鸭疫里默氏杆菌CH-1株37℃和42℃的转录组测序及比较分析

Transcriptome sequencing and comparison of Riemerella anatipestifer CH-1 at 37°C and 42°C

摘要:

【目的】鸭疫里默氏杆菌(Riemerella anatipestifer)是一种感染鸭的革兰氏阴性菌，给养鸭业造成较大经济损失。由于鸭体内温度为42 ℃，当鸭疫里默氏杆菌感染鸭后必然会调节自身基因表达来适应鸭体内温度。为鉴定鸭疫里默氏杆菌CH-1株感染鸭的适应性机制，本研究测定和比较了鸭疫里默氏杆菌CH-1株在37 ℃和42 ℃下的转录组。【方法】将鸭疫里默氏杆菌RA-CH-1株在37 ℃培养至对数生长期后，分别在37 ℃和42 ℃热应激1 h，收集菌体，提取总RNA。利用转录组测序(RNA-seq)技术获得RA-CH-1在37 ℃和42 ℃下的转录组原始数据，筛选得到差异表达基因，通过基因本体论(gene ontology, GO)数据库和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)数据库对差异表达基因进行富集分析。选取dnaK作为热应激反应基因进行功能初步鉴定。【结果】共筛选获得234个显著差异表达基因，其中169个基因显著上调，65个基因显著下调。显著差异表达基因通过GO富集分析主要富集在核苷代谢过程、糖基化合物代谢过程和核心RNA聚合酶结合转录因子活性等；显著差异表达基因通过KEGG富集分析主要富集在氧化磷酸化、核糖体和细菌分泌系统等通路。与37 ℃条件下生长能力比较，dnaK缺失后导致鸭疫里默氏杆菌在42 ℃条件下生长能力受损。【结论】RA-CH-1在37 ℃和42 ℃下生长不受影响，通过热休克反应相关蛋白等因子表达上调或下调来应对温度升高后的应激状况。

Abstract:

[Objective] Riemerella anatipestifer is a Gram-negative bacterium infecting ducks, causing serious economic losses to the duck industry. After infection, R. anatipestifer regulates gene expression to adapt to the 42 ℃ body temperature of ducks. To identify the adaptation mechanism of R. anatipestifer CH-1 in ducks, we sequenced and compared the transcriptomes of R. anatipestifer CH-1 at 37 ℃ and 42 ℃. [Methods] R. anatipestifer CH-1 was cultured to the exponential growth phase at 37 ℃ and then subjected to heat stress at 37 ℃ and 42 ℃, respectively, for 1 h. The cells were then collected for the extraction of total RNA. The raw transcriptome data of the bacteria cultured at 37 ℃ and 42 ℃ were obtained by transcriptome sequencing, and differentially expressed genes (DEGs) were screened. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were carried out for the DEGs. The gene dnaK involved in the response to heat stress was selected for preliminary functional identification. [Results] A total of 234 DEGs were screened out, including 169 genes with up-regulated expression and 65 genes with down-regulated expression. The GO enrichment analysis showed that the DEGs were mainly enriched in the nucleotide metabolic process, glycosyl compound metabolic process, and core RNA polymerase binding transcription factor activity. The KEGG enrichment analysis indicated that the DEGs were mainly involved in oxidative phosphorylation, ribosomes, and bacterial secretion systems. The deletion of dnaK impaired the growth of R. anatipestifer CH-1 at 42 ℃, compared with that at 37 ℃. [Conclusion] Compared with that at 37 ℃, the growth of R. anatipestifer CH-1 was not affected at 42 ℃. The strain up-regulated or down-regulated the expression of heat shock response proteins and other factors to cope with heat stress.