【作者】 宋亚玲；

【导师】 熊立仲；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 植物激素研究的成果是人类进行作物遗传改良和育种的重要理论源头,对作物遗传改良和育种的实践起到了很重要的作用。生长素调控很多复杂的生理过程,影响植物的顶端优势,侧根的形成,重力反应,胚胎发生,微管分化,也影响营养器官和生殖器官的生长、成熟和衰老。油菜素内酯是与植物细胞的生长、分裂、分化和生殖有关的多羟基化的类固醇植物激素。在拟南芥中,对这两类激素的信号传递已经有了比较详尽的研究,但是在水稻这种作为全球最重要的粮食作物以及禾谷类作物的模式植物中,研究相对较少。本研究旨在对水稻中分别在生长素和油菜素内酯信号途径发挥重要作用的两个基因家族(OsIAA和OsGSK)进行系统的分析,同时鉴定其中一些成员的功能。主要内容包括三个方面:一是分析OsIAA基因家族在各个发育阶段以及在各种激素和逆境处理下的表达模式;二是系统分析水稻Glycogen shaggy-like kinase基因家族在水稻中的情况,并对OsGSK在各个发育阶段以及各种激素和逆境处理下的表达模式进行了分析。三是对3个OsIAA基因进行了初步的功能分析,较深入的研究了OsIAA1在油菜素内酯和生长素信号中的相互作用的功能。主要结果如下:OsIAA家族分析结果:1.对31个OsIAA家族基因在水稻全生育期的表达分析发现多数OsIAAs是组成型表达的。同时分析了与OsIAA互作的OsARF家族基因的表达谱,发现多数OsARF具有组织特异性的表达模式,并且在幼穗中有比较高的表达。聚类分析发现有8对OsIAA-OsARFs存在类似的表达模式,可能共同参与某一特定发育过程。2.对OsIAAs在不同激素和逆境处理下的表达分析发现,多数OsIAAs不仅受激素诱导同时也受逆境诱导,暗示OsIAAs可能即参与激素应答,也参与逆境反应。3.分析甘露醇处理下DR5-GUS转基因植株根的表型以及DR5-GUS表达模式的变化发现逆境信号(甘露醇处理)引起生长素的再分布,进一步说明逆境和生长素信号传递途径可能存在相互作用。OsIAA基因的功能研究1.通过RT-PCR,OsIAA1-Promoter-GFP,real-time PCR以及芯片数据分析OsIAA1的表达模式,发现该基因是一个组成型表达的基因,并受多种激素的诱导。2.用ubiquitin启动子驱动OsIAA1超表达发现,转基因植株对生长素的反应下降,对油菜素内酯的感应上升。超表达植株出现叶夹角增大,植株变矮的表型,说明OsIAA1可能同时参与了生长素和油菜素内酯的信号传导。3.对OsIAA1的互作蛋白分析发现,OsIAA1与OsARF1互作,分析OsARF1的突变体发现,osarf1对油菜素内酯的反应与超表达OsIAA1的植株相类似,说明OsIAA1与OsARF1共同参与生长素和油菜素内酯的信号传导。4.分析OsIAA1超表达植株中一些生长素和油菜素内酯的应答基因的表达情况发现,转基因植株同时改变了这两种激素应答的基因的表达模式。进一步证明了OsIAA1介导了生长素和油菜素内酯的信号传导。5.超表达OsIAA4表现出分蘖角增大,植株变矮,对生长素反应下降等表型。6.对OsIAA9自身启动子的分析发现,OsIAA9为根和胚芽鞘特异表达基因,在根中表达模式与DR5-GUS相似。超表达OsIAA9表现出对生长素反应下降等表型。对OsGSK家族的分析结果:1.序列分析发现水稻中有9个OsGSK成员,分别定位在第1,3,5,6和9染色体上。蛋白序列分析发现所有成员都含有一个非常保守的激酶结构域。2.对OsGSKs在水稻全生育期的表达分析发现OsGSKs的表达水平都很高,说明该基因家族在水稻的生长发育中发挥着重要作用。3.对OsGSKs在各种激素和逆境的表达谱分析发现,多数OsGSKs参与激素和逆境应答,暗示该基因家族参与多种激素的途径,以及可能在逆境中发挥功能。更多还原

【Abstract】 Rice(Oryza Sativa L.) is one of the most important food crops in the world. Phytohormones play critical roles in plant growth,development and response to environmental stimuli.Auxin regulates arrays of growth and developmental processes including apical dominance,lateral/adventitious root formation,tropisms,fruit set and development,vascular differentiation,and embryogenesis.Brassinosteroid(BR) also regulates a wide range of cellular and development processes such as elongation, senescence,seed germination,reproductive development,and photomorphogenesis.The signaling pathways of these two phytohormones have been intensively studied in Arabidopsis,however,relatively limited information is available in rice.This study aimed to systematically analyze two gene families in rice,OsIAA and OsGSK that plays critical roles in auxin and brassinosteroid signaling pathways respectively,and to characterize the functions of some members of the two families.The research components of this study include three parts:1) sequence analysis and expression profiling of OsIAA gene family in developmental process and in response to phytohormone and stress treatments;2) systematic analysis of glycogen shaggy-like kinase gene family in rice;and 3) characterization of a few members of OsIAA gene family including OsIAA1 gene which was identified with roles in auxin and BR responses and plant morphogenesis.The main results of OsIAA gene family analysis are as follows:1.Expression profiles for the genes of OsIAA and OsARF families have been investigated in the tissues and organs from an entire life cycle of rice.Results indicated that most of OsIAA members exhibited tissue-specific expression patterns while most of OsARF members showed constitutive expression patterns.By hierarchical analysis,eight OsIAA-OsARF co-expression gene pairs were identified. These gene pairs are possibly involved in specific developmental processes.2.Expression pattern analyses of OsLAA family genes(OsIAAs) under different phytohormones and stress treatments showed that most of the OsIAAs were induced not only by phytohormone treatment but also by abiotic stress,indicating possible involvement of OsIAAs in phytohormone and abiotic stress responses.3.Analysis of the DR5-GUS transgenic rice plant revealed that root phenotypic changes induced by mannitol is associated with the expression change of DR5-GUS,which provides direct evidence for the link between abiotic stress and auxin signaling. The main results of functional analysis of OsIAA genes are as follows:1.Expression profiling analyses by RT-PCR,reporter gene(GFP driven by OsIAA1 promoter),and real-time PCR indicated that OsIAA1 is a constitutively expressed gene.2.Over-expression of OsIAA1 in rice resulted in decreased auxin sensitivity and increased BR sensitivity in root growth.Moreover,transgenic plant exhibited dwarfism and loose plant architecture,suggesting an involvement of OsIAA1 in auxin and BR responses.3.Analysis of OsIAA1-interacting protein implied that OsIAA1 could interact with OsARF1.OsARF1 T-DNA insertion mutant(osarf1) showed similar phenotype to that of OsIAA1-overexpression plant,indicating a critical role of OsIAA1 in mediating auxin and BR signal pathway by interacting with OsARF1.4.Overexpression of OsIAA4 resulted in increased tiller angle and dwarfism,and decreased sensitivity to auxin.5.OsIAA9 is expressed only in roots and coleoptiles,and the expression pattern in the roots is similar to DR5-GUS.OsIAA9-overexpressing rice plants also showed decreased sensitivity to auxin.The main results of OsGSK gene family analysis are as follows:1.Nine members of OsGSK family,distributing on chromosome 1,3,5,6 and 9,were identified by sequence analysis.Protein sequence alignment revealed a highly conserved "kinase domain" in OsGSKs.2.High expression level was detected for most OsGSKs at different developmental stages,suggesting that different OsGSKs may play important roles at different developmental stages of rice.3.Expression profile analysis of OsGSKs under different type of phytohormones and abiotic stresses suggested that some members of OsGSK family are also involved in phytohormone and abiotic stress responses.Taken together,this study provided not only plenty of basic information on the expression profiles of OsIAA and OsGSK gene families,but also some evidence on the involvement of these two families in stress responses with crosstalk to auxin and/or BR signaling.更多还原