油菜附加系EE抗根结线虫的蛋白质组学初步研究

Preliminary Proteomics Study on Rapeseed Addition Line EE Against Root-knot Nematode

【目的】通过蛋白质组技术寻找抗线虫油菜附加系EE和感病油菜接种根结线虫后根系表达蛋白的差异,筛选出抗根结线虫相关蛋白。【方法】以抗根结线虫的油菜附加系EE和感根结线虫的油菜根系为材料,通过蛋白质组学双向电泳技术和Image Master 5.0软件分析筛选差异表达蛋白,经MALDI-TOF-TOF/MS质谱测序鉴定蛋白。另外,通过Gene Ontology,KEGG,WoLF PSORT和STRING生物信息学分析对鉴定蛋白进行功能分类、代谢途径分析、亚细胞定位和蛋白互作分析。【结果】Madora和油菜附加系EE分别有1184和1163个蛋白点。21个差异蛋白质经MALDI-TOF/TOF MS质谱测序成功鉴定,其中一半以上定位于细胞外(52.38%),其次为细胞质(28.57%),叶绿体(4.76%),线粒体(4.76%),内质网(4.76%),液泡(4.76%)。蛋白质相互作用分析表明,与抗病相关的上调蛋白内源性α-淀粉酶/枯草杆菌蛋白酶抑制剂与3-异丙基苹果酸脱水酶大亚基,微管蛋白α-6链,表皮特异性分泌糖蛋白EP1,BnaC04g03560D和BnaC06g34210D 5个蛋白质具有一定的相关性。【结论】筛选的抗线虫相关蛋白对于将来克隆抗性基因和用分子标记培育新的抗性品种奠定基础。

【Objective】In this study,we would search for the differentially expressed protein between the rapeseed addition line EE and rapeseed inoculated with the root-knot nematode by proteomic technique to screen the resistance-related protein against root-knot nematodes.【Method】Roots of the the resistant rapeseed addition line EE and susceptible rapeseed were used as materials.Differentially expressed proteins were screened by proteomic two-dimensional electrophoresis and Image Master 5.0 software.Proteins were identified by MALDI-TOFTOF/MS mass spectrometry.In addition,functional classification,metabolic pathway analysis,subcellular localization and proteins interaction analysis of identified proteins were carried using Gene Ontology,KEGG,WoLF PSORT and STRING.【Result】Madora and rapeseed addition line EE had 1184 and 1163 protein spots,respectively.21 differentially expressed proteins were successfully identified by MALDITOF/TOF MS.Among these proteins,more than half of them were localized extracellularly(52.38%),followed by cytoplasm(28.57%),chloroplasts(4.76%),mitochondria(4.76%),and internal plasma reticulum(4.76%),vacuoles(4.76%).Analysis of protein interactions showed that endogenousα-amylase/subtilisin inhibitors was connected wisth 3-isopropylmalate dehydratase large subunits,tubulinα-6 chain,epidermis-specific secreted glycoprotein EP1,BnaC04g03560D and BnaC06g34210D.【Conclusion】The screened resistance-related proteins lay the foundation for cloning of resistance genes and breeding of new resistant varieties with molecular markers in future.