碱性条件下苏云金芽胞杆菌基础代谢分析

The analysis of major metabolic pathways in Bacillus thuringiensis under alkaline stress

摘要:

[目的]探索苏云金芽胞杆菌(Bacillus thuringiensis)形成转录差异的碱性条件,明确B.thuringiensis在该条件下的基础代谢途径变化。[方法]采用半定量RT-PCR技术及实时荧光定量PCR技术,确定碱刺激下参考基因pspA存在表达差异的碱性处理条件。在该条件下提取RNA进行Agilent定制B.thuringiensis表达谱芯片杂交,对芯片数据进行差异表达分析、GO富集分析及生物途径富集分析等。[结果]通过检测pspA表达变化,将对数生长中期的菌体加入终浓度为28 mmol/L的NaOH并诱导培养10min,作为B. thuringiensis响应碱刺激的研究条件。富集分析表明碳代谢、脂肪酸合成代谢、氨基酸合成代谢途径变化明显。细胞糖酵解途径至少19个酶促基因上调表达,三羧酸循环中催化α-酮戊二酸转化为苹果酸的大部分酶蛋白编码基因上调2倍以上。[结论]本研究发现在碱性条件下B. thuringiensis基础代谢明显增强,细胞可能通过大量合成酸性物质如乳酸、苹果酸等来提高细胞对于碱性环境的适应能力。

Abstract:

[Objective] The study aimed to determine the appropriate stage for exploring the response of Bacillus thuringiensis to the alkaline stress, to profile the metabolic pathways under this stress. [Methods] Using semiquantitative RT-PCR and qRT-PCR, the proper stage was defined by monitoring the transcriptional changes of marker gene pspA, which was known as a responsive gene under the alkaline stress. The total RNA was then extracted to perform the microarray hybridizations for samples under stress and control, respectively. Gene Ontology and pathway enrichments were conducted to analyze the global changes of carbon metabolism, metabolism of fatty acid synthesis and amino acid. [Results] For B. thuringiensis in the mid-log growth phase, treatment of 28 mmol/L NaOH for 10 mins is the feasible approach to analyze the response of B. thuringiensis to this stress. More than twenty genes encoding important enzymes in glycolytic pathway were up-regulated and majority of genes involved in catalyzing alpha-ketoglutarate into malic acid were also found to up-regulated more than two folds. [Conclusion] By analyzing the gene expression profile, the major metabolisms of B. thuringiensis were found to be clearly enhanced under alkaline stress. Large quantities of acid including malic acid and lactic acid may contribute a lot to the adaptation of alkaline condition.