Akbarabadi Ali, Ismaili Ahmad, Kahrizi Danial, Nazarian Firouzabadi Farhad
Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran.
Department of Agronomy and Plant Breeding, Razi University, Kermanshah, Iran.
Cell Mol Biol (Noisy-le-grand). 2018 Mar 31;64(4):113-118.
Weeds are serious problem in crop production and wild oat is a grass weed of economic and agronomic significance. We need to extend our basic knowledge of weeds especially in molecular genetics and gene expression. For study of gene expression by semi-quantitative and quantitative PCR, it is recommended that normalization of reference genes be carried out in order to select the most stable reference gene for a precise gene expression study. The purpose of this research was evaluation of four reference genes in response to treated and untreated (control) by herbicide in two tissues (stem and leaf) of non-target site resistance wild oat (A. ludoviciana). Four candidate reference genes including Actin, Ef1α (elongation factor 1 alpha), GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and TBP (TATA-box-binding protein) were used to determine stable reference gene exposed to the herbicide using the statistical methods of NormFinder, BestKeeper and delta-Ct. NormFinder indicated that TBP and Actin genes are the best combination of two genes for normalizing calculations (with a combined gene stability value of 0.012) for qPCR analysis under herbicide stress in different tissues of non-target site resistance wild oat. Based on the statistical results, the Ef1α gene was identified as the unstable reference gene. Totally, according to results of this study, TBP gene is the most stable reference gene and therefore, this gene can be used as a reference gene for future studies of quantitative PCR analysis of herbicide stress-responsive gene expression in wild oat and potentially in other grass weed species.
杂草是作物生产中的严重问题，野燕麦是一种具有经济和农艺重要性的禾本科杂草。我们需要扩展对杂草的基础知识，特别是在分子遗传学和基因表达方面。为了通过半定量和定量PCR研究基因表达，建议进行参考基因的标准化，以便选择最稳定的参考基因进行精确的基因表达研究。本研究的目的是评估四个参考基因在非靶标位点抗性野燕麦（A. ludoviciana）的两个组织（茎和叶）中对除草剂处理和未处理（对照）的反应。使用统计方法NormFinder、BestKeeper和delta-Ct，利用四个候选参考基因，即肌动蛋白、Ef1α（延伸因子1α）、GAPDH（甘油醛-3-磷酸脱氢酶）和TBP（TATA框结合蛋白）来确定暴露于除草剂下的稳定参考基因。NormFinder表明，在非靶标位点抗性野燕麦不同组织的除草剂胁迫下，TBP和肌动蛋白基因是用于qPCR分析标准化计算的两个基因的最佳组合（组合基因稳定性值为0.012）。根据统计结果，Ef1α基因被确定为不稳定的参考基因。总的来说，根据本研究结果，TBP基因是最稳定的参考基因，因此，该基因可作为未来野燕麦以及潜在其他禾本科杂草物种中除草剂胁迫响应基因表达定量PCR分析研究的参考基因。
