苜蓿中华根瘤菌代谢组学样品制备方法的研究

Research of metabolome sample preparation method of Sinorhizobium meliloti

代谢组样品制备是代谢组学研究的基础。本文以维生素B12生产菌株苜蓿中华根瘤菌Sinorhizobium meliloti 320为研究对象,通过检测细胞损伤、ATP泄漏、代谢物回收效率以及细胞代谢淬灭效率综合评价细胞淬灭方法,同时对5种提取试剂的提取效率进行比较优化胞内代谢物的提取方法。最终获得苜蓿中华根瘤菌S.meliloti 320的胞内代谢组学样品制备较佳条件:即-20℃40%甲醇淬灭细胞,过滤收集淬灭细胞,甲醇/乙腈/水(体积比为2∶2∶2,外加0.1%的甲酸)与50%甲醇相结合提取胞内代谢物。实验结果显示-20℃的40%甲醇(通过过滤收集细胞)对细胞膜的损伤较小,且细胞代谢淬灭效率和回收效率较高;甲醇/乙腈/水(体积比为2∶2∶2,外加0.1%的甲酸)与50%的甲醇对胞内代谢物的提取效率较高且有互补作用。

The sample preparation approach is the basis of metabolomics. The vitamin B12 producing strain Sinorhizobium meliloti 320 was applied in this paper. The method of cell quenching was evaluated comprehensively by detecting cell damage, ATP leakage, metabolite recovery efficiency and cell quenching efficiency. The extraction method of intracellular metabolites was optimized by comparing the extraction efficiency of five extraction solutions. Finally, the better optimal conditions for the metabolome sample preparation approach of S. meliloti 320 were obtained: 40% methanol at -20 ℃ for cell quenching, filtration for harvesting the quenched cells, the combining of methanol/acetonitrile/water(2∶2∶1, with 0.1% formic acid) and methanol/water(1∶1) for extracting intracellular metabolites. The experimental results showed that 40% methanol at -20 ℃(collected by filtration) caused less damage to cell membranes and higher quenching efficiency and recovery efficiency. Methanol/acetonitrile/water(2∶2∶1, with 0.1% formic acid) and methanol/water(1∶1) showed the higher extraction efficiency for most intracellular metabolites and supplementary effect.