离褶伞属菌株酯酶同工酶分析

对45个离褶伞属真菌菌株进行酯酶同工酶聚丙烯酰胺凝胶电泳(PAGE)分析,分析其酶谱多态性,并采用聚类分析方法对菌株间的亲缘关系进行鉴定。结果表明:在45个离褶伞属菌株酯酶同工酶酶谱中,共检测到了10条相对迁移率不同的酶带,多态性较高,并且在相似系数为0.75时,可将供试菌株分为4类,第一类包括17个菌株,第二类包括16个菌株,第三类包括11个菌株,第四类包括1个菌株。     

黑木耳菌株酯酶同工酶酶谱多样性研究

目的：为了探索黑木耳菌株之间的遗传距离,构建出供试菌株酯酶同工酶鉴别图。方法：采用垂直板聚丙烯酰胺凝胶电泳对21个黑木耳菌株酯酶同工酶的酶谱多样性进行了研究。结果：酯酶同工酶电泳各个菌株分别具有2~7条酶带,其中在Rf值为0.344处,21个菌株都有谱带出现。21个菌株之间的遗传相似系数在0.167-1.000之间,应用NTSYS软件进行聚类分析,当相似水平为0.73时,可将供试的21个黑木耳菌株分为6个不同的类群。结论：酯酶同工酶可以有效快捷的对黑木耳菌株进行菌种鉴定,是黑木耳菌株遗传多样性研究的理想手段。     

毛木耳种质资源的酯酶同工酶分析

采用垂直平板聚丙烯酰胺凝胶电泳技术对毛木耳56个菌株酯酶同工酶的酶谱多样性进行了研究。结果表明,56份材料有一定的遗传变异,共检测到迁移率不同的10条谱带,各菌株分别具有1~6条酶带,共有26种酶谱类型。菌株被分成了8大群:第一群包括黄耳zh等34个菌株;第二群包括白背木耳等8个菌株;第三、第四群分别为黑木耳和915两个单独的菌株;第五群包括99等4个菌株;第六群包括43等4个菌株;第七群包括50385和AP067两个菌株;第八群包括小上3和杂交34两个菌株。酯酶同工酶分析技术是鉴别种及品种以下菌株的有效手段。     

十字花科上的链格孢属真菌同工酶凝胶电泳分析

本文报道对从国内外收集到的生于十字花科植物上的3种链格孢属真菌21个菌株进行的10种同工酶的聚丙烯酰胺凝胶电泳分析结果。这10种酶分别是:酯酶(EST)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、多酚氧化酶(PPO)、过氧化氢酶(CAT)、谷氨酸脱氢酶(GDH)、甘露醇脱氢酶(MADH)、乙醇脱氢酶(ADH)、黄嘌呤脱氢酶(XDH)、过氧化物岐化酶(SOD)。对同工酶酶谱资料的聚类分析,在较高的相似性水平上将21个菌株归为3大类群,每一类群所包括的菌株与形态学的鉴定结果完全一致,各菌株的归类关系与其寄主植物、地理来源无关。表明同工酶凝胶电泳在Alternaria种级分类中是一个有用的工具,可以明确地将不同菌株鉴定到种级水平。     

木耳栽培菌株酯酶同工酶的酶谱多样性研究

采用聚丙烯酰胺凝胶电泳技术对木耳（Ａｕｒｉｃｕｌａｒｉａａｕｒｉｃｕｌａ（Ｈｏｏｋ）Ｕｎｄｅｒｗ）１０个栽培菌株酯酶同工酶的酶谱多样性进行了研究。１０个供试菌株的幼龄菌丝（７ｄ）中仅检测到２５条酶带,各个菌株分别具有２～３条酶带,１０个菌株仅有２种酶谱类型;老龄菌丝（７２ｄ）中共检测到４４条酶带,各个菌株分别具有３～６条酶带,１０菌株共有８种酶谱类型。研究表明,木耳双核体菌丝中某些酯酶同工酶基因位点在一定的发育时期才开始表达,老龄菌丝酯酶同工酶酶谱在木耳菌株鉴别和遗传育种研究中具有更大的应用价值。     

木耳栽培菌株酯酶同工酶的酶谱多样性研究

采用聚丙烯酰胺凝胶电泳技术对木耳（Ａｕｒｉｃｕｌａｒｉａ ａｕｒｉｃｕｌａ （Ｈｏｏｋ）Ｕｎｄｅｒｗ）１０个栽培菌株酯酶同工酶的酶谱多样性进行了研究。１０个供试菌株的幼龄菌丝（７ｄ）中仅检测到２５条酶带,各个菌株分别具有２～３条酶带,１０个菌株仅有２种酶谱类型;老龄菌丝（７２ｄ）中共检测到４４条酶带,各个菌株分别具有３～６条酶带,１０菌株共有８种酶谱类型。研究表明,木耳双核体菌丝中某些酯酶同工酶棋     

荞麦属植物三叶期幼叶酯酶同工酶研究

用聚丙烯酰胺凝胶电泳技术对荞麦属(Fagopyrum Mill)8个种(含大粒组7个种和小粒组1个种)29份栽培及野生荞麦植株三叶期幼叶的酯酶同工酶进行了研究.结果发现:酯酶同工酶酶带共31条,不同物种的酶带数4～9条,其中甜荞有8条带,而苦荞为9条.酶带分析及聚类分析表明:大粒组荞麦种的谱带与细野荞(F.gracilipes)等小粒组荞麦种间差异极大,甜荞(F.esculentum)和苦荞(F.tataricum)酶带分别与大野荞(F.megaspartanium)和毛野荞(F.pilus)相似,并分别与大野荞和毛野荞聚类最近, 提示大野荞和毛野荞可能分别是甜荞和苦荞的祖先种.     

低温处理对侧耳属栽培菌株菌丝体酯酶同工酶谱的影响

应用垂直板聚丙烯酰胺凝胶电泳对不同低温、不同缓化方式处理后的侧耳属(Pleurotus)不同栽培种12个菌株进行酯酶(EST)同工酶谱分析。结果表明:不同菌株对低温的耐受能力不同,缓化方式对于酯酶基因表达的影响不同,但低温并没有使其酯酶酶活力完全丧失。     

斑翅蝗科部分种类的酯酶同工酶研究：（直翅目：蝗总科）

本文采用聚丙烯酰胺凝胶电泳方法,对斑翅蝗科（Ｏｅｄｉｐｏｄｉｄａｅ）６属８个种进行了酯酶同工酶的研究,共显示３０条迁移率不同的酶带,根据酶带迁移距离的不同,可以分为５个泳动区。通过ＥＳＴ同工酶酶带的聚类分析,对斑翅蝗科部分种类的分类问题进行了探讨。     

铜陵牡丹和垫江牡丹的同工酶分析

采用垂直板聚丙烯酰胺凝胶电泳技术,对铜陵牡丹和垫江牡丹进行过氧化物酶（POD）和酯酶（EST）同工酶的测定分析。结果显示,两个品种牡丹的POD同工酶酶谱共栓出7条酶带,其中铜陵牡丹分离带为7条,垫江牡丹为3条,铜陵牡丹多出4条特有酶带,且活性较强;EST酯酶同工酶中,两种牡丹共有酶带为5条,活性上略有差异,铜陵牡丹特有2条酶带,垫江牡丹特有1条酶带。     

11个灵芝菌株的分子ID构建

[目的]收集11株灵芝菌种为材料,在分子水平上对其进行分类鉴定,并构建分子ID.[方法]采用ITS和SSR分子标记技术,对11株灵芝进行分子鉴定分析.[结果]通过内转录间隔区(ITS)序列测定分析表明,与GenBank上登录的灵芝(Ganoderma lucidum)菌株ITS序列相似度达到99％,在种的水平上证明实验所采用的供试菌株均属灵芝种(Ganoderma lucidum).利用SSR分子标记技术对菌株进行引物扩增,综合多态性条带,用NTSYS软件进行聚类分析,相似度在0.62水平上,1 1个灵芝菌种被分成4个类群,其中GL-2与GL-4各自聚为一类.用ID Analysis 1.0软件进行数据分析表明,用5对SSR引物可将11株灵芝供试菌种完全区分开,并构建其分子身份证.[结论]基于SSR分子标记构建灵芝菌属的分子ID是可行的.     

An assessment of the genetic diversity within Ganoderma strains with AFLP and ITS PCR-RFLP

Ganoderma lucidum is one of the most important medicinal materials and plant pathogens. Because of its specific interhybridization, the genetic background, however, is relatively unclear. It made identification of Ganoderma strains, especially closely related strains difficulty. Amplified fragment length polymorphism (AFLP) using 14 primer combinations and internal transcribed spacer (ITS) PCR-RFLP were used in a comparative study which was designed to investigate the closely related Ganoderma strains genetic relations at molecular level. The analysis of 37 Ganoderma strains showed there were 177 polymorphic AFLP markers and 12 ITS PCR-RFLP markers, and all accessions could be uniquely identified. Among the Ganoderma accessions, similarity coefficients ranged from 0.07692 to 0.99194 in AFLP. The Ganoderma strains formed a tight cluster in nine groups in AFLP whereas seven groups in ITS PCR-RFLP. The cluster analysis revealed that the taxonomical system of subgenus Ganoderma is composed of Sect. Ganoderma and Sect. Phaeonema, and the strain 22 should be a variant form of strain 21. All methods delineated the Ganoderma strains from the different regions seeming to show a greater level of genetic diversity. It indicated that the genotype study at molecular level is a useful complement method to the current classification system of Ganoderma strains based on morphological traits. The congruency of the experiments was analyzed using the biostatistical software DPS V3.01.     

Intrastrain internal transcribed spacer heterogeneity in Ganoderma species

Intrastrain internal transcribed spacer (ITS) heterogeneity is first reported from Ganoderma, a fungal genus within Basidiomycetes. ITS amplification products from 4 strains, representing 4 Ganoderma species, were cloned and sequenced. Two to five different ITS types were found within a single strain. The clone sequences were analyzed along with other sequences from Ganoderma retrieved from GenBank. The results show that sequence variation within strains varies considerably with species and the heterogeneity may occur in the 3 parts (ITS1, ITS2, and 5.8S) of the ITS region.     

Phylogenetic analysis of Ganoderma based on nearly complete mitochondrial small-subunit ribosomal DNA sequences

Characteristics and structures of mt SSU rDNA were investigated for the phylogenetic study of Ganoderma. Phylogenetic information was concentrated mostly in the V1, V4, V5, V6 and V9 variable domains, but informative sites in conserved domains also significantly contributed in resolving phylogenetic relationships between Ganoderma groups. Secondary structure information of variable domains was found to be a useful marker in delineation of phylogenetic groups. Strains of Ganoderma species used in this study were divided into six monophyletic groups. Ganoderma colossus made a distinct basal lineage from other Ganoderma species and Tomophagus, created for G. colosuss, appeared to be a valid genus. Ganoderma applanatum and G. lobatum classified in subgenus Elfvingia made a monophyletic group. Ganoderma tsugae from North America and G. valesiacum from Europe, both living on conifers, were closely related. Ganoderma oregonense and strains labeled G. lucidum from Europe and Canada were grouped with G. tsugae and G. valesiacum. Strains labeled G. lucidum living on hardwoods from the United States and Taiwan were grouped with G. resinaceum, G. pfeifferi and G. subamboinense var. laevisporum, and they all produced chlamydospores. Two strains labeled G. lucidum and three strains labeled G. resinaceum from America were concluded to be conspecific. Strains labeled G. lucidum from Korea and Japan were monophyletic and were distinguished from strains labeled G. lucidum from Europe and North America. Host relationships and the presence of chlamydospores in culture proved to be important characteristics in the systematics as well as the phylogenetic relationships of Ganoderma.     

Development of strain-specific SCAR markers for authentication of Ganoderma lucidum

In this study we report the application of sequence-characterized amplified region (SCAR) markers in Ganoderma lucidum for strain identification, the first such study in this medicinal mushroom. One fragment unique to strain No. 9 was identified by inter-simple sequence repeats (ISSR), and then sequenced. Based on the specific fragment, one SCAR primer pair designated as GL₆₁₂F and GL₆₁₂R was designed to amplify a 612-bp DNA fragment within the sequenced region. Diagnostic PCR was performed using the primer pair. The results showed that this SCAR marker can clearly distinguish strain No. 9 from other related Ganoderma lucidum strains. Our data provided the foundation for a precise and rapid PCR-based strain-diagnostic system for Ganoderma lucidum.     

灵芝麦角硫因高通量检测方法研究

麦角硫因(ergothioneine,EGT)基于其强抗氧化及体内消耗率低的特点,在人体内拥有多种重要的生理功能。但是在高产EGT微生物筛选和新菌株的选育研究中,现有的EGT检测方法因其步骤繁琐、使用的试剂和设备昂贵而亟待改进。本研究基于EGT的理化性质,建立了EGT-硫氰酸铁高通量快速检测体系,同时选用不同EGT产量的灵芝菌株以及灵芝融合新菌株对该检测体系的准确性进行验证。结果表明该方法可以快速准确地比较出样本间EGT产量高低,使原本需3~4天的工作缩短至2~3 h,HPLC验证结果显示,EGT-硫氰酸铁高通量快速检测体系效果良好,体系稳定。本研究结果将为高产EGT微生物的高通量筛选及高产EGT新菌株的选育提供新的方法和思路。     

药用真菌子实体多糖SEC—HPLC图谱的相似度定量评价研究

目的：对灵芝属Ganoderma下4个种共7个菌株及灰树花Grifola ffondosa和云芝Trametes versicolor共9种子实体粗多糖进行了体积排阻HPLC色谱（SEC—HPLC）,并分析了相似度。方法：各样品水提物的HPLC图谱用平方欧氏距离法计算了相似度,用离差平方和法进行聚类分析。结果：灵芝属内各样品聚类距离为8,灰树花与云芝的聚类距离为16,它们与灵芝属的距离为25,说明同一灵芝属内的不同种及同种不同菌株所产生的多糖分子量分布近似度远高于来自树花菌属的灰树花或栓菌属的云芝。结论：此方法可量化描述不同多糖间SEC—HPLC的相似关系,有作为真菌多糖样品质量控制手段的潜在能力。     

14个灵芝菌株的酯酶同工酶分析

对14个灵芝菌株进行拮抗试验和酯酶同工酶聚丙烯酰胺凝胶电泳聚类分析,实验共检出迁移率不同的谱带18条,在50％相似水平上聚合为5类,第1类包括仙芝、大仙823、南韩、园芝6号、甜芝、韩芝;第2类包括赤芝10号、赤芝12号、赤芝、日本红芝;第3类包括紫芝和黑芝;第4类为血芝;第5类为白芝。聚类分析结果与形态观察及拮抗试验结果基本一致。实验还说明酯酶同工酶可作为灵芝良种选育的一种有效标记方法,在菌种鉴定、保藏和管理等方面具有重要作用。     

灵芝尿嘧啶营养缺陷型菌株的筛选和分子鉴定

【背景】营养缺陷型是一种应用广泛的分子标记,但是目前在灵芝中还未有研究和应用报道。【目的】为灵芝遗传转化研究、杂交育种和菌种鉴别提供亲本材料和技术支持。【方法】采用紫外光诱变、单单杂交、孢子单核化的方法从灵芝单核体菌株出发得到尿嘧啶营养缺陷型双核体菌株。【结果】获得8株稳定的尿嘧啶营养缺陷型单核体突变菌株和7株尿嘧啶营养缺陷型双核体菌株。【结论】灵芝尿嘧啶营养缺陷型菌株在添加外源营养物的基础上可恢复正常生长,可以为灵芝遗传转化体系的构建和灵芝育种提供材料。     

HPLC指纹图谱技术在灵芝组织分离试验中的应用

利用HPLC指纹图谱技术研究从同一灵芝子实体不同部位组织分离获得的菌株三萜化合物的差异。用常规组织分离方法获得不同菌株,在A、B、C、D四种培养基上进行出菇试验,运用HPLC指纹图谱技术获得各子实体三萜提取物HPLC指纹图谱,计算图谱间的相似度,分析三萜指纹的差异。结果表明,从子实体不同部位分离获得的四个菌株在同一培养基相同条件下培养获得的灵芝子实体的三萜指纹图谱相似度均大于0.99;同一部位分离获得的菌株在不同培养基相同培养条件下获得的子实体,其粗三萜HPLC图谱相似度均大于0.96;不同的组织分离部位和不同的培养基对灵芝菌株三萜指纹图谱的影响均不显著。18号菌株在C培养基上培养获得的子实体三萜含量最高,上层菌肉为最优组织分离部位,C培养基为最优培养基。灵芝的三萜组成不受生长环境的影响,而生长环境会对其三萜化合物含量产生一定的影响。本文首次应用HPLC指纹图谱方法对灵芝组织分离获得的菌株的差异性进行了研究。