| 杨雅云,张斐斐,张发美,等.利用RNA-seq技术筛选药用野生稻抗白叶枯病相关基因[J].广东农业科学,2024,(10-12):- |
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利用RNA-seq技术筛选药用野生稻抗白叶枯病相关基因 |
| Screening of genes related to resistance to bacterial leaf blight in Oryza officinalis using RNA-seq technology |
| 投稿时间：2024-07-12 修订日期：2024-08-03 |
| DOI： |
| 中文关键词: 药用野生稻 白叶枯病抗性 转录组测序 抗病相关基因 |
| 英文关键词: Oryza officinalis Resistance to bacterial leaf blight Transcriptome sequencing Disease resistance related genes |
| 基金项目:国家自然科学基金(32160455)；云南省科技计划重大专项(202302AE090011 )；云南省种子种业联合实验室(202205AR070001-07)；国家自然科学基金(31760375)； |
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| 摘要点击次数: 10 |
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| 中文摘要: |
| 【目的】药用野生稻基因组为CC染色体组野生稻,生长环境恶劣,积累了大量栽培稻所缺乏的抗性基因。 转录组测序(RNA-seq) 技术是在转录组水平上开展功能基因组学研究的强有力的工具,为非模式植物功能基因的发掘提供有效的方法。【方法】本研究利用2个水稻白叶枯病菌CX28-3和PXO99分别接种38份来自于云南省的药用野生稻居群,获得了不同的抗谱表型,挑选极抗极感的居群做转录组测序,分析了PXO99接种胁迫下,0h、24h,48h的药用野生稻36号(抗病)与药用野生稻37号(感病)的转录谱测序数据,获得差异表达基因,并对差异表达基因做深入的聚类分析和功能富集分析。【结果】(1)38份药用野生稻居群对CX-28和PXO99多出现典型的白叶枯病症状,但抗病等级存在差异,其中, 发现38份药用野生稻对CX28-3菌株感病的植株有5份,感病率为13.15%,对PXO99菌株的感病率为42.1%,说明PXO99的致病力强。同时抗两个菌株的药用野生稻材料有20份,占全部材料的66.67%,37号材料对两个菌株都表现为中感(MS),36号材料对两个菌株都表现为抗(R)。供试的云南药用野生稻居群对2个菌株表现为不同的抗病相关,但总体为中抗至抗以上,其中居群抗病率最高的是V居群为93.5%,最低的是III居群为62.5%。(2)通过对药用野生稻36(抗病)和37(感病)不同时间段的转录组测序,结果共鉴定得到 75650个差异表达基因(DEGs)。GO功能富集分析,PXO99处理后共包含45个显著富集的生物进程GO类别,其中富集DEGs数量最多的集中在细胞过程、代谢过程、细胞解剖实体、绑定、催化活性等。KEGG富集分析抗感材料共显著富集到19个 KEGG通路,其中与抗病作用相关的蛋白等KEGG通路显著富集上升,进一步明确抗病药用野生稻36号的抗病性受白叶枯病菌激发显著。(3)通过在抗感材料间不同时间点筛选共同差异表达基因获得256个,挑选出11个在抗感材料中存在差异表达且基因注释与抗病相关的转录本。应用qRT-PCR分析得知这11个基因在抗病和感病药用野生稻材料中PXO99相应处理后表达水平改变,Unigene1184、Unigene15669、CL1239、CL1421、CL4899、CL660、CL7463七个基因在呈在抗病品种中上调表达,在感病品种中下调表达,正调控趋势符合实验预期,Unigene18206、CL210、CL3554、CL9248五个基因呈抗病品种中下调表达,在感病品种中上调表达,负调控趋势符合实验预期。【结论】38份供试的云南药用野生稻居群对2个菌株表现为不同的抗病性,出现了抗感差异,但总体为中抗至抗以上。利用转录组学测序技术获得与药用野生稻抗白叶枯病性状相关的11个基因,研究结果将为药用野生稻抗白叶枯病性状相关基因的挖掘和功能研究提供参考。 |
| 英文摘要: |
| 【Objective】The genome of Oryza officinalis was the CC chromosome group. Wild rice growed in harsh environments and contained a large number of resistance genes that were lacking in cultivated rice. Transcriptome sequencing (RNA seq) technology is a powerful tool for conducting functional genomics research at the transcriptome level, providing an effective method for discovering functional genes in non model plants. 【Method】 In this study, two isolates of? Xanthomonas oryzae pv.oryzae CX28-3 and PXO99 were inoculated to 38 populations of Oryza officinalis wild rice from Yunnan Province, and different resistance phenotypes were obtained. The highly resistant and susceptible populations were selected for transcriptome sequencing. The transcriptome sequencing data of Oryza officinalis 36 (resistant) and Oryza officinalis 37 (susceptible) after PXO99 inoculation stress at 0h, 24h, and 48h were analyzed to obtain differentially expressed genes. In depth clustering analysis and functional enrichment analysis were performed on the differentially expressed genes. 【Results】 (1) Oryza officinalis 38 showed typical symptoms of rice bacterial leaf blight inoculated by CX-28 and PXO99, but there were differences in disease resistance levels. Among them, it was found that 5 accessions of Oryza officinalis populations were susceptible to CX28-3 isolate, with a disease incidence rate of 13.15%, and 42.1% to PXO99 isolate, indicating that PXO99 has strong pathogenicity. There were 20 Oryza officinalis materials that simultaneously resist two strains, accounting for 66.67% of all materials.37 shows moderate susceptibility (MS) to both strains, while 36 showed resistance (R) to both strains. The tested Oryza officinalis population from Yunnan showed different resistance to two strains, but most of them showed moderate resistance to resistance. The population with the highest resistance rate was V population with 93.5%, and the lowest was III population with 62.5%. (2) Through transcriptome sequencing of Oryza officinalis 36 (resistant) and 37 (susceptible) plant at different time periods, a total of 75650 differentially expressed genes (DEGs) were identified. GO functional enrichment analysis revealed that PXO99 treatment resulted in a total of 45 significantly enriched GO categories in biological processes. Among them, the most enriched DEGs were concentrated in cellular processes, metabolic processes, cellular anatomical entities, binding, catalytic activity, etc. KEGG enrichment analysis showed that 19 KEGG pathways were significantly enriched in the resistant plant, among which KEGG pathways related to disease resistance, such as resistant proteins, were significantly enriched and increased. This further clarified that the resistance to disease of the Oryza officinalis 36 was significantly stimulated by the rice leaf blight pathogen. (3) By screening for common differentially expressed genes between resistant materials at different time points, 256 genes were obtained, and 11 transcripts with differential expression and gene annotation related to disease resistance were selected from the resistant materials. The application of qRT-PCR analysis revealed that the expression levels of these 11 genes were altered after treatment with PXO99 in resistant and susceptible Oryza officinalis materials. Unigene1184, Unigene15669, CL1239, CL1421, CL4899, CL660, and CL7463 genes showed upregulation in resistant varieties and down regulation in susceptible varieties, with a positive regulatory trend consistent with experimental expectations. Unigene18206, CL210, CL3554, and CL9248 genes showed down regulation in resistant varieties and up regulation in susceptible varieties, with a negative regulatory trend consistent with experimental expectations. 【Conclusion】 38 accessions of Oryza officinalis from Yunnan tested showed different disease resistance to two strains, with differences in resistance and susceptibility, but overall they were moderate to above resistance. Using transcriptome sequencing technology, 11 genes related to the resistance to bacterial leaf blight in Oryza officinalis were obtained. The research results will provide reference for the exploration and functional study of genes related to the resistance to bacterial leaf blight in Oryza officinalis. |
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