转录组学分析盐单胞菌四氢嘧啶合成代谢相关的表达差异基因与qRT-PCR验证

Transcriptome analysis of differential genes associated with the ectoine synthesis pathway of Halomonas campaniensis under salt stress and expression verification by qRT-PCR

【目的】探究盐适应条件下坎帕尼亚盐单胞菌(Halomonas campaniensis)的差异基因表达水平,挖掘四氢嘧啶(ectoine)合成代谢相关联的差异基因。【方法】设置无盐组NS (0 mol/L NaCl)、中盐组MS (1.5 mol/L NaCl)和高盐组HS (2.5 mol/L NaCl),培养H. campaniensis XH26菌株,利用IlluminaHiSeq测序进行转录组学分析,筛选四氢嘧啶合成代谢关联的差异基因,并进行qRT-PCR验证。【结果】菌株XH26胞内四氢嘧啶的积累量与盐度变化密切相关,1.5mol/LNaCl时积聚量最大为419.2mg/L。转录组测序(n=3/组)能定位到基因组测序序列的数量统计为87.24%–95.87%,共注释操纵子748个(涉及2 182个基因),转录起始-终止位点941个,预测新转录本456个,上调基因385个和下调基因326个(涉及245个KEGG通路)。组间NSvsMS分析表明,合成基因ectABC和lysC表达上调以促进四氢嘧啶生成,关联基因gltB、gltD、davT、hisD、alh-9、betA、acnB、pckA以及gadA表达上调,参与四氢嘧啶合成关联通路的上游调控。比较组MSvsHS分析表明,基因ectA、acnB、pckA、gadA和gdhA表达下调致使四氢嘧啶产量减少。qRT-PCR验证结果与组学测序的表达趋势相一致。【结论】四氢嘧啶生物合成与Asp(或天冬氨酸半缩醛)、上游氨基酸代谢网络(Asn、Glu、Gln和His)以及三羧酸循环(琥珀酸、延胡索酸和草酰乙酸)密切相关,此为后续四氢嘧啶合成途径的优化和代谢通路的整合设计提供重要的参考依据。

[Objective] The study explored the differentially expressed genes of Halomonas campaniensis under salt stress and identified the differential genes associated with anabolism of ectoine. [Methods] Three experimental groups were set up: no-salt group(NS, 0 mol/L NaCl),medium-salt group(MS, 1.5 mol/L NaCl), and high-salt group(HS, 2.5 mol/L NaCl), and then H. campaniensis XH26 was respectively cultured. Illumina HiSeq was employed for transcriptome analysis, and differential genes associated with ectione metabolism were screened and then verified by qRT-PCR. [Results] The accumulation of ectione in XH26 was closely related to salinity and the maximum(419.2 mg/L) was achieved in MS(1.5 mol/L NaCl). According to the transcriptome sequencing(n=3) result, total mapped reads accounted for 87.24%–95.87% of the clean reads. A total of 748 operons(2 182 related genes) and 941 transcription start/stop sites were annotated and 456 new transcripts were predicted in addition to 385 up-regulated genes and 326 down-regulated genes(involving 245 KEGG pathways). For the differential genes between NS and MS, synthetic ectABC and lysC were up-regulated to promote the ectoine production, and associated genes gltB, gltD, davT, hisD,alh-9, betA, acnB, pckA, and gadA were up-regulated to modulate the upstream of ectoine synthesis pathway. As for the differential genes between MS and HS, ectA, acnB, pckA, gadA, and gdhA were down-regulated, thus decreasing ectoine production. qRT-PCR result supported the transcriptome sequencing result. [Conclusion] The biosynthesis of ectoine was closely related to the aspartic acid(or aspartic acid hemiacetal), the upstream amino acid metabolism networks(e.g., asparagine, glutamate,glutamine, and histidine), and tricarboxylic acid cycle(succinic acid, fumaric acid, and oxaloacetic acid). These results can serve as a reference for the optimization or integration design of ectoine synthesis pathway in the future.