大豆抗大豆疫霉根腐病的比较蛋白质组学研究
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摘要
由大豆疫霉引起的大豆疫霉根腐病是一种毁灭性的大豆病害,广泛分布于世界各大豆产区。目前该病已经在我国一些大豆主产区发生和为害,并在局部地区造成较大产量损失,对我国的大豆生产造成很大的危害。应用抗、耐病品种是防治该病害最经济、安全、有效的途径。
蛋白质是生理功能的执行者,是生命现象的直接体现者,对蛋白质结构和功能的研究将直接阐明生命的变化机制。基因转录水平的研究只能在一定程度上反映基因表达产物的变化,而真正发挥功能的蛋白质需要经过转录后加工、翻译调控以及翻译后加工等许多步骤和调控才能形成。因此,蛋白质组学已广泛应用于植物功能基因组学的研究。
本研究主要利用蛋白质组学技术对大豆和大豆疫霉互作过程中的蛋白质表达谱进行研究,并对鉴定蛋白点的功能进行分析。其目的主要是从蛋白表达水平上挖掘一些与大豆疫霉根腐病抗性有关的蛋白,进一步探讨这些蛋白在大豆—大豆疫霉互作过程中所参与的代谢途径以及大豆抗大豆疫霉根腐病的分子机制。主要研究结果如下：
1.大豆抗感种质接种大豆疫霉后的比较蛋白质组学研究
本实验采用下胚轴创伤接种法,利用2-DE结合MALDI-TOF/TOF技术探讨大豆抗病品种豫豆25和感病品种NG6255在接种大豆疫霉菌株PNJ1后大豆下胚轴蛋白在蛋白水平上的变化。结果表明,与未接种大豆疫霉的植株相比,共检测到46个差异表达蛋白点。在抗病品种豫豆25中检测到26个差异表达蛋白点,其中12个上调表达,14个下调表达；在感病品种NG6255中,检测到20个差异表达蛋白点,11个上调表达,9个下调表达。在这些差异表达蛋白中,26%与能量调控有关,15%涉及蛋白命运,11%涉及抗病防卫,11%与代谢有关,9%涉及蛋白合成,参与次生代谢的占4%,另外未知蛋白达到24%。
2.不同处理条件下大豆根、茎和叶的比较蛋白质组学分析
利用2-DE结合MALDI-TOF/TOF技术以感大豆疫霉菌株P6497的新沂小黑豆为材料,对SA、MeJA、ACC、H2O2、SNP、VB1以及P. sojae处理条件下大豆根、茎和叶进行蛋白质组学分析。结果表明：根中鉴定的35个蛋白点中,与抗病防卫及次生代谢有关各占23%,17%与代谢有关,其次是参与蛋白合成、蛋白命运、转录和代谢调控的,与细胞结构和信号转导有关的最少,各占3%,功能未知蛋白占8%。参与次生代谢有关的蛋白主要与乙烯、茉莉酸和类黄酮生物合成有关。茎中鉴定的58个蛋白点中,与能量和代谢有关的达到41%,参与抗病防卫和次生代谢的各占12%,10%与蛋白命运有关,与信号转导和转录有关的各占7%,与细胞结构有关的占4%,与蛋白合成和运输有关的各占2%。叶中鉴定到21个蛋白点,与能量有关的最多,达到62%,其次是与次生代谢、蛋白合成和代谢有关的,与蛋白命运和转录有关的最少,为5%。与能量有关的蛋白主要参与光合和光呼吸过程。
3.基于iTRAQ的大豆对大豆疫霉根腐病根的蛋白质表达谱分析
本实验采用根部创伤接种法,利用iTRAQ定量蛋白质组学技术研究抗大豆疫霉菌株Pm14材料苏88-M21接种后3d大豆根的蛋白质表达谱的变化。共鉴定出2136个蛋白,其中差异表达蛋白280个。这280个差异表达蛋白点中,与抗病防卫有关的最多,达到16%,14%与蛋白命运有关,12%参与代谢调控,与能量和蛋白合成有关的各为11%,8%与信号转导有关,6%与运输有关,参与次生代谢的占6%,4%与细胞结构有关,2%与转录有关,与细胞生长/分裂有关的最少,仅为1%,未知功能蛋白占9%。茉莉酸、乙烯、生长素以及泛素-蛋白酶体参与大豆抗大豆根腐病的抗性机制。
Phytophthora root and stem rot (PRR) caused by Phytophthora sojae M. J. Kaufmann&. J. W. Gerdemann (P. sojae), is one of the most devastating diseases in soybean [Glycine max (L.) Merr.] throughout soybean-growing regions all over the world. In China, P. sojae mainly exists in three soybean ecological regions including the Northeast region, the Huang-Huai-Hai region and the Southern region, and induces loss in yield and seed quality in main soybean production regions. Utilization of resistant varieties is the most economical and environmentally safe method for controlling this disease.
Protein is the physiological functions of the executive, and is also a direct manifestation of the phenomenon of life. The study of protein structure and function will directly clarify the mechanism of the change of life. Transcription levels only to a certain extent reflect the changes in product of gene expression, but the real function of the protein is formed, which need go through the steps of post-transcriptional processing, translation regulation and post-translational processing and regulation and so on. Thus, proteomics has been widely used in the study of plant functional genomics.
Using proteome, protein expression profiling and function of the differentially expressed proteins identified was analyzed in soybean-P. sojae interaction in this study. The main aims of this study were:(1) to mine the proteins involved in resistance-related P. sojae on the protein expression levels,(2) to further investigate the metabolic pathways of these proteins involved in the interaction of soybean-P. sojae,(3) to discover molecular mechanism of reisistance to P. sojae. The main results were as follows:
1-Proteomics study of changes in soybean lines resistant and sensitive to P. sojae
Using the hypocotyl inoculation technique, proteomics study of changes in soybean resistant Yudou25and sensitive NG6255to P. sojae strain PNJ1was analyzed. In the present study,46differentially expressed proteins were identified in soybean hypocotyls infected with P. sojae, using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight (MALDI-TOF/TOF). The expression levels of26proteins were significantly affected at various time points in the tolerant soybean line, Yudou25,(12up-regulated and14down-regulated). In contrast, in the sensitive soybean line, NG6255, only20proteins were significantly affected (11up-regulated and9down-regulated). Among these proteins,26%were related to energy regulation,15%to protein destination and storage,11%to defense against disease,11%to metabolism,9%to protein synthesis,4%to secondary metabolism, and24%were of unknown function.
2. Proteomics analysis of soybean roots, hypocotyls and leaves under different treatments
Using2-DE and MALDI-TOF/TOF, proteomics study of soybean roots, hypocotyls and leaves under different treatments of SA, MeJA, ACC, H2O2, SNP, VB1and P. sojae was analyzed. Xinyixiaoheidou was susceptible to P. sojae strain P6497. These findings were as follows:(1)35differently expressed proteins were identified from soybean roots by MALDI/TOF-TOF. Among these proteins,23%were relative to disease/defense and secondary metabolism, respectively;17%to energy, followed with protein synthesis, protein destination and storage, transcription and metabolism, and3%were of cell structure and signal transduction, respectively, and8%with unknown function. The proteins involved in secondary metabolism were relative to the biosynthesis of ethylene, jasmonic acid and flavonoids.(2)58differently expressed proteins were identified from soybean hypocotyls by MALDI/TOF-TOF. Among these58proteins,41%were relative to energy and metabolism,12%to disease/defense and secondary metabolism, respectively,10%to protein destination and storage,7%to signal transduction and transcription, respectively,4%to cell structure,2%were of protein synthesis and transporters, respectively.(3)21differently expressed proteins were idedtified from soybean leaves by MALDI-TOF/TOF. The21identified proteins involved in energy dominated62%, reapectively. Those involved in secondary metabolism, protein synthesis and metabolism categories followed. The proteins relative to energy involved in the process of photosynthesis and respiration.
3. iTRAQ-based protein profile analysis of soybean roots in response to P. sojae inoculation
Using the slant board technique, iTRAQ-based protein profile of soybean roots to P. sojae was analyzed. Su88-M21was resistant to P. sojae strain Pml4. A total of2136proteins were identified and a subset of280proteins was differentially expressed infected with P. sojae. Among of these280proteins, the proteins relative to defense against disease dominated16%,14%involved in protein destination and storage,12%involved in metabolism,11%to energy and protein synthesis, respectively,8%to signal transduction,6%involved in transporter and secondary metabolism, respectively,4%to cell structure. The last is relative to cell growth/division, only accounting for1%, and9%were of unknown function. The signal of jasmonid acid, ethylene, auxin and ubiquitin proteasome system is involved in the resistant mechanism of soybean to P. sojae.
蛋白质是生理功能的执行者,是生命现象的直接体现者,对蛋白质结构和功能的研究将直接阐明生命的变化机制。基因转录水平的研究只能在一定程度上反映基因表达产物的变化,而真正发挥功能的蛋白质需要经过转录后加工、翻译调控以及翻译后加工等许多步骤和调控才能形成。因此,蛋白质组学已广泛应用于植物功能基因组学的研究。
本研究主要利用蛋白质组学技术对大豆和大豆疫霉互作过程中的蛋白质表达谱进行研究,并对鉴定蛋白点的功能进行分析。其目的主要是从蛋白表达水平上挖掘一些与大豆疫霉根腐病抗性有关的蛋白,进一步探讨这些蛋白在大豆—大豆疫霉互作过程中所参与的代谢途径以及大豆抗大豆疫霉根腐病的分子机制。主要研究结果如下：
1.大豆抗感种质接种大豆疫霉后的比较蛋白质组学研究
本实验采用下胚轴创伤接种法,利用2-DE结合MALDI-TOF/TOF技术探讨大豆抗病品种豫豆25和感病品种NG6255在接种大豆疫霉菌株PNJ1后大豆下胚轴蛋白在蛋白水平上的变化。结果表明,与未接种大豆疫霉的植株相比,共检测到46个差异表达蛋白点。在抗病品种豫豆25中检测到26个差异表达蛋白点,其中12个上调表达,14个下调表达；在感病品种NG6255中,检测到20个差异表达蛋白点,11个上调表达,9个下调表达。在这些差异表达蛋白中,26%与能量调控有关,15%涉及蛋白命运,11%涉及抗病防卫,11%与代谢有关,9%涉及蛋白合成,参与次生代谢的占4%,另外未知蛋白达到24%。
2.不同处理条件下大豆根、茎和叶的比较蛋白质组学分析
利用2-DE结合MALDI-TOF/TOF技术以感大豆疫霉菌株P6497的新沂小黑豆为材料,对SA、MeJA、ACC、H2O2、SNP、VB1以及P. sojae处理条件下大豆根、茎和叶进行蛋白质组学分析。结果表明：根中鉴定的35个蛋白点中,与抗病防卫及次生代谢有关各占23%,17%与代谢有关,其次是参与蛋白合成、蛋白命运、转录和代谢调控的,与细胞结构和信号转导有关的最少,各占3%,功能未知蛋白占8%。参与次生代谢有关的蛋白主要与乙烯、茉莉酸和类黄酮生物合成有关。茎中鉴定的58个蛋白点中,与能量和代谢有关的达到41%,参与抗病防卫和次生代谢的各占12%,10%与蛋白命运有关,与信号转导和转录有关的各占7%,与细胞结构有关的占4%,与蛋白合成和运输有关的各占2%。叶中鉴定到21个蛋白点,与能量有关的最多,达到62%,其次是与次生代谢、蛋白合成和代谢有关的,与蛋白命运和转录有关的最少,为5%。与能量有关的蛋白主要参与光合和光呼吸过程。
3.基于iTRAQ的大豆对大豆疫霉根腐病根的蛋白质表达谱分析
本实验采用根部创伤接种法,利用iTRAQ定量蛋白质组学技术研究抗大豆疫霉菌株Pm14材料苏88-M21接种后3d大豆根的蛋白质表达谱的变化。共鉴定出2136个蛋白,其中差异表达蛋白280个。这280个差异表达蛋白点中,与抗病防卫有关的最多,达到16%,14%与蛋白命运有关,12%参与代谢调控,与能量和蛋白合成有关的各为11%,8%与信号转导有关,6%与运输有关,参与次生代谢的占6%,4%与细胞结构有关,2%与转录有关,与细胞生长/分裂有关的最少,仅为1%,未知功能蛋白占9%。茉莉酸、乙烯、生长素以及泛素-蛋白酶体参与大豆抗大豆根腐病的抗性机制。
Phytophthora root and stem rot (PRR) caused by Phytophthora sojae M. J. Kaufmann&. J. W. Gerdemann (P. sojae), is one of the most devastating diseases in soybean [Glycine max (L.) Merr.] throughout soybean-growing regions all over the world. In China, P. sojae mainly exists in three soybean ecological regions including the Northeast region, the Huang-Huai-Hai region and the Southern region, and induces loss in yield and seed quality in main soybean production regions. Utilization of resistant varieties is the most economical and environmentally safe method for controlling this disease.
Protein is the physiological functions of the executive, and is also a direct manifestation of the phenomenon of life. The study of protein structure and function will directly clarify the mechanism of the change of life. Transcription levels only to a certain extent reflect the changes in product of gene expression, but the real function of the protein is formed, which need go through the steps of post-transcriptional processing, translation regulation and post-translational processing and regulation and so on. Thus, proteomics has been widely used in the study of plant functional genomics.
Using proteome, protein expression profiling and function of the differentially expressed proteins identified was analyzed in soybean-P. sojae interaction in this study. The main aims of this study were:(1) to mine the proteins involved in resistance-related P. sojae on the protein expression levels,(2) to further investigate the metabolic pathways of these proteins involved in the interaction of soybean-P. sojae,(3) to discover molecular mechanism of reisistance to P. sojae. The main results were as follows:
1-Proteomics study of changes in soybean lines resistant and sensitive to P. sojae
Using the hypocotyl inoculation technique, proteomics study of changes in soybean resistant Yudou25and sensitive NG6255to P. sojae strain PNJ1was analyzed. In the present study,46differentially expressed proteins were identified in soybean hypocotyls infected with P. sojae, using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight (MALDI-TOF/TOF). The expression levels of26proteins were significantly affected at various time points in the tolerant soybean line, Yudou25,(12up-regulated and14down-regulated). In contrast, in the sensitive soybean line, NG6255, only20proteins were significantly affected (11up-regulated and9down-regulated). Among these proteins,26%were related to energy regulation,15%to protein destination and storage,11%to defense against disease,11%to metabolism,9%to protein synthesis,4%to secondary metabolism, and24%were of unknown function.
2. Proteomics analysis of soybean roots, hypocotyls and leaves under different treatments
Using2-DE and MALDI-TOF/TOF, proteomics study of soybean roots, hypocotyls and leaves under different treatments of SA, MeJA, ACC, H2O2, SNP, VB1and P. sojae was analyzed. Xinyixiaoheidou was susceptible to P. sojae strain P6497. These findings were as follows:(1)35differently expressed proteins were identified from soybean roots by MALDI/TOF-TOF. Among these proteins,23%were relative to disease/defense and secondary metabolism, respectively;17%to energy, followed with protein synthesis, protein destination and storage, transcription and metabolism, and3%were of cell structure and signal transduction, respectively, and8%with unknown function. The proteins involved in secondary metabolism were relative to the biosynthesis of ethylene, jasmonic acid and flavonoids.(2)58differently expressed proteins were identified from soybean hypocotyls by MALDI/TOF-TOF. Among these58proteins,41%were relative to energy and metabolism,12%to disease/defense and secondary metabolism, respectively,10%to protein destination and storage,7%to signal transduction and transcription, respectively,4%to cell structure,2%were of protein synthesis and transporters, respectively.(3)21differently expressed proteins were idedtified from soybean leaves by MALDI-TOF/TOF. The21identified proteins involved in energy dominated62%, reapectively. Those involved in secondary metabolism, protein synthesis and metabolism categories followed. The proteins relative to energy involved in the process of photosynthesis and respiration.
3. iTRAQ-based protein profile analysis of soybean roots in response to P. sojae inoculation
Using the slant board technique, iTRAQ-based protein profile of soybean roots to P. sojae was analyzed. Su88-M21was resistant to P. sojae strain Pml4. A total of2136proteins were identified and a subset of280proteins was differentially expressed infected with P. sojae. Among of these280proteins, the proteins relative to defense against disease dominated16%,14%involved in protein destination and storage,12%involved in metabolism,11%to energy and protein synthesis, respectively,8%to signal transduction,6%involved in transporter and secondary metabolism, respectively,4%to cell structure. The last is relative to cell growth/division, only accounting for1%, and9%were of unknown function. The signal of jasmonid acid, ethylene, auxin and ubiquitin proteasome system is involved in the resistant mechanism of soybean to P. sojae.
引文
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