| 中国3个主栽烟草品种的差异蛋白质组学研究 |
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| 引用本文： | 徐 莹,晏国全,张 扬,余红秀.中国3个主栽烟草品种的差异蛋白质组学研究[J].中国农业科学,2016,49(16):3084-3097. |
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| 作者姓名： | 徐 莹 晏国全 张 扬 余红秀 |
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| 基金项目： | 国家重点基础研究发展计划（“973”计划）（2012AA020201）、烟草化学安徽省重点实验室开放课题（0920140109008） |
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| 摘 要： | 【目的】从全蛋白质组学水平研究烟草,筛选具有重要特性的蛋白质,阐明其相关代谢通路,使得烟草的基础科学研究和品种培育工作有所突破。【方法】利用苯酚法提取中国3个主栽烟草品种红花大金元、K326和云烟87叶片的蛋白质,酶解后采用串联质量标签技术(tandem mass tag,TMT)标记,结合二维液相色谱串联质谱技术(2D LC-MS/MS)对3个烟草品种叶片的蛋白质组成进行分析,运用Proteome Discoverer软件提取谱图后在MASCOT搜索引擎上鉴定蛋白质和肽段,并对鉴定的蛋白质进行系统的生物信息学分析,包括数据相关性分析、分层聚类分析和主成分分析,同时按照2倍上下调的原则采用火山图筛选不同品种烟草中表达差异显著的蛋白质,最后利用KEGG通路分析烟草中重要蛋白质的分布及功能。【结果】鉴定红花大金元、K326和云烟87烟草样品的蛋白质Group总数为3 079,蛋白质ID数为10 343。从分层聚类分析和主成分分析中蛋白质的整体表达情况可见,K326和云烟87蛋白质表达情况较相似,而红花大金元与前两者相差较大。后续的差异蛋白质筛选也验证了该结果,表现为云烟87和K326之间仅存在29个差异表达的蛋白质,其中13个蛋白质覆盖8条代谢通路,包括类黄酮生物合成,参与类黄酮合成路径的查尔酮合成酶在K326中的相对含量显著高于云烟87。红花大金元和云烟87之间有160个蛋白质差异表达,其中103个蛋白质覆盖42条代谢通路,包括谷胱甘肽代谢,相对定量结果显示谷胱甘肽代谢途径相关的3种酶,谷胱甘肽过氧化物酶、磷脂过氧化氢物谷胱甘肽过氧化物酶和谷胱甘肽S-转移酶在红花大金元中的含量均显著低于云烟87。红花大金元和K326之间存在119个差异蛋白质,其中89个蛋白质覆盖41条代谢通路,包括外源物质细胞色素P450代谢。筛选获得的重要差异蛋白质的相对定量结果显示一些与抗性相关的蛋白质,如蛋白酶抑制剂,在云烟87中的表达量远低于K326和红花大金元,同时结果表明红花大金元中的超氧化物歧化酶含量处于较低水平。【结论】TMT技术结合2D LC-MS/MS可对不同品种烟草的叶片蛋白质进行有效地分离和鉴定,鉴定出的蛋白质大部分是参与光合作用、物质代谢或者与抗逆性相关。
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| 关 键 词： | 烟草 差异蛋白质组学 串联质量标签 二维液相色谱串联质谱 /> |
| 收稿时间： | 2016-03-14 |
Differential Proteomic Research of Three Varieties of Tobacco in China |
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| XU Ying,YAN Guo-quan,ZHANG Yang,YU Hong-xiu.Differential Proteomic Research of Three Varieties of Tobacco in China[J].Scientia Agricultura Sinica,2016,49(16):3084-3097. |
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| Authors: | XU Ying YAN Guo-quan ZHANG Yang YU Hong-xiu |
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| Institution: | Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 |
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| Abstract: | 【Objective】 In order to strengthen the basic scientific research and cultivation of tobacco, differential proteomics is used to select specific proteins and elucidate metabolic pathways in tobacco. 【Method】 Leaf proteins of three varieties of tobacco in China including Honghuadajinyuan, K326 and Yunyan 87 were extracted by phenol. After digestion the protein profiles of three varieties of tobacco were investigated by using tandem mass tag(TMT) coupled with two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS). The proteins and peptides from tobacco were identified with MASCOT search engine after spectra extraction in Proteome Discoverer Software. Then, bioinformatics analysis including data correlation analysis, hierarchical cluster analysis and principal component analysis (PCA) were conducted. Meanwhile, proteins with change ratio of more than 2 fold were defined as differentially expressed tobacco proteins, and they were screened out by volcano plot analysis. Finally, the distribution and function of important proteins in tobacco were analyzed using KEGG pathway analysis. 【Result】 A total of 3 079 proteins in Honghuadajinyuan, K326 and Yunyan 87 were identified and the number of protein ID was 10 343. Protein expression profiles of hierarchical cluster analysis and principal component analysis indicated that K326 and Yunyan 87 were relatively similar, and Honghuadajinyuan was significantly different from the other two. The subsequent protein screening also verified the results. Screening of differentially expressed proteins suggested that there were only 29 proteins differentially expressed between K326 and Yunyan 87. 13 proteins of them covered 8 metabolic pathways including flavonoids biosynthesis. Chalcone synthetase participating in the flavonoids synthesis was significantly higher in K326 than in Yunyan 87. Honghuadajinyuan and Yunyan 87 had 160 differentially expressed proteins. 103 proteins of them covered 42 metabolic pathways including glutathione metabolism. Three kinds of enzymes related to glutathione metabolic pathway including glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase and glutathione S-transferase were significantly lower in Honghuadajinyuan than in Yunyan 87. Honghuadajinyuan and K326 had 119 differentially expressed proteins. 89 proteins of them covered metabolic pathways such as metabolism of xenobiotics by cytochrome P450. It was indicated that several proteins related to stress resistance such as proteinase inhibitor was much less in Yunyan 87 than in K326 and Honghuadajinyuan, and superoxide dismutase in Honghuadajinyuan was the lowest among the three varieties of tobacco. 【Conclusion】Results of the study revealed that the TMT coupled with 2D LC-MS/MS is a powerful method for isolating and identifying differentially expressed proteins in various tobacco varieties. Most of them are involved in photosynthesis, metabolism or stress resistance. |
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| Keywords: | tobacco differential proteomics tandem mass tag 2D LC-MS/MS |
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