An improved method for RNA extraction from urediniospores of and wheat leaves infected by an obligate fungal pathogen, Puccinia striiformis f. sp. tritici

作     者：MA Li-Jie QIAO Jia-xing KONG Xin-yu WANG Jun-juan XU Xiang-ming HU Xiao-ping 

作者机构：State Key Laboratory of Crop Stress Biology for Arid Areas College of Plant Protection Northwest A&F University Yangling 712100 P.R.China Genetics and Crop Improvement East Mailing Research East Mailing ME19 6B J UK Erdos Ecological Environment of Career Academy Ordos 017010 P.R. China 

出 版 物：《Journal of Integrative Agriculture》 (农业科学学报（英文版）)

年 卷 期：2016年第15卷第6期

页      面：1293-1303页

核心收录：

学科分类：09[农学] 0904[农学-植物保护] 090401[农学-植物病理学] 090402[农学-农业昆虫与害虫防治] 

基　　金：supported by the National Key Basic Research Program of China (2013CB127700) the Na-tional Natural Science Foundation of China (31071640 and 31271985) partially supported by the 111 Project from Education Ministry of China (B07049) 

主　　题：Puccinia striiformis f. sp. tritici RNA extraction RT-PCR urediniospore mycelium 

摘      要：Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important wheat disease in China, seriously threatening wheat production. Understanding the winter survival of the fungus is a key for predicting the spring epidemics of the disease, which determines the crop loss. Estimation of P. striiformis f. sp. tritici winter survival requires processing a large number of samples for sensitive detection of the pathogen in wheat leaf tissue using real-time quantitative reverse transcription PCR （qRT-PCR）. A bottleneck for the analysis is the acquisition of a good yield of high quality RNA suitable for qRT-PCR to distinguish dead and alive fungal hyphae inside leaves. Although several methods have been described in the literatures and commercial kits are available for RNA extraction, these methods are mostly too complicated, expensive and inefficient. Thus, we modified three previously reported RNA extraction methods with common and low-cost reagents （LiCI, SDS and NaCI） to solve the problems and selected the best to obtain high quality and quantity RNA for use in qRT-PCR. In the three improved methods, the NaCI method was proven to be the best for extracting RNAfrom urediniospores of and wheat leaves infected by P. striiformis f. sp. tritici, although the modified LiCI and SDS methods also increased yield of RNA compared to the previous methods. The improved NaCI method has the following advantages： 1） Complete transfer of urediniospores of P. striiformis f. sp. tritici from the mortar and pestle can ensure the initial amount of RNA for the qRT-PCR analysis; 2） the use of low-cost NaCI to replace more expensive Trizol can reduce the cost; 3） the yield and quality of RNA can be increased; 4） the improved method is more suitable for a large number and high quantity of samples from fields. Using the improved NaCI method, the amount of RNA was increased three times from urediniospores of P. striiformis f. sp. tritici compared from the extraction kit. Approximately, 1