Identification and Gene Mapping of a Lesion Mimic Mutant lm8015-2 in Rice

doi:10.16819/j.1001-7216.2021.200908

Abstract

Abstract:

【Objective】 To substantiate the genetic mechanisms behind the disease resistance of rice, we identified a rice lesion mimic mutant lm8015-2. 【Method】 lm8015-2 was obtained from an EMS-induced Zhonghui 8015 (ZH8015) mutant library. The SOD, CAT, POD activities, H₂O₂, MDA, SP, photosynthetic pigments contents, the expression level of defense genes were analyzed in lm8015-2 and its wild-type leaves. The population derived from 02428/ lm8015-2 was used for genetic analysis and fine mapping. 【Result】 Its red rust-like spots were first observed on the tips of the mutant leaves three weeks after sowing, then spread gradually to the whole leaf and throughout the plant at tillering stage, causing the death of whole leaf at heading stage. The shading assay showed that the lesions on the leaves of lm8015-2 were induced by natural light. Meanwhile, photosynthetic pigment contents of lm8015-2 decreased significantly. The staining experiments of EB and DAB showed the excessive H₂O₂deposition and cell death of lm8015-2. Compared with the wild type, the activities of SOD and POD, the MDA content increased, while the content of SP decreased in the mutant lm8015-2. Genetic analysis suggested that the phenotype of lm8015-2 was controlled by a single nuclear recessive gene, which was located in a 104 kb interval between the markers W32-85 and C32-8 on chromosome 5. Sequencing analysis revealed that a single base substitution (T to A) of the initiation codon (ATG) occurred in the DNA sequence of LOC_Os05g48390, resulting in the absence of initiation codon. The qRT-PCR results showed the expression of partial defense genes were up-regulated obviously in lm8015-2. 【Conclusion】 The results reveal the target gene is allelic to LTN1, and the mutation of LM8015-2 likely enhances the expression of partial defense genes.

Key words: rice, lesion mimic mutant, fine mapping

摘要：

【目的】为进一步解析水稻抗病分子机制,对类病斑突变体lm8015-2进行了鉴定。【方法】利用EMS诱变籼稻中恢8015,从其后代中鉴定出一个类病斑突变体lm8015-2。对突变体lm8015-2及其野生型的叶片进行超氧化物歧化酶活性、过氧化氢酶活性、过氧化物酶活性、过氧化氢含量、丙二醛含量、可溶性蛋白含量、光合色素含量、防御基因表达量的测定。将粳稻02428与突变体lm8015-2杂交获得的F₂群体用于遗传分析,采用图位克隆的方法对目的基因进行精细定位。【结果】突变体的红锈状斑点自播种3周后于叶尖出现,分蘖期缓慢扩散至全叶及整个植株,至抽穗期可致整个叶片枯亡。lm8015-2类病斑的产生受自然光的诱导,其叶片的光合色素含量明显低于野生型。EB染色与DAB染色结果表明,lm8015-2中发生了大量的过氧化氢沉积与细胞死亡。与野生型相比,lm8015-2中超氧化物歧化酶和过氧化物酶的活性显著上升,同时伴有可溶性蛋白含量下降和丙二醛含量上升。遗传分析表明,lm8015-2类病斑表型由一对隐性核基因控制,该基因位于第5染色体的W32-85和C32-8两个引物之间,物理区间为104 kb。测序结果表明该区间内候选基因LOC_Os05g48390的起始密码子(ATG)发生了单碱基替换突变(T变为A),导致起始密码子缺失。qRT-PCR结果表明,lm8015-2中部分防御基因被激活,表达显著上调。【结论】LM8015-2与LTN1互为等位基因,其突变可能增强了部分防御基因的表达。

关键词: 水稻, 类病斑突变体, 精细定位

Chen WANG, Beifang WANG, Yingxin ZHANG, Yongrun CAO, Yue ZHANG, Min JIANG, Kangji BIAN, Xiaohui ZHANG, Qunen LIU. Identification and Gene Mapping of a Lesion Mimic Mutant lm8015-2 in Rice[J]. Chinese Journal OF Rice Science, 2021, 35(4): 352-358.

王晨, 王备芳, 张迎信, 曹永润, 张越, 江敏, 边康吉, 张小惠, 刘群恩. 水稻类病斑突变体lm8015-2的鉴定与基因的精细定位[J]. 中国水稻科学, 2021, 35(4): 352-358.

Figures/Tables 6

References 23

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标记 Marker	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
W32-2	CGGGAAGCTGAACCGGC	GTACCGGAATTTCCACACCTT
W32-14	GTGTGCTTGCTGCGGATATG	GCTAACACACCCAACGAATGA
W32-44	TGGCTTTGTAGATGGCCGATT	ATCCAAAGTGTTGTGGGCCT
W32-85	CGGAAATCCCCTCACGCAC	CTGTTGGAGATGCTCTAACCCT
C32-8	CCAAGAGGTCTGGAGATTGC	TGGAGCTCCTGGAACAACAT
PR1B	CATTGCTTTGGCCATGGTAG	GAACCCCAGAAGAGGTTCTC
PR5	CTTCTGCCCATAATGCATCATC	TGATTATCGATCAAGGTGTCGT
PR10	GGTGTGGGAAGCACATACAA	GTCTCCGTCGAGTGTGACTTG
AOS2	AAGCTGCTGCAATACGTGTACTGG	CGACGAGCAACAGCCTTCCG
UBQ	TGGTCAGTAATCAGCCAGTTTGG	GCACCACAAATACTTGACGAACAG

标记 Marker	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
W32-2	CGGGAAGCTGAACCGGC	GTACCGGAATTTCCACACCTT
W32-14	GTGTGCTTGCTGCGGATATG	GCTAACACACCCAACGAATGA
W32-44	TGGCTTTGTAGATGGCCGATT	ATCCAAAGTGTTGTGGGCCT
W32-85	CGGAAATCCCCTCACGCAC	CTGTTGGAGATGCTCTAACCCT
C32-8	CCAAGAGGTCTGGAGATTGC	TGGAGCTCCTGGAACAACAT
PR1B	CATTGCTTTGGCCATGGTAG	GAACCCCAGAAGAGGTTCTC
PR5	CTTCTGCCCATAATGCATCATC	TGATTATCGATCAAGGTGTCGT
PR10	GGTGTGGGAAGCACATACAA	GTCTCCGTCGAGTGTGACTTG
AOS2	AAGCTGCTGCAATACGTGTACTGG	CGACGAGCAACAGCCTTCCG
UBQ	TGGTCAGTAATCAGCCAGTTTGG	GCACCACAAATACTTGACGAACAG

组合Cross	F₁	F₂		比例 Ratio	χ²(3:1)
组合Cross	F₁	无斑点株数 No. of normal plants	F₂有斑点株数 No. of spotted plants	比例 Ratio	χ²(3:1)
02428/lm8015-2	无斑点 Normal	3 796	1 217	3.11∶1	0.40

组合Cross	F₁	F₂		比例 Ratio	χ²(3:1)
组合Cross	F₁	无斑点株数 No. of normal plants	F₂有斑点株数 No. of spotted plants	比例 Ratio	χ²(3:1)
02428/lm8015-2	无斑点 Normal	3 796	1 217	3.11∶1	0.40