鸭疫里默氏杆菌RiaR基因缺失株的构建及其生物学特性分析

Construction and biological characteristics analysis ofRiaR gene deletion strain ofRiemerella anatipestifer

全基因组测序及生物信息学分析表明,鸭疫里默氏杆菌RA-LZ01株中存在1对新的双组分调控系统(two-component regulatory systems,TCS)RiaR/RiaS,且在黄杆菌目中高度保守。为探讨RiaR/RiaS在鸭疫里默氏杆菌中的作用,采用同源重组和结合转移的方法构建反应调节器RiaR的基因缺失株RA^(ΔRiaR)和回补株RAcΔRia R,并比较了它们与亲本株RA-LZ01在生长、抗生素敏感性和毒力方面的生物学特性差异;通过RA-LZ01和RA^(ΔRiaR)的原核转录组测序,进一步分析RiaR所发挥的生物学功能。结果显示,与亲本株相比,RA^(ΔRiaR)在其生长、抗生素敏感性和毒力方面并无明显差异。转录组分析表明,从RA^(ΔRiaR)中筛选到203个差异基因[lb(FC)值的差异倍数≥2且P<0.05]均为显著上调的基因,提示RiaR/RiaS是1对负调控下游基因表达的TCS。COG分析结果显示,富集程度最高的差异基因为功能未知的基因(50个,19%),其次为氨基酸运输和代谢相关基因(29个,11%)。KEGG的分析结果显示,差异基因主要富集于代谢相关的通路(116个,77.8%),包括氨基酸代谢(29个,19.4%)、核苷酸代谢(14个,9.3%)、碳水化合物代谢(23个,15.4%)等14个代谢相关的子通路。因此,验证了鸭疫里默氏杆菌中RiaR基因的生物学功能,为深入探究该TCS参与鸭疫里默氏杆菌的代谢调控提供依据。

Whole genome sequencing and bioinformatics analysis showed that a new pair of two-component regulatory systems(TCS)RiaR/RiaS was found in Riemerella anatipestifer RA-LZ01,which was highly conserved in the family Flavobacteriales.To investigate the effect of RiaR/RiaS on the biological characteristics of R.anatipestifer,the response regulator RiaR gene deletion strain RA^(ΔRiaR)and the complemented strain RAcΔRiaR were obtained by homologous recombination and combination transfer,and compared their biological characteristics with the parental strain RA-LZ01 in terms of growth,antibiotic sensitivity and virulence.The prokaryotic transcriptome sequencing results of RA-LZ01 and RA^(ΔRiaR)was analyzed to explore the biological function of RiaR.The results showed that the absence of RiaR did not significantly affect the biological characteristics of R.anatipestifer in terms of growth,antibiotic sensitivity and virulence.Transcriptome analysis showed that all of screened 203 differential genes[lb(FC)≥2,P<0.05]were significantly upregulated,indicating that RiaR/RiaS was a TCS acting negatively on downstream-gene expression.COG enrichment results showed that the genes with unknown function had the highest enrichment degree(50,19%),followed by genes related to amino acid transport and metabolism(29,11%).KEGG analysis showed that differentially expressed genes were mainly enriched in metabolism-related pathways(116,77.8%),including 14 metabolism-related pathways such as amino acid metabolism(29,19.4%),nucleotide metabolism(14,9.3%)and carbohydrate metabolism(23,15.4%).In conclusion,this study explored the biological function of RiaR gene in R.anatipestifer,providing a basis for further exploring the role of TCS in the metabolic regulation of R.anatipestifer.