小麦抗条锈菌基因Yr10抗病通路的初步解析
发布时间：2018-10-29 18:56
【摘要】：小麦条锈病是由条形柄锈菌(Puccinia striiformis f.sp.tritici)引起的一类真菌病害,严重威胁我国小麦的生产。条形柄锈菌气流传播并且小种变异频繁,所以小麦条锈病的防治一直处于被动状态。大量的生产实践和科研表明,培育和种植对条锈病具有抗性的小麦是最经济安全和有效的方法。Yr10作为一个重要的抗病基因,表现为全生育期抗性,对国内大部分的条锈生理小种表现抗性。因此探究Yr10介导的抗病机制,明晰Yr10的抗病信号通路为小麦抗条锈品种的遗传改良和选育提供理论基础。本实验通过实时荧光定量PCR技术分析Yr10在不同诱导下的表达模式及病程相关基因的表达模式,初步明确Yr10介导的抗病通路中相关基因的表达变化;通过测定小麦内源水杨酸变化,探究Yr10的抗病信号通路与SA信号通路之间的联系;通过组织学分析研究Yr10介导的非亲和互作体系和亲和互作体系中寄主及病原菌的发育变化;对实验室前期酵母双杂交筛选出的与Yr10互作的小麦基因TaMYB29,我们进一步验证其在小麦与条锈菌互作中的功能。研究结果如下:(1)Yr10首先具有组织特异性,在根中积累最多,小穗中最少。受条锈菌的诱导表达水平上下微浮动,前12h表达下调,24h表达上调达到峰值接着回复至将近原来的水平。病程相关基因PR1、PR2、PR5,在24h出现第一次表达上调的峰值,72h以后在非亲和的寄主体内迅速大量积累,这些PR基因可能位于Yr10抗病信号通路的下游发挥重要作用。(2)Yr10受外源激素ABA、SA、JA、ET的诱导2h、6h均下调表达,ABA、SA处理48h上调表达较明显。内源SA受外源ABA诱导含量增加,受条锈菌诱导非亲和寄主内源SA的变化趋势与Yr10相反,并且含量低于亲和组合,推测SA信号通路与Yr10的抗病信号通路关系密切,辅助Yr10共同调控着寄主的抗病反应。(3)不同互作体系中的组织学分析发现寄主受条锈菌侵染的24-48h迸发活性氧,细胞坏死快速增加,至96h达到峰值,亲和寄主中DAB染色面积和坏死面积较少。条锈菌在非亲和寄主体内生长扩展受限,至侵染后期分支数、分支长度、吸器数和菌落面积极显著小于亲和组合。(4)同源克隆出小麦TaMYB29,通过亚细胞定位发现TaMYB29在细胞核中发挥功能。酵母自激活验证TaMYB29具有较弱的自激活活性。TaMYB29受外源激素诱导表达变化,在12-48h表达上调。VIGS瞬时沉默TaMYB29,接菌处理后非亲和小麦AVS+Yr10的坏死斑减少;PVX介导的病毒表达系统过表达TaMYB29,能引起烟草叶片的PCD,说明TaMYB29能够促进植物细胞的坏死。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.121.42
本文编号：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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.121.42
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