Evaluation of suitable reference genes for normalization of quantitative real-time PCR analysis in rice plants under Xan
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(2020) 2:21
Food Production, Processing and Nutrition
RESEARCH
Open Access
Evaluation of suitable reference genes for normalization of quantitative real-time PCR analysis in rice plants under Xanthomonas oryzae pv. oryzae--infection and melatonin supplementation Xian Chen1,2, Pedro Laborda3, Yan Dong1 and Fengquan Liu1*
Abstract Exogenous melatonin (MT) was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv. oryzae (Xoo)-caused bacterial blight (BB). However, the accurate comparison of the expression levels across samples was a challenging task. In this work, the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR (qRT-PCR), and algorithms geNorm, NormFinder and BestKeeper. Our results indicated that most reference genes remained stable in Xoo-infected rice plants, while a number of reference genes were affected by MT supplementation. Among all studied genes, the transcript levels of 18S(18S ribosomal RNA) and UBC (Ubiquitin-conjugating enzyme E2) remained unaltered by Xoo infection, while UBC and UBQ5(Ubiquitin 5) were the most stable genes when examining simultaneous Xoo-infection and MT supplementation, demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation. Keywords: Rice, Xanthomonas oryzae pv. oryzae, Melatonin, qRT-PCR, Reference genes
Introduction Rice is one of the most important crops worldwide, providing essential food supply to most of the world's population. However, rice is highly susceptible to a wide range of pathogens that limit its production and quality. Among rice pathogens, Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight (BB), is a member of γ-proteobacteria, and is able to invade plant through wounds and hydathodes to colonize the xylem (White and Yang 2009). This pathogen can rapidly spread through the vascular system, leading to systemic infection (Yuan et al. 2010). Its pathogenicity is partially * Correspondence: [email protected] 1 Institute of Plant Protection,Jiangsu Key Laboratory for Food Quality and Safety–State Key Laboratory Cultivation Base of Ministry of Science and Technology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China Full list of author information is available at the end of the article
dependent on a type III protein secretion system (TTSS) (Pfeilmeier et al. 2016). During the early infection stages, the expression levels of 541 genes were altered in response to Xoo in susceptible rice JG30 (Tariq et al. 2019), while the expression levels of 115 genes were altered in resistant hybrid rice Y73 in response to Xoo infection, which corresponds to 0.22% of the rice genome (Wang et al. 2012). Many of these differential expressed genes (DEGs) were related to signaling, transcription and metabolic processes. Melatonin (N–acetyl–5–methoxytryptamine) consists of an indole structure with low molecular weight, and has bee
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