水稻矮化少蘖突变体dlt3的基因定位和蛋白质组学分析

目的 株高是农作物的重要农艺性状之一。导致株高变矮的原因很多,最受关注的是赤霉素(GA)和油菜素内酯(BR)对株高的影响,其调控机制的阐明对于植物科学基础研究及遗传育种研究均具有重要意义。方法 利用γ射线诱变水稻材料9311,获得一个矮化少蘖突变体,命名为dlt3 (dwarf and low-tillering 3),通过形态学调查手段分析dlt3突变体的株高、分蘖数、叶夹角、叶形态和结实率等性状,通过叶枕部位的伸直情况和α-淀粉酶活性检测分析其对外源BR和GA应答的敏感性,通过构建遗传群体和筛选分子标记对其进行基因定位,并利用基于iTRAQ的定量蛋白质组学技术分析dlt3突变体的蛋白质组表达谱。结果 表型分析显示dlt3突变体具有半矮化、少分蘖、叶夹角减小、叶表面皱褶、叶形变短变宽和结实率降低等多个突变表型;突变体对GA正常应答,而对BR处理表现为不应答。遗传分析显示dlt3突变因单个基因隐性突变导致;利用分子标记将dlt3基因定位在第6染色体标记RM2615和R6M14之间。定量蛋白质组学分析在dlt3突变体中鉴定到330个差异表达蛋白,包括222个上调和108个下调表达蛋白。其中,4个差异表达蛋白与BR信号途径直接相关;多个差异表达蛋白与水稻株高或生长发育调控直接相关;此外,多个差异表达蛋白,如丝氨酸/苏氨酸蛋白激酶、Ca²⁺结合蛋白和锌指结构域蛋白等在dlt3突变体中大量富集。结论 dlt3是一个BR不敏感的矮化少蘖突变体,DLT3基因突变引起BR信号途径的异常,进而可能导致胞内蛋白磷酸化信号转导和转录激活途径受到广泛影响,从而引起植株生长发育等多方面性状异常。

Genetic Mapping and Proteomic Analysis of the Dwarf and Low-tillering Mutant dlt3 in Rice

【Objective】Plant height is an important agronomic trait for crops because dwarf cultivars are more resistant to lodging. Many factors are responsible for dwarfism in plants, but gibberellin (GA) and brassinosteroid (BR) are the most intensely studied factors, since their molecular mechanism in regulating plant height is important for both basic and applied researches of plant breeding. 【Method】In the present study, we reported the characterization and genetic mapping of a dwarf and low-tillering mutant, dlt3, gained through gamma ray mutagenesis of O. sativa indica 9311. The agronomic traits of the dlt3 mutant including plant height, tiller number, lamina inclination, leaf length and width, and seed-setting rate were analyzed by morphological observation and statistics. The responses to GA and BR were detected based on the changes of leaf angle by exogenous application of BR and GA-induced α-amylase activity. The F2 population and molecular markers were used for genetic mapping of the dlt3 gene. Furthermore, isobaric tags for the relative and absolute quantitation (iTRAQ)-based proteomic method were applied to determine the proteomics of the dlt3 mutant as compared with wild type. 【Result】The plant height, tiller number, leaf angle, leaf length and seed-setting rate were significantly reduced in dlt3. The mutant also showed increased width of leaf blades, dark green and crinkled leaves. The mutant showed normal response to GA, but no response to exogenous BR. Genetic analysis revealed that the dlt3 phenotype was controlled by a single recessive nuclear gene. The dlt3 gene was mapped on the short arm of chromosome 6 between molecular markers RM2615 and R6M14. The iTRAQ-based proteomic analysis revealed that a total of 330 proteins were differentially expressed in the dlt3 mutant, including 222 up-regulated proteins and 108 down-regulated proteins. It showed that four proteins were directly involved in BR signaling pathway and several proteins involved in plant height and developmental regulation were differentially expressed in the mutant. Furthermore, many proteins especially some kinase and phosphatase related proteins, Ca2+ binding related proteins and zinc finger containing proteins were significantly enriched in the mutant. 【Conclusion】These results indicate that the dlt3 is a BR-insensitive dwarf and low-tillering mutant. The DLT3 locus, whose mutation results in abnormal BR signaling, could play important roles in regulating both plant height and other aspects of rice growth and development.