【作者】 邹良平；

【导师】 路铁刚；

【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】 水稻是我国的主要粮食作物。由于人口的增加和可耕地面积的减少，稳定和提高水稻总产量只有依靠单产的提高，而水稻株型改良对提高单产具有重要的作用。在水稻株型改良中特别注重叶片的直立性和叶片的适度卷曲。叶片适度卷曲能保持叶片直立不披垂，改善水稻生长中后期群体基部的受光条件，提高光能利用率，从而有利于水稻产量的提高，其作用已在高产和超高产育种中得到充分的体现。因此，对水稻卷叶突变体的发现、控制相应性状基因的挖掘并将其应用到理想株型的改良越来越受到遗传育种家的重视和关注。但是，利用传统遗传育种方法将卷叶性状转移到没有该性状的主栽品种中去存在着周期长，且有时还不能选育到理想性状的缺点。随着现代分子生物学技术的崛起和进步，克隆和研究控制水稻卷叶性状的基因，希望在分子水平上通过人为操作构建符合遗传育种家理想株型的水稻就成为了很多国内外分子生物学家追求的目标。现在揭示水稻基因功能的一个通用策略就是构建其T-DNA插入突变体库。本实验室通过6年的努力，建立了一个大容量的T-DNA插入突变体库，并且克隆了它们大部分的插入位点侧翼序列，为克隆功能基因奠定了良好的基础。为了研究水稻卷叶突变体，我们从增强子捕获水稻突变体库中筛选得到了300多份具有不同卷叶性状的独立转基因株系。次年，我们将种植于廊坊试验田中的所有卷叶突变体成熟叶片进行石蜡切片观察，从中克隆了一个控制水稻叶片反卷的基因并对其功能进行了详细的研究。具体研究结果如下：一、卷叶性状的细胞学分类根据观察结果我们将卷叶性状从细胞学上分成8种类型：1、泡状细胞的变化。具体有3种表型，第I类型是泡状细胞数量和面积增加可以导致叶片的反卷；第II类型是泡状细胞数量和面积减少能够促使叶片发生正卷；第III类型的泡状细胞数量和面积变化无规律，叶片是正卷还是反卷由泡状细胞最终的大小决定。2、部分厚壁细胞缺失（第IV类型）。厚壁细胞对维持水稻叶片正常的形态结构具有重要作用，叶片远轴面厚壁细胞缺失会丧失其所提供的支撑力而导致叶片正卷，反之，则可能促使叶片反卷。3、位于维管束中的韧皮部变化也能导致叶片的卷曲（第V类型）。韧皮部细胞增加，面积增大，从而使叶脉远轴面皱缩，进而导致叶片反卷。4、第VI类型是维管束鞘延伸区和主脉薄壁细胞数量改变促使叶片发生卷曲。5、第VII类型是叶片中的多种细胞类型同时发生变化，叶片最终的卷曲方向和程度由这些细胞共同决定。6、部分叶肉细胞变成了薄壁样细胞也能改变叶片的形态（第VIII类型）。二、OUL1基因的克隆及功能分析1、oul1突变体表型及OUL1基因的克隆。我们挑选细胞学典型的卷叶突变体，根据已克隆的插入位点序列信息设计相应的特异性引物进行共分离验证，最后确定了一个泡状细胞增加、叶片反卷成接近筒状的突变体oul1的T-DNA插入与表型共分离。将该侧翼序列在NCBI中BLAST出其全长序列，基因注释为Rice outermost cell-specific gene5(Roc5)基因，属于HD-ZipIV家族成员。2、功能互补验证证明了oul1叶片发卷是由于Roc5基因功能缺失所致。将Roc5基因cDNA全长序列转入到oul1突变体所诱导的愈伤中得到的植株叶片表现为平展，由此初步验证了oul1叶片卷曲可能是Roc5由于T-DNA的插入，破坏了其功能所致。并且亚细胞定位表明Roc5编码产物为一个转录因子。3、Roc5过量表达使叶片泡状细胞减少，从而发生正卷；而在Roc5的共抑制水稻植株中发现的叶片与oul1突变体的叶片表型相似。由此更进一步证明了Roc5负调控水稻叶片泡状细胞的命运和发育。4、基因表达谱以及RNA干涉（RAN interference, RNAi）分析结果表明，Protodermal FactorLike（PFL）基因可能是Roc5下游直接作用的位点，但具体的直接相互作用还需要进一步的研究证明。更多还原

【Abstract】 Rice is a leading cereal crop and main staple food for over half of the world population. Withaccretion of population and arable land decrease, steady increase in rice production only depends onincreasing per unit area yield. Nevertheless, improvement of plant type in rice plays an important rolein it. Leaf shape has long been considered an important agronomic trait in rice. Moderate leaf rollingin rice leads to erect leaf canopies and higher photosynthetic efficiency, improving stress response byreducing transpirational water loss and radiant heat absorption, thereby increasing grain yield.Therefore, moderate leaf rolling is an ideal trait for rice breeding.It is possible that leaf rollingphenotypes are resulted from changes of any cell types making up leaf blade. During screening ofrice T-DNA insertion lines, more than300stable mutants showing various leaf rolling phenotypeswere obtained. Cytological analysis of those mutants indicated that changes of number, size andpattern of bulliform cells, sclerenchymatous cells, parenchyma cells, mesophyll cells as well asvascular bundle could lead to leaf rolling. Those mutants have been categorized into8groupsaccording to their cytological characteristics: increased bulliform cell number and area, decreasedbulliform cell number and area, disordered bulliform cell number and area, partial sclerenchymatouscell absence, phloem expansion of vascular system, increased parenchyma cell, a few cell typeschange, partial mesophyll cells become parenchyma cell-like.To understand the molecular mechanism controlling leaf rolling, we screened a rice T-DNAinsertion population and isolated the outcurved leaf1(oul1) mutant showing abaxial leaf rolling. Thephenotypes were caused by knockout of Roc5, an ortholog of the Arabidopsis homeodomain leucinezipper (HD-Zip) class IV gene GLABRA2. Interestingly, overexpression of Roc5led to adaxiallyrolled leaves, whereas cosuppression of Roc5resulted in abaxial leaf rolling. Bulliform cell numberand size increased in oul1and Roc5cosuppression plants but were reduced in Roc5-overexpressinglines. The data indicate that Roc5negatively regulates bulliform cell fate and development. Geneexpression profiling, quantitative PCR, and RNA interference analyses revealed that ProtodermalFactor Like was probably downregulated in oul1. The mRNA level of PFL was increased inRoc5-overexpressing lines, and PFL-RNAi transgenic plants exhibit reversely rolling leaves byreason of increase of bulliform cell number and size, indicating that Roc5may have a conservedfunction. These are the first functional data for a gene encoding an HD-Zip IV transcriptional factorin rice that modulates leaf rolling.更多还原