Chinese)
[18] NIU J, YAN M, XU J, et al. The resistance mechanism of Mycoplasma bovis from Yaks in Tibet to fluoroquinolones and aminoglycosides[J]. Front Vet Sci, 2022, 9:840981.
[19] 严明帅. 牛支原体西藏分离株生物学特性研究[D]. 林芝:西藏农牧学院, 2021.
YAN M S. Study on biological characteristics of Mycoplasma bovis Xizang isolate[D]. Linzhi:Xizang Institute of Agriculture and Animal Husbandry, 2021. (in Chinese)
[20] 牛家强. 西藏牦牛源牛支原体生物学特性及感染兔肺脏转录组学研究[D]. 武汉:华中农业大学, 2021.
NIU J Q. Biological characteristics of isolated Mycoplasma bovis from yaks in Tibet and transcriptomics of lungs in infected rabbits[D]. Wuhan:Huazhong Agricultural University, 2021. (in Chinese)
[21] 徐晋花. 西藏牦牛源牛支原体比较基因组学及其致病性研究[D]. 林芝:西藏农牧学院, 2023.
XU J H. Comparative genomics and pathogenicity of Mycoplasma bovis isolated from yak in Tibet[D]. Linzhi:Xizang Institute of Agriculture and Animal Husbandry, 2023. (in Chinese)
[22] BESEMER J, LOMSADZE A, BORODOVSKY M. GeneMarkS:a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions[J]. Nucleic Acids Res, 2001, 29(12):2607-2618.
[23] SAHA S, BRIDGES S, MAGBANUA Z V, et al. Empirical comparison of ab initio repeat finding programs[J]. Nucleic Acids Res, 2008, 36(7):2284-2294.
[24] BENSON G. Tandem repeats finder:a program to analyze DNA sequences[J]. Nucleic Acids Res, 1999, 27(2):573-580.
[25] LOWE T M, EDDY S R. tRNAscan-SE:a program for improved detection of transfer RNA genes in genomic sequence[J]. Nucleic Acids Res, 1997, 25(5):955-964.
[26] LAGESEN K, HALLIN P, RØDLAND E A, et al. RNAmmer:consistent and rapid annotation of ribosomal RNA genes[J]. Nucleic Acids Res, 2007, 35(9):3100-3108.
[27] HSIAO W, WAN I, JONES S J, et al. IslandPath:aiding detection of genomic islands in prokaryotes[J]. Bioinformatics, 2003, 19(3):418-420.
[28] ZHOU Y, LIANG Y J, LYNCH K H, et al. PHAST:a fast phage search tool[J]. Nucleic Acids Res, 2011, 39(Web server issue 2):W347-W532.
[29] ASHBURNER M, BALL C A, BLAKE J A, et al. Gene Ontology:tool for the unification of biology[J]. Nat Genet, 2000, 25(1):25-29.
[30] KANEHISA M, GOTO S, KAWASHIMA S, et al. The KEGG resource for deciphering the genome[J]. Nucleic Acids Res, 2004, 32(Datebase issue 1):D277-D280.
[31] GALPERIN M Y, MAKAROVA K S, WOLF Y I, et al. Expanded microbial genome coverage and improved protein family annotation in the COG database[J]. Nucleic Acids Res, 2015, 43(Datebase issue 1):D261-D269.
[32] SAIER M H Jr, REDDY V S, TAMANG D G, et al. The transporter classification database[J]. Nucleic Acids Res, 2014, 42(Datebase issue 1):D251-D258.
[33] CANTAREL B L, COUTINHO P M, RANCUREL C, et al. The carbohydrate-active EnZymes database (CAZy):an expert resource for Glycogenomics[J]. Nucleic Acids Res, 2009, 37(Datebase issue 1):D233-D238.
[34] URBAN M, PANT R, RAGHUNATH A, et al. The Pathogen-Host Interactions database (PHI-base):additions and future developments[J]. Nucleic Acids Res, 2015, 43(Datebase issue 1):D645-D655.
[35] CHEN L H, XIONG Z H, SUN L L, et al. VFDB 2012 update:toward the genetic diversity and molecular evolution of bacterial virulence factors[J]. Nucleic Acids Res, 2012, 40(Datebase issue 1):D641-D645.
[36] GRISSA I, VERGNAUD G, POURCEL C. CRISPRFinder:a web tool to identify clustered regularly interspaced short palindromic repeats[J]. Nucleic Acids Res, 2007, 35(Web server issue 2):W52-W57.
[37] KURTZ S, PHILLIPPY A, DELCHER A L, et al. Versatile and open software for comparing large genomes[J]. Genome Biol, 2004, 5(2):R12.
[38] HARRIS R S. Improved pairwise alignment of genomic DNA[D]. University Park:Pennsylvania State University, 2007.
[39] EMMS D M, KELLY S. OrthoFinder:phylogenetic orthology inference for comparative genomics[J]. Genome Biol, 2019, 20(1):238.
[40] LETUNIC I, BORK P. Interactive Tree Of Life (iTOL) v5:an online tool for phylogenetic tree display and annotation[J]. Nucleic Acids Res, 2021, 49(W1):W293-W296.
[41] WISE K S, CALCUTT M J, FOECKING M F, et al. Complete genome sequence of Mycoplasma bovis type strain PG45 (ATCC 25523)[J]. Infect Immun, 2011, 79(2):982-983.
[42] 王艳杰, 李平安, 吴太平, 等. 牛支原体分离株全基因组序列测定及初步分析[J]. 动物医学进展, 2014, 35(8):44-49.
WANG Y J, LI P A, WU T P, et al. Complete genome sequence and preliminary analysis of Mycoplasma bovis isolated strain[J]. Progress in Veterinary Medicine, 2014, 35(8):44-49. (in Chinese)
[43] CHEN S L, HAO H F, ZHAO P, et al. Complete genome sequence of Mycoplasma bovis strain 08M[J]. Genome Announc, 2017, 5(19):e00324-17.
[44] ROMERO-GARCÍA J, FRANCISCO C, BIARNÉS X, et al. Structure-function features of a Mycoplasma glycolipid synthase derived from structural data integration, molecular simulations, and mutational analysis[J]. PLoS One, 2013, 8(12):e81990.
[45] SIROVER M A. On the functional diversity of glyceraldehyde-3-phosphate dehydrogenase:biochemical mechanisms and regulatory control[J]. Biochim Biophys Acta (BBA)-Gen Subj, 2011, 1810(8):741-751.
[46] VAN DER MERWE J, PRYSLIAK T, GERDTS V, et al. Protein chimeras containing the Mycoplasma bovis GAPDH protein and bovine host-defence peptides retain the properties of the individual components[J]. Microb Pathog, 2011, 50(6):269-277.
[47] PEREZ-CASAL J, PRYSLIAK T. Detection of antibodies against the Mycoplasma bovis glyceraldehyde-3-phosphate dehydrogenase protein in beef cattle[J]. Microb Pathog, 2007, 43(5-6):189-197.
[48] ZHENG L, WHITE R H, CASH V L, et al. Mechanism for the desulfurization of L-cysteine catalyzed by the nifS gene product[J]. Biochemistry, 1994, 33(15):4714-4720.
[49] PFLOCK M, BATHON M, SCHÄR J, et al. The orphan response regulator HP1021 of Helicobacter pylori regulates transcription of a gene cluster presumably involved in acetone metabolism[J]. J Bacteriol, 2007, 189(6):2339-2349.
[50] DALLO S F, KANNAN T R, BLAYLOCK M W, et al. Elongation factor Tu and E1 β subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae[J]. Mol Microbiol, 2002, 46(4):1041-1051.
[51] WANG H, LIU L, CAO Q, et al. Haemophilus parasuis α-2, 3-sialyltransferase-mediated lipooligosaccharide sialylation contributes to bacterial pathogenicity[J]. Virulence, 2018, 9(1):1247-1262.
[52] QI J J, GUO A Z, CUI P, et al. Comparative geno-plasticity analysis of Mycoplasma bovis HB0801 (Chinese Isolate)[J]. PLoS One, 2012, 7(5):e38239.
[53] LI Y, ZHENG H J, LIU Y, et al. The complete genome sequence of Mycoplasma bovis strain Hubei-1[J]. PLoS One, 2011, 6(6):e20999.
[54] 季文恒, 储岳峰, 赵萍, 等. 牛支原体逃避宿主免疫的研究进展[J]. 畜牧兽医学报, 2017, 48(3):393-402.
JI W H, CHU Y F, ZHAO P, et al. Advances in the research of immune evasion by Mycoplasma bovis[J]. Acta Veterinaria et Zootechnica Sinica, 2017, 48(3):393-402. (in Chinese)
[55] MARIA-SOLANO M A, ROMERO-RIVERA A, OSUNA S. Exploring the reversal of enantioselectivity on a zinc-dependent alcohol dehydrogenase[J]. Org Biomol Chem, 2017, 15(19):4122-4129.