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吉林省和山东省鹅膏属的分类及肽类毒素的检测
The Taxonomy of Amanita from Jilin and Shandong Provinces and Detection of Peptide Toxins
【作者】 王玉玲;
【导师】 图力古尔;
【作者基本信息】 吉林农业大学 , 生药学, 2011, 硕士
【摘要】 本论文通过对吉林农业大学菌物标本馆(HMJAU)、中国科学院微生物研究所菌物标本馆(HMAS)及鲁东大学菌物标本馆(HMLD)的共169份鹅膏菌标本的宏观形态及显微结构的观察,描述了吉林省及山东省分布的鹅膏菌27种和2变种,包括3个鹅膏属待定种。吉林省描述了19种(包括变种及待定种)是:毒蝇鹅膏A.muscaria、毒蝇鹅膏黄色变种A. muscaria var. formosa、亚球基鹅膏A.subglobosa、短棱鹅膏A.imazekii、长棱鹅膏A.longistriata、红黄鹅膏A.hemibapha、杏黄鹅膏A.crocea、灰鹅膏A.vaginata、东方褐盖鹅膏A.orientifulva、红褐鹅膏A. orsonii、赭盖鹅膏A.rubescens、黄柄鹅膏A. flavipes、黄盖鹅膏A.subjunquillea、黄盖鹅膏白色变种A.subjunquillea var.alba、鳞柄白毒鹅膏A.virosa、玫瑰红鹅膏A.pallidorosea、雀斑鳞鹅膏A.avellaneosquamosa、待定种2个。山东省16种:可食鹅膏A.esculenta、卵孢鹅膏A.ovalispora、假灰鹅膏A.pseudovaginata、杏黄鹅膏A.crocea、灰褶鹅膏A.griseofolia、灰鹅膏A.vaginata、红褐鹅膏A. orsonii、赭盖鹅膏A.rubescens、格纹鹅膏A.fritilaria、暗盖淡鳞鹅膏A.sepiacea、黄盖鹅膏A.subjunquillea、锥鳞白鹅膏A.virgineoides、日本鹅膏A.japonica、雀斑鳞鹅膏A. avellaneosquamosa和待定种2个。其中《中国真菌志.鹅膏科》中未描述的有玫瑰红鹅膏A.pallidorosea、赭盖鹅膏A.rubescens、鳞柄白毒鹅膏A.virosa、杏黄鹅膏A.crocea及3个待定种。采用基于LSU序列的贝叶斯分析法进行了本论文中形态学上存在疑惑的10份鹅膏菌的分子系统学研究,其结果基本上与形态分类学研究结果相吻合。通过在GenBank中序列比对及系统发育树的构建及形态学研究得出,山东省的待定种HMLD0565与吉林省的HMJAU20443、HMJAU20469、HMJAU20482是同一个种。A.sp.2是杏黄鹅膏A.crocea的一个近似种。标本鳞柄白毒鹅膏A.virosa及格纹鹅膏A.fritilaria与GenBank中已有的序列相似度达99%及以上。A.sp.1未能扩出DNA。采用HPLC法,对13种15个鹅膏材料进行了主要肽类毒素即α-鹅膏毒肽(α-AMA)、β-鹅膏毒肽(β-AMA)、二羟鬼笔毒肽(PHD)的检测,并分别测定了剧毒鹅膏玫瑰红鹅膏完整子实体、菌盖、菌柄和菌托混合物中3种肽类毒素的含量。结果显示:含α-AMA的鹅膏菌有暗盖淡鳞鹅膏A.sepiacea、玫瑰红鹅膏A.pallisorosea和A.sp.3;含β-AMA的鹅膏菌有格纹鹅膏A.fritilaria、暗盖淡鳞鹅膏A.sepiacea、攻瑰红鹅膏A.pallisorosea及A.sp.3;含PHD的鹅膏菌有灰褶鹅膏A.griseofolia、格纹鹅膏A.fritilaria、暗盖淡鳞鹅膏A.sepisacea、欧氏鹅膏A. oberwinklerana及玫瑰红鹅膏A.pallidorosea。从HPLC谱图分析,格纹鹅膏A.fritilaria (HMLD0332)和A.sp.3子实体中β-AMA含量较高,可考虑做制备的原料。测定玫瑰红鹅膏A.pallidorosea中α-AMA、β-AMA和PHD三种毒素的含量,三种毒素的含量分别为,子实体中30.3168mg/g、6.9932mg/g和9.9459mg/g;菌盖中44.9573mg/g、11.0798mg/g和11.3025mg/g;菌柄和菌托混合部分中:α-AM A 11.6904mg/g和PHD 7.9775mg/g。可作为制备毒素的原料。还发现玫瑰红鹅膏不同部位的各毒素的含量有所差异,α-AMA在菌盖中的含量为菌柄和菌托混合物中含量的4.43倍,β-AMA则主要存在于菌盖部分,PHD在菌盖中的含量仍高于菌柄和菌托混合部分中的含量。
【Abstract】 Through to the obserbations of 169 specimens deposited in the Academy of Science (HMAS), Mycological Herbarium of Jilin Agricultural University (HMJAU) and Mycological Herbarium of Ludong University,27 species and 2 variations (include four undetermined species) were conducted to the morphological descriptions, illustrations and line drawings.19 species described from Jilin province (include variation and undetermined):A.muscaria, A. muscaria var. formosa, A.subglobosa, A.imazekii, A.longistriata, A. hemibapha, A.crocea, A.vaginata, A.orienfifulva, A.orsonii, A. flavipes, A.subjunquillea, A.subjunquillea var.alba, A.virosa, A.pallidorosea, A. avellaneosquamosa,3 undetermined species.16 species described from Shandong province:A.esculenta, A.ovalispora, A.pseudovaginata, A.crocea, A.griseofolia, A. vagwata, A.orsonii, A. fritilaria, A.sepiacea, A.subjunquillea, A.virgineoides, A.japonica, A.avellaneosquamosa, and undetermined 2 species. A.pallidorosea, A.rubescens,A.virosa,A.crocea and 3 undetermined species are undiscribed in <Flora Fungoroom Sinicorum. Amanitaceae>The molecular biology research were conducted to ten Amanitas exists morphological doubt, Based on LSU sequence for the bayesian analysis. Molecular biology results was basically consistent with the taxonomic results. After sequence alignment in GenBank, construction of Phylogenetic Tree and Morphological research,we know that A.sp.3 from shandong province and HMJAU20443, HMJAU 20469,HMJAU20482 from Jilin province were the same species; A.sp.2 is an approximate species of A.crocea. Specimens A.virosa and A.fritilaria and GenBank known sequence was similar and above to 99%. A.sp.1 failed to enlarge the DNA.Using HPLC for detected the main peptide toxins (α-AMA,β-AMA and PHD) of 13 species 15 Amanitas, and the contents of of them in A.pallidorosea basidiocap, cap, the mixture of stipe and volva were determined separately. The result showed that, A. sepiacea, A.pallisorosea and A.sp.3 containsα-AMA, A.fritilaria, A.sepiacea, A.pallisorosea and A.sp.3 containsβ-AMA; A.griseofolia, A.fritilaria, A.sepisacea, A.oberwinklerana and A.pallidorosea contains PHD. HPLC analysis showed that, the contents ofβ-AMA in A.frililaria (HMLD0332) and A.sp.3 basidiocap can considered to preparation of a raw material.The contents ofα-AMA,β-AMA and PHD in A.pallidorosea were determined, they were 30.3168 mg/g,6.9932 mg/g and 9.9459 mg/g in fruit body, and 44.9573 mg/g,11.0798 mg/g and 11.3025 mg/g in pileus, respectively. The contents ofα-AMA and PHD in the mixture of stipe and volva were 11.6904 mg/g and 7.9775 mg/g, but theβ-AMA was not detected in this part.Can be used as the raw material preparation of toxins. In A.pallisorosea also found the contents of each toxin but contents was different, the contents ofα-AMA in cap was 4.43 times of the contents in the mixture of stipe and volva,β-AMA was mainly exists in cap, the content of PHD in cap still higher than the mixture of stipe and volva.
【Key words】 Amanita; morphology; Molecular biology; HPLC methed; Amanita pallidorosea;