低温胁迫下高山离子芥差异蛋白质组学研究
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摘要
低温是一个至关重要的环境因子,能够影响植物的生长,发育以及地理分布。高山离子芥是一种典型的高山冰缘植物,主要分布于亚洲中部海拔3800米以上的大陆冰川附近。在高山离子芥的生活周期中,经常忍受从15℃到-4℃的剧烈的温度变化。形态学观察发现高山离子芥不具备外在的抗低温结构,推测其内部生理及分子机制起主要的抗低温功能。本文以高山离子芥为研究对象,分析了低温下高山离子芥叶片的蛋白质组变化,鉴定了许多低温胁迫下差异表达的蛋白,并克隆了其中一个低温下上调表达蛋白的基因,对其功能进行了初步研究。我们的研究为分析植物响应低温胁迫的分子机制提供了基础资料。具体的研究结果如下：
1.建立了一套适合高山离子芥叶片总蛋白质分离的双向电泳体系,获得了高分辨率和高重复性的双向电泳图谱。在Ph3-10和Ph4-7的胶图上,分别可以检测到,1000±50和1400±50个蛋白点。
2.研究发现低温下高山离子芥叶片中156个蛋白点的表达出现了显著差异(变化量超过2倍,P≤0.05)。这156个点中,72个点低温下均出现了上调表达,82个点低温下均出现了下调表达。通过MALDI-TOF MS和Q-TOF MS方法,从156个蛋白点中,鉴定了84个蛋白。
3.根据蛋白质功能的不同,82个蛋白可以归类为13个功能组,主要包括：光合作用,呼吸作用,信号转导和转录因子,蛋白质合成、运输、降解相关蛋白,抗逆相关蛋白,氨基酸代谢,脂代谢,能量代谢,细胞骨架以及其他代谢相关蛋白等。除了一些已经报道过的低温相关蛋白,很多未知蛋白也被发现。这些新发现的低温诱导蛋白给我们进一步研究低温响应机制提供了基础。
4.研究中发现一个新的冷诱导的DEAD-box RNA解旋酶。该蛋白在低温下表达量上调七倍。根据该蛋白的质谱信息,我们从离子芥中克隆到一个新的DEAD-box RNA解旋酶基因(CbDRH)。该基因全长为2111bp,包含一个1788bp的开放阅读框,预测编码含有595个氨基酸的蛋白质。该基因与拟南芥一个功能未知的乙烯应答的DEAD box RNA解旋酶基因直系同源,含有RNA解旋酶基因家族全部的9个保守motif。
5. CbDRH在冷胁迫和冻胁迫下,转录水平上均上调,UV-B和ABA也能导致其转录水平上调,但是NaCl胁迫下,其转录水平下调。CbDRH表达无器官特异性,在植物的根茎叶中均有表达。通过亚细胞定位发现,该基因定位在细胞核中。这些结果验证了我们蛋白质组学的结果,也说明了DEAD box RNA解旋酶基因是在细胞核中起作用,参与了植物多种抗性反应。
Low temperature is one of major environmental factors that affect plant growth, development and geographical distribution. Chorispora Bungeana Fisch&CA.Mey. (C.bungeana) is a representative alpine subnival plant. It mainly distributes in continental glacier nearby of central Asia with a 3800-3900m height above sea level. During life cycle, it has to often undergo drastic temperature fluctuations from 15℃to-4℃. Through morphological research, we find Chorispora Bungeana has no special morphological characteristics that help to survive under low temperature. So we deduce physiological and molecular mechanisms mainly contribute to adapting to the freezing environment. The present study is to investigate the proteome changes of Chrispora bungeana leaves under low temperature based on two dimensional electrophoresis. Many differently expressed proteins under low temperature were successfully identified in our research. A new gene, named DEAD-box RNA helicase, was cloned based on an up-regulated protein under low temperature and its functions were further explored in present research. Our research provides basic data for analyzing the molecular mechanisms of low temperature response. The detailed research results are as followings:
1, we developed a most suitable system of 2-DE for separating the total proteins from Chrispora bungeana leaves and obtained the high-resolution and reproducible gel maps. About 1000±50 and 1400±50 protein spots were detected in gels of pH 3-10 and pH4-7 respectively.
2, Based on comparison and statistical analysis, we found 156 protein spots had significant changes (P≤0.05). Of these 156 spots,72 spots were up-regulated under low temperature,84 spots were down-regulated under low temperature.82 proteins were successfully identified from these 156 spots by MALDI-TOF MS and Q-TOF MS. According to functional features of identified proteins, we classified them into 13 major categories, including:photosynthesis, cell rescue/defense, protein biosynthesis, folding and degradation, transcription factors and cell signal transduction, respiratory metabolism, amino acid metabolism, lipid metabolism, energy, cytoskeleton and other metabolisms. Most of identified proteins were well known low temperature responsive proteins, but some are novel low temperature induced proteins, such as DEAD box RNA helicase. These novel low temperature induced proteins provide new insights into the low temperature stress defense mechanism in plants and deserve to be further researched.
3, An identified protein named DEAD-box RNA helicase, wwas up-regulated by 7 times under low temperature, which was a novel cold induced protein and was never reported in the past. Based on the information of this protein, we cloned a new gene from Chorispora Bungeana, named CbDRH. The full-length cDNA of CbDRH was 2111bp and contained an ORF (open reading frame) of 1788bp, which was predicted to encode a protein consisting of 595 amino acids. CbDRH was an orthologous protein of Arabidopsis ethylene-responsive DEAD-box RNA helicase, AtRH30, whose functions were to date largely unknown. Structurally, CbDRH possesses all the nine conserved motifs characteristic of DEAD-box protein family in its central region.
4, Transcriptional expression of CbDRH was up regulated by both chilling and freezing treatment. UV-B and ABA treatment also led to the accumulation of CbDRH transcript. However, NaCl treatment down regulated the transcriptional expression level of CbDRH. Besides, CbDRH transcript was detected in all of the tissues tested (root, stem and leaf) with little tissue specificity. Subcellular localization suggested that CbDRH is localized in the nucleus. These data supported our proteomics results and also suggested that CbDRH may function in the nucleus and be involved in several cellular stress responses in Chorispora bungeana.
1.建立了一套适合高山离子芥叶片总蛋白质分离的双向电泳体系,获得了高分辨率和高重复性的双向电泳图谱。在Ph3-10和Ph4-7的胶图上,分别可以检测到,1000±50和1400±50个蛋白点。
2.研究发现低温下高山离子芥叶片中156个蛋白点的表达出现了显著差异(变化量超过2倍,P≤0.05)。这156个点中,72个点低温下均出现了上调表达,82个点低温下均出现了下调表达。通过MALDI-TOF MS和Q-TOF MS方法,从156个蛋白点中,鉴定了84个蛋白。
3.根据蛋白质功能的不同,82个蛋白可以归类为13个功能组,主要包括：光合作用,呼吸作用,信号转导和转录因子,蛋白质合成、运输、降解相关蛋白,抗逆相关蛋白,氨基酸代谢,脂代谢,能量代谢,细胞骨架以及其他代谢相关蛋白等。除了一些已经报道过的低温相关蛋白,很多未知蛋白也被发现。这些新发现的低温诱导蛋白给我们进一步研究低温响应机制提供了基础。
4.研究中发现一个新的冷诱导的DEAD-box RNA解旋酶。该蛋白在低温下表达量上调七倍。根据该蛋白的质谱信息,我们从离子芥中克隆到一个新的DEAD-box RNA解旋酶基因(CbDRH)。该基因全长为2111bp,包含一个1788bp的开放阅读框,预测编码含有595个氨基酸的蛋白质。该基因与拟南芥一个功能未知的乙烯应答的DEAD box RNA解旋酶基因直系同源,含有RNA解旋酶基因家族全部的9个保守motif。
5. CbDRH在冷胁迫和冻胁迫下,转录水平上均上调,UV-B和ABA也能导致其转录水平上调,但是NaCl胁迫下,其转录水平下调。CbDRH表达无器官特异性,在植物的根茎叶中均有表达。通过亚细胞定位发现,该基因定位在细胞核中。这些结果验证了我们蛋白质组学的结果,也说明了DEAD box RNA解旋酶基因是在细胞核中起作用,参与了植物多种抗性反应。
Low temperature is one of major environmental factors that affect plant growth, development and geographical distribution. Chorispora Bungeana Fisch&CA.Mey. (C.bungeana) is a representative alpine subnival plant. It mainly distributes in continental glacier nearby of central Asia with a 3800-3900m height above sea level. During life cycle, it has to often undergo drastic temperature fluctuations from 15℃to-4℃. Through morphological research, we find Chorispora Bungeana has no special morphological characteristics that help to survive under low temperature. So we deduce physiological and molecular mechanisms mainly contribute to adapting to the freezing environment. The present study is to investigate the proteome changes of Chrispora bungeana leaves under low temperature based on two dimensional electrophoresis. Many differently expressed proteins under low temperature were successfully identified in our research. A new gene, named DEAD-box RNA helicase, was cloned based on an up-regulated protein under low temperature and its functions were further explored in present research. Our research provides basic data for analyzing the molecular mechanisms of low temperature response. The detailed research results are as followings:
1, we developed a most suitable system of 2-DE for separating the total proteins from Chrispora bungeana leaves and obtained the high-resolution and reproducible gel maps. About 1000±50 and 1400±50 protein spots were detected in gels of pH 3-10 and pH4-7 respectively.
2, Based on comparison and statistical analysis, we found 156 protein spots had significant changes (P≤0.05). Of these 156 spots,72 spots were up-regulated under low temperature,84 spots were down-regulated under low temperature.82 proteins were successfully identified from these 156 spots by MALDI-TOF MS and Q-TOF MS. According to functional features of identified proteins, we classified them into 13 major categories, including:photosynthesis, cell rescue/defense, protein biosynthesis, folding and degradation, transcription factors and cell signal transduction, respiratory metabolism, amino acid metabolism, lipid metabolism, energy, cytoskeleton and other metabolisms. Most of identified proteins were well known low temperature responsive proteins, but some are novel low temperature induced proteins, such as DEAD box RNA helicase. These novel low temperature induced proteins provide new insights into the low temperature stress defense mechanism in plants and deserve to be further researched.
3, An identified protein named DEAD-box RNA helicase, wwas up-regulated by 7 times under low temperature, which was a novel cold induced protein and was never reported in the past. Based on the information of this protein, we cloned a new gene from Chorispora Bungeana, named CbDRH. The full-length cDNA of CbDRH was 2111bp and contained an ORF (open reading frame) of 1788bp, which was predicted to encode a protein consisting of 595 amino acids. CbDRH was an orthologous protein of Arabidopsis ethylene-responsive DEAD-box RNA helicase, AtRH30, whose functions were to date largely unknown. Structurally, CbDRH possesses all the nine conserved motifs characteristic of DEAD-box protein family in its central region.
4, Transcriptional expression of CbDRH was up regulated by both chilling and freezing treatment. UV-B and ABA treatment also led to the accumulation of CbDRH transcript. However, NaCl treatment down regulated the transcriptional expression level of CbDRH. Besides, CbDRH transcript was detected in all of the tissues tested (root, stem and leaf) with little tissue specificity. Subcellular localization suggested that CbDRH is localized in the nucleus. These data supported our proteomics results and also suggested that CbDRH may function in the nucleus and be involved in several cellular stress responses in Chorispora bungeana.
引文
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