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恶性疟原虫感染期间的最佳内参基因选择及潜在靶基因鉴定

Optimal Reference Gene Selection and Potential Target Gene Identification During pv. - Infection.

作者信息

Chuang Shu-Cheng, Dobhal Shefali, Amore Teresita D, Alvarez Anne M, Arif Mohammad

机构信息

Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Honolulu, HI 96822, USA.

Department of Tropical Plant and Soil Sciences, University of Hawaii at Manoa, Honolulu, HI 96822, USA.

出版信息

Methods Protoc. 2025 Jul 4;8(4):72. doi: 10.3390/mps8040072.

Abstract

pv. (Xpd), the causal agent of bacterial blight in within the Araceae family, is listed as an EPPO A2 quarantine organism. Although the whole genome of Xpd has been sequenced, the molecular mechanisms underlying anthurium bacterial blight (ABB) remain unknown. Selecting an optimal reference gene is crucial for obtaining accurate and reliable gene expression profiles during the initial interactions between Xpd and . The stability of four reference genes was evaluated by applying three statistical methods-BestKeeper, geNorm, and delta Ct (ΔCt)-using reverse-transcription quantitative PCR (RT-qPCR) data. The and genes exhibited the most consistent gene expression profiles, whereas and were less stable at four time points (0, 0.5, 1, and 2 h) during the interactions between Xpd and susceptible cultivar 'Marian Seefurth.' The suitability of these reference gene candidates was validated by normalizing the gene expression levels of four pathogenicity-related genes. The highly upregulated expression of , which encodes xanthan biosynthesis glycosyltransferase, observed after 1 h of interaction, suggests it may be a key virulence determinant in the Xpd- pathosystem. The stable reference genes identified here will facilitate more accurate and comprehensive gene expression studies in the Xpd- pathosystem going forward.

摘要

青枯雷尔氏菌(Xanthomonas phaseoli pv. (Xpd))是天南星科细菌性叶枯病的病原体,被列为欧洲和地中海植物保护组织(EPPO)A2类检疫性有害生物。尽管Xpd的全基因组已被测序,但红掌细菌性叶枯病(ABB)的分子机制仍不清楚。选择一个最佳的内参基因对于在Xpd与[未提及的对象]初始相互作用期间获得准确可靠的基因表达谱至关重要。通过应用三种统计方法——BestKeeper、geNorm和ΔCt,利用逆转录定量PCR(RT-qPCR)数据评估了四个内参基因的稳定性。[未提及的两个基因]基因表现出最一致的基因表达谱,而[未提及的两个基因]在Xpd与感病[未提及的品种名称]品种‘Marian Seefurth’相互作用的四个时间点(0、0.5、1和2小时)稳定性较差。通过对四个致病相关基因的基因表达水平进行标准化,验证了这些候选内参基因的适用性。在相互作用1小时后观察到的编码黄原胶生物合成糖基转移酶的[未提及的基因]的高度上调表达,表明它可能是Xpd-[未提及的对象]病理系统中的关键毒力决定因素。这里鉴定出的稳定内参基因将有助于今后在Xpd-[未提及的对象]病理系统中进行更准确和全面的基因表达研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7817/12286264/aff49448aea4/mps-08-00072-g001.jpg

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