Phenotypic Identification and Gene Fine Mapping of the Lesion Mimic and Early Senescence Mutant lmes7 in Rice

doi:10.16819/j.1001-7216.2026.240718

Abstract

Abstract:

【Objective】To study the molecular mechanisms of defense response and leaf premature senescence in rice,【Method】we screened a lesion mimic and early senescence mutant, designated lmes7 (lesion mimic and early senescence 7), from a mutant library of the japonica rice variety Wuyunjing 7. The main agronomic traits of the mutant and the photosynthetic pigment content in its leaves were measured. The target gene was identified through fine mapping and sequencing, and its encoded protein was analyzed and aligned.【Result】Physiological analysis showed that the photosynthetic pigment content in the mutant leaves was significantly lower than that of the WT, along with excessive accumulation of reactive oxygen species (ROS). In the lmes7 mutant, the effective tiller number, plant height, flag leaf length, flag leaf width, panicle length, grain number per panicle, seed setting rate, and grain yield per plant decreased significantly, while grain thickness increased significantly. Genetic analysis indicated that the mutant phenotype was controlled by a single recessive nuclear gene. Using map-based cloning, the target gene was fine-mapped to a 90-kb region between RM28486 and RM28489 on chromosome 12. PCR and sequencing revealed a 7-bp deletion in OsACL-A2, a gene encoding ATP-citrate lyase, in the lmes7 mutant, resulting in a frameshift mutation and premature termination of translation. Phylogenetic analysis and sequence alignment indicated that the mutation site of lmes7 lies within the succinyl-CoA synthetase domain, identifying lmes7 as a novel allelic variant of OsACL-A2.

Key words: rice, lesion mimic and early senescence, lmes7, phenotypic identification, fine mapping

摘要：

【目的】研究水稻防御反应和叶片早衰相关基因的分子机理。【方法】从粳稻武运粳7号诱变库中筛选出一个类病变早衰突变体lmes7 (lesion mimic and early senescence 7)。调查统计了该突变体的主要农艺性状，对其叶片中的光合色素含量进行测定，利用图位克隆技术对目的基因进行精细定位和测序鉴定，并对其编码蛋白进行分析与序列比对。【结果】突变体叶片中的光合色素含量比WT极显著降低，且细胞中存在活性氧(reactive oxygen species, ROS)的过量积累；突变体植株的生长发育受到严重影响，有效分蘖数、株高、剑叶长、剑叶宽、穗长、每穗粒数、结实率及单株产量等均显著下降，但粒厚显著增加；遗传分析表明，lmes7的突变表型受单隐性核基因控制；利用图位克隆技术将目的基因精细定位在水稻12号染色体上RM28486和RM28489之间的90 kb区域内；PCR测序分析表明，lmes7突变体中一个编码ATP-柠檬酸裂解酶的目标基因OsACL-A2发生了7个碱基缺失，导致该基因发生移码突变，蛋白翻译提前终止；对水稻OsACL-A2蛋白的进化分析与序列比对可知，lmes7突变位点发生在琥珀酰辅酶A合成酶结构域，lmes7是OsACL-A2新的等位变异。

关键词: 水稻, 类病变早衰, lmes7突变体, 表型鉴定, 精细定位

LIU Yaping, DONG Yici, ZHENG Junyan, QIU Xuan, LIU Pengcheng, YE Yafeng, LIU Binmei, CHEN Xifeng, MA Bojun. Phenotypic Identification and Gene Fine Mapping of the Lesion Mimic and Early Senescence Mutant lmes7 in Rice[J]. Chinese Journal OF Rice Science, 2026, 40(1): 85-94.

刘亚萍, 董译词, 郑君妍, 邱绚, 刘鹏程, 叶亚峰, 刘斌美, 陈析丰, 马伯军. 水稻类病变早衰突变体lmes7的鉴定与基因精细定位[J]. 中国水稻科学, 2026, 40(1): 85-94.

Figures/Tables 6

Table 1. PCR molecular marker primers used in this study

引物名称 Primer name	正向引物 Former primer(5’ →3’)	反向引物 Reverse primer(5’ →3’)
Indel1	CGATTTCTCGTCGAGGTTCC	TCCTTCAAGAATCTCCATCTGC
lmes7-1	CGCTGCCTCACTAACGCCTAT	ACCTCCGAGTCCAAAACA
lmes7-2	TTAGGTCACGGAATCAAC	TCCAGAATGGGAGAAAAG
lmes7-3	GGAGAACTGGGATAAGGT	TTAGACTTACAGTATCAGCAT
lmes7-4	GGTTGGAGATTTGGGAT	TCTTTGAGTTTGGTGGAT

Fig. 1. Phenotype of the lesion mimic and early senescence mutant lmes7 A, Phenotype of plants during the tillering stage, bar=10 cm; B, Phenotype of plants at maturity, bar=20 cm; C, Flag leaf phenotype, bar=2 cm; D, WT and lmes7 leaf phenotype before staining; E, NBT staining of leaves; F, DAB staining of leaves, bar=1 cm; G, Content of photosynthetic pigments in leaves. H and J, Grain length, grain width and grain thickness of WT and lmes7, bar=1 cm.

Fig. 2. Comparison of agronomic traits between WT and rice mutant lmes7 A, Effective tiller number; B, Plant height; C, Flag-leaf length; D, Flag-leaf width; E, Panicle length; F, Grain number per panicle; G, Setting percentage; H, Yield per plant; I, 1000-grain weight; J, Grain length; K, Grain width; L, Grain thickness. t-test showed significant difference (P< 0.05) and extremely significant difference (P< 0.01).

Fig. 3. Fine mapping and candidate gene sequencing of rice lmes7 A, Genetic map of the lmes7 gene by fine mapping, ‘n’ refers to the number of F2 mutants used for mapping, the number under each marker represents the number of recombination detected by the corresponding marker; B, Annotation genes in the localization interval; C, Sequencing results of candidate gene LOC_Os12g37870 in the mutation sites.

Fig. 4. Phylogenetic tree analysis of OsACL-A2 protein

Fig. 5. Sequence alignment of OsACL-A2 protein between mutant lmes7, spl30 and WT

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