Proteomics research for plant self defense proteins

为了研究其蛋白质组学分析和固有的自卫蛋白，对诱导自卫蛋白进行了蛋白质组学分析。建立了植物组织提取液的二维电泳、凝胶消化和质谱分析方法，并将其应用于胁迫诱导的几丁质酶检测，然后用质谱法对蛋白质进行鉴定。建立了植物几丁质酶的酶谱分析方法，并采用优化后的质谱分析方法进行了分析。对铝胁迫下水培培养的金穗草的诱导蛋白进行了二维电泳分析。用已建立的方法对蛋白进行凝胶消化后，用质谱法分析检测到的蛋白斑点。该蛋白被鉴定为1类几丁质酶家族，但其分子量高于正常的1类几丁质酶。这表明检测到的诱导几丁质酶是新发现的携带多个几丁质结合域的1类几丁质酶。然后将细穗草根提取物用Chitipearl涂于亲和柱上，分离吸附的几丁质酶组分。为阐明几丁质酶在真菌侵染和昆虫侵染薯蓣薯蓣块茎中诱导型几丁质酶的鉴定，对真菌侵染和昆虫侵染的薯蓣块茎进行了底物凝胶SDS电泳酶谱分析。结果表明，几丁质酶明显受到真菌感染和损伤的诱导。

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.