【作者】 马琳；

【导师】 李刚；

【作者基本信息】 山东农业大学 ， 植物学， 2015， 硕士

【摘要】 光是控制植物生长发育最重要也是最基本的环境因素之一。不仅为植物的光合作用提供能量,同时也可作为信号调控植物的生长发育。高等植物在进化过程中已形成一套由多个光受体组成的网络,用于感受环境中时刻变化的光信号。不同光受体感受光信号变化后,蛋白构象或定位发生改变,并通过与其相互作用的信号蛋白,把外界环境的光信号变化传递到体内下游信号转导途径中,从而影响着植物的生长发育,促使植物更好的适应外界环境。FHY3 (Far-red elongated hypocotyl 3)和FAR1 (Far-red impaired response 1)是远红光信号转导途径中的两个重要组分(Wang and Deng,2002),是一类从古老的Mutator like转座酶衍变而来的新型转录因子,以同源或异源二聚体的形式特异结合在下游靶基因的FBS (FHY3/FAR1 DNA Binding sites, FBS)顺式调控元件上调控基因表达(Lin et al.,2007)。已有文章报道,FHY3/FAR1参与幼苗光形态建成、开花调节以及细胞器发育等生长发育过程。本研究以拟南芥中fhy3far1双突变体细胞死亡表型为出发点,通过分子生物学与反向遗传学的方法部分解析了FHY3/FAR1调控细胞死亡的分子机理。主要结果如下所示：(1)表型观察发现fhy3far1双突变体在短日照条件下具有严重细胞死亡表型,推测FHY3/FAR1是细胞死亡过程的负向调控因子。进一步通过DAB及NBT染色,fhy3far1双突变体体内ROS大量积累；且外源施加MV处理,FHY3功能缺失突变体更加敏感,证明FHY3/FAR1提高植物对外界氧化胁迫的适应性。(2)通过在fhy3far1双突变体背景下组成型表达SA分解相关基因S3H (SA 3-hydroxylase),可明显缓解fhy3far1双突变体短日照条件下的细胞死亡表型,表明fhy3far1的细胞死亡表型依赖SA积累。(3) FHY3/FAR1是远红光信号途径中重要的转录因子,可通过调控多个下游靶基因的表达介导植物生长发育各个过程。通过FHY3蛋白的ChIP-seq及短日照条件下fhy3far1双突变体基因芯片结果分析,对比发现25个FHY3候选下游靶基因。基于mips1具有与fhy3far1双突变体相类似的细胞死亡表型,最终推测肌醇合成途径关键酶基因MIPS1可能介导FHY3调控的氧化胁迫响应。通过研究FHY3对MIPS1的DNA启动子结合及fhy3far1中MIPS1的基因表达量检测,证明FHY3直接正向调控MIPS1的基因表达。另外,fhy3farl双突变体中,肌醇含量降低,而组成型表达MIPS1, fhy3farl双突变体中肌醇含量得到部分恢复,表明光信号蛋白FHY3通过直接激活MIPS1的基因表达促进植物体内肌醇合成。(4)FHY3对维持肌醇合成途径关键基因MIPS1的转录水平至关重要。为进一步研究FHY3对肌醇合成的重要性,通过RT-PCR检测发现肌醇代谢途径中多个相关基因如MIK、MIOX2、VTC4以及INT1的表达也受到FHY3的调控,推测FHY3介导整个肌醇代谢途径。(5)肌醇途径是抗坏血酸合成途径之一,而VTC4作为一个双功能酶可同时参与肌醇与抗坏血酸合成。通过RT-PCR检测fhy3far1双突变体中抗坏血酸合成相关基因VTC1、VTC2、VTC3、VTC4以及VTC5的表达量,结果表明FHY3正向调控VTC1-5的基因表达。通过检测抗坏血酸含量,FHY3功能缺失突变体中抗坏血酸含量明显降低,而fhy3互补株系中抗坏血酸含量增加,表明FHY3通过间接调控VTC类相关基因的表达介导植物体内抗坏血酸合成。(6)肌醇代谢对维持植物正常生长发育,抵御外界氧化胁迫是必不可少的。为探究肌醇代谢对fhy3far1双突变体生长发育的作用。通过将MIPS1-OX/fhy3far1转强光处理,结果表明MIPS1过量表达缓解fhy3far1双突变体由胁迫引起的提前衰老,证明FHY3通过促进肌醇合成以此来提高植物对外界氧化胁迫的抗逆性。综上所述,我们的研究证实细胞死亡过程负向调控子FHY3/FAR1通过直接激活MIPS1/2的转录水平,促进植物体内光调控的肌醇合成,并最终提高植物对外界氧化胁迫的适应性。为进一步解析植物抗逆分子机制奠定了科研基础和理论依据。更多还原

【Abstract】 Light is one of the most important and basic environment cues affecting plant growth and development. Light acts not only as a source of energy for photosynthesis, but also a signal to regulate plant growth and development. Light signals are percepted by a series of photoreceptors in higher plants. When photoreceptors recieved the light changes of external environment, the protein conformation and location of photoreceptors was changed, and transmit the light signal to the downstream pathways by interaction with other signal proteins to regulate plant growth and deveiopment and acclimatize the environment exchange.FHY3 and FAR1 are two important transcription factors involved in phyA signaling, derived from a kind of Mutator-like transposase. They bind the FBS cis-elements by homogenous or heterogenous dimers to control the expression of target genes. Previous studies have proved that FHY3/FAR1 involved in the photomorphogenesis of seedlings, flowering time regulation and organelles development. Here, based on the phenotype of cell death on fhy3far 1, we parsed the molecular mechanism on FHY3 regulating cell death using molecular biology and reverse genetics method.(1) According to the different levels of cell death phenotypes under short-day condition, FHY3/FAR1 are the negative regulators of cell death. Based on the DAB and NBT staining, ROS is highly accumulated in fhy3farl double mutant and fhy3farl is more sensitive to exogenous MV treatment. we vertify that FHY3/FAR1 are important to acclimatize the oxidative stress.(2) Because of constitutive expression of S3H under fhy3farl double mutant easing the cell death under SD condition obviously, then we confirmed that the cell death of fhy3far1 is dependent on the accumulation of SA.(3) FHY3/FAR1 are the important transcription factors in phyA signaling, and they participate in controlling multiple genes to regulate plant growth and development. According to the ChIP-seq of FHY3 and gene chip on fhy3farl double mutant, we blasted 25 downstream target genes and finally because of the similar cell death phenotype between fhy3farl and mipsl, we confirmed myo-inositol 1-phosphate synthase 1(MIPS1) as candidate gene. Eventurally, we proved that FHY3 activates the expression of MIPS1 directly, and over-expression of MIPS1 rescues the inositol level of fhy3farl. So we proved that FHY3/FAR1 is essential to the de novo of myo-inositol.(4) FHY3 is impotant to the transcriptional level of MIPS1. To further investigate the importance of FHY3 to the synthesis of inositol, we detect the expression of multiple genes associated with the synthesis of inositol. For example, the expression of MIK, MIOX2, VTC4 and INT1 are changed remarkably in fhy3farl. The result indicated that FHY3 mediate the whole mechanism of inositol.(5) Inositol pathway is one of the ways to synthesize ascorbic acid (AsA) and VTC4 as a bi-functional enzyme involved in the synthesis of inositol and ascorbic acid at the same time. By RT-PCR, the expression of VTC1-5 and the level of AsA decreased in fhy3farl, and the content of AsA was improved in the complementary stain of fhy3. At last, we confirmed that FHY3 facilitated the synthesis of AsA by regulating positively the expression of VTC indirectly.(6) Inositol is essential to maintain normal growth and development of plants and resist the oxidative stress. To study the function of inositol to fhy3farl double mutant, using high light treatment we proved that MIPS1-OX1fhy3farl not only improved the level of inositol, but also released the premature senescence of fhy3farl double mutant under high light condition.All in all, our date indicated FHY3/FAR1 are negative regulators of cell death, and FHY3/FAR1 directly activate the gene expression of MIPS1/2 to promote the biosynthesis of inositol and strengthen the adaptation of oxidative stress in plants finally. Establish the basis and evidiences to further resolve the molecular mechanism of plant resistance to stress.更多还原