【作者】 庞秋颖；

【导师】 王洋；

【作者基本信息】 东北林业大学 ， 细胞生物学， 2010， 博士

【摘要】 本文以盐生模式植物盐芥(Thellungiella halophila)和模式植物拟南芥(Arabidopsis thaliana)为研究对象,系统地分析了盐胁迫下二者芥子油苷组成和含量的变化规律,并对二者进行了盐胁迫比较蛋白质组学研究,分别鉴定并比较了盐芥、拟南芥盐胁迫差异表达的可溶性蛋白和疏水膜蛋白,为分析盐胁迫调控芥子油苷代谢机制、比较盐生植物与非盐生植物响应盐胁迫的分子机制提供了基础资料。(1)利用高效液相色谱-质谱首次在盐芥中准确鉴定出7种芥子油苷,包括脂肪族的Allyl、3MSOP、10MSD、3MTP和吲哚族的I3M、4MOI3M,以及芳香族的2PE。盐芥花、角果、子叶和根中含有全部的7种芥子油苷,茎、茎生叶和叶柄中含有6种(缺少I3M),莲座叶中含有5种(缺少2PE和I3M),而种子中只含有4种(Allyl、3MSOP、10MSD和3MTP)且全部属于脂肪族。芥子油苷总量在不同器官、不同发育时期存在显著差异。(2)以50、150mmol/LNaCl处理拟南芥(生长4周)和盐芥(生长6周)5天,芥子油苷组成没有改变。芥子油苷总量、脂肪族芥子油苷总量、吲哚族芥子油苷总量在拟南芥中受盐胁迫的影响均不显著,而在盐芥中则随盐胁迫增强先减少、后增加并高于对照水平。拟南芥脂肪族的3MSOP、5MSOP和吲哚族的4OHI3M、4MOI3M随盐胁迫增强而含量降低,而脂肪族的6MSOH、吲哚族的I3M以及盐芥脂肪族的3MSOP则随盐胁迫增强有含量增加的趋势。拟南芥脂肪族的8MS00和吲哚族的1MOI3M,盐芥脂肪族的3MTP、Allyl、10MSD和吲哚族的4MOI3M,在盐胁迫下的含量变化与盐芥芥子油苷总量的变化趋势一致。(3)利用双向电泳(2D-E)分离可溶性蛋白,在拟南芥和盐芥中分别得到88和37个受盐胁迫影响而变化显著的蛋白质点。经质谱分析及MASCOT数据库检索,有意义的差异蛋白分别为79和32个。拟南芥差异表达显著的蛋白大部分在150 mmol/L NaCl胁迫下,而盐芥则是在50mmol/LNaCl胁迫下。(4)利用同位素标记相对和绝对定量(iTRAQ)及二维液相串联质谱(2DLC-MS/MS)分析疏水膜蛋白,拟南芥中差异表达的膜蛋白31个(21个上调,10个下调),盐芥中差异表达的膜蛋白32个(11个上调,21个下调)。这些蛋白大部分为叶绿体、线粒体和质膜蛋白及膜相关蛋白。(5)根据差异表达蛋白所参与的生物学过程,对非冗余可溶性蛋白和膜蛋白进行了分类和功能分析。拟南芥划分为11类,比例最大的是物质代谢相关蛋白(22%)；盐芥划分为13类,蛋白合成相关蛋白的比例最大(22%)。这表明,代谢相关的蛋白对维持盐逆境下拟南芥的生长与代谢具有重要的作用,而蛋白合成相关蛋白可能在盐芥的抗盐机制中发挥重要的作用。更多还原

【Abstract】 Here we report glucosinolate profile and comparative proteomics of Arabidopsis thaliana, a glycophyte, and its close relative Thellungiella halophila, a halophyte, under different salt stress conditions. Soluble proteins from control and NaCl treated samples were extracted and separated by two-dimensional gel electrophoresis. As a complementary approach, isobaric tag for relative and absolute quantification (iTRAQ) LC-MS was used to identify crude microsomal proteins. Collectively, this work represents the most extensive proteomic description of salinity responses of A. thaliana and T. halophila. The studies may provide the basis for the future research of the effect of salt stress on glucosinolate metabolism and salt tolerance molecular mechanism in glycophytes and halophytes.First, seven glucosinolates in T. halophila were unambiguously identified by high performance liquid chromatography and mass spectrometry (HPLC-MS) for the first time, including aliphatic glucosinolates Allyl,3MSOP,10MSD and 3MTP, indole glucosinolates I3M and 4MO13M, aromatic glucosinolate 2PE. The composition of glucosinolates varied considerably among different tissues, with seven compounds identified in flowers, siliques, cotyledons and roots, six in stems, cauline leaves and petioles (I3M was not detected), five in rosette leaves (2PE and I3M were not detected), only four in seeds and all four glucosinolates belong to aliphatic (including Allyl,3MSOP,10MSD and 3MTP). The total contents of glucosinolates in different organs at various developmental stages also displayed significant variationsSecond, the composition of glucosinolates was not varied in the rosette leaves of four-week-old A. thaliana and 6-week-old T. halophila, which were irrigated with 50 mM and 150 mM NaCl. The contents of total, aromatic and indole glucosinolates were not varied significantly in A. thaliana after treatment. However, the contents of total, aromatic and indole glucosinolates decreased after 50 mM NaCl treated and increased higher than control after 150 mM NaCl treated. Aliphatic glucosinolates 3MSOP,5MSOP and indole glucosinolates 4OHI3M and 4MOI3M in A. thaliana decreased with increasing NaCl concentrations, while aliphatic glucosinolates 6MSOH, indole glucosinolates I3M in A. thaliana and aliphatic glucosinolates 3MSOP in T. halophila increased with increasing NaCl concentrations. The content of 8MSOO and 1MOI3M in A. thaliana,3MTP, Allyl,10MSD and 4MOI3M in T. halophila displayed a similar pattern of variation.Third, soluble proteins from control and NaCl treated samples were separated by two-dimensional gel electrophoresis. A total of 88 protein spots from A. thaliana gels and 37 protein spots from T. halophila gels showed significant changes. Out of these spots, a total of 79 and 32 proteins, which were significantly regulated by salt stress, were identified by mass spectrometry in A. thaliana and T. halophila, respectively. Most of the changes were observed in A. thaliana samples treated with 150 mM NaCl. In contrast, most proteins were found to exhibit significantly changes in 50 mM NaCl treated T. halophila samples.Fourth, isobaric tag for relative and absolute quantification (iTRAQ) LC-MS was used to identify crude microsomal proteins. There were 31 differentially expressed (21 increased and 10 decreased) in A. thaliana. In T. halophila, there were 32 differentially expressed proteins (11 increased and 21 decreased). Most of the differentially expressed proteins are membrane proteins or membrane associated proteins in chloroplasts, mitochondria and plasma membraneFifth, the non-redundant identified proteins were grouped according to functional categories. In T. halophila the proteins were grouped into 11 categories, the largest group is the protein synthesis (22%). In A. thaliana, the salt-responsive proteins were classified into 13 categories, metabolic proteins (22%) formed the largest group. This finding suggests that although proteins involved in plant metabolism play an important role in A. thaliana salt tolerance, the protein synthesis-related proteins play an important role in T. halophila salt tolerance.更多还原