Abstract:

【Objective】Rice blast caused by Magnaporthe oryzae and bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) seriously affect rice yield and quality. The transgenic materials of OsLOX10 were created to evaluate the resistance to M. oryzae and Xoo. The possible mechanism of OsLOX10 in regulating the defense responses to rice blast and bacterial blight were elucidated. 【Method】The knockout vector of OsLOX10 was constructed using CRISPR/Cas9 system, and the overexpression vector of OsLOX10 was constructed by linearizing pCXUN-HA with restriction enzyme XcmⅠ and linking with TA ligation. Transgenic rice was obtained by genetic transformation, and the homozygous transgenic lines were screened for fungal and bacterial resistance analysis. The expression dynamics of SA (salicylic acid) and JA (jasmonic acid) pathway marker genes were analyzed by qRT-PCR after infection with M. oryzae. The outbreak of reactive oxygen species (ROS) induced by chitin and flg22 in rice was also observed. 【Result】qRT-PCR analysis showed that the OsLOX10 expression was up-regulated 24 h after inoculation with M. oryzae and Xoo. The OsLOX10 knockout lines were more susceptible to the rice blast than that of the wild type (Nipponbare) when inoculated with the spore suspension of M. oryzae Guy11, while the overexpressed lines had no typical disease symptoms. At 6, 12, 24 and 36 hours post inoculation, the transcriptional levels of three pathogenesis-related genes, OsPBZ1, OsPR1a and OsPR1b, and two JA pathway genes, OsAOS2 and OsLOX5, and SA pathway gene OsPAL1, were significantly down-regulated in the knockout transgenic lines. However, they were significantly up-regulated in overexpressed transgenic lines. The OsLOX10 transgenic rice was inoculated with Xoo (PXO99A), and the OsLOX10 knockout lines were more susceptible to bacterial blight. qRT-PCR analysis showed that the relative expression levels of OsPR1b, OsPAL1 and the three genes (OsAOS2, OsAOC and OsJAZ) involved in the JA synthesis pathway were up-regulated in OsLOX10 overexpression lines, significantly down-regulated in OsLOX10 knockout lines seven days after Xoo inoculation. OsLOX10 knockout also significantly reduced the ROS level induced by chitin and flg22 in rice, delayed the peaking time of ROS induced by chitin, and reduced the sensitivity of rice to chitin and flg22.【Conclusion】OsLOX10 could be induced by infection with M. oryzae and Xoo. OsLOX10 may be involved in pathogen-associated molecular patterns triggered immunity, and play a positive regulatory role in rice resistance to rice blast and bacterial blight. Moreover, the OsLOX10 may regulate rice resistance to M. oryzae and Xoo through SA and JA mediated signaling pathways.

Key words: OsLOX10, CRISPR/Cas9, disease resistance, rice blast, bacterial blight, signal pathway

摘要：

【目的】由稻瘟病菌（Magnaporthe oryzae）引起的稻瘟病和由水稻黄单胞菌（Xanthomonas oryzae pv. oryzae，Xoo）引起的白叶枯病严重影响水稻的产量和品质。创制转OsLOX10基因水稻材料，进行稻瘟菌和白叶枯菌的抗病性分析，有助于揭示其调控水稻对稻瘟病和白叶枯病的抗性机制。【方法】采用CRISPR/Cas9系统构建OsLOX10的敲除载体，利用限制性内切酶XcmⅠ线性化pCXUN-HA，TA连接构建OsLOX10的过表达载体，遗传转化获得OsLOX10转基因水稻，筛选过表达株系和纯合敲除株系进行真菌和细菌的抗病性分析。在稻瘟菌（Guy11）侵染水稻后，对水杨酸（salicylic acid，SA）、茉莉酸（jasmonic acid，JA）途径的标志基因进行qRT-PCR分析;在几丁质（chitin）和flg22诱导下，观测水稻活性氧（reactive oxygen species，ROS）的暴发情况。【结果】 qRT-PCR分析表明，接种稻瘟菌和白叶枯菌24 h后，OsLOX10表达量上调;OsLOX10的纯合敲除和过表达水稻转基因株系接种稻瘟病菌Guy11孢子悬浮液，与野生型（日本晴）相比，OsLOX10敲除株系更易感病，过表达株系则无典型的病斑症状;接种 6、12、24和36 h时，3个病程相关蛋白基因OsPBZ1、OsPR1a、OsPR1b和SA通路基因OsPAL1，以及JA合成通路上的2个基因OsAOS2、OsLOX5的转录水平在敲除转基因株系中显著下调，而在过表达转基因株系中显著上调。对转OsLOX10基因水稻接种白叶枯菌（PXO99A），发现敲除OsLOX10的转基因水稻对白叶枯菌更易感病。qRT-PCR分析OsPR1b和OsPAL1以及JA合成通路上的3个基因OsAOS2、OsAOC和OsJAZ在OsLOX10过表达基因水稻中表达量明显上调，而在敲除OsLOX10的转基因水稻中却保持在较低水平，在接种7 d后表现出显著性差异。在几丁质和flg22诱导下，OsLOX10敲除株系的ROS水平显著性降低，而且在几丁质诱导下，ROS的起峰时间推迟。【结论】稻瘟病菌和白叶枯病菌能够诱导OsLOX10的表达，OsLOX10通过病原菌分子模式触发的免疫途径（PTI）参与抗病反应，其在水稻抵御稻瘟病和白叶枯病中起着正调控作用。同时，OsLOX10可能通过调节SA和JA介导的信号通路来正调控水稻对稻瘟病和白叶枯病的抗性。

关键词: OsLOX10, CRISPR/Cas9基因编辑技术, 抗病性, 稻瘟病, 白叶枯病, 信号途径