【作者】 翁小煜；

【导师】 张启发；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 植物通过感知复杂的环境信号,从而调整各种生长发育的过程,最终完成物种的存活和延续。而在水稻中,这种因为环境信号而产生的生长发育过程的改变往往导致了重要农艺性状的巨大变化,最终深刻的影响到水稻产量。因此研究水稻如何响应环境变化调节生长发育同时具有理论和应用的意义。该课题对水稻第七染色体着丝粒附近一个同时控制抽穗期、株高和穗粒数的主效QTL基因Ghd7进行克隆和功能研究,首次在水稻中成功克隆到Ghd7这样一个一因多效的重要农艺性状基因。利用遗传学和分子生物学等手段初步阐明Ghd7在水稻中处于信号枢纽的中心地位,通过整合多种环境信号(光质,日长,温度,胁迫等),调节自身转录和蛋白水平,最终达到控制水稻时期转换,株型建成和胁迫反应等过程的目的。本研究主要结果如下：1.多效基因Ghd7的克隆利用RACE的方法分离到Ghd7的全长cDNA,发现Ghd7为一个新的CCT结构域基因,与拟南芥中已发表的CCT基因不具有显著的同源关系。亚细胞定位结果表明Ghd7定位于细胞核,并且具有转录抑制的活性。表达谱分析显示,Ghd7主要在叶片表达,并且从叶尖向叶基梯度递减。同时该基因又受到光周期和昼夜节律的调控,即特异在长日照条件下的白天表达,表达峰值处于黎明时分。长日照条件下,Ghd7的表达直接导致对水稻抽穗期基因的强烈抑制,从而导致抽穗延迟的表型。Ghd7-Ehd1-Hd3a代表了水稻中独有的开花调控途径,并且Ghd7-Ehd1-Hd3a在表达量上具有一种级联放大的特性。通过对五个品种中Ghd7位点的比较测序,鉴定出四种Ghd7的等位基因型。其中HR5和TQ在Ghd7位点分别具有强等位基因型Ghd7-1和Ghd7-3; ZH11在Ghd7位点包含弱等位基因型Ghd7-2；而MDJ8和HJ19则在Ghd7位点发生终止突变,定义为Ghd7-0a。此外我们还发现不同的Ghd7等位基因在3’UTR区域存在AT重复序列的多态性。以上结果说明Ghd7基因参与了水稻自然变异和选择育种的过程,在增加水稻产量潜力和适应性方面具有重要作用。2.Ghd7在水稻中的功能遗传结果表明,Ghd7的表达量的高低与抽穗期,株高和穗粒数的表型程度呈正相关,说明Ghd7存在剂量效应。然而在ZH11背景中,超量表达Ghd7虽然能够显著延迟抽穗期,但是对株高和穗粒数的促进作用只在短日照条件下才能显著的表现出来,说明Ghd7对穗型和株高的调控依赖于遗传背景和环境的变化。遗传和分子的结果表明Ghd7介导PHYB-OsTBl的途径调控分蘖,其中GHD7蛋白含量在phyB突变体中显著降低。此外ABA、JA、干旱以及高温均可以抑制Ghd7的表达量,而低温则能够促进Ghd7的表达。同时我们发现超量表达Ghd7和抑制Ghd7分别能够增强和降低水稻幼苗对于旱胁迫的敏感性。说明Ghd7在水稻中处于信号传导的中心枢纽,整合来自环境的各种信号,参与水稻的时期转换,株型调控以及胁迫反应。3.Ghd7分子机制的研究为了研究Ghd7的下游途径,我们利用RNA-seq的策略,在黎明、正午、黄昏和午夜四个时间点,对phyB突变体、Ghd7超量表达和抑制的材料进行差异表达基因分析,得到PHYB和Ghd7在一天中不同时间点的调控基因。为了研究Ghd7的互作蛋白,我们将GHD7与3×Flag标签融合并在转化,通过gel filtration的方法,发现GHD7在体内存在440-kDa左右的复合体形式。同时,Ghd7可以和HAP家族的多个亚家族成员发生蛋白互作,而和Ghd7互作的部分HAP成员又可以和生物钟基因OsTOCl发生蛋白互作,说明这些HAP成员联接了外源信号和内源生物钟。最后我们发现HAP2亚家族和生物钟基因的表达量均受到PHYB的调控。以上结果初步表明Ghd7, HAP家族和生物钟三者之间可能通过蛋白互作的方式形成一个缓冲系统,调节水稻的鲁棒性(robustness)、而PHYB则通过多种方式对其进行调控,使得水稻能够响应复杂多变的环境。4.Ghd7参与激素调控我们对Ghd7和激素的关系进行了详细分析：1)超量表达Ghd7增加了内源ABA的含量,增强了植株对ABA的敏感性,同时延迟了种子的萌发；2)Ghd7改变了顶端生长素极性运输基因OsPIN1的表达,超量表达Ghd7抑制了内源IAA的含量；3)Ghd7特异在黄昏时分调节GA2ox基因的表达。而在超量表达Ghd7的背景下抑制内源GA合成酶GA20ox1,植株仍然表现为半矮化表型,抽穗期也不再延迟,说明Ghd7对株高和抽穗期的调控需要GA。以上结果说明Ghd7综合参与多条激素途径,其机制还有待深入研究。更多还原

【Abstract】 To survival, plants adjust the process of growth and development to adapt complex environmental signals. In rice, this process often leads to great change of important agronomic traits, which affect final yield. So the issue about the interaction between rice plant and environmental signal has the theory and application significance. In this project, we show that the quantitative trait locus (QTL) Ghd7has major effects on an array of traits in rice, including number of grains per panicle, plant height and heading date. And we reveal that Ghd7functions to integrate the dynamic environmental inputs (such as light quality, day length, temperature, stress) with phase transition, architecture regulation, and stress response to maximize the reproductive success of the rice plant. Our results are performed as follow:1. Cloning of pleiotropic gene Ghd7We obtained the complete transcribed sequence of Ghd7by RACE and found that Ghd7is a new CCT domain gene, which has no significant homology in Arabidopsis. Subcellular localization result confirmed that GHD7is a nuclear protein and represses the transcriptional activity. Using quantitative RT-PCR analysis, we performed that the expression of Ghd7was mainly detected in the emerged leaf blade and displayed a gradient with much higher transcript accumulation in the leaf tip than the leaf base. Moreover, Ghd7was affected by photoperiodic and diurnal rhythm. The Ghd7transcript was much more abundant during the light period than in dark period, especially under long day conditions. And the expression peak performed at dawn under long day conditions. Ghd7suppressed the expression of flowering time genes under long day conditions and delay heading. Ghd7-Ehdl-Hd3a represents a distinct pathway that does not exist in Arabidopsis. We also identified four Ghd7alleles from five varieties by comparative sequencing analysis. Among them, the alleles from HR5and TQ are strong function alleles, ZH11has the weak function allele and the alleles from MDJ8and HJ19has a premature termination and consider as the nonfunctional allele. Meanwhile, we found alleles of Ghd7also associated with the polymorphism of AT-repeat at3’UTR region. We concluded that Ghd7played crucial roles for increasing productivity and adaptability of rice globally.2. The role of Ghd7in riceThe degree of phenotypic effect of Ghd7on heading date and yield traits was quantitatively related to the transcript level, and was also influenced by both environmental conditions and genetic backgrounds. Ghd7regulated plasticity of tiller branching by mediating the PHYB-OsTB1pathway as adaption to shade signal and the protein level of Ghd7was decrease in phyB mutant. Meanwhile, the expression of Ghd7was regulated by various environmental cues and this gene was also involved in regulation of drought stress response. The results suggested that Ghd7serves as a link between the dynamic environmental inputs with phase transition, architecture regulation and stress response to maximize the reproductive success of the rice plant.3. The mechanism study of Ghd7To detect the downstream network of Ghd7, we used OX-Ghd7ZH11, Ami-Ghd7, phyB mutant and wild type plants to perform the RNA-seq analysis in a diurnal approach (dawn, mid-day, dusk and mid-night). To understand the protein interaction of Ghd7, we fusion the GHD7to3xFlag tag and overexpressed in HJ19. Using this transgenetic plants, we identified that Ghd7formed as a450-kDa protein complex in vivo by gel filtration. Meanwhile, Ghd7can interact with HAP genes in yeast, which also have the ability to interact with OsTOC1. Finally, both HAP genes and clock genes were regulated by PHYB in transcript level. The results indicated that the selective of interaction between HAPs, Ghd7and OsTOCl form a robustness system to mediate the crosstalk between the photoperiod and circadian clock. And PHYB modulate this robustness system in transcript and post-translational levels.4. Ghd7affect hormone pathway1) Overexpressed of Ghd7increase the endogenous ABA content, have more sensitive to exogenous ABA and inhibit seed germination.2) Ghd7controlled the OsPIN genes in SAM and repress the endogenous IAA content.3) Ghd7repressed GA2ox genes expression and its function about the plant height and heading date dependent on endogenous GA contents. The results suggested that Ghd7involve in multiple hormone pathways.更多还原