【作者】 王静；

【导师】 祁智；

【作者基本信息】 内蒙古大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 本实验室以模式植物拟南芥为材料,研究植物对矿质养分贫瘠响应和适应的分子机制。本论文由两个相对独立的课题组成：一方面通过突变体筛选比较深入的研究了RHD3基因在低氮诱导花青素过量积累过程中的分子功能；另一方面结合对活体细胞内细胞质Ca浓度的动态检测和基因表达谱芯片技术,对植物感应低Ca环境的细胞和分子生物学机制进行了初探。(1)本研究筛选到一株在低氮条件下过量积累花青素的、隐性单基因突变体,表现主根短,根毛短而弯曲和植株矮小的表型。结合图位克隆与以二代测序技术为基础的DNA多态性分析,确定突变基因为ROOT HAIR DEFECTIVE3(RHD3),突变位点为第16个内含子的最后一个碱基,导致RNA剪接错误,命名为rhd3-10,其所有表型同已知的rhd3-1以及rhd3-10/rhd3-1杂交F1代植物完全一致,证明我们观察到的表型确实为RHD3基因突变造成。RHD3定位于内质网,基因启动子活性集中在叶子和根尖。在rhd3-10突变体体内引入花青素合成关键基因TT4的突变,完全阻断在低氮条件下rhd3-10过量积累花青素的表型。在低氮条件下,rhd3-10相对于野生型植物(Co1),体内花青素合成相关基因表达上调,花青素含量过量积累。证明在Col植物体内,RHD3是低氮诱导花青素积累的负调控因子。乙烯是高光强诱导花青素积累的负调控因子。我们假设RHD3通过介导乙烯途径间接负调控花青素积累。该假设得到如下四个结果的支持：(a)RHD3基因突变部分阻断低氮对乙烯反应基因表达的诱导；(b)在低氮条件下,乙烯信号转导阻断突变体,etrl、ein2、ein3/eill表现出过量积累花青素的表型；而乙烯过量产生突变体eto1则表现出花青素积累显著降低的表型。这证明,乙烯是低氮诱导花青素积累的负调控因子;(c)对于Co1.7烯合成前体ACC强烈抑制低氮诱导的花青素积累；对于乙烯信号转导阻断突变体和rhd3-10, ACC的这种抑制作用显著降低；(d)在乙烯过量产生突变体eto1体内进一步突变RHD3基因可以显著提高突变体花青素的积累。尽管RHD3和ETR1, EIN2和CTR1都位于或同内质网关联,但是通过酵母双杂交实验没有发现RHD3的N和C端同ETR1, EIN2和CTR1相互作用。RHD3调节乙烯信号转导的分子机制还需要进一步研究。(2)生态学证据表明,酸雨导致全球范围土壤中Ca含量降低。植物应对低Ca的分子和细胞学机制未知。本研究发现低Ca环境诱导大量基因表达发生显著变化,其中很多基因是以前证明参与植物响应其他逆境信号的基因。一类编码细胞质Ca2+([Ca2+]cyt)感知蛋白的基因显著上调,我们假设[Ca2+]cyt参与植物细胞感应外界低Ca环境。研究证实,降低细胞外Ca2+浓度([Ca2+]ext)确实会激发[Ca2+]cyt瞬时升高,而且[Ca2+]cyt升高的峰值与[Ca2+]ext降低的程度呈正相关。通过提高细胞外K+浓度使细胞质膜电势相对去极化,显著抑制[Ca+2]cxt降低对[Ca2+]cyt的诱导作用,说明Ca2+可能通过受膜电势调节的Ca通道进入细胞内。Gd3+,一个Ca2+通道抑制剂确实强烈抑制这个反应。动物细胞质膜存在对细胞外低Ca感知的受体蛋白。受体调节的信号事件经常表现脱敏现象：细胞在对第一次信号刺激作出反应以后,需要一段时间的恢复才能对连续的第二次同样信号刺激作出反应。我们发现植物细胞对低Ca的反应有显著的时间依赖性的脱敏现象,但是对动物细胞Ca感知蛋白拮抗剂不敏感。磷脂酶C拮抗剂neomycin可以抑制细胞外低Ca对[Ca2+]cyt的诱导作用。进一步研究发现,当用Ca2+通道抑制剂Gd3+抑制低Ca引起的[Ca2+]cyt升高以后,可以抑制一部分低Ca诱导的下游基因表达,这说明植物细胞对低Ca环境在基因表达层面的响应有依赖[Ca2+]cyt和不依赖[Ca2+]cyt两条途径。综上所述,本研究一方面在植物响应低氮逆境方面鉴定到RHD3基因的新功能,并进一步证明RHD3通过调节乙烯信号转导通路实现对花青素合成的负调控功能。另一方面,本研究建立了植物响应低钙的基本细胞和基因转录机理。为实验室进一步研究植物响应矿质营养贫瘠信号奠定了基础。更多还原

【Abstract】 Our lab studies molecular mechanism of plants to mineral nutrient deprivation with Arabidopsis as material. This thesis is composed of two relatively independent sections:by screening genetic mutants, we conducted analysis on molecular role of RHD3in low nitrogen-induced anthocyanin accumulation; by combining measuring cytosolic Ca dynamics change in vivo and microarray-based transcriptional analysis, we initiated investigation of cellular and molecular mechanism of plant cell response to low calcium.(1) We isolated a single gene and recessive mutant showing anthocyanin overly accumulation phenotype under low nitrogen, which displayed short primary root, short and wavy root hair, dwarf phenotypes. Combination of positional cloning and next generation sequencing-based DNA polymorphisms analysis identified an single base mutation in the last base of16th intro of ROOT HAIR DEFECTIVE3(RHD3) leading truncated RNA product, which was named as rhd3-10. The mutant’s phenotype was copied by rhd3-1and rhd3-1/rhd3-10F1plants. It demonstrated that RHD3indeed is the gene responsible for all observed phenotype. RHD3locates in ER and its promoter activity primarily is in the leaf and root tip. Mutating TT4, an essential gene for anthocyanin biosynthesis, completely blocked the low nitrogen-induced anthocyanin accumulation in rhd3-10.Under low nitrogen, rhd3-10had higher expression of anthocyanin biosynthesis related genes and accumulation of anthocyanin itself than those of wild type (Col). It demonstrated that RHD3is a negative regulator of the low nitrogen-induced anthocyanin accumulation. Ethylene negatively regulates high light-induced anthocyanin accumulation. We hypothesized that RHD3negatively regulates anthocyanin biosynthesis through modulating ethylene signaling pathway, which was supported by following four evidences:(a) RHD3mutation partially blocked the low nitrogen-induced ethylene responsive gene expression;(b) Under low nitrogen, ethylene signaling blocking mutants etrl、ein2、ein3/eill accumulated more, but ethylene over-producing mutant etol accumulated less anthocyanin. It demonstrated that ethylene indeed is the negative regulator of this process;(c) Ethylene precursor ACC strongly suppressed the low nitrogen-induced anthocyanin accumulation in Col. The suppressive effect of ACC was significantly attenuated in ethylene signaling mutants and rhd3-10;(d) Introducing rhd3-10into etol increased low nitrogen induced anthocyanin accumulation. Though RHD3, ETR1, EIN2and CTR1locate or associate with ER, we didn’t detect protein-protein interaction between RHD3N or C terminals and ETR1, EIN2and CTR1. It implies further investigation needed for exploring molecular mechanism of RHD3-regulated ethylene signaling. (2) Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca2+levels caused by increasing acid deposition and massive timber harvesting. Little is known about the genetic and cellular mechanism of plants’responses to this decrease. In this study, transcriptional profiling analysis identified many extracellular Ca ([Ca2+]ext) deprivation-responsive genes in Arabidopsis thaliana, many of which are involved in plant responses to other environmental stresses. Interestingly, a group of putative cytosolic Ca2+([Ca2+]cyt) sensor-encoding genes were significantly up-regulated, which implied that [Ca2+]cyt has a role in sensing the [Ca2+]ext deprivation. Supportively, decreasing [Ca2+]ext evoked a transient rise in [Ca2+]cyt, the amplitude of which positively correlated with decreasing [Ca2+]ext and negatively [K+]ext.The response was insensitive to an animal Ca sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. The [Ca2+]cyt response to [Ca2+]ext deprivation was significantly desensitized after the initial treatment, which was typical of a receptor-mediated signaling event. Gd3+, an inhibitor of Ca2+channels, suppressed the [Ca2+]ext triggered [Ca2+]cyt rise and the downstream gene expressions. Taken together, this study demonstrated that [Ca2+]cyt plays an important role in the putative receptor-mediated cellular and transcriptional sensing of [Ca2+]ext deprivation of plant cells.In summary, this study identified a new function of RHD3in plant response to low nitrogen and demonstrated that RHD3negatively regulated low nitrogen-induced anthocyanin biosynthesis through modulating ethylene signaling. On the other hand, this study established the cellular and molecular mechanism of plant response to low calcium. It will pave foundation for conducting further investigation of plant response to mineral nutrition deprivation signals.更多还原