【作者】 张光恒；

【导师】 钱前；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2007， 博士

【摘要】 水稻是世界上最重要的粮食作物之一，叶片形态研究一直备受关注。叶片是植物体进行光合作用的最基本营养器官，大部分叶片形态改变都会影响到植物体的光合作用、蒸腾作用和抗逆性等生理功能，从而极大的影响到植株的生长和发育。为了研究水稻叶形态建成的控制机制和植物叶片结构组成的分子机理，我们从EMS化学诱变粳稻日本晴种子的M₂代中分离了两个水稻叶片极度卷曲的突变体H77、H28。同样，我们从⁶⁰Coγ-射线诱变的籼稻品种双科早处理的M₂代中获得的2份卷叶突变体材料K27和K92。本研究对这些卷叶突变体进行了等位性检测、叶片细胞形态学切片观察，突变性状遗传分析，卷叶控制基因的图位克隆及功能初步研究，主要结果如下：一、水稻卷叶突变体sll1的鉴定、遗传分析及基因精细定位1、等位性检测：对4份卷叶突变体进行正反交，并对F1植株进行表型鉴定。结果表明H77和H28两个卷叶突变体等位。暂时把这些卷叶突变类型称统为葱卷突变（shallot like leaf,sll），分别命名为sll1-1和sll1-2。2、从叶片细胞形态学切片观察和生理生化测定结果显示，SLL1基因的突变造成叶片维管束远轴面厚壁细胞的缺失，叶片叶绿素含量增加和纤维素含量的下降。3、遗传分析：sll1-1与3个正常平展叶籼稻品种NJ6、广四和粳稻品种日本晴作父本分别进行正反交，并对所有的杂交组合的F₁、F₂代的植株叶片表型进行分析。结果表明，在所有组合的F₁的全部植株均为正常平展叶，F₂代分离群体中的叶片正常株与突变株的分离比符合3：1。由此我们认为sil1-1的突变体的表型受隐性单基因控制。4、利用卷叶性状能稳定遗传的sill1-1突变体（粳稻）与籼稻品种南京6号（NJ6）杂交构建定位群体，运用SSR和STS标记，最终将SLL1基因精细定位在第9染色体长臂上物理距离为29.57Kb的区间内，共包含3个开放阅读框，预测分别编码一个合成蛋白、类似En/Spm转座子和MYB蛋白。二、水稻卷叶控制基因SSL1的克隆和初步功能研究1、测序结果表明突变体sill1-1、sll1-2基因组序列都发生了单碱基的替换（G到A）。根据cDNA水平检测发现sill1-1、sll1-2的突变事件分别发生在同一基因内部两个不同的剪接位点上，导致mRNA不能正常剪接。2、明确SLL1基因位于BAC克隆B1040D06的103061-108515的位置，其gDNA长度为5455bp，cDNA全长为1134bp，共编码377个氨基酸。共包含6个外显子（Exon）和5个内含子（Intron）。3、从RT-PCR的结果表明，SLL1基因在整个植株个器官中都有不同程度的表达。其中，叶、花和颖壳中的表达量最高，其次是根、最弱的是茎秆和叶鞘部分。4、我们通过构建植物表达载体SLL1::GUS转化水稻，在SLL1::GUS转基因植株的整个生长发育阶段，所有部位都能检测到GUS活性，但在各组织器官表达范围存在一定差异。在叶片泡状细胞中没有表达，而在维管束、气孔、导管等疏导组织中都有较强表达。在种子幼芽、叶片（包括叶鞘）、茎秆和种子颖壳中表达范围较广，主要在脉络系统中有较强的表达；而在根中只有中柱鞘、籽粒中只用疏导组织和花中只有雄蕊能检测到蓝色。5、通过互补试验，结果发现转入SLL1基因的卷叶突变体sll1叶片表型恢复正常。三、水稻卷叶突变体alm的遗传分析及基因精细定位1、等位性检测结果表明，卷叶突变体K27与K92等位。由于其卷曲程度没有sll1严重，为区别起见，我们将K92、K27突变体（adaxialized leaf mutant）分别命名为alm-1和alm-2。2、从叶片细胞形态学切片观察发现，ALM1基因功能的丧失造成了维管束两侧、靠近叶片近轴面的泡状薄壁细胞的增加，其维管束细胞结构正常。3、alm-1经过连续多代自交种植，确认突变性状能稳定遗传。对alm-1/NPB、alm-1/TN12个组合的F₁和F₂代植株的叶片表型调查，明确alm-1突变性状受1对隐性基因控制。4、通过构建alm-1/TN1定位群体，在F₂群体中共选出1,200多株卷叶表型个体，通过设计SSR和STS分子标记，最终将ALM1基因精细定位在第2染色体端粒附近物理距离为91Kb区域内，用GENSCAN软件预测认为该区域内包含14个开放阅读框（ORF）。更多还原

【Abstract】 Rice is one of the most important crops in the world, and has become the model crop in plant for genetics and genomic studies. Leaf is a major vegetative organ, which plays an important role in photosynthesis, transpiration and resistance to the stress and ultimately affecting development and growth.In the present study, in order to systematically dissect the molecular mechanism of leaf morphogenesis and development, two ethyl methane sulfonate （EMS）-induced rice （Oryza sativa L.） mutants with rolling leaf from the progeny of Nippobare （O. sativa ssp. japonica）, namely H77 and H28, were used. We also isolated another two rolling leaf mutants K27 and K92 from Shuang Kezao （O. sativa ssp. indica） which were induced by ⁶⁰Coγ-ray. We performed anatomical analysis of all these mutants and the genetic analysis; allelic test and mapping caused genes through map-based cloning strategy were also be conducted. The main results were as follows:The identification, genetic analysis and fine mapping of the rice mutant sll11、The allelic test was performed between these four rolling leaf mutants. The identification of the phenotype of the F₁ plants that was derived from the reciprocal cross between these mutants mutually, showed that they were controlled by the same gene, which were designated as sll1-1 （shallot like leaf1-1, sll-l） and sil1-2.2、Through the anatomical and histological analysis, we have draw the conclusion that the mutation of gene SLL1, which lead to the increase of chlorophyll and reduction of the cellulose contents in rice leaf, caused the absence of the prothenchyma in the abaxial vascular bundle.3、Genetic analysis: we have constructed four segregated populations from the cross between sll1-1 and three other parents NJ6, GuangSi and nipponbare with normal leaf, and the analysis on the phenotype of the F₁ and F₂ was performed. The results showed that all the F₁ plants had normal leaf and the ratio of normal rice plants to mutant rice plants tallied with 3:1 rule. Therefore, the phenotype of the mutant sill1-1 was controlled by single recessive gene.4、The progeny from the cross between sll1-1 and a O. sativa ssp. indica parent NJ6 were used as the mapping population. The caused gene SLL1 was finely located into a region of 29.57Kb on the long arm of chromosome 9, which had 3 ORF and were presumed to be a synthetic enzyme, alike En/Spm transposon and MYB-domain containing protein, respectively, according to the annotation.The isolation of SLL1 and the gene function analysis1.The results of sequence analysis showed that there is a single substitution in the sequence of mutant Sll-1 or sll-2 （G to A）. Amplification of the cDNA showed that the substitution occurred at different splice sites of the same gene, which lead to the abnormal splice.2. The SLL1 gene was located at 103061-108515 of the BAC clone, and the length of gDNA was 5455 bp. The analysis of gene structure and the amplification of the cDNA showed the length of FL-cDNA was 1134bp, with 6 exons and 5 introns, which coding a 377 amino acids protein.3. The results of RT-PCR showed that the SLL1 gene expressed to different extents in the different organs of the whole plants, which expressed highly in flower, leaf and glume, then inferiorly at root and poorly at stem and sheath.4. The transformation of SLL1::GUS was conducted. All the organ were detected to have different GUS activity in all the growth and development period, and the high level expression was detected in vascular bundle, stoma and conduit in contrast to no expression in vesicular cell. The GUS expression was detected in yang plant, leaf, stem and glume of seed, and highly at venation system, but poorly at rooting and flower which only the culm sheath, kernel and stamen could be stained.5. The result of complement test showed that SLL1 gene could recover the leaf phenotype of the mutants.The genetic analysis and fine mapping of ALM11、Allelic test showed the mutant K27 and K92 which exhibits a phenotype of rolling leaf was controlled by the same allele. Due to the gentle phenotype when compared with slll, we nominated K92、K27as alm-1（adaxialized leaf mutant-1） and alm-2, repectively.2、Anatomical and histological analysis showed that the lose of function of ALM1 caused the increases of the cell number of vesicle parenchymatous cell, though the cells in vascular bundle were normal.3、Investigation on the phenotype of the cross of alm-1/NPB、alm-1/TN1 showed that the mutant was controlled by one recessive gene.4、The population for gene mapping was established from alm-1/TN1, and about 1,200 plants with mutant phenotype were selected in the F₂ generation. The gene was finely located in 91 Kb on the chromosome 2, which was forecasted to have fourteen ORF by GENSCAN software.更多还原