应用GDPs转录物标记法进行水稻白叶枯病菌早期侵染表达谱分析

DNA Microarray Expression Analysis of Xanthomonas oryzae pv. oryzae in Rice Leaves at Early Infection Stages Using Selective Bacterial Transcript Labeling with Genome-Directed Primers

【目的】揭示水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)在侵染水稻叶组织早期的基因表达谱差异。【方法】采用GDPs-Finder计算法,设计了能够覆盖KACC10331基因组全部ORFs的定向引物(GDPs),对Xoo与水稻互作1、3和7d的总RNA进行反转录,对病菌cDNA探针进行Cy3/Cy5选择性标记,与含有371个Xoo致病相关基因的芯片进行杂交。【结果】在水稻侵染的不同时期,Xoo致病相关基因表达谱存在差异。与在NBY中生长相比,Xoo在侵染1、3和7d时分别有42个(18个上调和23个下调)、51个(29个上调和22个下调)和33个(16个上调和17个下调)基因发生了差异表达;其中5个基因是共有上调表达的,5个是共有下调表达的。推测这些基因的差异表达可能与病菌适应寄主体内环境、生长与定殖以及为害与显症过程有关。【结论】应用GDPs转录物标记技术可以成功地进行Xoo侵染过程的表达谱分析,鉴定出的差异表达的基因可以作为功能研究的靶标基因。

【Objective】 The objective of this study is to reveal the bacterial gene expression profiling of Xanthomonas oryzae pv. oryzae （Xoo） in rice leaves at early infection stages via DNA expression microarray analysis. 【Method】 A computer-based algorithm for prediction of the minimal number of primers to specifically anneal to all genes in the genome （genome-directed primers,GDPs） of KACC10331,a genome sequenced strain of Xoo was used. Seventy-four oligonucleotides （8 mer in length each） priming all genes in the genome were predicted and used. A set of microarray of 371 pathogenicity-related genes was hybridized with the GDPs-primed and fluorescent Cy3/Cy5-labeled cDNAs,which were reverse-transcribed from RNAs isolated from rice leaves 1d,3d and 7d post-inoculation of Xoo. 【Result】 The differential expression profiling of pathogenicity-related genes of Xoo at different early infection stages was found. Forty-two,fifty-one and thirty-three genes of Xoo were differentially expressed in the rice leaf tissues at 1 d,3 d and 7 d after bacterial inoculation relative to the nutrition rich media NBY,respectively. Among them,5 up-regulated genes and 5 down-regulated genes were observed in Xoo at all early infection stages. The expression of Xoo genes might be related to bacterial invasion,adaptation,proliferation and appearance of symptom. 【Conclusion】 The approach of selective transcript labeling with GDPs for in planta gene expression profiling of Xoo is reported for the first time. The differentially-expressed genes of Xoo might be the candidate targets for the further functional analysis.