| 摘要: |
| 【目的】建立苹果树腐烂病菌菌丝蛋白双向电泳体系，鉴定不同致病力材料的差异表达蛋白，为从蛋白质组学角度揭示苹果树腐烂病菌致病机理奠定基础。【方法】以苹果树腐烂病菌野生型03 8菌丝为材料，从蛋白提取方法、IPG胶条长度及胶条的pH范围3个方面对双向电泳体系进行优化，并利用该优化体系对苹果树腐烂病菌野生型03-8和致病力丧失突变体X900的菌丝蛋白进行双向电泳分析和比较，对二者的差异表达蛋白进行鉴定和分析。【结果】采用TCA/丙酮法 SDS/酚蛋白抽提法及17 cm pH 4.0~7.0 IPG胶条，对苹果树腐烂病菌菌丝蛋白进行双向电泳可以得到理想的蛋白图谱。利用该体系对苹果树腐烂病菌野生型菌株03-8及其致病力丧失突变体菌株X900的菌丝总蛋白进行双向电泳分析发现，2个菌株共存在27个差异表达蛋白点，其中有15个显著差异表达蛋白点；质谱分析结果表明，突变体X900中缺失的2个蛋白点4633和8518分别为丝氨酸苏氨酸蛋白激酶和假想蛋白，其他13个显著差异表达蛋白点分别为1个微管蛋白、1个线粒体内膜转移酶亚基、1个丙酮酸脱羧氧化酶、1个腺苷高半胱氨酸激酶、2个压力诱导的磷蛋白、1个己糖磷酸肌醇激酶、1个热激蛋白和5个假想蛋白。GO分析表明，该差异蛋白主要参与能量代谢、胁迫应答、细胞结构组成和未知功能。【结论】建立了适合苹果树腐烂病菌菌丝蛋白的双向电泳体系，并通过该体系分析获得了不同致病力材料的一系列差异表达蛋白。 |
| 关键词: 蛋白提取 双向电泳 蛋白质组 质谱鉴定 苹果树腐烂病菌 |
| DOI： |
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| 基金项目:教育部高校博士点基金项目(20120204110002);公益性行业（农业）科研专项(201203034) |
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| Development of two dimensional electrophoresis for hyphal proteins of Valsa mali |
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FENG Hao,WANG Haiying,FENG Yaqiong,et al
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| Abstract: |
| 【Objective】The research established a 2-DE gel electrophoresis system for identification of differentially expressed proteins from different virulent strains to provide theoretical basis for studyingpathogenesis from proteome level.【Method】The protein extraction method,IPG strip type for 2-DE gel electrophoresis,and pH of mycelia protein were optimized.Then the differences in intracellular proteins between wild strain 03-8 and a no pathogenicity mutant X900 were analyzed unde rthe optimized conditions and identified by mass spectrometry.【Result】The TCA/acetone-SDS phenol method for mycelia protein extraction with 17 cm IPG strip at pH of 4.0-7.0 were suitable for 2-DE gel electrophoresis to obtain ideal protein profile.With the seconditions,the intracellular proteins of wild strain 03-8 and mutant X900 were analyzed,and 27 proteins were differentially expressed,among which 15 showed significant difference in expression.Mass spectrometry showed that the protein points 4633 and 8518 lost in mutant X900 were serine threonine protein kinase and hypothetical protein.The other 13 proteins included one tubulin β,one mitochondrial membrane transferase subunit,one pyruvate decarboxylase,one adenosylhomocysteinase,2 stress induced phosphor proteins,one inositol hexakisphosphate kinase,one heat shock protein,and 5 hypothetical proteins.GO analysis showed that the proteins mainly involved in energy metabolism,stress responses,cell structure and unknown functions. 【Conclusion】A suitable 2-DE gel electrophoresis system was established for mycelia protein of Valsa mali and a series of different proteins were determined from different virulent strains. |
| Key words: protein extraction 2-DE proteome LC-MS/MS mass spectrometry apple tree valsa canker |
