水稻长护颖小粒突变体lsg8的表型鉴定与基因克隆
收稿日期: 2024-10-11
修回日期: 2024-12-02
网络出版日期: 2025-11-19
基金资助
国家重点研发计划资助项目(2022YFD1200103);江苏省重点研发计划资助项目(BE2022336)
Identification and Gene Cloning of a Long Sterile Lemma and Small Grain Mutant lsg8 in Rice (Oryza sativa L.)
Received date: 2024-10-11
Revised date: 2024-12-02
Online published: 2025-11-19
【目的】通过对一个水稻长护颖、小粒突变体的表型鉴定与基因克隆，探明控制该性状的遗传基础和分子机制。【方法】利用甲基磺酸乙酯(EMS)诱变粳稻品种武育粳27(WYJ27)，从中获得一个稳定遗传的长护颖、小粒突变体，将该突变体命名为lsg8 ( long sterile lemma and small grain on chromosome 8)。观察并统计野生型和突变体的农艺性状变化；利用透射电镜观察颖壳外表皮细胞的变化；将突变体lsg8与籼稻品种IR36进行杂交，构建F2群体并进行遗传分析，利用图位克隆对LSG8基因进行定位，通过对候选基因测序和表达分析进一步确定候选基因；利用RT-qPCR分析细胞扩展相关基因和花器官特征基因的表达量。【结果】与野生型相比，突变体lsg8护颖长度明显变长，粒宽和粒厚显著下降，从而导致千粒重下降。此外，突变体lsg8的株高、穗长、倒1节间长、倒2节间长、倒4节间长、一次枝梗数、二次枝梗数、每穗粒数和结实率均较野生型WYJ27显著降低。颖壳外表皮扫描电镜观察结果表明，突变体lsg8的细胞长度较野生型显著变短，细胞宽度较野生型显著变窄。遗传分析表明，突变体lsg8受一对隐性核基因控制。通过图位克隆将LSG8基因定位在8号染色体标记M5和标记M6之间，物理距离约为276 kb，该区间包含42个开放阅读框。通过对候选基因测序分析，发现ORF18(LOC_Os08g06480)在野生型和突变体之间出现了一个碱基的差异，认为该基因可能是控制长护颖、小粒表型的候选基因。RT-qPCR分析表明，LSG8在不同时期各个组织中均有表达，其中在成熟期的穗中表达量最高，而在成熟期的叶鞘中表达量最低。此外，突变体lsg8在细胞扩展相关基因、护颖发育调控基因和颖壳特征基因的表达量也发生了显著的变化。【结论】水稻长护颖、小粒突变体lsg8是已报道基因ASP1的新等位基因，该基因突变导致护颖变长、种子变小，对于维持水稻护颖的形态建成及籽粒形态起到重要的作用。
陆帅, 陶涛, 刘冉, 周文玉, 曹蕾, 杨青青, 张明秋, 任鑫哲, 杨芝笛, 徐福祥, 环海东, 龚远航, 张皓程, 金素奎, 蔡秀玲, 高继平, 冷语佳 . 水稻长护颖小粒突变体lsg8的表型鉴定与基因克隆[J]. 中国水稻科学, 2025 , 39(6) : 813 -824 . DOI: 10.16819/j.1001-7216.2025.241003
【Objective】 The aim of this study was to identify and clone a gene responsible for long sterile lemma and small grain size in rice, and to explore the genetic basis and molecular mechanism controlling these traits. 【Method】 A stably inherited mutant displaying long sterile lemmata and small grains was obtained from the japonica rice WYJ27 through ethyl methanesulfonate (EMS) mutagenesis. This mutant was named as lsg8 (long sterile lemma and small grain on chromosome 8). The agronomic traits of both the wild-type and the mutant were observed and recorded. The epidermal cells of the glume were observed using scanning electron microscopy (SEM). An F2 segregating population was generated by crossing lsg8 with the indica rice variety IR36, and used for genetic analysis and gene mapping. Candidate genes were further identified through gene sequencing and expression analysis. RT-qPCR was performed to analyze the relative expression levels of genes regulating cell expansion and floral development. 【Result】 Compared with the wild type, the mutant lsg8 exhibited a significantly longer sterile lemma, significantly decreased grain width and grain thickness, and consequently a reduced 1000-grain weight. In addition, the plant height, panicle length, the length of the first, second, and fourth internodes from the top, the number of primary branches, the number of secondary branches, the number of grains per panicle, and the seed setting rate of lsg8 were significantly lower than those of the wild type WYJ27. SEM observation of the outer epidermis of the glume showed that the cell length and width of the mutant lsg8 were significantly shorter and narrower than those of the wild type. Genetic analysis suggested that the mutant phenotype is controlled by a single recessive nuclear gene. Through map-based cloning, the LSG8 gene was mapped to a region between markers M5 and M6 on chromosome 8. The physical distance between these two markers is approximately 276 kb, containing 42 open reading frames (ORFs). Sequencing analysis revealed a single nucleotide difference in ORF18 (LOC_Os08g06480) between the wild type and the lsg8 mutant, indicating that ORF18 is likely the candidate gene controlling the long sterile lemma and small grain phenotype. RT-qPCR analysis revealed that LSG8 is expressed in various tissues at different developmental stages, with the highest expression in panicles at the mature stage and the lowest in leaf sheaths at the mature stage. Furthermore, significant alterations were observed in the relative expression levels of genes related to cell expansion, sterile lemma development, and floral organ identity in the lsg8 mutant. 【Conclusion】 The lsg8 mutant is a new allele of the previously reported ASP1 gene. The mutation in OsLSG8 results in a long sterile lemma and small grain phenotype, playing an important role in maintaining the morphogenesis of rice sterile lemma and grain morphology.
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