小麥抗條銹菌基因Yr10抗病通路的初步解析
發(fā)布時間：2018-10-29 18:56
【摘要】：小麥條銹病是由條形柄銹菌(Puccinia striiformis f.sp.tritici)引起的一類真菌病害,嚴重威脅我國小麥的生產(chǎn)。條形柄銹菌氣流傳播并且小種變異頻繁,所以小麥條銹病的防治一直處于被動狀態(tài)。大量的生產(chǎn)實踐和科研表明,培育和種植對條銹病具有抗性的小麥是最經(jīng)濟安全和有效的方法。Yr10作為一個重要的抗病基因,表現(xiàn)為全生育期抗性,對國內(nèi)大部分的條銹生理小種表現(xiàn)抗性。因此探究Yr10介導的抗病機制,明晰Yr10的抗病信號通路為小麥抗條銹品種的遺傳改良和選育提供理論基礎。本實驗通過實時熒光定量PCR技術分析Yr10在不同誘導下的表達模式及病程相關基因的表達模式,初步明確Yr10介導的抗病通路中相關基因的表達變化;通過測定小麥內(nèi)源水楊酸變化,探究Yr10的抗病信號通路與SA信號通路之間的聯(lián)系;通過組織學分析研究Yr10介導的非親和互作體系和親和互作體系中寄主及病原菌的發(fā)育變化;對實驗室前期酵母雙雜交篩選出的與Yr10互作的小麥基因TaMYB29,我們進一步驗證其在小麥與條銹菌互作中的功能。研究結果如下:(1)Yr10首先具有組織特異性,在根中積累最多,小穗中最少。受條銹菌的誘導表達水平上下微浮動,前12h表達下調(diào),24h表達上調(diào)達到峰值接著回復至將近原來的水平。病程相關基因PR1、PR2、PR5,在24h出現(xiàn)第一次表達上調(diào)的峰值,72h以后在非親和的寄主體內(nèi)迅速大量積累,這些PR基因可能位于Yr10抗病信號通路的下游發(fā)揮重要作用。(2)Yr10受外源激素ABA、SA、JA、ET的誘導2h、6h均下調(diào)表達,ABA、SA處理48h上調(diào)表達較明顯。內(nèi)源SA受外源ABA誘導含量增加,受條銹菌誘導非親和寄主內(nèi)源SA的變化趨勢與Yr10相反,并且含量低于親和組合,推測SA信號通路與Yr10的抗病信號通路關系密切,輔助Yr10共同調(diào)控著寄主的抗病反應。(3)不同互作體系中的組織學分析發(fā)現(xiàn)寄主受條銹菌侵染的24-48h迸發(fā)活性氧,細胞壞死快速增加,至96h達到峰值,親和寄主中DAB染色面積和壞死面積較少。條銹菌在非親和寄主體內(nèi)生長擴展受限,至侵染后期分支數(shù)、分支長度、吸器數(shù)和菌落面積極顯著小于親和組合。(4)同源克隆出小麥TaMYB29,通過亞細胞定位發(fā)現(xiàn)TaMYB29在細胞核中發(fā)揮功能。酵母自激活驗證TaMYB29具有較弱的自激活活性。TaMYB29受外源激素誘導表達變化,在12-48h表達上調(diào)。VIGS瞬時沉默TaMYB29,接菌處理后非親和小麥AVS+Yr10的壞死斑減少;PVX介導的病毒表達系統(tǒng)過表達TaMYB29,能引起煙草葉片的PCD,說明TaMYB29能夠促進植物細胞的壞死。
[Abstract]:Wheat stripe rust is a kind of fungal disease caused by stripe stem rust (Puccinia striiformis f.sp.tritici), which is a serious threat to wheat production in China. The control of wheat stripe rust was always passive because of the airflow spread of stripe rust and frequent variation of wheat species. A large number of production practices and scientific research have shown that breeding and planting wheat resistant to stripe rust is the most economical and safe and effective method. As an important disease resistance gene, Yr10 is resistant to the whole growth period. It was resistant to most of the physiological races of stripe rust in China. Therefore, exploring the mechanism of disease resistance mediated by Yr10 and clarifying the signal pathway of disease resistance of Yr10 provide a theoretical basis for genetic improvement and breeding of wheat stripe rust resistant varieties. In this study, real-time fluorescent quantitative PCR was used to analyze the expression patterns of Yr10 and pathogenesis-related genes under different induction conditions, and the expression changes of related genes in disease-resistance pathway mediated by Yr10 were preliminarily determined. By measuring the changes of endogenous salicylic acid in wheat, the relationship between Yr10 signal pathway and SA signaling pathway was explored, and the development of host and pathogen in Yr10 mediated non-affinity interaction system and affinity interaction system was studied by histological analysis. For the wheat gene TaMYB29, which was screened by yeast two-hybrid and interacting with Yr10 in laboratory we further verify its function in the interaction between wheat and stripe rust. The results were as follows: (1) Yr10 had tissue specificity and accumulated the most in root and the least in spikelet. The induced expression level of Stripe rust fluctuated slightly, the expression was down-regulated at the first 12 hours, and reached the peak at 24 h, and then returned to the same level. The expression of pathogenesis-related gene PR1,PR2,PR5, was up regulated for the first time at 24 h, and accumulated rapidly in the non-compatible host after 72 h. These PR genes may play an important role in the downstream of the Yr10 signal pathway. (2) the expression of Yr10 was down-regulated at 2 h or 6 h induced by exogenous hormone ABA,SA,JA,ET, and increased significantly at 48 h after ABA,SA treatment. The content of endogenous SA was increased by exogenous ABA, and the change trend of endogenous SA of non-compatible host induced by Stripe rust was opposite to that of Yr10, and the content of endogenous SA was lower than that of affinity combination. It was inferred that the SA signaling pathway was closely related to Yr10 resistance signaling pathway. (3) histological analysis in different interaction systems showed that Ros burst from 24 to 48 hours after infection by Stripe rust, cell necrosis increased rapidly and reached its peak at 96 h. DAB staining area and necrotic area were less in affinity host. The number of branches, the length of branches, the number of haustens and the area of colony were significantly lower than those of compatible combinations. (4) Wheat TaMYB29, was cloned by homologous cloning. TaMYB29 was found to function in the nucleus by subcellular localization. Yeast self-activation showed that TaMYB29 had weak self-activation activity. TaMYB29 was induced by exogenous hormone and up-regulated at 12-48 h. VIGS transient silencing of TaMYB29, decreased the necrotic spot of AVS Yr10 in non-compatible wheat. Overexpression of TaMYB29, by PVX mediated virus expression system could induce PCD, in tobacco leaves, suggesting that TaMYB29 could promote the necrosis of plant cells.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S435.121.42
本文編號：2298513
[Abstract]:Wheat stripe rust is a kind of fungal disease caused by stripe stem rust (Puccinia striiformis f.sp.tritici), which is a serious threat to wheat production in China. The control of wheat stripe rust was always passive because of the airflow spread of stripe rust and frequent variation of wheat species. A large number of production practices and scientific research have shown that breeding and planting wheat resistant to stripe rust is the most economical and safe and effective method. As an important disease resistance gene, Yr10 is resistant to the whole growth period. It was resistant to most of the physiological races of stripe rust in China. Therefore, exploring the mechanism of disease resistance mediated by Yr10 and clarifying the signal pathway of disease resistance of Yr10 provide a theoretical basis for genetic improvement and breeding of wheat stripe rust resistant varieties. In this study, real-time fluorescent quantitative PCR was used to analyze the expression patterns of Yr10 and pathogenesis-related genes under different induction conditions, and the expression changes of related genes in disease-resistance pathway mediated by Yr10 were preliminarily determined. By measuring the changes of endogenous salicylic acid in wheat, the relationship between Yr10 signal pathway and SA signaling pathway was explored, and the development of host and pathogen in Yr10 mediated non-affinity interaction system and affinity interaction system was studied by histological analysis. For the wheat gene TaMYB29, which was screened by yeast two-hybrid and interacting with Yr10 in laboratory we further verify its function in the interaction between wheat and stripe rust. The results were as follows: (1) Yr10 had tissue specificity and accumulated the most in root and the least in spikelet. The induced expression level of Stripe rust fluctuated slightly, the expression was down-regulated at the first 12 hours, and reached the peak at 24 h, and then returned to the same level. The expression of pathogenesis-related gene PR1,PR2,PR5, was up regulated for the first time at 24 h, and accumulated rapidly in the non-compatible host after 72 h. These PR genes may play an important role in the downstream of the Yr10 signal pathway. (2) the expression of Yr10 was down-regulated at 2 h or 6 h induced by exogenous hormone ABA,SA,JA,ET, and increased significantly at 48 h after ABA,SA treatment. The content of endogenous SA was increased by exogenous ABA, and the change trend of endogenous SA of non-compatible host induced by Stripe rust was opposite to that of Yr10, and the content of endogenous SA was lower than that of affinity combination. It was inferred that the SA signaling pathway was closely related to Yr10 resistance signaling pathway. (3) histological analysis in different interaction systems showed that Ros burst from 24 to 48 hours after infection by Stripe rust, cell necrosis increased rapidly and reached its peak at 96 h. DAB staining area and necrotic area were less in affinity host. The number of branches, the length of branches, the number of haustens and the area of colony were significantly lower than those of compatible combinations. (4) Wheat TaMYB29, was cloned by homologous cloning. TaMYB29 was found to function in the nucleus by subcellular localization. Yeast self-activation showed that TaMYB29 had weak self-activation activity. TaMYB29 was induced by exogenous hormone and up-regulated at 12-48 h. VIGS transient silencing of TaMYB29, decreased the necrotic spot of AVS Yr10 in non-compatible wheat. Overexpression of TaMYB29, by PVX mediated virus expression system could induce PCD, in tobacco leaves, suggesting that TaMYB29 could promote the necrosis of plant cells.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S435.121.42
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