关键词: 水稻（Oryza sativa L.）, 籽粒, 千粒重, 油菜素内酯, AP2类转录因子

Abstract:

【Objective】Phenotypic identification, genetic analysis, gene mapping and candidate gene analysis of grain width mutant gw87 (grain width87) were performed in rice, so as to lay a good foundation for further understanding molecular mechanism and utilization potentiality of this gene regulating rice grain size.【Method】Through ethyl methanesulfonate mutagenesis, we isolated gw87 mutant showing significant increase of grain width and 1000-grain weight from indica restorer line material 676R. To identify phenotypic and physiological characteristics of the mutant, we performed observation of phenotypes, investigation of agronomic traits, sensitivity analysis of exogenous brassinolide (BL), determination of chlorophyll contents and photosynthetic parameters by using gw87 and its wild type. To analyze its genetic behavior, we investigated F₁ phenotypes and F₂ segregation of the crossing combination between gw87 and its wild-type parent 676R. To find candidate gene of gw87, we conducted high-throughput sequencing and MutMap analysis of 30 mutant plants from the F₂ population of gw87×676R, and molecular mapping using the F₂ population crossing gw87 and japonica cultivar Nipponbare. Meanwhile, we confirmed the candidate gene by sequencing its DNA and cDNA sequences. In addition, we detected expression levels of brassinolide synthesis pathway genes OsDWARF4, D11 and D2 using qRT-PCR analysis.【Result】Compared with the wild-type parent 676R, the gw87 mutant showed significant reduction of productive panicle number per plant, main panicle length and seed setting rate, but significant increase of grain width and 1000-grain weight. Meanwhile, the mutant displayed decreased plant height and shortened internodes, in which the first internode was distorted and its length is shortened the most. Besides, leaf length of gw87 was decreased but the width was increased. Sensitivity assays of exogenous BL suggested that the sensitivity of gw87 seedlings to exogenous BL was reduced. Determination of photosynthetic pigments and photosynthetic parameters showed that chlorophyll content and net photosynthetic rate were decreased. Genetic analysis revealed that the mutant phenotype of gw87 was governed by a single recessive nuclear gene. High-throughput sequencing and MutMap analysis suggested that the gw87 locus was located in the middle of chromosome 5, and only one base mutation causing amino acid change of the encoded protein occurred in this chromosome region. Molecular marker linkage analysis demonstrated that the gw87 gene was mapped to a 101 kb genomic region between InDel markers X2 and X3. The MutMap and molecular mapping revealed that the candidate gene was LOC_Os05g32270 that encodes a transcription factor containing the AP2/EREBP DNA-binding domain. Subsequently, we confirmed the candidate gene by sequencing its DNA and cDNA. In the gw87 mutant, a single nucleotide G-to-A substitution occurred at position 1041 in its DNA sequence, and the 76-bp intron sequence adjacent to this mutated nucleotide was spliced into exon in its cDNA, which caused shift of the reading frame and then premature termination of the protein translation. In addition, qRT-PCR analysis showed that the expression of BR synthetic genes were significantly up-regulated in gw87, indicating that BR signal was weakened in the mutant.【Conclusion】gw87 is a new allelic mutant of smos1, shb, rla1, and ngr5. However, unlike these mutants, gw87 exhibited significant increase of grain width and 1000-grain weight relative to its wild type. The reason for the differentiation could be that different mutation sites in the LOC_Os05g32270 gene, resulting in different functional activities of the encoded protein.

Key words: rice (Oryza sativa L.), grain, 1000-grain weight, brassinosteroid, AP2-type transcription factor