Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR.
Selection of endogenous genes for gene expression studies in Eucalyptus under biotic (Puccinia psidii) and abiotic (acibenzolar-S-methyl) stresses using RT-qPCR.
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DOI：
10.1186/1756-0500-3-43
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发表时间：
2010-02-24
影响因子：
1.8
通讯作者：
Furtado EL
中科院分区：
文献类型：
--
作者：
Boava LP;Laia ML;Jacob TR;Dabbas KM;Gonçalves JF;Ferro JA;Ferro MI;Furtado EL
由冬季锈菌引起的锈病一直限制着新桉树种植园的建立，以及敏感遗传物质的重新萌发。识别参与防御反应的寄主基因对于阐明抗性机制很重要。逆转录-定量聚合酶链式反应是基因表达分析中最常用的mRNA定量方法。这种方法通常使用一个参考基因作为内对照，以使结果标准化。由于实验条件的影响，一个好的内源对照转录本的差异很小。我们分析了两个桉树无性系(抗锈和感病)60d龄幼苗在生物胁迫(P.psidii)和非生物胁迫(酸性苯并茂-S甲基，ASM)胁迫下的13个基因的表达，以确定叶片中变异最小的基因。对于没有接受任何刺激的克隆的组织样本，eEF2和EgldH基因的组合是正常化的最佳对照。比较接种病原菌和未接种病原菌的植株样品，eEF2和UBQ一起作为正规剂更合适。在ASM处理和未处理的两个无性系的叶片中，CYP和elF4B基因的转录本是变异最小的。最后，当比较ASM处理的叶片、接种P.psidii的叶片、ASM处理+P.psidii接种的叶片及其各自对照的两个无性系的表达时，最稳定表达的基因是EgIDH和UBQ。几丁质酶基因在植物对植物病原菌的抗性研究中高度表达，它被用来证实基因表达的差异。
Rust caused by Puccinia psidii Winter has been limiting for the establishment of new Eucalyptus plantations, as well as for resprouting of susceptible genetic materials. Identifying host genes involved in defense responses is important to elucidate resistance mechanisms. Reverse transcription-quantitative PCR is the most common method of mRNA quantitation for gene expression analysis. This method generally employs a reference gene as an internal control to normalize results. A good endogenous control transcript shows minimal variation due to experimental conditions. We analyzed the expression of 13 genes to identify transcripts with minimal variation in leaves of 60-day-old clonal seedlings of two Eucalyptus clones (rust-resistant and susceptible) subjected to biotic (P. psidii) and abiotic (acibenzolar-S-methyl, ASM) stresses. For tissue samples of clones that did not receive any stimulus, a combination of the eEF2 and EglDH genes was the best control for normalization. When pathogen-inoculated and uninoculated plant samples were compared, eEF2 and UBQ together were more appropriate as normalizers. In ASM-treated and untreated leaves of both clones, transcripts of the CYP and elF4B genes combined were the ones with minimal variation. Finally, when comparing expression in both clones for ASM-treated leaves, P. psidii-inoculated leaves, ASM-treated plus P. psidii-inoculated leaves, and their respective controls, the genes with the most stable expression were EgIDH and UBQ. The chitinase gene, which is highly expressed in studies on plant resistance to phytopathogens, was used to confirm variation in gene expression due to the treatments.