一种简便、快速测定鹅膏多肽毒素的方法——抑芽法  

BUD-INHIBITED ASSAY, A SIMPLE AND QUICK METHOD FOR DETECTING AMATOXINS IN AMANITA MUSHROOM

作　　者：张志光[1] 刘建强[1] 张晓元[1] 陈作红[1] 张在海[1] 

机构地区：[1]湖南师范大学生命科学学院,长沙410081

出　　处：《菌物系统》2001年第3期381-386,共6页Mycosystema

基　　金：国家科技部重点课题资助项目:96-C02-03-07

摘　　要：低浓度（5-10ngml-1）的鹅膏毒肽(amatoxins) 能专一性抑制RNA聚合酶Ⅱ的活性，高浓度（为RNA聚合酶Ⅱ103-104倍的浓度）也能抑制RNA聚合酶Ⅲ的活性，因而影响细胞mRNA的转录和蛋白质的合成，抑制种子的萌发和生长。根据此原理我们建立了一种以植物种子萌发试验检测鹅膏毒肽的方法，称之为“抑芽法”（bud-inhibited assay）。该方法简便、实用、准确。操作流程：45℃干燥至恒重的鹅膏菌等子实体菌盖；水提法提取毒素并用氯仿沉淀蛋白质等；粗毒液浓度为0.025-0.05gml-1干子实体；萝卜或绿豆种子培养温度为28℃，培养时间为24-72h。绿豆芽下胚轴生长被抑制率在95%以上，可能为鹅膏毒肽含量高的剧毒鹅膏菌；当被抑制率在60-80%，可能为鹅膏肽含量较低的微毒鹅膏菌；当被抑制率在30%以下时，可能是不含鹅膏菌肽的鹅膏菌。RNA polymerase II is inhibited specifically by amantoxins at 1~10μg/ml. Therefore the transcription of mRNAs and syntheses of proteins are restrained.It resulted in the inhibition of germination and growth of plant seeds and thus a simple and quick method, bud-inhibition assay,was developed for detecting amantoxins in Amanita mushrooms. The main procedures of the method included: (1)Extraction for amantoxins:the dried Amanita mushrooms were extracted with water and then the extracted solution was extracted with chloroform to remove proteins and liqids; (2) Seeds germination:the selected seeds were incubated in crude extraction solution at 28℃ for 72 hours. Result evaluation: The content of amantoxin in Amanita mushroom can be approximately estimated by the inhibited percentage of hypocotyle of seedlings. If the Amanita species contain high concentration of amantoxins, the inhibited percentage of the length of hypocoty of mung beans was over 95%. If the Amanita species was less poisonous , the inhibited percentage of the length of hypocoty of mung beans was between 60~80%. If the mushroom was slightly poisonous or non-toxic, the inhibited percentage of the length of hypocoty of mung beans was less than 30%. The bud-inhibied assay is concise, practical and especial for preliminary examination of Amanita mushrooms.

关 键 词：抑芽法 鹅膏多肽毒素 鹅膏菌 绿豆种子 RNA聚合酶Ⅱ 野生菌 食用菌 

分 类 号：Q949.32[生物学—植物学]