水稻类病斑突变体lm52的基因克隆及其广谱抗病性分析

doi:10.16819/j.1001-7216.2025.240304

中国水稻科学 ›› 2025, Vol. 39 ›› Issue (3): 322-330.DOI: 10.16819/j.1001-7216.2025.240304

水稻类病斑突变体lm52的基因克隆及其广谱抗病性分析

黄涛¹, 魏兆根¹, 陈玘¹, 程泽¹, 刘欣¹, 王广达¹, 胡珂鸣¹^,², 谢文亚¹^,², 陈宗祥¹^,²^,³, 冯志明¹^,²^,³^,^*(), 左示敏¹^,²^,³^,^*()

¹扬州大学农学院/江苏省作物基因组学和分子育种重点实验室/生物育种钟山实验室/植物功能基因组学教育部重点实验室，江苏扬州 225009
²扬州大学江苏省粮食作物现代产业技术协同创新中心/江苏省作物遗传生理重点实验室，江苏扬州 225009
³扬州现代种子创新研究所, 江苏高邮 225600

收稿日期:2024-03-04 修回日期:2024-05-06 出版日期:2025-05-10 发布日期:2025-05-21
通讯作者: *email: fengzm@yzu.edu.cn;smzuo@yzu.edu.cn
基金资助:
江苏省政府种业振兴计划资助项目[JBGS(2021);国家自然科学基金资助项目(32272110);扬州大学大学生创新创业训练计划项目(XCX20230650);扬州大学“青蓝工程”资助项目

Gene Cloning and Broad-spectrum Disease Resistance Analysis of Rice Lesion Mimic Mutant lm52

HUANG Tao¹, WEI Zhaogen¹, CHENG Qi¹, CHENG Ze¹, LIU Xin¹, WANG Guangda¹, HU Keming¹^,², XIE Wenya¹^,², CHEN Zongxiang¹^,²^,³, FENG Zhiming¹^,²^,³^,^*(), ZUO Shimin¹^,²^,³^,^*()

¹Agricultural College, Yangzhou University/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding/Zhongshan Biological Breeding Laboratory/Key Laboratory of Plant Functional Genomics of Ministry of Education, Yangzhou 225009, China
²Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops/Jiangsu Key Laboratory of Crop Genetics and Physiology, Yangzhou University, Yangzhou 225009, China
³Yangzhou Modern Seed Innovation Research Institute, Gaoyou 225600, China

Received:2024-03-04 Revised:2024-05-06 Online:2025-05-10 Published:2025-05-21
Contact: *email: fengzm@yzu.edu.cn;smzuo@yzu.edu.cn
About author:^#These authors contributed equally to this work

摘要/Abstract

摘要：

【目的】类病斑突变体(Lesion mimic mutant, LMM)是研究植物细胞死亡和抗病机制的理想材料，可为作物抗病育种提供新的分子靶点和育种材料。【方法】利用图位克隆技术对水稻类病斑突变体lm52的突变基因进行了定位，并利用CRISPR/Cas9敲除技术对候选基因进行了功能验证，同时鉴定了lm52对水稻对主要病害(稻瘟病、纹枯病和白叶枯病)的抗性，并分析其抗性机制。【结果】突变体lm52从4叶期开始逐渐出现褐色点状病斑，成熟期病斑扩展至全叶；ATP酶基因LOC_Os06g03940上的单碱基突变(G₁₄₄₀A)是lm52产生类病斑表型的原因；lm52显著增强了对水稻三大主要病害(稻瘟病、纹枯病和白叶枯病)的抗性，且这与突变体中活氧性(ROS)的过度积累和防御基因的激活表达有关。【结论】ATP酶基因LM52的突变增强了水稻对不同类型病原菌的抗性，为水稻抗病分子育种提供潜在的靶标基因。

关键词: 水稻, 类病斑突变体, 图位克隆, 抗病性

Abstract:

【Objective】Lesion mimic mutants are ideal materials to study the mechanisms of plant cell death and disease resistance, offering new strategies for plant disease resistance breeding.【Method】The lm52 mutant gene was cloned by the map-based cloning method, and the function of the candidate gene was verified by CRISPR/Cas9 knockout technology. Meanwhile, the resistance of lm52 to multiple rice pathogens and the possible resistance mechanisms were analyzed.【Result】The mutant lm52 gradually developed progressive brown spot lesions from the 4th leaf stage, with whole leaf necrosis at maturity stage. A single base mutation (G₁₄₄₀A) on the ATPase encoding gene LOC_Os06g03940 caused the lesion mimic phenotype of lm52. lm52 is characterized by significantly enhanced resistance to blast, sheath blight and bacterial blight, which was found to be related to the overaccumulation of ROS and the activation of defense-related genes.【Conclusion】The mutation of the ATPase gene LM52 enhances rice resistance against diverse pathogens, and provides potential target genes for molecular breeding of rice disease resistance.

Key words: rice, lesion mimic mutant, map-based cloning, disease-resistance

黄涛, 魏兆根, 陈玘, 程泽, 刘欣, 王广达, 胡珂鸣, 谢文亚, 陈宗祥, 冯志明, 左示敏. 水稻类病斑突变体lm52的基因克隆及其广谱抗病性分析[J]. 中国水稻科学, 2025, 39(3): 322-330.

HUANG Tao, WEI Zhaogen, CHENG Qi, CHENG Ze, LIU Xin, WANG Guangda, HU Keming, XIE Wenya, CHEN Zongxiang, FENG Zhiming, ZUO Shimin. Gene Cloning and Broad-spectrum Disease Resistance Analysis of Rice Lesion Mimic Mutant lm52[J]. Chinese Journal OF Rice Science, 2025, 39(3): 322-330.

图/表 7

图1 野生型(日本晴)与突变体lm52表型对比 A: 野生型(日本晴)与突变体lm52叶片；B: 野生型与lm52整株对比；C: 野生型与lm52穗部对比。Nip: 日本晴。

Fig. 1. Comparison between wild type(Nipponbare) and mutant lm52 A, Leaf phenotypes of wild type (Nipponbare) and lm52 mutant; B, Comparison between wild type and lm52; C, Comparison of panicle of wild type and lm52. Nip, Niponbare.

表1 野生型(日本晴)和突变体lm52的农艺性状

Table 1. Agronomic traits of wild type(Nipponbare, WT) and lm52

材料 Material	株高 Plant height(cm)	分蘖数 Number of tillers	穗长 Panicle length(cm)	一次枝梗数 Primary rachis branch number	每穗粒数 Grains per panicle	千粒重 1000-grain weight(g)
WT lm52	102.70±3.35 85.75±1.21**	13.8±1.0 10.4±0.5**	22.20±1.23 20.30±0.46**	9.5±1.3 8.8±0.9	134.5±6.1 91.1±5.1**	24.40±0.26 21.33±0.31**

图2 lm52的图位克隆 A：在6号染色体上通过RM19319和RM3805两个标记进行初步定位，定位在S4和S6之间，物理距离分别为1.54 Mb和1.64 Mb；B：17个候选基因分布情况，在LOC_Os06g03940上发现突变位点；C：lm52与野生型氨基酸序列比对。*表示比对结果一致。

Fig. 2. Map-based cloning of lm52 A, LM52 was preliminarily located between S4 and S6 on chromosome 6 with RM19319 and RM3805, with a physical distance of 1.54 Mb and 1.64 Mb, respectively; B, Distribution of 17 candidate genes, and mutation sites on LOC_Os06g03940; C, lm52 and wild-type amino acid sequence comparison. * indicates that the comparison results are consistent.

图3 候选基因的功能验证 A: Nip与lm52-ko1和lm52-ko2 LOC_Os06g03940基因上的cDNA序列比对；B: 野生型Nip、突变体lm52、敲除系lm52-ko1和敲除系lm52-ko2叶片对比；C: 野生型Nip、突变体lm52、敲除系lm52-ko1和敲除系lm52-ko2整株对比。Nip: 日本晴。

Fig. 3. Functional verification of candidate genes A, cDNA sequence comparison between Nip and lm52-ko1 (lm52-ko2) on LOC_Os06g03940 gene; B, Comparison of leaves of wild-type Nip, mutant lm52, and knockout lines lm52-ko1 and lm52-ko2; C, Comparison of wild-type Nip, mutant lm52, and knockout lines lm52-ko1 and lm52-ko2.

图4 lm52对不同病原菌的抗性 A: 日本晴与lm52的纹枯病表型和相应病级; B：日本晴与lm52的稻瘟病表型和相应病级; C：日本晴与lm52的白叶枯病表型和相应病级。

Fig. 4. Resistance of lm52 to diverse pathogens A, Sheath blight symptoms and corresponding disease grade histogram of Nipponbare and lm52; B, Rice blast symptoms and corresponding disease grade histogram of Nipponbare and lm52; C, Bacterial blight symptoms and disease grades of Nipponbare and lm52.

图5 lm52体内ROS富集情况 A，B分别为日本晴和lm52 DAB染色前(A)后(B)对比；C：过氧化氢(H2O2)活性含量检测与超氧阴离子含量检测。

Fig. 5. ROS accumulation in lm52 A and B, Comparison between Nipponbare and lm52 before (A) and after (B) DAB-staining; C, Detection of hydrogen peroxide (H2O2) content and superoxide anion content.

图6 防御相关基因(PR)的相对表达量

Fig. 6. Relative expression levels of defense-related gene (PR)

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水稻类病斑突变体lm52的基因克隆及其广谱抗病性分析

Gene Cloning and Broad-spectrum Disease Resistance Analysis of Rice Lesion Mimic Mutant lm52

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