【作者】 马小定；

【导师】 翟虎渠； 万建民；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2012， 博士

【摘要】 水稻等禾本科植物具有与双子叶植物不同的花器官结构，典型的双子叶植物花器官从外到内依次为花萼、花瓣、雄蕊和雌蕊，水稻雄蕊和雌蕊与单子叶植物相似，而外层的内外稃和浆片是禾本科植物特有的。目前，关于水稻内外稃起源和发育的分子机理还知之甚少。本研究以从T-DNA插入突变体库和自发突变体库中获得的具有鹰嘴（beak like spikelet，bls1）表型的水稻内外稃发育突变体bls1-1与bls1-2为材料，通过图位克隆和基因功能分析，揭示了引起鹰嘴表型的遗传与分子机理，加深了我们对水稻内外稃发育分子机制的认识。主要结果如下：1. bls1突变体内外稃变小，特别是内外稃上部明显变窄、变尖，整朵小花形成类似鹰嘴的形状，而其它花器官，包括退化颖片、护颖及内部浆片、雄蕊和雌蕊的形态正常，没有发生花器官同源转化现象。石蜡切片观察发现，突变体内外稃从外到内四种类型细胞（硅化细胞、纤维状厚壁组织、薄壁细胞、未硅化细胞）都明显变小，统计未硅化细胞数目和细胞面积时发现，突变体内外稃细胞面积显著减小。扫描电镜观察发现，突变体花器官原基的起始与花器官轮生模式的形成过程正常，只是在胚珠和花粉形成期（Sp8期）内外稃不膨大，横向生长受到抑制。2.通过Real-time PCR分析，发现内外稃发育相关的花器官特征基因，包括AP1-like基因、SEP-like基因、AGL6-like基因，在突变体中的表达不受影响。由此推测，BLS1基因可能位于这些花器官特征基因下游，与突变体bls1没有发生同源转化的表型结果一致。此外，其它内外稃发育相关基因，包括REP1、DP1、DL基因在突变体中正常也表达，BLS1可能是从新的途径调控内外稃的发育。3.遗传分析表明，鹰嘴表型由一对隐性核基因控制。以bls1-1×Dular杂交组合获得的定位群体，将bls1定位在第2染色体长臂约110kb的区间内，在该区间内，bls1-1突变体大约有87kb的染色体片段缺失。再利用bls1-1等位突变体bls1-2与培矮64配置的群体进行精细定位，最终将bls1位点定位在大约65kb的区间内，bls1-2突变体在定位区间内也存在50kb染色体片段缺失。定位区间内包括7个基因，经过测序和RT-PCR表达分析发现，只有LOC_Os02g56610能够在水稻幼穗中表达，将该基因确定为候选基因BLS1。互补实验和干扰实验结果证明bls1突变体的鹰嘴表型是由BLS1基因缺失造成的。4. BLS1基因编码287个氨基酸，包含一个DUF640结构域。通过数据库搜索发现，DUF640结构域可能是陆生植物特有的。转基因植株GUS组织染色结果表明，GUS活性在幼穗中最强，特别是幼穗的内外稃，该结果与Real-time PCR基因组织特异性表达分析结果一致。瞬时表达和转基因结果都表明，BLS1蛋白定位在细胞核上。更多还原

【Abstract】 Grass species, like rice (Oryza sativa L), have highly specialized flowers that are different fromthose of eudicots. A typical eudicot flower comprises of sepals, petals, stamens, and carpel from outer toinner, whereas rice flower consists of stamens and carpel like eudicots, but the surroundingstructures, lodicules, lemma and palea, are unique to grasses. At present, the origin and molecularmechanism of lemma and palea development are poorly understood. In this study, we characterized tworice mutants, bls1-1and bls1-2(beak like spikelet1) with deformed lemmas and paleas, were obtainedby screening a T-DNA insertion population and spontaneous mutant population, respectively.Map-based cloning and functional analysis of the bls1mutant revealed the genetic and molecularmechanism that caused the mutant phenotype of bls1. These results enhanced our understanding of themolecular mechanisms of lemma and palea development in rice spikelet. Results from our experimentare summarized as follows:1. bls1lemma and palea exhibited slender phenotype, especially at the top where it displayed abeak like structure. However, the morphology of other floral organs, including rudimentary glumes,sterile lemmas, lodicules, stamens and pistils was normal. This suggested that the alterations in themutant were due to shape changes, rather than homeotic transformation of the lemma and palea. In ahistological analysis, the bls1cell sizes of four types of the lemma and palea, including silicified cell,fibrous sclerenchyma, spongy parenchymatous cell and nonsilicified cell were obviously smaller thanthose of WT. With the statistic analysis of the innermost nonsilicified cell in lemma and palea, there wasno difference in the cell number between bls1and WT, while the average cell area reduced significantlyin bls1. Scanning electron microscope analysis showed that the floral-organ primordia initiation andfloral organ patterning of bls1mutant were normal. By Sp8with formation of ovule and pollen, thelemma and palea of WT started to develop laterally, whereas the developmental process of bls1seemedto be delayed and the lemma and palea were prevented from growing horizontally.2. Real-time PCR analysis revealed that the expression of several floral organ identity genes relatedlemma and palea, including AP1-like, SEP-like and AGL6-like gene were apparently not affected in bls1mutant. In addition, in bls1, palea-related genes REP1and DP1or lemma-related gene DL displayedsimilar expression level as in WT. No significant alterations in these genes expression suggested thateither BLS1functions as the downstream of genes measured or BLS1represents a new pathway relatedto lemma and palea development.3. Genetic analysis revealed that bls1phenotype is controlled by a single recessive nuclear locus.Through mapping the F2population produced by bls1-1crossing Dular, the bls1locus was mappedwithin a region of about110kb on the long arm of chromosome2. A chromosome segment ofapproximately87kb was deleted within the restricted region in bls1-1. Then we used another mutantbls1-2, which is an allele mutant with bsl1-1to cross with Peiai64, and refined the bls1locus to a65kbregion. There was also an approximately50kb deletion within the restricted region in bls1-2. Sevenputative genes were including, among them, mere LOC_Os02g56610expressed in the young inflorescence of WT. Complement test and RNA interference experiment confirmed that the beak likespikelet of bls1mutant was caused by the deletion of gene LOC_Os02g56610, which was subsequentlydesignated as BLS1.4. BLS1encodes a protein containing287amino acids with a conserved DUF640domain withunknown function. A GenBank database search suggested that DUF640domain may be specific to landplants. Histochemical staining of the transgenic plants showed that GUS activity was strong in theyoung inflorescence, specifically the young lemmas and paleas of spikelets, corresponding to the resultsrevealed by Real-time PCR analysis. Both the results of transiently expression in rice leaf protoplastsand transgenic rice revealed that BLS1localizes in the nucleus.更多还原