茄青枯拉尔氏菌Rs-T02在3,4,5-三羟基苯甲酸甲酯作用下的转录组分析

Transcriptome profiling in Ralstonia solanacearum Rs-T02 treated with methyl gallate

本文利用Illumina Hi Seq测序技术,对在3,4,5-三羟基苯甲酸甲酯(methyl gallate,MG)作用下和对照条件下的茄青枯拉尔氏菌(Ralstonia solanacearum) Rs-T02菌株的转录组进行测序分析,并用GO和KEGG Pathway富集化分析的方法分析差异表达基因(differentially expressed genes,DEGs)的功能和代谢通路,以初步了解MG对R. solanacearum作用的分子机制。结果表明,MG处理组和对照组的差异表达的基因有2 172个,其中1 037个基因上调,1 135个基因下调。随意挑选10个DEGs进行qRT-PCR验证,其基因表达趋势与转录组测序结果一致。通过对差异表达基因的功能和代谢通路分析发现,与细胞结构相关的肽聚糖水解酶、3-磷酸甘油酰基转移酶和UDP-N-乙酰胞壁酰丙氨酸-D-谷氨酸连接酶等蛋白的编码基因上调表达,蛋白YeaQ、外膜蛋白W和外膜蛋白Bam E等蛋白的编码基因下调表达;与能量代谢相关的ATP合成酶结构蛋白、辅酶Q亚基和细胞色素等蛋白的编码基因上调表达,甘油醛-3-磷酸脱氢酶、甘油醛脱氢酶和异柠檬酸裂解酶等蛋白的编码基因下调表达;与致病性相关的gspD、gspE和gspF等基因上调表达,hrpY、hrpB和gspG等基因下调表达;与细菌抗药性相关的氨基酸的氨酰-tRNA连接酶、核糖体RNA小亚单位甲基转移酶G基因和多重药物外排泵亚基AcrA等蛋白的编码基因上调表达;与细菌运动性相关的flg B、flg C和flg D等基因上调表达。推测MG对Rs-T02菌株的抑菌机制可能与MG影响病菌的细胞结构和能量代谢相关基因的差异表达有关;同时,MG影响病菌T3SS和T2SS相关基因的差异表达,从而影响细菌的致病力。此外,3-磷酸甘油酰基转移酶基因、核糖体RNA小亚单位甲基转移酶G和多重药物外排泵亚基AcrA基因等上调表达,可能与病菌抵抗MG的机制有关。

In order to gain new insights into molecular mechanisms of methyl gallate(MG)in Ralstonia solanacearum,transcriptome sequencing of MG-treated and non-treated R.solanacearum Rs-T02 was performed via Illumina Hiseq technology.Differentially-expressed genes(DEGs)were then sorted using GO terms and KEGG pathways analyses.As a result,1 037 genes were up-regulated and 1 135 genes were down-regulated in a total of2 172 DEGs.To validate the findings,10 DEGs were selected randomly for further analyses using quantitative real-time PCR(qRT-PCR)where the results were fairly consistent with that of transcriptional sequencing.Specially,the findings showed that the genes associated with cytoarchitecture including the encoding genes of peptidoglycan hydrolase,3-phosphate glyceryl transferase and UDP-N-acetyl muramoylalanine-D-glutamate ligase,etc.were up-regulated,and the coding gene of YeaQ,outer membrane protein W and outer membrane protein Bam E,etc.were down-regulated in the Rs-T02 strain treated with MG.Genes associated with energy metabolism including the encoding genes of ATP synthetase structural protein,coenzyme Q subunit and cytochrome,etc.were up-regulated,the encoding genes of glyceraldehyde-3-phosphate dehydrogenase,glyceraldehyde dehydrogenase and isocitrate lyase,etc.were downregulated.Genes associated with pathogenicity such as gspD,gspE and gspF,etc.were up-regulated,whereas hrpY,hrpB and gspG,etc.were down-regulated.Furthermore,the encoding genes of the aminoacyl-tRNA ligase,the small subunit of ribosome RNA methyltransferase G and the multidrug efflux pump subunit AcrA etc.related to bacterial resistance were up-regulated.Genes associated with bacterial motility such as Flg B,flg C and flg D,etc.were up-regulated.It is speculated that the bacteriostasis mechanism of MG on R.solanacearum Rs-T02 strain may be related to the DEGs that associated with cytoarchitecture and energy metabolism.Meanwhile,MG affected the differential expression of T3SS and T2SS related genes,which had an impact to the pathogenicity of bacteria.Last but not least,the up-regulated expression of 3-phospho glyceryl transferase,small subunit of ribosome RNA methyltransferase G and the multidrug efflux subunit AcrA,etc.might be related to the mechanism of the bacterial resistance to MG.