玉米抗白斑病菌侵染的主要生理机制及其谷胱甘肽过氧化物酶相关基因响应模式分析

Main physiological mechanisms of maize resistance to white spot pathogen infection and the response patterns of glutathione peroxidase related genes

  • 摘要: 【目的】探究玉米(Zea mays L.)白斑病对玉米叶片光合性状及生理指标的影响,以及谷胱甘肽过氧化物酶相关基因(GSH-Px)在白斑病病原菌胁迫下的表达模式,为玉米在白斑病菌胁迫下的功能和调控机制研究提供理论依据。【方法】在2个不同白斑病抗性玉米自交系(高抗ZHL908、高感QB2816)抽雄吐丝期进行人工接种白斑病病原菌试验,设无菌蒸馏水接种(CK)和病原菌接种(T)2个处理,于接种后0、48、72和168 h进行光合相关性状分析,测定过氧化氢酶(CAT)、过氧化物酶(POD)活性和谷胱甘肽(GSH)含量,实时荧光定量PCR检测ZmGSH-Px基因家族6个成员(GPX1GPX2GPX3GPX4GPX6GPX7基因)的表达模式,并对其进行生物信息学分析。【结果】白斑病菌侵染导致玉米叶片的叶绿素相对含量(SPAD值)、最大荧光(Fm)、潜在光化学量子效率(Fv/Fm)、非光化学淬灭系数(NPQ)明显下降,胞间二氧化碳浓度(Ci)、GSH含量、CAT和POD活性明显上升。高抗自交系和高感自交系感病叶片的GSH含量和POD活性对白斑病菌的响应时间存在差异,分别在接种后72和48 h出现更明显的响应。白斑病菌侵染72 h后,2个自交系的净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)均较CK显著(P<0.05)或极显著(P<0.01)下降。实时荧光定量PCR检测结果显示,接种病原菌后,6个基因呈现不同的响应模式,GPX1GPX3GPX4基因的表达模式和表达峰出现时间大体一致;GPX2GPX6GPX7基因在2个自交系中的表达模式存在明显差异。2个自交系中6个基因的相对表达量与各项理化指标的相关性存在差异,其中PnCi和NPQ分别与2个自交系中不同的基因有较高的相关系数。生物信息学分析结果显示,6个基因分别分布在玉米的6条染色体上,并且具有不同的理化性质。【结论】白斑病导致玉米的光合性能受损,高感自交系较高抗自交系更早表现损伤现象,相关ROS清除酶和小分子抗氧化剂也更早做出响应,推测GSH和POD在玉米对白斑病的抗性差异中发挥重要作用。ZmGSH-Px基因家族6个成员对白斑病菌胁迫的敏感程度存在差异,推测GPX1GPX3GPX4基因是玉米系统性获得白斑病抗性的参与基因,GPX2GPX6GPX7基因是玉米特异性抗病性的参与基因。

     

    Abstract: 【Objective】To study the effects of maize white spot disease on the photosynthetic traits and physiological indexes of maize leaves, and to explore the expression pattern of glutathione peroxidase related genes(GSH-Px) under the stress of white spot pathogen, which could provide theoretical basis for the study of the function and regulatory mechanism of maize under the stress of white spot pathogen. 【Method】Artificial inoculation of white spot pathogen(Epicoccum sorghinum) was carried out on 2 maize inbred lines with different resistances to white spot disease(highly resistant ZHL908 and highly susceptible QB2816) during the tasseling and silking stage. Two treatments were set up:inoculated with sterile distilled water(CK) and inoculated with pathogenic bacteria(T). Photosynthetic related traits were analyzed at 0, 48, 72 and 168 h after inoculation. The activities of catalase(CAT) and peroxidase(POD) as well as the content of glutathione(GSH), were determined. The expression patterns of 6 members(GPX1, GPX2, GPX3, GPX4, GPX6, GPX7 genes) of the ZmGSH-Px gene family were detected by real-time fluorescence quantitative PCR, and their bioinformatics analysis was conducted. 【Result】The infection of white spot pathogen led to obvious decrease in relative chlorophyll content(SPAD value), maximum fluorescence(Fm), potential photochemical quantum efficiency(Fv/Fm), nonphotochemical quenching coefficient(NPQ), but obvious increase in intercellular CO2 concentration(Ci), GSH content, CAT activity and POD activity in maize leaves. Notably, the response times of GSH content and POD activity in the infected leaves of the highly resistant and highly susceptible inbred lines to the white spot pathogen were different, and showed more obvious response at 72 and 48 h after inoculation respectively. After 72 h of white spot pathogen infection, net photosynthetic rate(Pn), stomatal conductance(Gs) and transpiration rate(Tr) in both inbred lines significantly(P<0.05) or extremely significantly(P<0.01) decreased compared to CK. The results of real-time fluorescence quantitative PCR showed that the 6 genes exhibited different response patterns after pathogen inoculation. The expression patterns and expression peak timings of GPX1, GPX3 and GPX4 genes were generally consistent, while the expression patterns of GPX2, GPX6 and GPX7 genes in the 2 inbred lines were greatly different. The correlation between relative expression and physiological indexes of the 6 genes in the 2 inbred lines were different. Among them, Pn, Ci and NPQ had high correlation coefficients with different genes in the 2 inbred lines respectively. The bioinformatics analysis results showed that the 6 genes were distributed on 6 different chromosomes of maize and had different physicochemical properties. 【Conclusion】White spot disease causes damage to the photosynthetic performance of maize, and the highly susceptible inbred line shows damage earlier than the highly resistant inbred line. Related ROS scavenging enzymes and small-molecule antioxidants also respond earlier. GSH and POD may play key roles in different resistance responses of maize to white spot disease. The 6 members of the ZmGSH-Px gene family show different degrees of sensitivity to white spot pathogen stress. It is speculated that GPX1, GPX3 and GPX4 genes are the genes involved in the systemic acquired resistance of maize to white spot disease, while GPX2, GPX6 and GPX7 genes are the genes involved in the specific disease resistance of maize.

     

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