Alkan Noam, Gadkar Vijay, Coburn Joel, Yarden Oded, Kapulnik Yoram
Department of Agronomy and Natural Resources, Institute of Field and Garden Crops, ARO, the Volcani Center, PO Box 6, Bet Dagan 50 250, Israel.
These authors contributed equally to this work.
New Phytol. 2004 Mar;161(3):877-885. doi: 10.1046/j.1469-8137.2004.00975.x. Epub 2004 Jan 8.
• A rapid method to quantify the colonization of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices in planta using quantitative real-time polymerase chain reaction (qRT-PCR) technique. • Specific PCR primers for the fungus (28S rDNA sequence) and host root tissue (chitinase and chalcone synthase gene) were developed and their respective specificity determined. • The plant specific primers for Lycopersicon esculentum, Medicago truncatula amplified linearly over a concentration range of: 6.4 pg to 20 ng. The G. intraradices-specific primer amplified as low as 1 pg of its target DNA, which allowed us to detect a single spore of the fungus. High degrees of correlation were obtained when threshold cycle (Ct) was plotted against vesicular, hyphal and total colonization using microscopically quantified host roots. • This is the first report of the application of the qRT-PCR technique for quantification of AMF colonization in planta. The success of its application should open up the possibility of its wider application in AM research.
• 一种使用定量实时聚合酶链反应(qRT-PCR)技术在植物中定量丛枝菌根真菌(AMF)根内球囊霉定殖的快速方法。
• 开发了针对该真菌(28S rDNA序列)和宿主根组织(几丁质酶和查尔酮合酶基因)的特异性PCR引物,并确定了它们各自的特异性。
• 番茄、蒺藜苜蓿的植物特异性引物在6.4 pg至20 ng的浓度范围内呈线性扩增。根内球囊霉特异性引物能扩增低至1 pg的目标DNA,这使我们能够检测到该真菌的单个孢子。当使用显微镜定量的宿主根将阈值循环(Ct)与泡囊、菌丝和总定殖进行绘图时,获得了高度相关性。
• 这是关于qRT-PCR技术用于定量植物中AMF定殖的首次报道。其应用的成功应会为其在AM研究中的更广泛应用开辟可能性。