發(fā)布時間：2018-05-11 14:09

  本文選題：擬南芥 + 組蛋白H2B單泛素化�。� 參考：《中國農(nóng)業(yè)大學》2016年博士論文

【摘要】：組蛋白H2B單泛素化(H2Bub)是組蛋白翻譯后修飾的一種類型。H2Bub通過影響組蛋白與DNA雙鏈的親和性,改變?nèi)旧�質(zhì)的構(gòu)象,進而調(diào)控基因轉(zhuǎn)錄。研究表明,H2Bub通過調(diào)控擬南芥的基因轉(zhuǎn)錄,從而調(diào)控種子萌發(fā)、根和葉的發(fā)育、開花時間、生物鐘等生理過程,同時,H2Bub在擬南芥抗真菌防衛(wèi)反應(yīng)中起關(guān)鍵作用。植物受到病原菌侵染時,微管骨架發(fā)生動態(tài)變化并在抗病信號轉(zhuǎn)導(dǎo)中起重要作用。植物細胞內(nèi),微管骨架動態(tài)受精細而復(fù)雜的調(diào)控,目前,人們對微管骨架動態(tài)的調(diào)控機制認識有限。本研究以擬南芥野生型和H2Bub功能缺失突變體hub1-4、hub2-2、hub1-4 hub2-2和ubc1-1ubc2-1為實驗材料,研究H2Bub參與擬南芥抗大麗輪枝菌毒素(Vd toxins)防衛(wèi)反應(yīng)的分子機制,并探討防衛(wèi)反應(yīng)中微管骨架動態(tài)的調(diào)控機制。用大麗輪枝菌孢子處理擬南芥野生型和H2Bub功能缺失突變體,發(fā)現(xiàn)突變體對大麗輪枝菌的抗性比野生型弱；用Vd toxins處理,突變體的細胞死亡比野生型少,并驗證Vd toxins處理引起的細胞死亡是細胞程序性死亡(programmed cell death,PCD);此外,Vd toxins處理后,突變體的花青素積累比野生型少。這些結(jié)果表明：H2Bub參與擬南芥抗Vdtoxins防衛(wèi)反應(yīng)。采用激光共聚焦顯微掃描(confocal laser scanning microscopy,CLSM)方法研究Vd toxins處理對擬南芥鋪板細胞的微管骨架動態(tài)的影響,發(fā)現(xiàn)H2Bub功能缺失使微管解聚速度變慢,程度變輕,證明H2Bub參與調(diào)控擬南芥抗Vd toxins防衛(wèi)反應(yīng)中的微管骨架動態(tài)。同時,在Vdtoxins中分別加入酪氨酸激酶(PTKs)抑制劑染料木素(genistein,GN)和酪氨酸磷酸酶(PTPs)抑制劑氧化苯砷(phenylarsine oxide,PAO)處理擬南芥,結(jié)果顯示,GN使微管骨架更穩(wěn)定,而PAO則使微管骨架更不穩(wěn)定,證明酪氨酸磷酸化參與調(diào)控擬南芥在防衛(wèi)反應(yīng)中的微管骨架動態(tài)變化。用Real-time PCR方法定量分析基因表達,發(fā)現(xiàn)H2Bub影響PTKs基因CRK41以及PTPs基因PTP1和PFA-DSP5的表達,推測H2Bub可能通過調(diào)控與微管骨架動態(tài)相關(guān)的因子的酪氨酸磷酸化水平進而影響微管骨架動態(tài)。用基因芯片方法,發(fā)現(xiàn)擬南芥抗Vd toxins防衛(wèi)反應(yīng)中有1500多個基因受H2Bub調(diào)控。對這些基因進行功能聚類分析,發(fā)現(xiàn)這些基因主要與防衛(wèi)反應(yīng)、非親和性防衛(wèi)反應(yīng)、對細菌防衛(wèi)反應(yīng)、活性氧響應(yīng)、細胞內(nèi)氧化還原平衡、毒素分解代謝、生物脅迫響應(yīng)、免疫系統(tǒng)、真菌響應(yīng)和水楊酸刺激響應(yīng)等生理過程有關(guān),表明受H2Bub調(diào)控的基因分布在多種防衛(wèi)反應(yīng)途徑中。綜上所述：H2Bub通過調(diào)控與防衛(wèi)反應(yīng)相關(guān)的基因的表達進而調(diào)控擬南芥抗Vd toxins防衛(wèi)反應(yīng),并在這一過程中調(diào)控微管骨架動態(tài),H2Bub正向調(diào)控PTKs基因CRK41以及PTPs基因PTP1和PFA-DSP5的表達,PTKs和PTPs參與調(diào)控微管骨架動態(tài)。研究結(jié)果為闡明植物抗大麗輪枝菌防衛(wèi)反應(yīng)的分子機理提供基礎(chǔ)數(shù)據(jù),為最終控制棉花黃萎病的發(fā)生提供理論依據(jù)。
[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.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S432.2
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本文編號：1874277