水稻卷叶突变体rl15(t)的生理学分析及基因定位

【目的】对环境诱导卷叶突变体开展生理学特性分析，并对候选的突变基因开展初步定位，为下一步的基因克隆与功能分析提供研究基础。【方法】用60Coγ射线诱变粳稻品种日本晴（Nipponbare）种子，发现了一份叶片在晴朗天的正午时分高度内卷的突变体，命名为rl15(t)（rolled leaf 15）。通过田间种植鉴定，对该突变体进行表型观察及主要农艺性状调查。采用不同温度和相对湿度处理rl15(t)和野生型，以揭示影响突变体叶片卷曲的环境因素。试验设置3个处理温度（24℃、29℃、34℃）和2个相对湿度（RH=60%或95%），在人工气候箱处理抽穗期的rl15(t)和野生型，以处理1.5 h后的剑叶测定叶片卷曲度（RLI）。自清晨6:00时至下午18:00时，每隔2 h用便携式气体交换系统Li-6400测定rl15(t)和野生型剑叶的净光合速率（Pn）、蒸腾速率（Tr）和气孔导度（Gs）等指标，同时用WP4露点水势仪测定剑叶的叶片水势，分析并比较突变体和野生型的上述生理表现的异同。将rl15(t)与野生型日本晴杂交，观察F1植株和F2群体的叶片表型，对F2表型分离进行χ2测验，分析突变体的遗传行为。以rl15(t)×珍汕97B的F2群体为材料，利用BSA法对候选基因进行定位。【结果】与野生型亲本日本晴相比，rl15(t)突变体植株变矮、分蘖减少、穗长变短、籽粒变小、生育期延迟；rl15(t)突变体叶片短窄且在阴雨天气或晴天的清晨和黄昏时表现为正常的平展或轻微内卷，但在晴朗天的正午时分表现高度内卷。温度和湿度梯度处理试验表明，rl15(t)突变体叶片卷曲行为受环境诱导，湿度是诱导突变体叶片卷曲的主要因素，高温可促进该表型的表现。rl15(t)突变体剑叶的净光合速率、蒸腾速率和气孔导度等光合参数以及叶片水势在清晨和黄昏同野生型亲本较接近，但在正午时分均显著低于野生型；而rl15(t)突变体剑叶的水分利用效率（WUE）在清晨、正午时分和黄昏与野生型接近，但在其他时段显著高于野生型。rl15(t)与野生型亲本日本晴的F1表现叶片正常的平展，F2群体中平展叶与卷叶表型株符合3﹕1分离比，表明rl15(t)突变体的卷叶突变性状受1对隐性核基因控制。RL15(t)初步定位于水稻第10染色体长臂端SSR标记RM25302和RM25343之间，与两标记的遗传距离分别为0.8和2.0 cM。【结论】突变体rl15(t)的卷叶表型是受环境诱导的，候选基因定位于SSR标记RM25302和RM25343之间，该区段内未见同类表型基因的报道，推测RL15(t)可能是一个新的卷叶调控基因。

Molecular Mapping and Physiological Characterization of a Novel Mutant rl15(t) in Rice

【Objective】Phenotypic and physiological characteristics of an environmentally induced rolled leaf mutant were studied in present paper. Meanwhile, the mutant gene was preliminarily mapped on rice chromosome. 【Method】A rolled leaf mutant, named rl15 (t) (rolled leaf 15), was obtained by 60Coγ-ray mutagenesis from japonica cv. Nipponbare. By field identification, the phenotypes and main agronomic traits of the mutant were investigated. Different temperature and relative humidity treatments could reveal the environment factors that affect leaves rolling in mutant. Both of rl15(t) and wild type at heading stage were divided into 6 groups and were treated with temperature at 24℃, 29℃ and 34℃, and relative humidity at 60% and 95%. After treatment for 1.5 h, flag leaves were used to measure leaf rolling index (LRI). From 6:00 AM to 18:00 PM, the photosynthesis rate (Pn), transpiration rate (Tr), stamatal conductance (Gs) of flag leaves in rl15(t) and wild type were measured at 2-hour intervals by using the portable gas exchange system Li-6400, meanwhile, leaf water potential was measured by WP4 dewpoint potential meter. These physiological characteristics were analyzed and compared between rl15(t) and wild type. The rl15(t) mutant was crossed with the wild type Nipponbare, leaf phenotype of the F1 progeny and segregation ratio of flat leaf plants and rolled leaf plants in F2 population were investigated. On the basis of BSA method according to Michelmore et al., preliminarily mapping of the candidate mutant gene were conducted using a F2 population derived from rl15(t) crossed with indica line Zhenshan97B.【Result】Compared with the wild type, the mutant plants have shortened plant height, reduced tiller numbers, shorter panicle, smaller grains, delayed heading duration, shorter and narrowed leaves. All of the leaves in rl15(t) were observed to highly inward roll at midday hours under sunny conditions, whereas were flat or slightly inward rolled under rainy conditions or at early morning and sunset under sunny conditions. Experiments of different temperature and relative humidity treatments showed that leaf rolling index in rl15(t) mutant were mainly depended on air humidity and could be promoted by high temperature. The photosynthesis rate, transpiration rate, stamatal conductance and leaf water potential of flag leaves in mutant were extremely lower than those in the wild type at midday hours under sunny conditions. However, the instantaneous water use efficiency (WUE) was similar to that of wild type at 6:00, 12:00, 18:00 o’clock, whereas in other time of a day, WUE was dramatically higher than that of the wild type. F1 plants derived from crossing rl15(t) with wild Nipponbare showed normal flat leaves, The segregation ratio of flat leaf plants to rolled leaf plants in F2 population was consistent with the inheritance of single recessive nuclear locus. Further molecular genetics studies revealed that RL15(t) was mapped on the long arm of rice chromosome 10 between SSR markers RM25302 and RM25343, with genetic distances of 0.8 cM and 2.0 cM, respectively. 【Conclusion】Mutant rl15 (t) was environmentally induced rolled leaf in phenotype. RL15(t) gene is located between SSR markers RM25302 and RM25343. In these distance segment, it hasn’t any similar phenotype genes reported up to now. So, RL15(t) gene would be a putative novel rolled leaf gene.