Bayogenin 3-O-Cellobioside is a novel anti-blast metabolite produced in rice in response to Pyricularia oryzae infection

Susceptible (CO39 and NPB) and resistant (Pi_gm) rice cultivars were spray inoculated with P. oryzae spore and incubated for 12-hours. Total RNA was extracted from the inoculated rice seedlings (T_Co39, T_NPB, and T_gm) along with their non-inoculated control group C_Co39, C_NPB, and C_gm. The extraction of total RNA from inoculated and non-inoculated control samples was carried-out with RNAprep pure Plant Kit (Tiangen, Beijing) by following processes recommended by the manufacturer. To observe the level of RNA degradation and contamination, the extracted RNA was run on 1% agarose gels. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA concentration was measured with RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, CA, USA). The cDNA library was sequenced on the Illumina sequencing platform (Illumina HiSeq™ 2000) with 150 bp pair-end reads length and 300 bp insert size by Gene Denovo Co. (Guangzhou, China). Novogene in-house Perl script was used to select clean reads by removing adaptor sequences, low quality sequences (reads with more than 50% of bases quality lower than 20) and reads with more than 5% N bases. mRNA profile of susceptible and resistant rice seedlings inoculated with P. oryzae for 12-hours and non-inoculated control groups were generated through deep sequencing, data was obtained from one biological experiment with three technical replicates using Illumina HiSeq™ 2000.

供试材料涵盖感病水稻品种CO39、NPB与抗病水稻品种Pi_gm，采用稻瘟病菌（P. oryzae）孢子对其进行喷雾接种，随后置于保湿条件下培养12小时。分别从接种后的水稻幼苗（标记为T_Co39、T_NPB与T_gm）及其未接种的对照组（标记为C_Co39、C_NPB与C_gm）中提取总RNA。总RNA提取采用北京天根生化科技（Tiangen, Beijing）的RNAprep pure植物总RNA提取试剂盒，严格遵循厂商推荐的操作流程完成。为评估提取RNA的降解程度与污染状况，将RNA样品在1%琼脂糖凝胶上进行电泳检测。借助美国安捷伦科技（Agilent Technologies, CA, USA）的2100生物分析仪系统（Bioanalyzer 2100 system）配套RNA Nano 6000检测试剂盒，对RNA完整性进行质检。使用美国Life Technologies公司的Qubit 2.0荧光定量仪（Qubit 2.0 Fluorometer）搭配RNA检测试剂盒（RNA Assay Kit），测定RNA的浓度。由中国广州的Gene Denovo公司使用Illumina HiSeq™ 2000测序平台对构建完成的cDNA文库进行测序，测序策略为150 bp双端读长，插入片段长度为300 bp。采用诺禾致源（Novogene）内部开发的Perl脚本，通过移除接头序列、低质量读段（质量值低于20的碱基占比超过50%的读段）以及含N碱基占比超过5%的读段，筛选得到清洁读段（clean reads）。本研究通过深度测序，获得了接种稻瘟病菌12小时的感病、抗病水稻幼苗以及未接种对照组的mRNA表达谱；测序数据来自1次生物学实验，包含3次技术重复，测序平台为Illumina HiSeq™ 2000。