关键词: 水稻, 短根突变体, 遗传分析, 图位克隆, 功能互补

Abstract:

【Objective】The root system of rice is an important agronomic trait closely related to shoot growth and yield. Identifying new root development-related genes in rice will help further clarification of the underlying molecular mechanisms.【Method】In the present study, a mutant with significantly shorter roots was isolated from an EMS (ethyl methane sulfonate)-generated mutant library of rice and designated as Osksr7 (Oryza sativa kasalath short root 7 ). By using solution culture and field planting, analysis of young seedling phenotype and main agronomic traits of mature plants was conducted. The F₂ populations from crossing of Osksr7 with indica Kasalath and japonica Nipponbare were used for genetic analysis and map-based cloning, respectively. Candidate genes were examined by DNA sequencing. Complementation analysis of the Osksr7 mutant with the protein-coding region of OsKSR7 driven by the 35S promoter was performed using Agrobacterium tumefaciens -mediated transformation. 【Result】 At the seedling stage, the elongation of primary roots, adventitious roots, lateral roots and root hairs in Osksr7 was severely impaired. The length of primary roots, adventitious roots and lateral roots of Osksr7 was only 33%, 38.9% and 35.3% of those of the wild type, respectively. Nevertheless, the number of adventitious roots of Osksr7 was significantly increased when compared with the wild type. At the maturation stage, the agronomic traits of Osksr7 were also significantly compromized, including the shoot height, panicle number, clum thickness, seed setting rate, 1000-grain weight and length and width of flag leaves. Among them, the panicle number and seed setting rate of Osksr7 dramatically decreased to only 56.3% and 37.3% of those of the wild type, respectively. Genetic analysis showed that the growth of F₁ plants from the crossing of Osksr7 with indica Kasalath was similar to the wild type and the segregation ratio of wild type and mutant phenotype plants in the corresponding F₂ population fitted a ratio of 3:1, indicating that the mutant trait of Osksr7 was controlled by a single recessive nuclear gene. The OsKSR7 locus was further mapped between InDel markers IND1 and IND2 on chromosome 11 with a physical distance of 143 kb, where there were 25 predicted genes with annotation. Sequencing analysis found a point mutation (T ⁷³ to A) in the first exon of the gene LOC_Os11g24560 within this region in Osksr7 , resulting in an amino acid substitution (Trp ²⁵ to Arg). The gene encodes a putative rice homolog of the SEC23 subunit of the coat protein complex II (COPII) involved in ER-to-Golgi transport. RT-PCR analysis revealed no significant difference in the expression level of LOC_Os11g24560 between the wild type and Osksr7 . Transformation of Osksr7 with the coding sequence of LOC_Os11g24560 driven by the 35S promoter could successfully restore its growth defects, confirming that the mutation in LOC_Os11g24560 was responsible for the mutant phenotype of Osksr7 .【Conclusion】 Osksr7 is a rice short root mutant, and yield-related agronomic traits are significantly suppressed in Osksr7 . OsKSR7 is confirmed to be within the locus LOC_Os11g24560 , which encodes the SEC23 subunit of the coat protein complex II (COPII). OsKSR7 is not allelic to any previously reported rice root gene and is a newly identified regulator of root development in rice.

Key words: Oryza sativa L., short root mutant, genetic analysis, map-based cloning, functional complementation