水稻草状矮化病毒侵染寄主水稻差异表达蛋白的鉴定和分析

doi:10.3864/j.issn.0578-1752.2014.09.007

摘要/Abstract

摘要： 【目的】对水稻草状矮化病毒（Rice grassy stunt virus，RGSV）侵染寄主水稻后其叶片的差异表达蛋白进行筛选、鉴定和生物信息学分析，为进一步研究RGSV和寄主水稻互作的分子机制提供线索。【方法】采用双向荧光差异凝胶电泳（Two-dimensional fluorescence difference in gel electrophoresis，2D-DIGE）和Image Master 2D platinum 7.0分析软件寻找差异表达蛋白，MALDI-TOF-MS（Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry）鉴定差异蛋白，利用BioTools软件搜索NCBI数据库，寻找匹配的相关蛋白质和功能查询。采用GOminer软件对差异蛋白进行GO（Gene ontology）聚类分析，KEGG（Kyoto encyclopedia of genes and genomes）数据库分析差异蛋白所参与的生物通路。【结果】建立了RGSV侵染与未侵染水稻的叶片双向荧光差异凝胶电泳图谱，RGSV病株与健株相比，差异倍数大于1.5的差异蛋白质点有173个，其中表达丰度升高的点有72个，表达丰度下降的点有101个，质谱鉴定了44个差异蛋白质点，25个获得成功鉴定，分属于24种蛋白质，包括RGSV的2种蛋白20.6K非结构蛋白和P5蛋白，水稻中的蛋白推定的核酮糖-1,5-二磷酸羧化酶/加氧酶大亚基、景天庚酮糖-1,7-二磷酸酶前体、推定的酪氨酸磷酸酶、HAD超家族水解酶-亚族IA蛋白变体3蛋白、水稻核酮糖-1,5-二磷酸羧化酶/加氧酶小亚基结合2-羧基阿拉伯糖醇-1,5-二磷酸复合体、类似C1结构域蛋白以及其他一些功能未知蛋白和假定蛋白。对已鉴定蛋白的生物信息分析显示，差异蛋白涉及氮素固定、氮循环代谢过程、含氮化合物代谢过程、单一生物体代谢过程、代谢过程和生物过程等6个生物学过程，在生物功能上分属7类，包括分子功能、酸胺连接酶活性、酰胺连接酶活性、催化活性、谷氨酸氨连接酶、连接酶活性和形成C-N键的连接酶活性，在细胞组件上，差异蛋白分布于不同的细胞位置，涉及细胞组分、细胞器、细胞、膜结合细胞器、囊（泡）、细胞部分、膜结合囊（泡）、胞内、胞内部分、胞内细胞器、细胞质、胞内膜结合细胞器、细胞质部分、质体、细胞质囊（泡）和细胞质膜结合囊（泡）。KEGG通路分析显示，差异蛋白参与了代谢途径、光合生物固碳反应、碳代谢、乙醛酸和二羧酸代谢、丙氨酸、天门冬氨酸和谷氨酸代谢、氨基酸生物合成、丙酮酸盐代谢、精氨酸和脯氨酸代谢、谷胱甘肽代谢和氮代谢途径等10个KEGG代谢通路。【结论】RGSV侵染诱导了水稻差异蛋白质表达，获得了一些与RGSV侵染水稻相关的蛋白质分子，差异蛋白涉及到多个功能类别。其中与氮有关的生物学过程和光合作用过程变化较为明显。

关键词: 水稻草状矮化病毒 , 差异表达蛋白 , 2D-DIGE , MALDI-TOF-MS

Abstract: 【Objective】The objective of this study is to screen, identify and analyze the differentially expressed proteins in rice seedlings after infection with Rice grassy stunt virus (RGSV), and to shed new lights on the understanding of molecular mechanisms of RGSV pathogenesis.【Method】Two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) was used to analyze and identify differentially expressed protein spots. BioTools was used to search the matched proteins in NCBI database. Bioinformatics included GO (Gene ontology) terms and KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis.【Result】The proteomic profile of the leaves of rice cultivar Xianyou 63 infected with RGSV compared with the control healthy plants was explored by 2D-DIGE. One-hundred and seventy-three protein spots (differential ratio＞1.5) were differentially expressed by RGSV infection over the healthy rice plants, including 72 up-regulated and 101 down-regulated protein spots. Among them, 25 differentially expressed protein spots were identified, including RuBisCO large subunit, sedoheptulose-1,7-bisphosphatase precursor, putative tyrosine phosphatase, HAD-superfamily hydrolase, subfamily IA, variant 3 containing protein, chain S, crystal structure of activated rice Rubisco complexed with 2-carboxyarabinitol-1,5-bisphosphate, C1-like domain containing protein, 20.6 K nonstructural protein (RGSV), P5 protein (RGSV) and some hypothetical proteins. GO function analysis revealed that the proteins’ biological processes were mainly involved in biological process, metabolic process, single-organism metabolic process, nitrogen compound metabolic process, nitrogen cycle metabolic process and nitrogen fixation, and the proteins’ molecular functions included molecular function, catalytic activity, ligase activity, ligase activity-forming carbon-nitrogen bonds, acid-ammonia ligase activity, ammonia ligase activity and glutamate-ammonia ligase activity. The proteins located different cell parts including cell, cell part, cellular component, cytoplasm, cytoplasmic membrane-bounded vesicle, cytoplasmic part, cytoplasmic vesicle, intracellular, intracellular membrane- bounded organelle, intracellular organelle, intracellular part, membrane, membrane-bounded organelle, membrane-bounded vesicle, mitochondrion, organelle, plastid and vesicle. KEGG pathway analysis suggested that the metabolic pathways of these proteins were those of metabolic pathways, carbon fixation in photosynthetic organisms, carbon metabolism, glyoxylate and dicarboxylate metabolism, alanine, aspartate and glutamate metabolism, biosynthesis of amino acids, pyruvate metabolism, arginine and proline metabolism, glutathione metabolism and nitrogen metabolism.【Conclusion】According to the proteomic profile of rice seedling infected with RGSV, 24 proteins identified might be related to the pathogenicity of RGSV which provided an insight into the interactive relationship between RGSV and the host rice. The biological process and photosynthesis process related to nitrogen changed more obviously.

Key words: Rice grassy stunt virus , differentially expressed proteins , 2D-DIGE , MALDI-TOF-MS

丁新伦, 谢荔岩, 吴祖建. 水稻草状矮化病毒侵染寄主水稻差异表达蛋白的鉴定和分析[J]. 中国农业科学, 2014, 47(9): 1725-1734.

DING Xin-Lun, XIE Li-Yan, WU Zu-Jian. Identification and Analysis of Differentially Expressed Proteins of Host Rice (Oryza sativa) Infected with Rice Grassy Stunt Virus[J]. Scientia Agricultura Sinica, 2014, 47(9): 1725-1734.

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