基于转录组学分析Xenorhabdus bovienii肽基脯氨酰异构酶ppia-l20基因的表达调控

Regulation of gene expression of the peptidyl-prolyl isomerase ppia-l20 in Xenorhabdus bovienii based on transcriptomic analysis

[目的]前期从广谱抗菌的Xenorhabdus bovienii NN6菌株的胞外蛋白中分离出一种新型杀菌蛋白PPIA-L20,为肽基脯氨酰异构酶A类似物,该蛋白能够抑制尖孢镰刀菌西瓜专化型的孢子的萌发,致使孢子死亡。经过多次试验验证ppia-l20基因的表达水平不稳定,与发酵时间没有明显的相关性。本文旨在探寻调控ppia-l20基因表达的调控因子,进而推测ppia-l20基因表达调控的分子机制,为进一步提高抗菌蛋白的产量提供参考。[方法]对ppia-l20的转录水平进行Real-time PCR分析并与菌株生长曲线进行相关性分析,选择ppia-l20基因表达量差异显著的2个发酵时间点进行转录组学比较,并对表达差异显著的DEG(differential expressed gene)进行GO功能注释、KEGG富集通路和蛋白网络互作分析。[结果]4~84 h发酵菌体中的ppia-l20基因表达趋势不稳定,其转录水平与时间不相关。与ppia-l20低表达量的8 h发酵菌体相比,ppia-l20高表达量的12 h发酵菌体中有466个DEG,包括193个上调基因和273个下调基因。Real-time PCR验证表明基因表达差异趋势与转录组测序结果一致,证明测序结果可信。对差异表达的基因进行GO功能注释、KEGG富集分析,发现DEG的功能与膜形成相关,富集通路与双组分调节系统、磷酸转移酶系统和阳离子抗菌肽抵制通路相关。其中,双组分调节系统中的qseC基因和opmR基因表达量均上调2倍,ppia-l20基因位于阳离子抗菌肽抵制通路,该通路上的nlpE基因(负责编码外膜脂蛋白)显著下调。蛋白互作网络中,PPIA-L20蛋白与位于外膜的AMP结合蛋白TycC、GrsB有互作关系,NlpE蛋白与双组分调节系统中cpxR基因、cpxA基因表达的蛋白有互作关系。[结论]Xenorhabdus bovienii NN6菌株可能通过双组分调节系统通路、阳离子抗菌肽抵制通路上的关键基因nlpE和磷酸转移酶系统共同调控ppia-l20的表达。

[Objectives]A novel fungicidal protein,PPIA-L20,was isolated from extracellular proteins of the broad-spectrum anti-microbial strain Xenorhabdus bovienii NN6.It was a peptidyl-prolyl isomerase A like,and could kill the spore of Fusarium oxysporum f.sp.niveum.The expression level of ppia-l20 gene was unstable and had no significant correlation with fermentation time.The paper aimed to explore the regulatory factors regulating the expression of ppia-l20 gene,and then speculate the molecular mechanism of ppia-l20 gene differential expression,so as to provide reference for further improving the production of the antifungal protein.[Methods]Real-time PCR analysis was performed on the transcription level of ppia-l20 and correlation analysis was performed with the growth curve of NN6 strains.Two fermentation time points with significant differences in ppia-l20 gene expression were selected for transcriptomic comparison.GO functional annotation,KEGG enrichment pathway and protein network interaction analysis were performed for DEG(differential expressed genes)with significant expression differences.[Results]The expression trend of ppia-l20 gene in 4-84 h fermentation bacteria was unstable,and the transcription level was not correlated with time.There were 466 DEG in 12 h fermentation bacteria with high expression of ppia-l20,including 193 up-regulated genes and 273 down-regulated genes,compared with 8 h fermentation bacteria with low expression of ppia-l20.Real-time PCR showed that the trend of gene expression difference was consistent with the results of transcriptome sequencing,which proved that the sequencing results were reliable.GO functional annotation and KEGG enrichment analysis showed that the function of DEG was mainly related to membrane formation,and the enrichment pathway was related to the two-component regulatory system,phosphotransferase system and cationic antimicrobial peptide resistance pathway.The expression levels of qseC gene and opmR gene in the two-component regulatory system were up-regulated by 2-fold.The ppia-l20 gene was located in the cationic antimicrobial peptide resistance pathway,and the nlpE gene(responsible for encoding outer membrane lipoproteins)in this pathway was significantly down-regulated.In the protein interaction network,PPIA-L20 protein interacted with AMP-binding proteins TycC and GrsB located in the outer membrane,and NlpE protein interacted with cpxR gene and cpxA gene expression proteins in the two-component regulatory system.[Conclusions]X.bovienii NN6 strain might co-regulate the expression of ppia-l20 through two-component regulatory system pathway,the key genes nlpE on the cationic antimicrobial peptide resistance pathway and phosphotransferase system.