本文選題：辣椒疫霉 + 效應分子��； 參考：《揚州大學》2015年碩士論文

【摘要】：辣椒疫霉是一種寄主范圍廣的半活體營養(yǎng)型植物病原卵菌,常危害茄科、葫蘆科和豆類等蔬菜的安全生產。由于疫霉菌在進化上與真菌相差甚遠,現有的殺菌劑大多對植物疫病防治無效,加之品種抗病性利用尚不理想,一直以來還沒有有效的辣椒疫霉防控手段。因此,在傳統(tǒng)的防治措施之外,從疫霉菌與植物互作的角度出發(fā)深入了解疫霉菌致病的分子機理,對開發(fā)新的殺菌劑作用靶標、設計植物疫病控制策略具有重要價值。本論文首先研究了辣椒疫霉在侵染前期差異表達的基因種類。通過對侵染前期三個關鍵階段(菌絲、游動孢子和萌發(fā)休止孢)的轉錄組測序及生物信息學分析,篩選出98個在這些階段差異表達的效應分子編碼基因,其中包含25個RXLR、9個CRN和16個Elicitin編碼基因。利用半定量RT-PCR技術,分析獲得的效應分子編碼基因在辣椒疫霉侵染階段(本氏煙接種后1.5、3、6、12、24、36、72h)和侵染前期階段(菌絲、游動孢子囊、游動孢子和萌發(fā)休止孢)的表達水平。結果發(fā)現35個基因在辣椒疫霉侵染本氏煙的過程中差異表達,包括RXLR(12個)、CRN(5個)、Elicitin(13個)、PcF/SCR(2個)和NLP(3個)基因。不同類型效應分子在侵染過程中的轉錄模式不同,同一類型的不同效應分子的轉錄模式也存在差異。通過高保真擴增技術將這35個侵染階段差異表達的效應基因克隆到馬鈴薯X病毒(Potato virus X, PVX)表達載體中,利用農桿菌介導的瞬時表達法分析這些基因是否能夠引起本氏煙植物的細胞死亡(Plant cell death, PCD)。結果表明,4個Elicitin、1個NLP以及1個RXLR效應分子均能誘導本氏煙的PCD。為進一步明確效應分子引起PCD的功能區(qū)域,對其中的RXLR效應分子Pc129113進行了缺失和替換突變分析,構建了13個突變體。結果顯示,第1和第2個W-Y-L domain對其觸發(fā)本氏煙PCD的功能是必需的,第81-312位氨基酸殘基對其維持觸發(fā)PCD的功能很重要。除上述6個基因之外,其余的基因不能誘導本氏煙產生PCD。本研究分析了RXLR和CRN效應分子對本氏煙PCD的抑制情況,分析其是否具有毒性因子的作用。所選取的觸發(fā)本氏煙PCD的效應分子包括INF1、BAX、PsCRN63、PsojNIP、PsAvh241、R3a/AVR3a。結果表明,大多數效應分子(15/17)具有抑制寄主植物PCD的毒性功能。其中,效應分子中64.7%能夠抑制INF1所誘導的PCD,82.4%能夠抑制BAX誘導的PCD,76.5%能夠抑制PsCRN63誘導的PCD,88.2%能夠抑制PsAvh241和PsojNIP所誘導的PCD,而只有17.6%能夠抑制AVR3a/R3a組合所誘導的PCD。預測含有典型的核定位信號(NLS)的RXLR效應分子Pc503142能夠抑制本氏煙產生的PCD。為明確該效應分子的功能區(qū)域,對其進行了缺失和替換突變分析,構建了4個突變體。結果顯示其NLS的缺失導致其抑制PCD毒力功能的喪失,表明該效應分子可能是在寄主細胞核內發(fā)揮作用。為進一步明晰辣椒疫霉效應分子是否具有毒性功能,本研究利用辣椒疫霉菌株接種已經表達效應分子的本氏煙葉片,測量在其表達的情況下,病原菌的侵染是否更加劇烈。結果表明,兩個CRN效應分子(Pc559084和Pc563418)在本氏煙上的表達能夠顯著促進辣椒疫霉侵染本氏煙。為明確效應分子在進化過程中面臨的寄主選擇壓力,分析了來自全國不同地區(qū)的36個辣椒疫霉菌株中RXLR和PcF/SCR效應分子的序列多態(tài)性。結果發(fā)現,三個RXLR效應分子(Pe107349、Pc503142和Pc129113)均有豐富的序列多態(tài)性,PcF/SCR效應分子Pc96045家族至少含有4個成員。結果說明,這些效應分子可能在侵染寄主植物的過程中起著重要的作用,在進化的過程中面臨著很大的寄主選擇壓力。為進一步分析上述效應分子的功能,在辣椒疫霉中進行基因沉默分析,測定轉化子的致病力和生物學性狀是否產生變化。對3個RXLR效應分子(Pe107349、Pc503142和Pc129113)進行基因沉默,結果發(fā)現這三個基因的沉默均導致辣椒疫霉對本氏煙的致病力降低。而且,基因Pc503142的沉默轉化子對外源活性氧的耐受性降低,表明Pc503142可能在抑制本氏煙抗病相關的活性氧途徑中發(fā)揮作用。類似地,基因沉默實驗發(fā)現PcF/SCR效應分子Pc96045對辣椒疫霉的致病性具有重要作用。綜上所述,基因沉默結果表明,RXLR和PcF/SCR效應分子在辣椒疫霉侵染寄主植物的過程中起著重要的作用。本論文通過轉錄組測序及RT-PCR分析,獲得了35個在生長發(fā)育和侵染階段差異表達的辣椒疫霉效應分子；利用農桿菌浸潤法在本氏煙上瞬時表達這些效應分子,明確了效應分子抑制植物免疫反應的毒性功能,或者觸發(fā)植物免疫反應的無毒性功能；進一步利用CaCl2-PEG介導的原生質體轉化技術和dsRNA介導的基因瞬時沉默技術分析效應分子的功能,明確了4個效應分子(Pc107349、Pc503142、Pc129113和Pc96045)對辣椒疫霉的致病性具有重要作用。本論文通過差異表達基因篩選、克隆和功能分析,較為系統(tǒng)地分析了重要效應分子在辣椒疫霉侵染過程中的作用,為全面解析辣椒疫霉的致病機理、有效防控由辣椒疫霉引起的植物疫病提供了堅實的基礎。
[Abstract]:Phytophthora capsici is a kind of semi living vegetative oobacteria with wide host range, which often endangering the safe production of vegetables such as Solanaceae, cucurbit and legumes. Because the Phytophthora has evolved far away from fungi, most of the existing fungicides are not effective for plant disease prevention and control, and the use of the disease resistance is still not ideal, and has not yet been found. Effective means of prevention and control of Phytophthora capsici, therefore, in addition to the traditional prevention and control measures, from the angle of Phytophthora and plant mutual understanding of the molecular mechanism of Phytophthora pathogenicity, the development of a new bactericide target target, the design of plant disease control strategy is of great value. This paper first studied the poor early infection of Phytophthora capsici in the early infection By sequencing and bioinformatics analysis of the three critical stages (mycelia, spores and germinating spore), 98 effector genes, including 25 RXLR, 9 CRN and 16 Elicitin encoding genes, were screened by the sequencing of the transcriptional group of the early infecting stage (mycelia, spore and germinating spore), and the semi quantitative RT-PCR technique was used. The expression level of the effector molecular coding gene was analyzed in the infecting stage of Phytophthora capsici (1.5,3,6,12,24,36,72h after inoculation of benthia paprika) and the stage of early infection (mycelium, sporo sporsa, spores and germinating spore). The results showed that the 35 genes were expressed differently in the process of infection by Phytophthora capsici, including RXLR (12), C RN (5), Elicitin (13), PcF/SCR (2) and NLP (3) genes. The transcriptional patterns of different types of effector molecules are different in the infection process, and the transcription patterns of the same type of different effector molecules are also different. By high fidelity amplification, the effect genes of the 35 infection stages are cloned to the potato X virus (Potato viru). S X, PVX) expression vector, using Agrobacterium mediated transient expression to analyze whether these genes can cause cell death of Plant cell death (PCD). The results show that the PCD. of 4 Elicitin, 1 NLP and 1 RXLR effector molecules can induce the functional region of PCD. 13 mutants were constructed by the deletion and replacement mutation analysis of the RXLR effect molecule Pc129113. The results showed that first and second W-Y-L domain were necessary to trigger the function of the PCD, and the 81-312 amino acid residue was important for its maintenance of the function of triggering PCD. In addition to the above 6 genes, the remaining genes could not be induced. The effect of RXLR and CRN effect molecules on the PCD of benfic smoke was analyzed and the effects of the toxic factors were analyzed. The selected effector molecules that trigger the PCD of PCD. include INF1, BAX, PsCRN63, PsojNIP, PsAvh241, and R3a/AVR3a. results show that most of the effector molecules (15/17) have inhibition of host plants. Among them, 64.7% of the effector molecules can inhibit the PCD induced by INF1, 82.4% can inhibit BAX induced PCD, 76.5% can inhibit PCD induced by PsCRN63, 88.2% can inhibit PCD induced by PsAvh241 and PsojNIP, and only 17.6% can inhibit PCD. prediction induced by AVR3a/R3a combination containing typical nuclear positioning signal (NLS) The effector molecule Pc503142 can inhibit the PCD. produced by Benedict smoke to clarify the functional region of the effector molecule, and the deletion and substitution mutation analysis of the effector molecule is used to construct 4 mutants. The result shows that the deletion of NLS leads to the loss of its inhibition of PCD toxicity, indicating that the effector molecule may play a role in the host nucleus. One step is to clarify whether the Phytophthora capsicum has a toxic function. This study uses the Phytophthora capsici strain to inoculate the tobacco leaf slices that have expressed the effector molecules. In the case of its expression, the infection of the pathogen is more intense. The results show that the expression of the two CRN effect molecules (Pc559084 and Pc563418) on the tobacco can be shown to be significant. In order to promote the infection of Phytophthora capsici, the sequence polymorphism of RXLR and PcF/SCR effect molecules from 36 Phytophthora capsici strains from different regions of the country were analyzed. The results showed that three RXLR effector molecules (Pe107349, Pc503142 and Pc129113) had a rich sequence. Polymorphisms, the PcF/SCR effect molecule Pc96045 family contains at least 4 members. The results indicate that these effector molecules may play an important role in the process of infecting host plants and face great host selection pressure during the process of evolution. In order to further analyze the function of the above effect, gene silencing in Phytophthora capsici Analysis of the changes in the pathogenicity and biological characters of the transformants. Gene silencing of 3 RXLR effect molecules (Pe107349, Pc503142 and Pc129113) was carried out. The results showed that the silence of the three genes resulted in the decrease of the pathogenicity of Phytophthora capsici and the tolerance to exogenous reactive oxygen species by the silenced transformant of Pc503142. The decrease in sex indicates that Pc503142 may play a role in inhibiting the active oxygen pathway related to the disease resistance of benchic tobacco. Similar, gene silencing experiments have found that the PcF/SCR effect molecule Pc96045 plays an important role in the pathogenicity of Phytophthora capsici. To sum up, the gene silencing results indicate that the RXLR and PcF/SCR effectors infected host plants in Phytophthora capsici. In this paper, 35 Phytophthora capsicum effector molecules were obtained by transcriptional sequencing and RT-PCR analysis, and the effector molecules were instantaneously expressed on the tobacco by Agrobacterium tumefacience method, and the toxic function of the effector molecules to inhibit the plant immune response was clearly defined. Or triggering the nontoxic function of the plant immune response; further using CaCl2-PEG mediated protoplast transformation and dsRNA mediated gene transient silence technique to analyze the function of the effector molecules, it is clear that 4 effector molecules (Pc107349, Pc503142, Pc129113 and Pc96045) have an important role in the pathogenicity of Phytophthora capsici. Through the screening of differentially expressed genes, cloning and functional analysis, the role of important effector molecules in the infection process of Phytophthora capsici was systematically analyzed, which provided a solid foundation for the comprehensive analysis of the pathogenic mechanism of Phytophthora capsici and the effective prevention and control of Phytophthora plague caused by Phytophthora capsici.

【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S432.4

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