Zhou Yanyan, Liu Huan, Wu Ting, Zheng Yu, Wang Ruimin, Xue Dong, Yan Qiang, Yuan Xingxing, Chen Xin
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China.
Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, China.
Genes (Basel). 2023 Aug 30;14(9):1739. doi: 10.3390/genes14091739.
Mung bean () production has been greatly threatened by numerous diseases. Infection with these pathogens causes extensive changes in gene expression and the activation of hormone signal transduction. Quantitative real-time PCR (qRT-PCR) is the most common technique used for gene expression validation. Screening proper reference genes for mung bean under pathogen infection and hormone treatment is a prerequisite for ensuring the accuracy of qRT-PCR data in mung bean disease-resistance research. In this study, six candidate reference genes (, , , , , and ) were selected to evaluate the expression stability under four soil-borne disease pathogens (, , , and ) and five hormone treatments (SA, MeJA, ETH, ABA, and GA). In the samples from different treatments, the Ct value distribution of the six candidate reference genes was different. Under the condition of hormone treatment, the Ct value ranged from a minimum of 17.87 for to a maximum of 29.63 for . Under the condition of pathogen infection, the Ct value ranged from a minimum of 19.43 for to a maximum of 31.82 for . After primer specificity analysis, it was found that was not specific, so the five reference genes , , , and were used in subsequent experiments. The software products GeNorm, NormFinder, BestKeeper and RefFinder were used for qRT-PCR data analysis. In general, the best candidates reference genes were: for SA, ABA, GA3, and treatment; for ETH treatment; for MeJA and treatment; and for and treatment. The most stably expressed genes in all samples were , while was the least stable reference gene. Finally, the reliability of the reference gene was further validated by analysis of the expression profiles of four mung bean genes (, , , and ) selected from transcriptome data. Our results provide more accurate information for the normalization of qRT-PCR data in mung bean response to pathogen interaction.
绿豆()的生产受到多种病害的严重威胁。这些病原体的感染会导致基因表达发生广泛变化以及激素信号转导的激活。定量实时聚合酶链反应(qRT-PCR)是用于基因表达验证的最常用技术。在病原体感染和激素处理条件下筛选合适的绿豆内参基因是确保绿豆抗病性研究中qRT-PCR数据准确性的前提。本研究选择了6个候选内参基因(、、、、和),以评估在4种土传病害病原体(、、、和)和5种激素处理(SA、MeJA、ETH、ABA和GA)下的表达稳定性。在不同处理的样本中,6个候选内参基因的Ct值分布不同。在激素处理条件下,Ct值范围从的最小值17.87到的最大值29.63。在病原体感染条件下,Ct值范围从的最小值19.43到的最大值31.82。经过引物特异性分析,发现不具有特异性,因此在后续实验中使用了5个内参基因、、、和。使用GeNorm、NormFinder、BestKeeper和RefFinder软件产品进行qRT-PCR数据分析。总体而言,最佳候选内参基因是:SA、ABA、GA3和处理时为;ETH处理时为;MeJA和处理时为;和处理时为。所有样本中表达最稳定的基因是,而最不稳定的内参基因是。最后,通过对从转录组数据中选择的4个绿豆基因(、、、和)的表达谱分析,进一步验证了内参基因的可靠性。我们的结果为绿豆对病原体相互作用反应中qRT-PCR数据的标准化提供了更准确的信息。