水稻抗稻瘟病菌质膜蛋白的蛋白质组学分析
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摘要
稻瘟病是由子囊菌Magnaporthe oryzae (Hebert) Barr[无性世代为Pyricularia grisea (Cooke) Sacc]引起的最严重的水稻三大病害之一,严重影响水稻的产量和品质。稻瘟病菌在侵入寄主细胞时,初始侵染菌丝和扩展菌丝在侵染过程中都不直接与侵染细胞的胞质接触,而是通过一层来源于侵入细胞的细胞质膜膜状结构(EIHM)与侵染细胞的胞质隔离。植物质膜蛋白是一类具有独特结构的蛋白质,镶嵌于膜脂的特性使这一类蛋白处于细胞与外界的交界部位,介导细胞与外界之间的信号传导,并执行很多基本的和重要的细胞生物学功能,在植物抵御病原物侵染防卫反应信号传导途径中也起着重要的作用。
为了研究水稻叶片质膜蛋白在水稻抗稻瘟病过程中的作用,本文采用改进的质膜蛋白提取、纯化和双向电泳方法,分析了太湖流域抗稻瘟病品种黑壳子粳在稻瘟病菌菌株北1接种前和接种后24h的叶片质膜蛋白表达变化,发现有28个蛋白的表达在接种前后存在差异。其中21个为上调表达,7个为下调表达。通过对这28个蛋白点进行质谱鉴定和二级串联质谱MS/MS鉴定,结果表明28个诱导表达蛋白可分为五类：(1)参与植物生长代谢相关的酶类：二磷酸核酮糖羧化酶/加氧酶(RuBisCO)、Y衰老相关蛋白、ATP酶；(2)参与植物信号转导的相关蛋白：磷脂酶C(PLC)、膜连蛋白(Annexin)和谷胱甘肽-s-转移酶(GST)；(3)与植物物质合成分解调节有关的蛋白：ACC氧化酶(ACO)、调控质膜多肽(DREPP)、ATP合成酶(ATP synthase)、磺基转移酶(SULT);(4)与抗氧化系统相关的蛋白：抗坏血酸过氧化物酶(APX)、酪氨酸激酶(PTKs);(5)功能未知的蛋白：14-3-3类似蛋白。
针对质谱鉴定与前人已有的研究结果,我们选取了一个Remorin蛋白对其进行克隆和相关的功能验证,已有的研究表明Remorin蛋白是一个植物专一性蛋白,其广泛地参与拟南芥,烟草等植物的生物胁迫和非生物胁迫过程,在豆科和茄科植物中都参与植物的抗病过程,但是目前对于其具体的抗病机理还不清楚。我们通过对稻瘟病抗感水稻品种在接种稻瘟病不同时间段Remorin蛋白的表达分析发现：OsREM1.2蛋白在抗感品种的表达方式存在明显差异,在抗病品种的表达水平明显高于感病品种中的表达水平,说明OsREM1.2蛋白可能在水稻品种黑壳子粳的抗稻瘟病过程中发挥重要作用。
Blast, caused by the filamentous ascomycete Mangnaporthe oryzae (Hebert) Barr,is one of the three most devastating of rice diseases, and Serious impact on rice yield and quality. when the Mangnaporthe oryzae infect the Epidermal cells of rice, both the bulbous invasive hyphae and the extensional hyphae are outside of the host cells. they all surrounded by a plant-derived extra-invasive hyphae membrane(EIHM).
Plant plasma membrane protein is one of the unique structure of the protein. Features embeded in the membrane of cells so that this class of protein sin the junction area with the outside world. Mediated signal transduction between the outside world and perform many basic and important cell biological functions, also plays an important role in plant defense responses against pathogen infection through take part in the signal transduction pathway.
In order to study role of the plasma membrane proteins in the process of the rice blast resistance. In this paper, we used an improved membrane protein extraction method and two-dimensional electrophoresis method to analyze the differences of the plasma membrane proteins expression, which extract from the leaves of the Heikezijing that a Japonica rice variety from Taihu Lake region. By comparing before and 24 hours after inoculation,we found that 28 proteins expression was different.which 20 were up-regulated expression and 8 were down-regulated expression.28 protein spots with predicted functions in plant defense were identified by MS and MS/MS. All of the induced proteins can be divided in to five types:first associate with metabolizing, such as ribulose-1,5-bisphosphate carboxylase/oxygenaselarge (RuBisCO),Y putative senescence associated protein; second, associate with signal transduction, such as annexin and glutathione-S-transferase (GST); third,associate with regulating peptide,such as ACC oxidase (ACO),Developmentaly regulated plasma membrane polypeptide (DREPP);fourth, associate with detoxification enzymes, such as Ascorbate peroxidase (APX),Tyrosine kinase (PTKs); fifth, hypothetical protein, such as 14-3-3 like protein.
Considering the MS identification results and previous studies, we cloned a remorin protein from the rice and do some research for its function. Previous studies showed that remorin protein is a plant specific protein. It is take part in Arabidopsis, tobacco and other plants in the biotic stress and abiotic stress process extensively. In leguminous and solanaceous plants are involved in plant disease resistance, But now for the specific resistance mechanism is still unclear. By through analysis the impression level of the remorin protein in resistant and susceptible varieties between different time segment.We found that the expression of OsREM1.2 inresistant and susceptible varieties are obvious differences. The expression in the resistant varieties was significantly higher than the expression level of susceptible. These means OsREM1.2 protein may play an important role in rice blast resistance.
为了研究水稻叶片质膜蛋白在水稻抗稻瘟病过程中的作用,本文采用改进的质膜蛋白提取、纯化和双向电泳方法,分析了太湖流域抗稻瘟病品种黑壳子粳在稻瘟病菌菌株北1接种前和接种后24h的叶片质膜蛋白表达变化,发现有28个蛋白的表达在接种前后存在差异。其中21个为上调表达,7个为下调表达。通过对这28个蛋白点进行质谱鉴定和二级串联质谱MS/MS鉴定,结果表明28个诱导表达蛋白可分为五类：(1)参与植物生长代谢相关的酶类：二磷酸核酮糖羧化酶/加氧酶(RuBisCO)、Y衰老相关蛋白、ATP酶；(2)参与植物信号转导的相关蛋白：磷脂酶C(PLC)、膜连蛋白(Annexin)和谷胱甘肽-s-转移酶(GST)；(3)与植物物质合成分解调节有关的蛋白：ACC氧化酶(ACO)、调控质膜多肽(DREPP)、ATP合成酶(ATP synthase)、磺基转移酶(SULT);(4)与抗氧化系统相关的蛋白：抗坏血酸过氧化物酶(APX)、酪氨酸激酶(PTKs);(5)功能未知的蛋白：14-3-3类似蛋白。
针对质谱鉴定与前人已有的研究结果,我们选取了一个Remorin蛋白对其进行克隆和相关的功能验证,已有的研究表明Remorin蛋白是一个植物专一性蛋白,其广泛地参与拟南芥,烟草等植物的生物胁迫和非生物胁迫过程,在豆科和茄科植物中都参与植物的抗病过程,但是目前对于其具体的抗病机理还不清楚。我们通过对稻瘟病抗感水稻品种在接种稻瘟病不同时间段Remorin蛋白的表达分析发现：OsREM1.2蛋白在抗感品种的表达方式存在明显差异,在抗病品种的表达水平明显高于感病品种中的表达水平,说明OsREM1.2蛋白可能在水稻品种黑壳子粳的抗稻瘟病过程中发挥重要作用。
Blast, caused by the filamentous ascomycete Mangnaporthe oryzae (Hebert) Barr,is one of the three most devastating of rice diseases, and Serious impact on rice yield and quality. when the Mangnaporthe oryzae infect the Epidermal cells of rice, both the bulbous invasive hyphae and the extensional hyphae are outside of the host cells. they all surrounded by a plant-derived extra-invasive hyphae membrane(EIHM).
Plant plasma membrane protein is one of the unique structure of the protein. Features embeded in the membrane of cells so that this class of protein sin the junction area with the outside world. Mediated signal transduction between the outside world and perform many basic and important cell biological functions, also plays an important role in plant defense responses against pathogen infection through take part in the signal transduction pathway.
In order to study role of the plasma membrane proteins in the process of the rice blast resistance. In this paper, we used an improved membrane protein extraction method and two-dimensional electrophoresis method to analyze the differences of the plasma membrane proteins expression, which extract from the leaves of the Heikezijing that a Japonica rice variety from Taihu Lake region. By comparing before and 24 hours after inoculation,we found that 28 proteins expression was different.which 20 were up-regulated expression and 8 were down-regulated expression.28 protein spots with predicted functions in plant defense were identified by MS and MS/MS. All of the induced proteins can be divided in to five types:first associate with metabolizing, such as ribulose-1,5-bisphosphate carboxylase/oxygenaselarge (RuBisCO),Y putative senescence associated protein; second, associate with signal transduction, such as annexin and glutathione-S-transferase (GST); third,associate with regulating peptide,such as ACC oxidase (ACO),Developmentaly regulated plasma membrane polypeptide (DREPP);fourth, associate with detoxification enzymes, such as Ascorbate peroxidase (APX),Tyrosine kinase (PTKs); fifth, hypothetical protein, such as 14-3-3 like protein.
Considering the MS identification results and previous studies, we cloned a remorin protein from the rice and do some research for its function. Previous studies showed that remorin protein is a plant specific protein. It is take part in Arabidopsis, tobacco and other plants in the biotic stress and abiotic stress process extensively. In leguminous and solanaceous plants are involved in plant disease resistance, But now for the specific resistance mechanism is still unclear. By through analysis the impression level of the remorin protein in resistant and susceptible varieties between different time segment.We found that the expression of OsREM1.2 inresistant and susceptible varieties are obvious differences. The expression in the resistant varieties was significantly higher than the expression level of susceptible. These means OsREM1.2 protein may play an important role in rice blast resistance.
引文
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