發(fā)布時(shí)間：2018-05-15 15:57

  本文選題：NB-LRR + Sw-5b��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：免疫受體蛋白(R蛋白)介導(dǎo)的對(duì)病原物效應(yīng)蛋白的特異性識(shí)別并激活級(jí)聯(lián)免疫反應(yīng)是植物主要的防疫體系。NB-LRR類受體蛋白作為最大的一類R蛋白其功能結(jié)構(gòu)域包括N端結(jié)構(gòu)域、核苷酸結(jié)合結(jié)構(gòu)域(NB-ARC)和C端富亮氨酸富集結(jié)構(gòu)域(LRR)�；�于N端結(jié)構(gòu)域的不同分為TIR-NB-LRR和CC-NB-LRR。為了減少R蛋白過(guò)度激活帶來(lái)的不必要成本,植物進(jìn)化出了相應(yīng)的機(jī)制來(lái)調(diào)控其自抑制和激活。番茄Sw-5b屬于具有額外的N端結(jié)構(gòu)域(NTD)的CC-NB-LRR類型R蛋白,但是額外的NTD、CC、NB-LRR如何調(diào)控其自抑制和激活的機(jī)制目前仍不清楚。此外,越來(lái)越多的研究表明,R蛋白的亞細(xì)胞定位在抗病信號(hào)通路中發(fā)揮著重要作用,然而帶有額外N端結(jié)構(gòu)域的植物R蛋白的亞細(xì)胞定位在抗病中的作用還沒(méi)有開(kāi)展過(guò)相關(guān)研究。本研究圍繞兩個(gè)重要科學(xué)問(wèn)題,以番茄免疫受體蛋白Sw-5b和番茄斑萎病毒(Tomatospotted wilt virus,TSWV)移動(dòng)蛋白NSm為研究模式,開(kāi)展了以下兩方面的研究:1.番茄免疫受體蛋白Sw-5b的自抑制和激活的多重調(diào)控機(jī)制。為了探究Sw-5b的自我調(diào)控機(jī)制,我們系統(tǒng)地剖析了 Sw-5b的各亞結(jié)構(gòu)域的功能以及他們之間的關(guān)系。在農(nóng)桿菌介導(dǎo)的本氏煙瞬時(shí)表達(dá)試驗(yàn)中,Sw-5b的NB-LRR片段能夠介導(dǎo)依賴于NSm的細(xì)胞死亡,且這種誘發(fā)死亡的特性具有和全長(zhǎng)Sw-5b 一樣的特異性。將同源建模技術(shù)和定點(diǎn)突變實(shí)驗(yàn)相結(jié)合,發(fā)現(xiàn)保守的中心區(qū)域NB-ARC是激活NB-LRR的重要分子開(kāi)關(guān)�；�于以上研究,首先我們探討了病毒激發(fā)子NSm不存在時(shí),Sw-5b自抑制的調(diào)控機(jī)制。在瞬時(shí)表達(dá)試驗(yàn)中,NB-ARC自激活誘導(dǎo)細(xì)胞死亡,而NB-ARC與LRR 一起表達(dá)或單獨(dú)表達(dá)NB-LRR則抑制細(xì)胞死亡,CC-NB-LRR或NTD-CC-NB-LRR單獨(dú)表達(dá)不能誘導(dǎo)死亡,組成型激活突變位點(diǎn)S594A, D642E和D857V引入到NB-LRR中會(huì)導(dǎo)致其自激活,引入到CC-NB-LRR中自激活減弱,引入到NTD-CC-NB-LRR中不再產(chǎn)生自激活。免疫共沉淀試驗(yàn)發(fā)現(xiàn):LRR, CC和NTD對(duì)NB-ARC的抑制作用與結(jié)構(gòu)域之間的互作有關(guān),LRR和NB-ARC發(fā)生物理互作,CC與LRR互作,而不與NB-ARC互作,NTD既可以和CC也可以和NB-LRR發(fā)生物理互作。因此,當(dāng)病毒激發(fā)子NSm不存在條件下,LRR, CC和NTD的三層負(fù)調(diào)控作用使得Sw-5b維持在一個(gè)很穩(wěn)定的自抑制狀態(tài);其次,我們探究了 Sw-5b激活的調(diào)控機(jī)制。在與NSm共同瞬時(shí)表達(dá)的試驗(yàn)中,NTD-CC-NB-LRR或其衍生突變體和NB-LRR 一樣誘導(dǎo)產(chǎn)生很強(qiáng)的細(xì)胞死亡反應(yīng),而CC-NB-LRR及其衍生突變體只呈現(xiàn)很弱的細(xì)胞死亡反應(yīng)。因此表明,當(dāng)病毒激發(fā)子NSm存在情況下,LRR釋放對(duì)NB-ARC的抑制,進(jìn)一步NTD解除CC對(duì)NB-LRR的抑制,最后Sw-5b被完全激活。此外,我們還發(fā)現(xiàn)NB-LRR這一層自抑制和激活調(diào)控并不能抵抗TSWV病毒,在轉(zhuǎn)基因植株的接病毒測(cè)試中,35S: NB-LRR轉(zhuǎn)基因植株雖然對(duì)番茄斑萎病毒產(chǎn)生HR反應(yīng),但不具有系統(tǒng)抗性。而全長(zhǎng)Sw-5b的轉(zhuǎn)基因植株無(wú)論是35S啟動(dòng)子下或是本身啟動(dòng)子下都對(duì)番茄斑萎病毒表現(xiàn)抗性。在本試驗(yàn)基礎(chǔ)上,我們提出如下假說(shuō):Sw-5b為了釋放CC的負(fù)調(diào)控作用,進(jìn)化出了額外NTD結(jié)構(gòu)域來(lái)協(xié)調(diào)CC的作用,最終Sw-5b進(jìn)化出了多重的調(diào)控機(jī)制來(lái)控制其自抑制和激活狀態(tài)。重要的是,這種多層的調(diào)控機(jī)制,對(duì)于Sw-5b蛋白抗性功能的發(fā)揮是必須的。2.番茄免疫蛋白受體Sw-5b的亞細(xì)胞定位與功能的關(guān)系。大多數(shù)的R蛋白沒(méi)有經(jīng)典的核定位信號(hào),因此最初人們認(rèn)為這類受體主要定位在細(xì)胞質(zhì)中。本研究首先測(cè)試Sw-5b的亞細(xì)胞定位,在其N端融合黃色熒光蛋白后,瞬時(shí)表達(dá)及轉(zhuǎn)基因抗性測(cè)試發(fā)現(xiàn),YFP-Sw-5b具有與Sw-5b一樣誘導(dǎo)細(xì)胞死亡和誘導(dǎo)抗性的能力。共聚焦觀察發(fā)現(xiàn)其在細(xì)胞質(zhì)和細(xì)胞核及核仁都有分布。進(jìn)一步探究Sw-5b進(jìn)出核與功能的關(guān)系,我們?cè)赮FP-Sw-5b的N端融合外源的核輸出信號(hào)(NES)將Sw-5b拉到細(xì)胞質(zhì),瞬時(shí)表達(dá)發(fā)現(xiàn),完全的細(xì)胞質(zhì)定位導(dǎo)致細(xì)胞死亡活性明顯增強(qiáng),但是,轉(zhuǎn)基因植株接毒測(cè)試表明出核導(dǎo)致其失去了對(duì)TSWV的抗性;融合核定位信號(hào)(NLS)將Sw-5b全部拉到細(xì)胞核內(nèi),其誘導(dǎo)的細(xì)胞死亡明顯減弱或消失但是卻足以抵抗病毒。同樣的方法改變NSm的亞細(xì)胞定位,發(fā)現(xiàn)NSm定位在細(xì)胞質(zhì)才能誘導(dǎo)細(xì)胞死亡。此外,我們發(fā)現(xiàn)Sw-5b的核定位特性與抗性激活與否無(wú)關(guān)。P-loop保守基序突變破壞Sw-5b核苷酸結(jié)合構(gòu)象后,YFP-Sw-5b誘導(dǎo)細(xì)胞死亡的功能喪失,但其核質(zhì)分布沒(méi)有受到影響。在重要分子伴侶SGT1被沉默的植株中,YFP-Sw-5b不能誘導(dǎo)細(xì)胞死亡,但依然能夠進(jìn)核。我們還發(fā)現(xiàn)Sw-5b的細(xì)胞核定位信號(hào)在N端的NTD結(jié)構(gòu)域,其中YFP-NTD1 (51.7 kDa)和全長(zhǎng)Sw-5b 一樣核質(zhì)都有分布,YFP-NTD2 (42kDa)能進(jìn)核但不進(jìn)核仁,細(xì)胞質(zhì)定位信號(hào)在NB-LRR結(jié)構(gòu)域�；�于以上發(fā)現(xiàn),我們認(rèn)為Sw-5b與NSm的識(shí)別發(fā)生在細(xì)胞質(zhì),且Sw-5b介導(dǎo)的細(xì)胞死亡和抗病功能可以分開(kāi)進(jìn)行。
[Abstract]:The specific identification of the immune receptor protein (R protein) and the activation of the cascade immunoreaction are the.NB-LRR receptor proteins in the main epidemic prevention system of plants as the largest class of R proteins as the largest class of protein, including the N domain domain, the nucleotide binding domain (NB-ARC) and the C terminal rich leucine enriched domain (LRR). The different N terminal domains are divided into TIR-NB-LRR and CC-NB-LRR. in order to reduce the unnecessary cost of R protein overactivation. Plants have evolved a corresponding mechanism to regulate their self suppression and activation. Tomato Sw-5b belongs to the CC-NB-LRR type R protein with an additional N terminal domain (NTD), but the additional NTD, CC, how NB-LRR regulates its self suppression. The mechanism of the system and activation is still unclear. In addition, more and more studies have shown that the subcellular localization of R protein plays an important role in the disease resistance signaling pathway. However, the role of the subcellular localization of the plant R protein with the additional N terminal domain in the disease resistance has not been studied. This study revolves around two important scientific questions. On the basis of the model of tomato immune receptor protein Sw-5b and Tomatospotted wilt virus (TSWV) mobile protein NSm as the research model, the following two aspects were carried out: 1. the self inhibition and activation of the immune receptor protein Sw-5b of tomato, the multiple regulation mechanism. In order to explore the self-regulation mechanism of Sw-5b, we systematically analyzed Sw-5b The function of each subdomain and their relationship. In the Agrobacterium mediated instant expression test of Benedict's smoke, the NB-LRR fragment of Sw-5b can mediate cell death dependent on NSm, and this characteristic of inducing death has the same specificity as the full length Sw-5b. The central region, NB-ARC, is an important molecular switch to activate NB-LRR. Based on the above study, we first explored the regulation mechanism of Sw-5b self suppression when the virus elicitor NSm does not exist. In the transient expression test, NB-ARC induces cell death by self activation, and NB-ARC and LRR are expressed together or individually expressed in NB-LRR to inhibit cell death, CC-NB-LRR Or NTD-CC-NB-LRR alone expressed the inability to induce death, the constituent activation mutation site S594A, D642E and D857V were introduced into NB-LRR to cause their self activation, and the introduction into CC-NB-LRR to reduce self activation and no longer produce self activation in NTD-CC-NB-LRR. The immunoprecipitation test found that LRR, CC and NTD have inhibitory effects on NB-ARC and the domain. Interaction between LRR and NB-ARC, CC and LRR interact with each other instead of NB-ARC, and NTD can and CC can interact with NB-LRR in physical interaction. Therefore, the three layer negative regulation of LRR, CC and NTD makes a stable self suppression state of LRR, CC and NTD; secondly, we explore The regulation mechanism of Sw-5b activation. In the experiment with NSm's common transient expression, NTD-CC-NB-LRR or its derivative mutant and NB-LRR induced a strong cell death response, while CC-NB-LRR and its derived mutants only showed a very weak cell death response. Therefore, LRR release to NB-ARC in the presence of the pathogen NSm. Inhibition, further NTD relieves the inhibition of CC to NB-LRR, and finally Sw-5b is fully activated. In addition, we also found that the self inhibition and activation regulation of NB-LRR is not resistant to TSWV virus. In the test of transgenic plants, the 35S: NB-LRR transgenic plants do not have systemic resistance to the tomato Verticillium virus, although they produce HR response to lycopene Verticillium virus. The transgenic plants with full length of Sw-5b are resistant to tomato Verticillium virus either under 35S promoter or under their own promoter. On the basis of this experiment, we propose the following hypothesis: Sw-5b has evolved an additional NTD domain to coordinate the role of CC in order to release the negative regulation of CC, and finally the Sw-5b has evolved multiple regulatory mechanisms. It is important to control the self inhibition and activation state. It is important that this multi-layer regulation mechanism, which plays a role in the subcellular localization and function of the.2. tomato immune protein receptor Sw-5b, is a necessary function of the Sw-5b protein resistance function. Most of the R proteins have no classical nuclear location signals. In the cytoplasm, this study first tested the subcellular localization of Sw-5b. After the fusion of yellow fluorescent protein at its N end, transient expression and transgenic resistance test found that YFP-Sw-5b had the ability to induce cell death and induced resistance as Sw-5b. Confocal observation found that the cytoplasm, nucleus and nucleolus were distributed in cytoplasm and nucleus and nucleolus. Further exploration of Sw-5b In the relationship between nuclear and function, we fused the exogenous nuclear output signal (NES) at the N end of YFP-Sw-5b to pull the Sw-5b into the cytoplasm. Instantaneous expression found that the complete cytoplasmic localization led to a significant increase in cell death activity. However, the transgenic plant toxicity test showed that the nucleus induced its loss of resistance to TSWV; fusion nuclear positioning signal (NLS) We pull all Sw-5b into the nucleus, and the induced cell death obviously weakened or disappeared but was enough to resist the virus. The same method changed the subcellular location of NSm and found that NSm was located in the cytoplasm to induce cell death. In addition, we found that the nuclear location of Sw-5b is not related to the anti sexual activation of the.P-loop conservative radical mutation. After the combination of bad Sw-5b nucleotides binding conformation, YFP-Sw-5b induces cell death function loss, but its nuclear distribution is not affected. In the silent plants of the important molecular chaperone, YFP-Sw-5b can not induce cell death, but still can enter the nucleus. We also found the Sw-5b cell nucleus localization signal in the NTD domain of the N terminal, in which YFP-NTD1 (Sw-5b) 51.7 kDa) are all distributed as the whole Sw-5b nucleolus, YFP-NTD2 (42kDa) can enter the nucleus but not nucleolus, and the cytoplasmic localization signal is in the NB-LRR domain. Based on the above findings, we think that the identification of Sw-5b and NSm occurs in the cytoplasm, and Sw-5b mediated cell death and disease resistance can be carried out separately.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S432.23

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