转录-蛋白组学关联分析坎帕尼亚盐单胞菌野生型与紫外突变型菌株的差异表达基因与蛋白质

Transcriptomics-proteomics conjoint analysis of differentially expressed genes/proteins between wild type and ultraviolet radiation-mutated Halomonas campaniensis strains

摘要:

野生型坎帕尼亚盐单胞菌(Halomonas campaniensis)经9轮紫外循环诱变，获得1株高产四氢嘧啶(ectoine)的突变菌株G₉-72，关于菌株差异表达基因、蛋白质以及四氢嘧啶产量暴发的分子机制有待深入探讨。目的 探讨野生菌株XH26和突变菌株G₉-72的差异表达基因或蛋白质(differential expression genes/proteins, DEGs/DEPs)，并关联分析四氢嘧啶高效积聚的分子响应机制。方法 在无盐和1.5 mol/L NaCl条件下培养菌株XH26和G₉-72，利用Illumina HiSeq高通量测序和定量质谱蛋白组学技术分析菌株转录组-蛋白质组学的差异变化，并采用逆转录定量PCR对显著DEGs的表达进行验证。结果 转录组学筛选出11条氨基酸代谢通路(涉及44个DEGs)与四氢嘧啶合成代谢相关；蛋白组学筛选出10条氨基酸代谢通路(涉及50个DEPs)与四氢嘧啶合成代谢相关。转录-蛋白关联分析筛选出15个显著DEGs，其中7个基因(ectB、betB、betA、asd、doeD、doeC、gabD)在2个组学中表达上调；4个基因(ItaE、gdhA、gabT、acnB)在2个组学中表达下调；3个基因(gltD、atoB、narG)在转录组学中下调，而在蛋白组学中表达上调；基因narK在转录组学中表达上调，但在蛋白组学中未检测到表达。RT-qPCR验证结果与RNA-seq测序分析一致。结论 突变菌株四氢嘧啶的积聚量暴发与代谢通路的关键基因有关(合成基因asd和ectB，分解基因doeD和doeC)，与代谢通路上游的参与基因间接相关(betB、betA、ItaE、gltD、gadA和acnB)，以及四氢嘧啶的生物合成与Ala/Asp/Glu/His代谢途径(gabD、gdhA、gabT、atoB)和氮源代谢(narK和narG)高度关联。

Abstract:

A mutant strain G₉-72 with a high yield of ectoine was obtained from wild type Halomonas campaniensis after nine rounds of ultraviolet mutagenesis. The differentially expressed genes/proteins (DEGs/DEPs) and the molecular mechanism underlying the excessive increase in the ectoine yield remain to be explored for the mutant strain.Objective To explore the DEGs/DEPs between the wild type strain XH26 and G₉-72 and decipher the molecular mechanism of efficient ectoine production by conjoint analysis.Methods A non-salt (NS, 0 mol/L NaCl) group and a high-salt (HS, 1.5 mol/L NaCl) group were designed for the culture of XH26 and G₉-72. Illumina HiSeq and quantitative mass spectrometry were employed to identify the DEGs/DEPs between the two strains by transcriptomics-proteomics conjoint analysis. Furthermore, RT-qPCR was carried out to verify the expression of significant DEGs.Results The transcriptomics analysis revealed 11 amino acid metabolic pathways (44 DEGs) associated with ectoine anabolism, and the proteomics analysis revealed ten amino acid metabolic pathways (50 DEPs) associated with ectoine anabolism. The transcriptomics-proteomics conjoint analysis identified 15 significant DEGs, including seven genes (ectB, betB, betA, asd, doeD, doeC, and gabD) with up-regulated mRNA and protein level, four genes (ItaE, gdhA, gabT, and acnB) with down-regulated mRNA and protein levels, three genes (gltD, atoB, and narG) with down-regulated mRNA levels and up-regulated protein levels, and one gene narK with up-regulated mRNA level and no protein level. Additionally, the RT-qPCR results were consistent with the transcriptomics analysis.Conclusion The excessive increase in the ectoine yield of the mutant strain was associated with key genes in the ectoine metabolic pathway (including the synthesis genes asd and ectB and the catabolism genes doeD and doeC) and indirectly associated with several genes (betB, betA, ItaE, gltD, gadA, and acnB) in the upstream metabolic pathway. Notably, ectoine biosynthesis was highly associated with the Ala/Asp/Glu/His metabolic pathway (gabD, gdhA, gabT, and atoB) and nitrogen source metabolism (narK and narG).