【作者】 张永华；

【导师】 孙丙耀；

【作者基本信息】 苏州大学 ， 植物学， 2010， 硕士

【摘要】 水稻(Oryza sativa L.)既是世界上最重要的粮食作物之一,也被公认为禾本科植物的分子生物学研究的模式植物。近年来,随着水稻基因组测序工作的基本完成,水稻基因组学研究由结构基因组学时代进入功能基因组学时代。开展水稻重要农艺性状相关基因的定位、克隆和功能分析等方面的研究,对于水稻和其它禾本科植物都具有十分重要的理论意义和应用价值。株高是水稻等禾本科作物的重要农艺性状之一。目前,有关水稻株高分子机制的研究成果,多数是通过对水稻矮秆和半矮秆突变体的基因克隆实现的。在筛选水稻Ds插入突变体的过程中,我们发现一株半矮秆突变体(sdwrky)。与野生型水稻相比,sdwrky突变体表现为种子萌发迟缓约1天、抽穗期提前约5天、叶色深绿、叶角张大、腋芽生长旺盛并形成有效分蘖、株高约为野生型的81%、对赤霉素敏感。突变体与野生型株高的差异来源于节间长度和穗长两个方面的不同。我们采用TAIL-PCR技术,从sdwrky突变体扩增并分离到Ds侧翼序列。以Ds侧翼序列为待查询序列进行NCBI-BLAST在线分析,发现Ds插入于sdwrky突变体4号染色体Os04g0597300(sdwrky)基因,导致sdwrky基因突变。以Os04g0597300(sdwrky)基因编码的氨基酸序列为查询序列,通过在线检索分析Pfam (http://pfam.sanger. ac.uk/); GRAMENE (http://gramene. agrinome.org/), RAP-DB (http://rapdb.dna.affrc.go.jp/), Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/)等数据库,表明sdwrky基因编码一种包含WRKY结构域的蛋白质(SDWRKY),推测SDWRKY可能存在两种机调控水稻种子萌发、幼苗生长以及节间伸长:(1)SDWRKY可能抑制其它WRKY转录因子(如OsWRKY71和OsWRKY51)的功能活性;(2)SDWRKY可能是水稻糊粉层细胞内ABA和GA信号传导途径中共同的抑制因子。在对sdwrky突变体的鉴定过程中,我们发现一个有趣的现象,sdwrky突变体的后代发生遗传分离,即:在467株T2代分离群体中,分离出现145株致死性黄化幼苗突变体(lysrcc1)。遗传分析表明,lysrcc1突变体受一对隐性核基因控制。采用TAIL-PCR技术,分离得到Ds插入位点的侧翼序列。以Ds侧翼序列为待查询序列进行NCBI-BLAST在线分析,发现Ds插入于sdwrky突变体3号染色体Os03g0599600(lysrcc1)基因,导致lysrcc1基因突变。以Os03g0599600(lysrcc1)基因编码的氨基酸序列为查询序列,通过在线检索分析Pfam (http://pfam.sanger. ac.uk/); GRAMENE (http://gramene. agrinome.org/), RAP-DB (http://rapdb.dna.affrc.go.jp/), Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/)等数据库,表明lysrcc1基因编码一个包含RCC1结构域的蛋白质(LYSRCC1)。推测LYSRCC1可能是一种Ran GEF,参与调控和质运输,从而影响叶绿体发育的某一环节。更多还原

【Abstract】 Rice (Oryza sativa L.) is one of the most important food crops for the people in the world, and it has also developed into a model species for molecular biology study within the monocotyledonous grasses. In recent years, with the completion of rice genome sequencing, the current trend in rice genome biology research has evolved from structural genomics to functional genomics. It is extremely meaningful in theoretical and practical respects to carry out mapping, cloning and functional analysis of genes associated with important agronomic traits in rice. Plant height is an important agronomic trait for morphogenesis and grain yield formation in rice and other crops in grass family. Currently, most understanding of molecular mechanism of plant height in rice attributes to the gene cloning from dwarf or semi-dwarf mutants.In this study, a rice semi-dwarf mutant (sdwrky) was isolated from a collection of Ds-inserted mutant lines. sdwrky mutant showed slow seed germination and growth, early heading, semi-dwarfism, black-green leaf color, large angle of leaf to stem, and its axillary buds showed faster growth and some of them were able to develop into tillers. Most significantly, the mutant was found to be susceptible to gibberellin treatment, and its plant height is about 81% of wild-type plant. Further analysis showed that the differences in plant height between the mutant and wild-type plants derived from both the intermodal length and ear length.The Ds-flanking sequences were cloned from the sdwrky mutant by using TAIL-PCR amplification technique. Analysis of the Ds-flanking sequences indicated that Ds element was inserted into the Os04g0597300 gene (sdwrky) of rice chromosome 4 in the sdwrky mutant. Other databases, such as Pfam (http://pfam.sanger. ac.uk/), GRAMENE (http://gramene. agrinome.org/), RAP-DB (http://rapdb.dna.affrc.go.jp/), and Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) searched online to find more molecular information, it was showed that the 04g0597300 (sdwrky) gene encoded a WRKY domain-containing SDWRKY protein. There are two possible mechanisms for SDWRKY to regulate seed germination, seedling growth and elongation: (1) SDWRKY may inhibit the functional activities of other WRKY transcription factors (such as OsWRKY71 and OsWRKY51); (2) SDWRKY may have the similar function of OsWRKY24, which is a negative regulator encoded by a rice WRKY gene represses both ABA and GA signaling in aleurone cells.Interestingly, among the 467 lines examined in the T2 generation, 145 lines showed a lethal yellow seeding mutant (lysrcc1) that segregated as a single recessive locus.The Ds-flanking sequences were cloned from the lysrcc1 mutant by using TAIL-PCR amplification technique. Analysis of the Ds-flanking sequences indicated that Ds element was inserted into the Os03g0599600 gene (lysrcc1) of rice chromosome 3 in the lysrcc1 mutant. Other databases, such as Pfam (http://pfam.sanger. ac.uk/), GRAMENE (http://gramene. agrinome.org/), RAP-DB (http://rapdb.dna.affrc.go.jp/), and Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) searched online to find more molecular information, it was showed that the Os03g0599600 (lysrcc1) gene encoded a RCC1 domain-containing LYSRCC1 protein. LYSRCC1 may be a Ran GEF, involving in regulation of nucleocytoplasmic transport, which regulates chloroplast development.更多还原