花生黄曲霉抗性研究
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摘要
以花生(Arachis hypogaea L.)汕油523种子为材料，研究其蛋白酶抑制剂和2S清蛋白的基本性质及其对黄曲霉的作用，并探讨MJ和黄曲霉菌细胞壁激发子对花生悬浮细胞抗性相关酶活性的影响。
采用凝胶层析、离子交换层析和电泳等方法，分别从花生种子醋酸提取液和磷酸缓冲液提取液中分离纯化胰蛋白酶抑制剂和2S清蛋白。研究表明，PI和2S清蛋白在花生中的含量很低，PI的最后得率为6％，2S的最后得率为1.2％，经PAGE、SDS-PAGE和HPLC分析表明，最后得到的PI为纯品，它是由一个单体组成的，2S清蛋白由5种低分子量的亚基组成，且部分以聚合体的形式存在。经SDS-PAGE和激光质谱测定其分子量，PI为7.07kDa，2S中含有与PI分子量相同的亚基(命名为2S-1)。PI的等电点为5.8，2S各组分的等电点相似，约为4.1，PI和2S都具有很高的热稳定性和酸碱稳定性，两者经100℃处理10min其抑制活性无明显变化，在pH2～11之间，活性也不发生改变。PI对胰蛋白酶、内肽酶和黄曲霉的生长具有较强的抑制作用，2S清蛋白在SDS处理前对胰蛋白酶、内肽酶和黄曲霉的生长无明显的抑制作用，但经SDS处理后其抑制活性大大增强。2S中含有对胰蛋白酶具有抑制作用的组分，具有与PI相似的性质，但蛋白N端序列和氨基酸分析表明，它可能属于一种病程相关蛋白，与PI不同。
用MJ和黄曲霉菌细胞壁激发子处理花生悬浮培养细胞，然后测定其抗病相关酶活性及抗病相关物质的含量。结果表明，MJ对苯丙氨酸解胺酶、脂氧酶-1、胰蛋白酶抑制活性、几丁质外切酶、几丁质内切酶具有较强的促进作用、对β-1，3葡聚糖苷酶、脂氧酶-3具有轻微的促进作用，略微抑制过氧化氢酶的活性，对活性氧含量无明显影响；激发子对苯丙氨酸解胺酶、胰蛋白酶抑制活性、几丁质外切酶、几丁质内切酶具有较强的促进作用，对活性氧含量具有轻微的促进作用，对脂氧酶-1、过氧化氢酶具有轻微的抑制作用，对β-1，3葡聚糖苷酶、脂氧酶-3无明显影响。MJ和激发子共同作用对不同的抗病相关物质的作用效果不同。因此可以认为，在MJ和激发子诱导的抗性相关物质的过程中，对不同的物质具有不同的作用方式和途径，它们之间既存在相互联系又相互独立。
Peamil(Arachis hypogaea L.Shanyou523)seeds were chosen as materials in this thesis to study the general characteristics of the natural proteinaceous protease inhibitors from them, and the effect of MJ and elicitor on the activities of some enzymes about resistance were investigated.
the crude PI and 2S fraction was separated from peanut seeds and purificated by Sephadex G-100 and DEAE cellulose(DE-32) chromatography . the properties of purified PI and 2S was determinded. The result showed that the content of PI and 2S in peanut seeds is very low and their last quantity is 6% and 1.2%seperately. PAGE, SDS-PAGE and HPLC analysis showed that the PI we gained are pure, which is a single polypetide with a molecular weight of 7.07kDa by SDS-PAGE and laser mass spectrum and a isoelectric point at pH5.8; The 2S protein is consist of five polypeptides with low molecular weight and the approximate isoelectric point at pH4.1and partly hang together, one of which has the same molecular weight as PI(named 2S-1).PI and 2S were stable below 100℃and between pH2.0-ll. PI can inhibite strongly proteinaceous protease,endopeptidase and A.flavus, while 2S before treatment had little inhibition on proteinaceous protease,endopeptidase and A.flavus, but after treatment, it had strong inhibition on proteinaceous protease,endopeptidase and A.flavus. Some of 2S had inhibiton on proteinaceous protease, which had the same properties as PI, but amino acid sequence and amino acid content analysis showed that it is different from PI.
Suspension cells of peanut were treated with MJ and elicitor to determine the activities of phenylalanine ammonialyase (PAL), # 1,3-glucanase, polyphenol oxidase, chitinase, lipoxygenase(LOX-l,LOX-3), catalase(CAT), inhibition activity and active oxygene. The result showed that MJ largely increased the activities of PAL, LOX-1, inhibition activity, chitinase and slightly increased the activities of # 1,3-glucanase and LOX-3 while it slightly inhibited the activity of CAT and had no effect on the activities of inhibition; while elicitor largely increased the activities of PAL, inhibition activity, chitinase and slightly increased the activities of active oxygene, while it slightly inhibited the activity of LOX-land CAT and have no effect on the activities of LOX-3 and # 1,3-glucanase .
Treatment with MJ and elicitor had different effect on different substance about resistance. It is inferred that there were various pathways in different substances on resistance induced by MJ and elicitor,which are both relateted and indenpendent
采用凝胶层析、离子交换层析和电泳等方法，分别从花生种子醋酸提取液和磷酸缓冲液提取液中分离纯化胰蛋白酶抑制剂和2S清蛋白。研究表明，PI和2S清蛋白在花生中的含量很低，PI的最后得率为6％，2S的最后得率为1.2％，经PAGE、SDS-PAGE和HPLC分析表明，最后得到的PI为纯品，它是由一个单体组成的，2S清蛋白由5种低分子量的亚基组成，且部分以聚合体的形式存在。经SDS-PAGE和激光质谱测定其分子量，PI为7.07kDa，2S中含有与PI分子量相同的亚基(命名为2S-1)。PI的等电点为5.8，2S各组分的等电点相似，约为4.1，PI和2S都具有很高的热稳定性和酸碱稳定性，两者经100℃处理10min其抑制活性无明显变化，在pH2～11之间，活性也不发生改变。PI对胰蛋白酶、内肽酶和黄曲霉的生长具有较强的抑制作用，2S清蛋白在SDS处理前对胰蛋白酶、内肽酶和黄曲霉的生长无明显的抑制作用，但经SDS处理后其抑制活性大大增强。2S中含有对胰蛋白酶具有抑制作用的组分，具有与PI相似的性质，但蛋白N端序列和氨基酸分析表明，它可能属于一种病程相关蛋白，与PI不同。
用MJ和黄曲霉菌细胞壁激发子处理花生悬浮培养细胞，然后测定其抗病相关酶活性及抗病相关物质的含量。结果表明，MJ对苯丙氨酸解胺酶、脂氧酶-1、胰蛋白酶抑制活性、几丁质外切酶、几丁质内切酶具有较强的促进作用、对β-1，3葡聚糖苷酶、脂氧酶-3具有轻微的促进作用，略微抑制过氧化氢酶的活性，对活性氧含量无明显影响；激发子对苯丙氨酸解胺酶、胰蛋白酶抑制活性、几丁质外切酶、几丁质内切酶具有较强的促进作用，对活性氧含量具有轻微的促进作用，对脂氧酶-1、过氧化氢酶具有轻微的抑制作用，对β-1，3葡聚糖苷酶、脂氧酶-3无明显影响。MJ和激发子共同作用对不同的抗病相关物质的作用效果不同。因此可以认为，在MJ和激发子诱导的抗性相关物质的过程中，对不同的物质具有不同的作用方式和途径，它们之间既存在相互联系又相互独立。
Peamil(Arachis hypogaea L.Shanyou523)seeds were chosen as materials in this thesis to study the general characteristics of the natural proteinaceous protease inhibitors from them, and the effect of MJ and elicitor on the activities of some enzymes about resistance were investigated.
the crude PI and 2S fraction was separated from peanut seeds and purificated by Sephadex G-100 and DEAE cellulose(DE-32) chromatography . the properties of purified PI and 2S was determinded. The result showed that the content of PI and 2S in peanut seeds is very low and their last quantity is 6% and 1.2%seperately. PAGE, SDS-PAGE and HPLC analysis showed that the PI we gained are pure, which is a single polypetide with a molecular weight of 7.07kDa by SDS-PAGE and laser mass spectrum and a isoelectric point at pH5.8; The 2S protein is consist of five polypeptides with low molecular weight and the approximate isoelectric point at pH4.1and partly hang together, one of which has the same molecular weight as PI(named 2S-1).PI and 2S were stable below 100℃and between pH2.0-ll. PI can inhibite strongly proteinaceous protease,endopeptidase and A.flavus, while 2S before treatment had little inhibition on proteinaceous protease,endopeptidase and A.flavus, but after treatment, it had strong inhibition on proteinaceous protease,endopeptidase and A.flavus. Some of 2S had inhibiton on proteinaceous protease, which had the same properties as PI, but amino acid sequence and amino acid content analysis showed that it is different from PI.
Suspension cells of peanut were treated with MJ and elicitor to determine the activities of phenylalanine ammonialyase (PAL), # 1,3-glucanase, polyphenol oxidase, chitinase, lipoxygenase(LOX-l,LOX-3), catalase(CAT), inhibition activity and active oxygene. The result showed that MJ largely increased the activities of PAL, LOX-1, inhibition activity, chitinase and slightly increased the activities of # 1,3-glucanase and LOX-3 while it slightly inhibited the activity of CAT and had no effect on the activities of inhibition; while elicitor largely increased the activities of PAL, inhibition activity, chitinase and slightly increased the activities of active oxygene, while it slightly inhibited the activity of LOX-land CAT and have no effect on the activities of LOX-3 and # 1,3-glucanase .
Treatment with MJ and elicitor had different effect on different substance about resistance. It is inferred that there were various pathways in different substances on resistance induced by MJ and elicitor,which are both relateted and indenpendent
引文
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