组蛋白H2B单泛素化调控微管骨架动态参与拟南芥抗大丽轮枝菌防卫反应的作用机制

发布时间：2018-05-11 14:09

  本文选题：拟南芥 + 组蛋白H2B单泛素化　； 参考：《中国农业大学》2016年博士论文

【摘要】：组蛋白H2B单泛素化(H2Bub)是组蛋白翻译后修饰的一种类型。H2Bub通过影响组蛋白与DNA双链的亲和性,改变染色质的构象,进而调控基因转录。研究表明,H2Bub通过调控拟南芥的基因转录,从而调控种子萌发、根和叶的发育、开花时间、生物钟等生理过程,同时,H2Bub在拟南芥抗真菌防卫反应中起关键作用。植物受到病原菌侵染时,微管骨架发生动态变化并在抗病信号转导中起重要作用。植物细胞内,微管骨架动态受精细而复杂的调控,目前,人们对微管骨架动态的调控机制认识有限。本研究以拟南芥野生型和H2Bub功能缺失突变体hub1-4、hub2-2、hub1-4 hub2-2和ubc1-1ubc2-1为实验材料,研究H2Bub参与拟南芥抗大丽轮枝菌毒素(Vd toxins)防卫反应的分子机制,并探讨防卫反应中微管骨架动态的调控机制。用大丽轮枝菌孢子处理拟南芥野生型和H2Bub功能缺失突变体,发现突变体对大丽轮枝菌的抗性比野生型弱；用Vd toxins处理,突变体的细胞死亡比野生型少,并验证Vd toxins处理引起的细胞死亡是细胞程序性死亡(programmed cell death,PCD);此外,Vd toxins处理后,突变体的花青素积累比野生型少。这些结果表明：H2Bub参与拟南芥抗Vdtoxins防卫反应。采用激光共聚焦显微扫描(confocal laser scanning microscopy,CLSM)方法研究Vd toxins处理对拟南芥铺板细胞的微管骨架动态的影响,发现H2Bub功能缺失使微管解聚速度变慢,程度变轻,证明H2Bub参与调控拟南芥抗Vd toxins防卫反应中的微管骨架动态。同时,在Vdtoxins中分别加入酪氨酸激酶(PTKs)抑制剂染料木素(genistein,GN)和酪氨酸磷酸酶(PTPs)抑制剂氧化苯砷(phenylarsine oxide,PAO)处理拟南芥,结果显示,GN使微管骨架更稳定,而PAO则使微管骨架更不稳定,证明酪氨酸磷酸化参与调控拟南芥在防卫反应中的微管骨架动态变化。用Real-time PCR方法定量分析基因表达,发现H2Bub影响PTKs基因CRK41以及PTPs基因PTP1和PFA-DSP5的表达,推测H2Bub可能通过调控与微管骨架动态相关的因子的酪氨酸磷酸化水平进而影响微管骨架动态。用基因芯片方法,发现拟南芥抗Vd toxins防卫反应中有1500多个基因受H2Bub调控。对这些基因进行功能聚类分析,发现这些基因主要与防卫反应、非亲和性防卫反应、对细菌防卫反应、活性氧响应、细胞内氧化还原平衡、毒素分解代谢、生物胁迫响应、免疫系统、真菌响应和水杨酸刺激响应等生理过程有关,表明受H2Bub调控的基因分布在多种防卫反应途径中。综上所述：H2Bub通过调控与防卫反应相关的基因的表达进而调控拟南芥抗Vd toxins防卫反应,并在这一过程中调控微管骨架动态,H2Bub正向调控PTKs基因CRK41以及PTPs基因PTP1和PFA-DSP5的表达,PTKs和PTPs参与调控微管骨架动态。研究结果为阐明植物抗大丽轮枝菌防卫反应的分子机理提供基础数据,为最终控制棉花黄萎病的发生提供理论依据。
[Abstract]:Histone H2B monoubiquitin (H2Bub) is a type of post-translational histone modification. H2Bub changes the conformation of chromatin and regulates gene transcription by affecting the affinity of histone to DNA double strands. It is suggested that H2Bub regulates the physiological processes of seed germination, root and leaf development, flowering time and biological clock by regulating the gene transcription of Arabidopsis thaliana, and that H2Bub plays a key role in the anti-fungal defense response of Arabidopsis thaliana. The microtubule cytoskeleton changes dynamically and plays an important role in the signal transduction of disease resistance. In plant cells, microtubule cytoskeleton dynamic fertilization and complex regulation, at present, people have limited understanding of the mechanism of microtubule cytoskeleton dynamic regulation. Using Arabidopsis wild-type and H2Bub deletion mutants hub1-4C hub2-2 hub1-4 hub2-2 and ubc1-1ubc2-1 as experimental materials, we studied the molecular mechanism of H2Bub involved in the defense response of Arabidopsis against Verticillium grandis toxin Vd toxin, and explored the mechanism of microtubule cytoskeleton dynamics in the defense response of Arabidopsis thaliana. The wild-type and H2Bub functional deletion mutants of Arabidopsis thaliana were treated with spores of Verticillium dahliensis. It was found that the resistance of the mutants to the wild-type and the wild-type of Arabidopsis was weaker than that of the wild type, and the cell death of the mutant was less than that of the wild type treated with VD toxins. The results showed that the cell death induced by VD toxins was programmed cell death, and the anthocyanin accumulation of the mutant was less than that of wild type after Vd toxins treatment. These results suggest that: h 2 Bub is involved in the anti Vdtoxins defense response of Arabidopsis thaliana. The effect of VD toxins treatment on the microtubule cytoskeleton dynamics of Arabidopsis thaliana paver cells was studied by confocal laser scanning microscopyCLSM. It was found that the depolymerization rate of microtubule was slower and the degree of microtubule depolymerization was lighter due to the absence of H2Bub function. The results showed that H2Bub was involved in regulating the dynamics of microtubule skeleton in Arabidopsis thaliana defense response to VD toxins. At the same time, Arabidopsis thaliana was treated with tyrosine kinase (PTKs) inhibitor genistein (GNN) and tyrosine phosphatase (PTPs) inhibitor (phenylarsine oxidePao) respectively. The results showed that GN made microtubule skeleton more stable, while PAO made microtubule skeleton more unstable. It was demonstrated that tyrosine phosphorylation was involved in regulating the dynamic changes of microtubule skeleton in Arabidopsis thaliana in defense response. Quantitative analysis of gene expression by Real-time PCR showed that H2Bub affected the expression of PTKs gene CRK41 and PTPs gene PTP1 and PFA-DSP5. It was speculated that H2Bub might affect the dynamics of microtubule skeleton by regulating the level of tyrosine phosphorylation of the factors related to the dynamics of microtubule skeleton. In Arabidopsis thaliana, more than 1500 genes were regulated by H2Bub in the defense response to VD toxins by microarray method. Functional cluster analysis showed that these genes were mainly related to defense response, non-affinity defense response, bacterial defense response, reactive oxygen species response, intracellular redox balance, toxin catabolism and biostress response. The immune system, fungal response and salicylic acid stimulatory response are related to physiological processes, indicating that genes regulated by H2Bub are distributed in multiple defense response pathways. To sum up, the resistance of Arabidopsis thaliana to VD toxins is regulated by regulating the expression of genes related to defense response, and then regulating the defense response of Arabidopsis thaliana to VD toxins. In this process, the microtubule cytoskeleton dynamics was regulated by H2Bub and the expression of PTKs gene CRK41 and PTPs gene PTP1 and PFA-DSP5. PTKs and PTPs were involved in the regulation of microtubule cytoskeletal dynamics. The results provide basic data for elucidating the molecular mechanism of plant defense response to Verticillium dahliae, and provide theoretical basis for controlling the occurrence of Verticillium dahliae in cotton.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S432.2
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本文编号：1874277