【摘要】：根據(jù)所涉植物是否為所涉病原物的寄主,植物的抗病性可分為寄主抗性和非寄主抗性(nonhost resistance)。兩類(lèi)抗病性的產(chǎn)生機(jī)理既有許多共同點(diǎn)也有一些明顯的區(qū)別。寄主抗性有病原相關(guān)分子模式觸發(fā)的免疫(PAMP triggered immunity, PTI)和病原物效應(yīng)蛋白觸發(fā)的免疫(Effector triggered immunity, ETI)兩個(gè)層次。番茄與葉霉菌(Cladosporium fulvum)的互作為典型的基因?qū)?gene-for-gene)寄主抗性,番茄抗葉霉菌基因Cf介導(dǎo)產(chǎn)生對(duì)攜帶互補(bǔ)Avr基因的葉霉菌的ETI抗性。水稻白葉枯病菌(Xanthomonas oryzae pv. oryzae, Xoo)寄主范圍窄,在非寄主本氏煙(Nicotiana benthamiana)中誘導(dǎo)產(chǎn)生強(qiáng)烈的過(guò)敏性反應(yīng)(hypersensitive response, HR)和非寄主抗性。本論文主要針對(duì)兩個(gè)問(wèn)題開(kāi)展研究,首先,傳統(tǒng)觀念認(rèn)為,介導(dǎo)產(chǎn)生ETI的植物抗病(resistance, R)基因不存在轉(zhuǎn)錄和轉(zhuǎn)錄后水平的基因表達(dá)調(diào)控。本論文通過(guò)對(duì)Cf介導(dǎo)的ETI研究發(fā)現(xiàn),番茄Cf基因存在轉(zhuǎn)錄和轉(zhuǎn)錄后水平的調(diào)控；另外,本氏煙中Xoo誘導(dǎo)的HR和非寄主抗性產(chǎn)生機(jī)理尚不明確。本研究采用多種策略篩選鑒定該非寄主抗性調(diào)控基因,并明確了農(nóng)桿菌對(duì)該非寄主抗性的抑制作用及機(jī)制。本研究獲得了以下主要結(jié)果： 1.初步明確了番茄抗葉霉病基因Cf-4和Cf-9基因表達(dá)調(diào)控機(jī)理。分別從轉(zhuǎn)錄水平—DNA甲基化以及轉(zhuǎn)錄后水平—miRNA兩個(gè)層次,對(duì)Cf-4和Cf-9基因表達(dá)調(diào)控機(jī)理進(jìn)行了分析。在轉(zhuǎn)錄水平上,高溫抑制Cf基因的表達(dá),Avr強(qiáng)烈誘導(dǎo)Cf基因的表達(dá),Cf-4的表達(dá)隨著番茄植株株齡增加而升高,而Cf-9不受顯著影響。啟動(dòng)子DNA甲基化參與高溫抑制的以及常溫下Avr9誘導(dǎo)的Cf-9基因的表達(dá)調(diào)控,另外在由高溫到常溫的降溫過(guò)程中Avr4和Avr9對(duì)Cf-4和Cf-9基因表達(dá)的誘導(dǎo)作用可能受到甲基化的調(diào)控。采用VIGS技術(shù),分析了番茄甲基化酶以及去甲基化酶基因?qū)f基因表達(dá)的影響,發(fā)現(xiàn)番茄個(gè)別甲基化酶和全部4個(gè)去甲基化酶基因單獨(dú)沉默顯著改變Cf-4和Cf-9基因的表達(dá),說(shuō)明這些番茄甲基化酶和去甲基化酶基因可能參與對(duì)Cf表達(dá)的調(diào)控。在轉(zhuǎn)錄后水平,我們預(yù)測(cè)到了一條能夠同時(shí)靶標(biāo)Cf-4和Cf-9基因的長(zhǎng)度為21nt的miRNA。通過(guò)該miRNA的靶標(biāo)序列與前體序列共表達(dá)等分析,證實(shí)該miRNA能有效降解Cf-4和Cf-9mRNA。 2.篩選獲得一批Cf-4/Avr4介導(dǎo)的HR的調(diào)控基因。通過(guò)差異表達(dá)蛋白對(duì)應(yīng)基因的功能分析及本氏煙cDNA文庫(kù)VIGS篩選,我們從500多個(gè)基因沉默結(jié)構(gòu)中,篩選得到了28個(gè)參與調(diào)控Cf-4/Avr4介導(dǎo)的HR的基因,其中核糖核蛋白R(shí)NP、一個(gè)DNA結(jié)合蛋白和一個(gè)Dof鋅指蛋白等正調(diào)控Cf-4/Avr4介導(dǎo)的HR,而一個(gè)環(huán)指蛋白、個(gè)含BTB/POZ和TAZ結(jié)構(gòu)域蛋白、CP結(jié)合蛋白、SUMO結(jié)合酶SCE1、紅色葉綠素代謝產(chǎn)物還原酶RCCR等22個(gè)蛋白負(fù)調(diào)控Cf-4/Avr4介導(dǎo)的HR。 3.建立和優(yōu)化了本氏煙—Xoo非寄主抗性互作體系并研究明確H2O2積累是Xoo誘導(dǎo)的HR和非寄主抗性產(chǎn)生所必需。研究結(jié)果表明,用1×108cfu/ml的Xoo接種本氏煙完全展開(kāi)的葉片,28℃下培養(yǎng)將產(chǎn)生最迅速最強(qiáng)烈的非寄主抗性。Xoo接種先在浸潤(rùn)區(qū)域內(nèi)誘導(dǎo)活性氧大量積累,而后導(dǎo)致產(chǎn)生強(qiáng)烈的HR,并強(qiáng)烈誘導(dǎo)PR和HR標(biāo)志基因的表達(dá),最終導(dǎo)致Xoo的菌量急劇下降。采用過(guò)氧化氫酶清除H2O2的藥理學(xué)實(shí)驗(yàn)結(jié)果表明,H2O2在Xoo誘導(dǎo)的HR和非寄主抗性中至關(guān)重要。 4.篩選獲得一批Xoo誘導(dǎo)的本氏煙非寄主抗性調(diào)控基因。通過(guò)差異表達(dá)蛋白對(duì)應(yīng)基因功能分析及本氏煙cDNA文庫(kù)VIGS篩選,我們從500多個(gè)基因沉默結(jié)構(gòu)中篩選獲得了8個(gè)調(diào)控Xoo誘導(dǎo)的本氏煙HR的基因,其中碳酸酐酶、硫氧還蛋白h1以及核糖核蛋白是該HR的負(fù)調(diào)控因子,而絲氨酸/蘇氨酸蛋白激酶、尿嘧啶特異的內(nèi)切酶DnaJ同源亞家族基因成員、GTP結(jié)合蛋白以及三生煙中的硫氧還蛋白h這五個(gè)基因則為正調(diào)控因子。本氏煙核糖核蛋白基因同時(shí)參與Xoo誘導(dǎo)的HR和Cf-4/Avr4介導(dǎo)的HR的調(diào)控,但作用相反。 5.闡明了農(nóng)桿菌對(duì)Xoo的抑菌作用以及對(duì)Xoo誘導(dǎo)的HR的抑制作用及其機(jī)制。結(jié)果顯示,四種農(nóng)桿菌菌株對(duì)Xoo誘導(dǎo)的HR的抑制作用有顯著差異,菌株GV3101,EHA105和LBA4404能強(qiáng)烈抑制,但菌株C58C1無(wú)此作用。三種農(nóng)桿菌對(duì)Xoo誘導(dǎo)的HR的抑制作用機(jī)制不同,菌株GV3101通過(guò)直接抑制Xoo的生長(zhǎng)來(lái)抑制HR,該抑制作用需要Xoo中三型分泌系統(tǒng)基因hrcU和農(nóng)桿菌Ti質(zhì)粒pTiC58的參與。菌株LBA4404和EHA105則無(wú)直接抑菌作用,而是通過(guò)抑制本氏煙上活性氧的積累來(lái)抑制HR的產(chǎn)生。 綜上所述,本研究首次揭示了Cf-4和Cf-9基因表達(dá)在轉(zhuǎn)錄及轉(zhuǎn)錄后水平的調(diào)控作用及其機(jī)理,鑒定了首個(gè)調(diào)節(jié)Cf基因表達(dá)的miRNA。此外,利用cDNA文庫(kù)VIGS篩選技術(shù),從500多個(gè)基因沉默結(jié)構(gòu)中篩選獲得一批參與Xoo誘導(dǎo)的本氏煙HR和Cf-4/Avr4介導(dǎo)的HR的新調(diào)控基因。另外,首次揭示農(nóng)桿菌對(duì)Xoo本身及其誘導(dǎo)的HR的抑制作用及其機(jī)理。這些研究結(jié)果增進(jìn)對(duì)植物寄主ETI抗性和非寄主抗性產(chǎn)生機(jī)理的理解。
[Abstract]:The disease resistance of plants can be divided into host resistance and non-host resistance according to whether the plant involved is the host of the pathogen involved. The mechanism of the two types of disease resistance has a lot of common ground and some obvious differences. Host resistance has two levels of immune (PI) triggered by pathogen-related molecular patterns and immune (ETI) triggered by pathogen-effector proteins. The interaction of tomato and leaf mold (Cladosporium fulvum) is a typical gene-to-gene host resistance, and the resistance to ETI of the leaf mold carrying the complementary Avr gene is mediated by the anti-leaf mold gene of tomato. Bacterial leaf blight of rice (Xanthoryzae pv.) oryzae, xoo), which is narrow in range, induces a strong allergic response (hr) and non-host resistance in non-host tobacco (nictiana bentahams). The paper mainly studies the two problems. Firstly, the traditional concept holds that the gene expression and regulation of the plant disease resistance (R) gene which mediated the ETI does not exist after transcription and post-transcriptional level. In addition, the mechanism of Xoo-induced HR and non-host resistance in the tobacco was unclear. In this study, a variety of strategies were used to identify the non-host resistant regulatory genes and to identify the inhibitory effects and mechanisms of Agrobacterium on the non-host resistance. The following main results were obtained in this study: 1. Preliminary study on the regulation and regulation of the gene expression of the anti-leaf genes of tomato and the expression of p27-9 gene Mechanism. The mechanism of gene expression and regulation of p27-4 and p27-9 was carried out from the two levels of transcription level, DNA methylation, and post-transcriptional gene expression. At the transcriptional level, the expression of survivin gene was inhibited by high temperature, and Avr strongly induced the expression of survivin gene. The expression of caspase-4 increased with the increase of plant age of tomato, while the expression of caspase-9 was not significant. The promoter DNA methylation is involved in the high temperature inhibition and the expression and regulation of the caspase-9 gene induced by Avr9 at normal temperature. In addition, the induction of Avr4 and Avr9 on the expression of p27-4 and p27-9 genes may be methylated during the cooling process from high temperature to normal temperature. The effects of methyl parathion and demethylase gene on the expression of parathion gene in tomato were analyzed by VGS technique. It is shown that these tomato parathion and demethylase genes may be involved in the expression of survivin. At the post-transcriptional level, we predicted that a miR gene with a length of 21nt at the same time, both for both the target caspase-4 and the caspase-9 gene NA. Through the analysis of the target sequence of the miRNA and the co-expression of the precursor sequence and the like, it is confirmed that the miRNA can effectively degrade the IL-4 and the IL-9mR. NA. 2. Screening of a batch of EHR-4/ Avr4-mediated HR According to the functional analysis of the differentially expressed protein corresponding gene and the VIGS screening of the cigarette cDNA library, 28 genes involved in the regulation of the R-4/ Avr4-mediated HR are screened from over 500 gene silencing structures, wherein the ribose core Protein RNP, a DNA binding protein and a Dof p27 protein are regulating the HR, while a ring finger protein, a BTB/ POZ and TAZ domain protein, a CP binding protein, a SUMO binding enzyme SCE1, a red chlorophyll metabolism product reductase RCCR, and the like, and has 22 protein negative regulatory proteins 4/ Avr4. The interaction system of Xoo's non-host resistance was established and optimized. The results showed that H2O2 accumulation was Xoo-induced HR and non-mail. The results of the study showed that the most rapid growth of the leaves at 28 鈩，

本文編號(hào)：2302958