Proteomics research for plant self defense proteins

为了研究蛋白质组分析和固有自卫蛋白，对诱导的自卫蛋白进行了蛋白质组学分析。建立了植物组织提取液双向电泳、凝胶内消化和质谱分析的方法，并将其应用于逆境诱导的几丁质酶的检测和蛋白质的质谱分析。建立了植物几丁质酶的酶谱分析程序，并用优化的方法进行了质谱分析。对铝胁迫下的水培草莓的诱导蛋白进行了双向电泳分析。用建立的方法对检测到的蛋白质进行凝胶内消化后，对检测到的蛋白质斑点进行质谱分析。该蛋白被鉴定为1类几丁质酶家族，但其相对分子质量高于正常的1类几丁质酶。这表明所检测到的诱导几丁质酶是新发现的携带多个几丁质结合区的I型几丁质酶。将山药根提取液应用于几丁质亲和层析柱上，分离吸附的几丁质酶组分。对感染真菌和虫害的山药地上块茎进行底物凝胶SDS电泳酶谱分析，以明确几丁质酶在侵染或伤害过程中的鉴定。结果表明，几丁质酶的表达明显受真菌感染和创伤的影响。

To investigate the proteome analysis and inherent self defense proteins, the inducible self defense proteins were subjected to proteomic analysis. The method for 2D electrophoresis of plant tissue extract, in-gel digestion, and mass spectrometry were established and applied to the detection of chitinase induced by stress followed by identification of the proteins using mass spectrometry. The zymographic procedure was also developed for plant chitinase and analyzed by mass spectrometry using optimized method.The hydroponic cultured ruzigrass under Al stress was analyzed its inducible proteins on 2D electrophoresis. The detected protein spot was analyzed on mass spectrometry after in-gel digestion of the protein using established protocol. The protein was identified as class 1 chitinase family, however, carried higher molecular weight than normal class 1 chitinase. This suggests that the detected induced chitinase was newly found class 1 type chitinase that carried multiple chitin binding domains. The extract of ruzigrass roots was then applied on to an affinity column using Chitipearl and separated adsorbed chitinase fractions.To elucidate the identification of inducible chitinase during infection or wounding, yam aerial tuber infected by fungi and wounded by insects were subjected zymographic analysis of substrate gel SDS electrophoresis. The results indicate that the chitinase apparently induced by fungal infection and wounding.