异三聚体G蛋白参与小麦抗叶锈病反应的研究
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摘要
由小麦叶锈菌(Puccinia triticina)引起的小麦叶锈病是世界范围内小麦上最重要的病害之一。应用抗病品种提高寄主植物的抗病性是控制小麦叶锈病的有效途径。而这一切又依赖于小麦抗叶锈病反应分子机制的全面了解。利用拟南芥、番茄、水稻等为材料研究表明,异三聚体G蛋白信号参与植物生长、发育以及对激素、光、病原物侵袭反应的调控。因此,研究异三聚体G蛋白在小麦抗叶锈病反应中的作用有助于了解小麦抗叶锈病反应的分子机制,对控制小麦叶锈病具有重要的意义。本实验以小麦抗叶锈病近等基因系Lr1和叶锈病菌典型的亲和互作组合与非亲和互作组合为材料,在小麦与叶锈病菌的互作表型、抗病生理水平、和基因表达水平对G蛋白的作用进行了研究,取得如下实验结果:
(1)用G蛋白的激活剂GTPγS、Mastoparan-7和抑制剂PTX预处理离体培养的小麦叶片后,分别接种毒力小种05-22-64和无毒力小种05-8-63①,观察表型发现与只接种毒力小种05-22-64的对照相比G蛋白的激活剂可以明显延长叶锈病的潜育期,从而延缓叶锈病的发生。用G蛋白的抑制剂PTX处理后发病情况与对照相比没有明显变化。
(2)用G蛋白的激活剂GTPγS、Mastoparan-7和抑制剂PTX预处理离体培养的小麦叶片后接种无毒小种05-8-63①,分别在接种后0h、24h、48h、72h、96h、120h、144h取样,测定小麦叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性,与单独接种毒力叶锈菌小种05-22-64和未处理直接接种无毒叶锈菌小种05-8-63①后这三种酶的活性比较,发现用G蛋白的激活剂(GTPγS,Mastoparan-7)处理后可诱导防卫反应酶的活性升高,与无毒小种接种处理后防卫酶活性有类似的变化趋势。而用抑制剂百日咳毒素(PTX)处理可以明显抑制无毒小种对防卫酶活性的诱导。
(3)采用半定量RT-PCR的方法研究了不同的互作组合中G蛋白α亚基基因的表达情况,结果显示无毒叶锈菌菌株05-8-63①可以诱导叶片中G蛋白基因表达量升高,在接种后48h表达量达到峰值。毒性菌株05-22-64抑制G蛋白基因的表达,在接种后6h表达量已有所下降,并在12h时达到最低点。
综合以上结果可以看出异三聚G蛋白同小麦的抗叶锈病反应密切相关,可能参与了小麦抗叶锈病的信号转导过程。
Wheat leaf rust caused by Puccinia triticina is one of the major diseases in wheat. Applying the disease-resistant varieties is a promising way to control wheat leaf rust if the molecular mechanism of wheat resistance are fully understood.Accumulating evidence from Arabidopsis thalina,tomato and rice demonstrated that heterotrimeric G protein play an essential role in regulating plant growth,development,and the response to hormone, light and pathogens.
The roles of heterotrimeric G protein in wheat resistance to its leaf rust disease were investigated at phenotype,bio-chemical and molecular level,respectively,using wheat NIL Lrl and P.triticina with different virulence as materials.The results were as follow:
(1)The detached leave were pretreated with heterotrimeric G protein activator(GTPγS, Mastoparan-7)or inhibitor PTX,and then were inoculated with virulent leaf rust race 05-22-64 or avirulent race 05-8-63①,respectively,the infection types were observed at the 9th day.It was found that the occurrence of leaf rust disease was delayed 4 days by pretreating the leaves with the activators when compared to the untreated leaves,whereas no difference in the development of wheat leaf rust disease was observed between the leaves pretreated with the inhibitor of heterotrimeric G protein and the untreated ones if both were inoculated with the avirulent race 05-8-63①.
(2)The profile of enzyme activities of POD,PAL,PPO were followed in wheat leaf after the detached leave were treated with GTPγS and Mastoparan-7,or inoculated with the avirulent race 05-8-63①alone,or the detached leave were first treated with and then inoculated with the avirulent race 05-8-63①.The results showed that the activities of these defensive enzymes were induced by treating wheat leave with heterotrimeric G protein activators,comparable to that of leave inoculated with the avirulent race;these enzyme activities were inhibited by heterotrimeric G protein inhibitor PTX,which could be induced by inoculation of the avirulent race.
(3)The expression of the heterotrimeric G protein a subunit gene was evaluated by semi-quantitative RT-PCR after inoculating the wheat with the virulent race and avirulent race,respectively.The results indicated that the avirulent race enhanced the expression of the heterotrimeric G proteinαsubunit gene,the expression level reached the peak at 48 h. However,the virulent race inhibited its expression,the expression began to decrease at 6 h and reached the lowest level at 12 h.
Taken together,these data demonstrated that heterotrimeric G protein might be involved in the signaling of wheat defense response to its leaf rust disease.
(1)用G蛋白的激活剂GTPγS、Mastoparan-7和抑制剂PTX预处理离体培养的小麦叶片后,分别接种毒力小种05-22-64和无毒力小种05-8-63①,观察表型发现与只接种毒力小种05-22-64的对照相比G蛋白的激活剂可以明显延长叶锈病的潜育期,从而延缓叶锈病的发生。用G蛋白的抑制剂PTX处理后发病情况与对照相比没有明显变化。
(2)用G蛋白的激活剂GTPγS、Mastoparan-7和抑制剂PTX预处理离体培养的小麦叶片后接种无毒小种05-8-63①,分别在接种后0h、24h、48h、72h、96h、120h、144h取样,测定小麦叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性,与单独接种毒力叶锈菌小种05-22-64和未处理直接接种无毒叶锈菌小种05-8-63①后这三种酶的活性比较,发现用G蛋白的激活剂(GTPγS,Mastoparan-7)处理后可诱导防卫反应酶的活性升高,与无毒小种接种处理后防卫酶活性有类似的变化趋势。而用抑制剂百日咳毒素(PTX)处理可以明显抑制无毒小种对防卫酶活性的诱导。
(3)采用半定量RT-PCR的方法研究了不同的互作组合中G蛋白α亚基基因的表达情况,结果显示无毒叶锈菌菌株05-8-63①可以诱导叶片中G蛋白基因表达量升高,在接种后48h表达量达到峰值。毒性菌株05-22-64抑制G蛋白基因的表达,在接种后6h表达量已有所下降,并在12h时达到最低点。
综合以上结果可以看出异三聚G蛋白同小麦的抗叶锈病反应密切相关,可能参与了小麦抗叶锈病的信号转导过程。
Wheat leaf rust caused by Puccinia triticina is one of the major diseases in wheat. Applying the disease-resistant varieties is a promising way to control wheat leaf rust if the molecular mechanism of wheat resistance are fully understood.Accumulating evidence from Arabidopsis thalina,tomato and rice demonstrated that heterotrimeric G protein play an essential role in regulating plant growth,development,and the response to hormone, light and pathogens.
The roles of heterotrimeric G protein in wheat resistance to its leaf rust disease were investigated at phenotype,bio-chemical and molecular level,respectively,using wheat NIL Lrl and P.triticina with different virulence as materials.The results were as follow:
(1)The detached leave were pretreated with heterotrimeric G protein activator(GTPγS, Mastoparan-7)or inhibitor PTX,and then were inoculated with virulent leaf rust race 05-22-64 or avirulent race 05-8-63①,respectively,the infection types were observed at the 9th day.It was found that the occurrence of leaf rust disease was delayed 4 days by pretreating the leaves with the activators when compared to the untreated leaves,whereas no difference in the development of wheat leaf rust disease was observed between the leaves pretreated with the inhibitor of heterotrimeric G protein and the untreated ones if both were inoculated with the avirulent race 05-8-63①.
(2)The profile of enzyme activities of POD,PAL,PPO were followed in wheat leaf after the detached leave were treated with GTPγS and Mastoparan-7,or inoculated with the avirulent race 05-8-63①alone,or the detached leave were first treated with and then inoculated with the avirulent race 05-8-63①.The results showed that the activities of these defensive enzymes were induced by treating wheat leave with heterotrimeric G protein activators,comparable to that of leave inoculated with the avirulent race;these enzyme activities were inhibited by heterotrimeric G protein inhibitor PTX,which could be induced by inoculation of the avirulent race.
(3)The expression of the heterotrimeric G protein a subunit gene was evaluated by semi-quantitative RT-PCR after inoculating the wheat with the virulent race and avirulent race,respectively.The results indicated that the avirulent race enhanced the expression of the heterotrimeric G proteinαsubunit gene,the expression level reached the peak at 48 h. However,the virulent race inhibited its expression,the expression began to decrease at 6 h and reached the lowest level at 12 h.
Taken together,these data demonstrated that heterotrimeric G protein might be involved in the signaling of wheat defense response to its leaf rust disease.
引文
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