《植物生理学报》 2014, 50(2): 223-228

一种高效提取杨树发病树皮总RNA的方法及应用

侯佳¹, 孙丰硕^2,3,4, 吴秋明^2,3,4, 杨玉巧⁵, 贺伟^1,*, 王延伟^2,3,4,*

北京林业大学¹森林培育与保护教育部重点实验室, ²林木育种国家工程实验室, ³林木花卉遗传育种教育部重点实验室, ⁴国家林业局树木花卉育种与生物工程重点开放实验室, 北京100083; ⁵濮阳市林业科学院, 河南濮阳457000

通信作者：贺伟；E-mail: hewei@bjfu.edu.cn和ywwang@bjfu.edu.cn；Tel: 13520517066和13718968958

摘　要：

对杨树发病树皮总RNA的高效提取是开展杨树抗溃疡病基因表达调控的基础, 为了探讨杨树发病树皮RNA的高效提取方法, 本研究以欧美杨细菌性溃疡病菌侵染后的‘中林46’杨为材料, 比较了包括本研究提出的RNA提取新方法(RNA试剂盒改良法)在内的6种方法提取的总RNA的质量和浓度。结果显示, 通过RNA试剂盒改良法提取的总RNA 28S rRNA条带亮度约为18S rRNA条带亮度的2倍且浓度高, 表明该方法更适合感染欧美杨细菌性溃疡病的‘中林46’杨发病树皮总RNA的提取。为了验证RNA试剂盒改良法对健康杨树树皮及不同胁迫处理、组织RNA提取的适用性, 进一步用该方法提取了‘中林46’杨、‘107’杨、‘北京’杨健康树皮, 低氮、低磷处理的毛白杨组培苗以及白玉兰花的总RNA。结果表明, 用RNA试剂盒改良法均能获取高质量的总RNA。所提取的总RNA已成功用于感染欧美杨细菌性溃疡病的杨树树皮转录组测序, 以及低氮处理的毛白杨组培苗RT-PCR和荧光定量PCR实验, 表明用RNA试剂盒改良法提取的总RNA可以用于后续分子实验。

关键词：RNA提取; 杨树; 侵染; 树皮

收稿：2013-11-18   修定：2013-12-18

资助：国家自然科学基金青年基金(31200511)、国家林业公益性行业科研专项(201104054)和教育部博士点基金新教师类基金(20110014120004)。

An Efficient Method for Total RNA Extraction of Poplar Bark Infected with Pathogen and the Application

HOU Jia¹, SUN Feng-Shuo^2,3,4, WU Qiu-Ming^2,3,4, Yang Yu-Qiao⁵, HE Wei^1,*, WANG Yan-Wei^2,3,4,*

¹Key Laboratory for Silviculture and Conservation of Ministry of Education, ²National Engineering Laboratory for Tree Breeding, ³Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, ⁴Tree and Ornamental Plant Breeding and Biotechnology Laboratory of State Forestry Administration, Beijing Forestry University, Beijing 100083, China; ⁵Forestry Research Institute of Puyang, Puyang, Henan 457000, China

Corresponding author: HE Wei; E-mail: hewei@bjfu.edu.cn和ywwang@bjfu.edu.cn; Tel: 13520517066和13718968958

Abstract:

The highly efficient total RNA extraction of poplar bark infected with pathogen is important for the expression and regulation research of resistance genes. To explore an efficient method for total RNA extraction from the lesion bark, 2-year-old seedling stem of Populus euramericana cv. ‘Zhonglin 46’ was inoculated with the pathogenic bacterium of Lonsdalea quercina subsp. populi and used for RNA extraction. A novel RNA extraction method was first proposed in this study and named “Improved method of RNA extraction kit”. We compared the extraction effects among this method and other five methods. The test results showed that RNA extracted using “Improved method of RNA extraction kit” was of high concentration and the band of 28S rRNA was nearly twice as bright as that of 18S rRNA. The analysis suggested that “Improved method of RNA extraction kit” was the most suitable for total RNA extraction from the lesion bark. To test the wider applicability, we further extracted total RNA from other tissues, including the healthy bark of P. euramericana cv. ‘Zhonglin 46’, P. euramericana cv. ‘74/76’ and Populus×beijingensis, tissue culture plantlets of Populus tomentosa treated with low nitrogen or phosphorus stress and the flower of Magnolia denudate. The testing showed that total RNA could be successfully extracted from all these plant tissues with high quality. The extracted RNA had been successfully used for transcriptome sequencing of poplar lesion bark, RT-PCR and qRT-PCR of tissue culture plantlets of P. tomentosa treated with low nitrogen stress. Consequently, the result showed that total RNA extracted using “Improved method of RNA extraction kit” could be further used in the subsequent molecular experiment.

Key words: RNA extraction; poplar; infection; bark