一株缺失pMT1质粒鼠疫菌的全基因组序列分析
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摘要
目的通过基因测序及生物信息学分析,确定O-1菌株基因组的一般特征及与参考菌株的差异,确定O-1菌株的pMT1质粒是否整合至染色体以及可能的整合机制。方法提取O-1鼠疫菌株的全基因组DNA,进行测序组装,获得全基因组序列及圈图;通过与参考菌株比较来描述基因组一般特征;通过序列比对(Blast/Mauve)确定整合位点,再通过引物设计和PCR扩增来验证整合位点。结果 O-1菌株基因组包括一个染色体,两个质粒(70.3Kb,9.6Kb);O-1菌株染色体基因数目与CO92菌株有差异,染色体的插入序列(尤其是IS100和IS1541)较CO92菌株多;O-1菌株质粒与CO92菌株很相似,但O-1菌株无独立存在的pMT1质粒。pMT1质粒序列位于O-1菌株染色体中,从1429315至1526240,序列长度为96926bp;且整合位点被PCR扩增证实;O-1菌株pMT1质粒序列含有5个IS序列;pMT1序列的插入位点处于O-1染色体的非编码区。结论 O-1菌株的pMT1质粒整合至染色体,而且是完整序列整合。O-1菌株染色体特征与CO92有差异,且插入序列较多。IS1541A介导的同源重组可能是pMT1质粒整合至染色体的原因。
Objective To determine the general characteristics of the genome of O-1 strain and the differences with the reference strains by gene sequencing and bioinformatics analysis,and to determine whether the pMT1 plasmid of O-1 strain was integrated into the chromosome and the possible integration mechanism.Methods Genomic DNA of O-1 strain was extracted and sequenced to obtain the whole genome sequence and circle graph.The general characteristics of the genome were described by comparison with the reference strains.The integration sites were identified by sequence alignment(Blast/Mauve),and then verified by primer design and PCR amplification.Results The genome of the O-1 strain consisted of a single circular chromosome and two plasmids(70.3 Kb,9.6 Kb).The number of chromosomal genes of O-1 strain was different from that of CO92 strain.The insertion sequence of chromosome(especially IS100 and IS1541) was more than that of CO92 strain.The plasmids of O-1 strain were similar to that of CO92 strain,while there was no independent pMT1 plasmid in O-1 strain.The pMT1 plasmid sequence was located in the chromosome of the O-1 strain ranging from 1429315 to 1526240 with a length of 96926 bp,which was similar to the pMT1 plasmid characteristics of the CO92 strain and contained five IS sequences.The integration site was confirmed by PCR amplification.The insertion site of the pMT1 sequence was in the non-coding region of the O-1 chromosome.Conclusion The pMT1 plasmid of the O-1 strain,the whole sequence,was integrated into the chromosome.The chromosomal characteristics of O-1 strain were different from CO92 strain,and the insertion sequence was more than that of CO92.The homologous recombination mediated by IS1541 A may be responsible for the integration of the pMT1 plasmid.
Objective To determine the general characteristics of the genome of O-1 strain and the differences with the reference strains by gene sequencing and bioinformatics analysis,and to determine whether the pMT1 plasmid of O-1 strain was integrated into the chromosome and the possible integration mechanism.Methods Genomic DNA of O-1 strain was extracted and sequenced to obtain the whole genome sequence and circle graph.The general characteristics of the genome were described by comparison with the reference strains.The integration sites were identified by sequence alignment(Blast/Mauve),and then verified by primer design and PCR amplification.Results The genome of the O-1 strain consisted of a single circular chromosome and two plasmids(70.3 Kb,9.6 Kb).The number of chromosomal genes of O-1 strain was different from that of CO92 strain.The insertion sequence of chromosome(especially IS100 and IS1541) was more than that of CO92 strain.The plasmids of O-1 strain were similar to that of CO92 strain,while there was no independent pMT1 plasmid in O-1 strain.The pMT1 plasmid sequence was located in the chromosome of the O-1 strain ranging from 1429315 to 1526240 with a length of 96926 bp,which was similar to the pMT1 plasmid characteristics of the CO92 strain and contained five IS sequences.The integration site was confirmed by PCR amplification.The insertion site of the pMT1 sequence was in the non-coding region of the O-1 chromosome.Conclusion The pMT1 plasmid of the O-1 strain,the whole sequence,was integrated into the chromosome.The chromosomal characteristics of O-1 strain were different from CO92 strain,and the insertion sequence was more than that of CO92.The homologous recombination mediated by IS1541 A may be responsible for the integration of the pMT1 plasmid.
引文
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[2] 龚震宇.2010-2015年全球鼠疫流行概况[J].疾病监测,2016,31(8):711-712.
[3] Ferber DM,Brubaker RR.Plasmids in Yersinia pestis[J].Infection and Immunity,1981,31(2):839-841.
[4] Du YD,Rosqvist R,Forsberg A.Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis[J].Infection and Immunity,2002,70(3):1453-1460.
[5] 张艳,郭英,董珊珊,等.caf1基因为靶标的鼠疫检测PCR法的改进[J].中华地方病学杂志,2018,37(3):203-206.
[6] 董兴齐,叶枫,彭何碧,等.云南省鼠疫菌质粒特征及地理分布[J].中华流行病学杂志,2001,22(5):30-33.
[7] 董兴齐,彭何碧,黄坚华,等.云南省鼠疫菌某些生物学特性及其流行病学意义[J].大理医学院学报,1995(1):40-45.
[8] Doll JM,Zeitz PS,Ettestad P,et al.Cat-transmitted fatal pneumonic plague in a person who traveled from Colorado to Arizona[J].American Journal of Tropical Medicine and Hygiene,1994,51(1):109-114.
[9] Parkhill J,Wren BW,Thomson NR,et al.Genome sequence of Yersinia pestis,the causative agent of plague[J].Nature,2001,413(6855):523-527.
[10] Cui Y,Song Y.Genome and Evolution of Yersinia pestis[M]//Yang R,Anisimov A.Yersinia pestis:retrospective and perspective,2016:171-192.
[11] Chain PS,Hu P,Malfatti SA,et al.Complete genome sequence of Yersinia pestis strains Antiqua and Nepal516:Evidence of gene reduction in an emerging pathogen[J].Journal of Bacteriology,2006,188(12):4453-4463.
[12] Zimbler DL,Schroeder JA,Eddy JL,et al.Early emergence of Yersinia pestis as a severe respiratory pathogen[J].Nature Communications,2015,6:7487.
[13] Friedlander AM,Welkos SL,Worsham PL,et al.Relationship between virulence and immunity as revealed in recent studies of the F1 capsule of Yersinia pestis[J].Clinical Infectious Diseases,1995,21(Suppl 2):S178-S181.
[14] 李俊勇,赵文红,董兴齐,等.1982-2005年云南省家鼠鼠疫流行状况分析[J].中国地方病学杂志,2006,25(6):654-657.
[15] 杨春光,赵文红,董兴齐.云南省家鼠鼠疫流行特征与强度分析[J].中国媒介生物学及控制杂志,2007,18(3):226-229.