An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7.
An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7.
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从感染大肠杆菌 O157:H7 的植物根中提取细菌 mRNA 的优化方法。
DOI：
10.3389/fmicb.2014.00286
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发表时间：
2014
影响因子：
5.2
通讯作者：
Holden NJ
中科院分区：
文献类型：
--
作者：
Holmes A;Birse L;Jackson RW;Holden NJ
在宿主定殖过程中微生物基因表达的分析提供了有价值的信息相互作用的性质，有益的或致病的，以及所涉及的适应过程。分离用于植物内分析的细菌mRNA可能具有挑战性，其中宿主核酸可能主导制备，或抑制性化合物影响下游分析，例如，定量逆转录酶PCR（qPCR）、微阵列或RNA-seq。这项工作的目标是优化从活植物中分离食源性病原体的细菌mRNA。报道的方法回收植物病原体感染的植物材料，使用热酚提取和高浓度的细菌接种或大量的感染组织，被认为是不合适的植物根接种大肠杆菌O 157：H7。细菌RNA产量太低，增加植物材料导致样品中植物RNA占主导地位。为了提高细菌RNA的产量并减少所需的植物数量，开发了一种优化的方法，该方法将珠击与使用SDS和溶菌酶的定向细菌裂解相结合。抑制植物化合物，如酚类和多糖，抵消了高分子量聚乙二醇和十六烷基三甲基溴化铵的加入。新方法大幅提高了细菌mRNA的总产量，并允许通过qPCR评估基因表达。这种方法可以应用于与植物根部相关的其他细菌物种，也可以应用于更广泛的食品安全背景下。
Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety.
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影响因子：
3.2
作者：
Roy D;Panchal S;Rosa BA;Melotto M
通讯作者：
Melotto M
影响因子：
4.2
作者：
Hernandez-Morales, Alejandro;De la Torre-Zavala, Susana;Alvarez-Morales, Ariel
通讯作者：
Alvarez-Morales, Ariel
影响因子：
4.4
作者：
Griffiths, RI;Whiteley, AS;Bailey, MJ
通讯作者：
Bailey, MJ
影响因子：
2.2
作者：
Leite, Goncalo M.;Magan, Naresh;Medina, Angel
通讯作者：
Medina, Angel
影响因子：
3.6
作者：
An SQ;Febrer M;McCarthy Y;Tang DJ;Clissold L;Kaithakottil G;Swarbreck D;Tang JL;Rogers J;Dow JM;Ryan RP
通讯作者：
Ryan RP