冬虫夏草及其相关类群的分子系统学分析

为了探明冬虫夏草及其相关类群的亲缘关系,以冬虫夏草、中国被毛孢及中华束丝孢(=冬虫夏草头孢=蝙蝠蛾多毛孢)共6株菌种作为内群和一株蛹草拟青霉作为外群进行了DNA随机多态型(RAPD)分析。此外,基于上述供试材料又在内群中增加了一株蝙蝠蛾拟青霉,并对内群和外群样品的nrDNA间隔区(ITS)碱基序列进行了测定;对于测定的8条序列连同来自GenBank中的4条相关序列进行了分子系统学分析。结果表明:中华束丝孢和中国被毛孢均系冬虫夏草菌的无性型。按照国际植物命名法规,中国被毛孢应为冬虫夏草菌无性型的正确名称。而蝙蝠蛾拟青霉为不同于冬虫夏草菌的另一种真菌;该名称由于不合格发表而不被国际植物命名法规所承认。     

虫草属分子系统学研究现状

近年来的分子系统学研究结果表明 ,麦角菌目 (Clavicipitales)与肉座菌目 (Hypocreales)以及虫草属[Cordyceps(Fr.)Link]与麦角菌科 (Clavicipitaceae)内的有关种属关系密切 ,但虫草属为一多系群 ,其中只有新虫草亚属的种类形成单系群。虫草属种类的进化与寄主有一定的关系 ,有关研究显示它们在亲缘关系相差很大的寄主之间多次转移。多个独立的ITS序列研究表明 ,阔孢虫草 (CordycepscrassisporaM .Zangetal.)、甘肃虫草(CordycepsgansuensisK .Y .Zhangetal.)、多轴虫草 (CordycepsmultiaxialisM .Zang&Kinjo)、尼泊尔虫草 (CordycepsnepalensisM .Zang&Kinjo)与冬虫夏草 [Cordycepssinensis(Berk.)Sacc.]为同一物种 ;RAPD和ITS序列分析还显示不同地理分布的冬虫夏草存在明显的遗传分化。分子生物学方法在确定有性型与无性型的关系上已经为多种虫草菌提供了有力的证据。本文主要对虫草属分子系统学研究现状进行了综述 ,同时就分子生物学研究中的取样问题、一些分子方法的适用范围以及有性型与无性型的关系等问题进行了讨论。     

丝虫草及其无性型关系的分子确证

目的对丝虫草（Ophiocordyceps filiform is）及从子囊孢子分离所得的无性型进行测序和比较分析,确证二者之间的对应关系。方法采用PCR技术,以rDNA-ITS区为分子指标。结果系统进化树显示丝虫草的无性型归为被毛孢属较为合理,同时通过形态特征比较的结果表明,其无性型为荔波被毛孢（Hirsutella liboensis）。结论本研究首次确立了丝虫草的有性型与无性型的对应关系,并且成功培育出了与天然丝虫草相同的虫草子实体。     

中国被毛孢和蝙蝠蛾拟青霉小鼠免疫功能调节作用的比较

目的观察中国被毛孢和蝙蝠蛾拟青霉对小鼠免疫功能的影响。方法分别给予中国被毛孢和蝙蝠蛾拟青霉后,测定观察对小鼠碳粒廓清功能、迟发性变态反应、溶血素抗体、淋巴细胞增殖和NK细胞活性等的影响。结果3．0g／kg中国被毛孢和蝙蝠蛾拟青霉可显著提高小鼠吞噬指数和半数溶血值（P〈0．01）、显著提高T、B淋巴细胞增殖能力（P〈0．01,P〈0．05）、显著提高NK细胞活性（P〈0．05）,0．5～1．5g／kg中国被毛孢都能显著抑制小鼠耳廓肿胀（P〈0．01,P〈0．05）,同时还能抑制脾脏和胸腺增大（P〈0．01,P〈0．05）。结论中国被毛孢和蝙蝠蛾拟青霉均具有免疫调节作用,而中国被毛孢免疫抑制及增强天然免疫系统作用要优于蝙蝠蛾拟青霉,而对适应性免疫系统增强作用蝙蝠蛾拟青霉优于中国被毛孢。     

台湾虫草及其无性型关系的分子确证

采用PCR技术,以rDNA-ITS区为分子指标,对台湾虫草Cordyceps formosana及其无性型黄山被毛孢Hirsutel lahuangshanensis进行测序和比较分析,以进一步确定两者的对应关系。结果表明:两者的序列完全相同,从而在分子水平上证明了黄山被毛孢是台湾虫草的无性阶段。系统进化树显示台湾虫草的无性型归为被毛孢属较为合理。     

台湾虫草及其无性型关系的分子确证

采用PCR技术,以rDNA-ITS区为分子指标,对台湾虫草Cordyceps formosana及其无性型黄山被毛孢Hirsutella huangshanensis进行测序和比较分析,以进一步确定两者的对应关系。结果表明：两者的序列完全相同,从而在分子水平上证明了黄山被毛孢是台湾虫草的无性阶段。系统进化树显示台湾虫草的无性型归为被毛孢属较为合理。     

虫草及其无性型中的抗肿瘤细胞活性成分

较系统地总结了目前在虫草及其无性型中发现的抗肿瘤细胞活性成分.其中冬虫夏草、蛹虫草、大团囊虫草、单侧虫草、戴氏虫草和拟蛹虫草等虫草以及它们的无性型蛹拟青霉、细脚拟青霉、金龟子绿僵菌和球孢白僵菌等虫生真菌代谢物中抗肿瘤细胞活性成分,其结构包括多糖类、甾醇类、含氮化合物和杂环化合物等多个类型.     

冬虫夏草无性型研究概况*

文献调查结果表明与冬虫夏草有关的无性型菌种已报道有22个学名,涉及13个属。针对这些名称上存在的命名问题本文展开了讨论,并评述了不同名称与冬虫夏草的关系及其有关菌种的研究进展。在已报道的分离菌种中,作为新种正式描述的名称有8个,其中6个为合格发表,其余2个未能满足名称合格发表的条件;还有4个名称在文献中作为新种提到,但没有合格发表的出处,也有一例只提到名称而无其他要素;另有5个名称则只有属名,尚未鉴定到种的水平。中国被毛孢为多个独立的研究证明为冬虫夏草的无性型,还有3个无性型名称已被证明不可能是冬虫夏草的无性型。其他无性型名称与冬虫夏草的关系有待进一步验证。文中对与冬虫夏草有关的菌种定名、无性型分离和验证方法、复无性型现象,以及有关学名问题作了讨论。     

冬虫夏草的微循环产孢及其无性型的分离*

从采自云南德钦白马雪山的冬虫夏草子实体上,利用改进的子囊孢子弹射法获得其无性型中国被毛孢,并利用冬虫夏草子囊孢子的微循环产孢和无性型菌丝的载片培养,证明我们获得纯培养是冬虫夏草的无性型。     

Morphological and genetic characterization of a cultivated Cordyceps sinensis fungus and its polysaccharide component possessing antioxidant property in H22 tumor-bearing mice

Cordyceps sinensis, one of the most precious traditional Chinese medicines, possesses the antitumor activity, antioxidant activity and the capability of modulating the immune system. In the present study, a fungus strain G1 isolated from wild C. sinensis was identified and initially characterized. A phylogenetic tree was generated based on the sequences of the internal transcribed spacer (ITS) region of related fungi. The analysis of ITS sequence showed that fungus G1 was clustered together with C. sinensis, Tolypocladium cylindrosporum and Tolypocladium inflatum in the phylogenetic tree. Both the morphological character and the ITS sequence analysis establish that fungus G1 is one of the anamorph strains of C. sinensis and belongs to Tolypocladium genus. Furthermore, the polysaccharide (PS) extracted from fungus G1 and its antioxidant activity on H22-bearing mice was investigated. H22 cells were hypodermically injected into the right oxter of each mouse after the ICR mice were treated with PS by means of gavage for 7 days. Then the same administration process continued for 9 days. At the end of the experiments, the tumor weight of each mouse was measured. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in mouse liver, brain and serum, as well as glutathione peroxidase (GSH-Px) activity in mouse liver and brain were assayed. The results showed that the H22 tumor growth was significantly inhibited by PS. Moreover, PS significantly enhanced SOD activity of liver, brain and serum as well as GSH-Px activity of liver and brain in tumor-bearing mice. PS also significantly reduced the level of MDA in liver and brain of tumor-bearing mice.     

安徽的虫草及其相关真菌I.

报道疣孢虫草Cordyceps phymatospora为新种;球孢虫草C. bassiana 及其无性型Beauveria bassiana, 长座虫草C. longissima 及其无性型 Hirsutella longissima, 台湾虫草C. formosana 及其无性型 Hirsutella sp., 拟细虫草C. gracilioides和丝虫草C. filiformis 等19种虫草及其部分相关真菌。模式标本保存在安徽农业大学虫生菌研究中心(RCEFAAU)。     

中国拟青霉新种及其与虫草的关系

从虫草[Cordyceps sinenisis(Berk.),Sacc.]上分离到中国拟青霉新种(Paecilomyces isnenisisp.nov.),对它进行了培养和形态学研究。中国拟青霉与蛹草[cordyceps militaris(L·)Liak]的无性阶段形态非常类似,但在分生孢子形态,分生孢子和瓶梗的量度上有所不同,且两者的寄主及生境不同,故定为新种。不仅可从长子座的殭虫获得其纯培养物,也可从不长子座而长孢梗束的虫草纯化培养获得。纯培养物的形态和天然虫体的孢梗束完全相同,故认为它可能是虫草的无性阶段。     

Genetic variation of Cordyceps sinensis, a fruit-body-producing entomopathogenic species from different geographical regions in China

Cordyceps sinensis is one of the most valuable medicinal fungi in the Orient. It is naturally distributed in the eastern extension area of the Qinghai-Tibet plateau, at an altitude over 4000 m high. In order to investigate genetic variation and evolutionary relationships of C. sinensis from different geographical regions, 17 isolates of C. sinensis were collected from different provinces and the complete sequences of rDNA ITS were determined. On the basis of 5.8S rDNA and ITS region analysis, it was clearly shown that the ITS sequences within C. sinensis are highly homologous regardless of geographical origin. The distance values between the sequences in this study were lower than 0.03. This implied that C. sinensis from different geographic regions are the same species; they are not different species or a species complex. The results also showed that distance values between C. sinensis and Hirsutella sinensis are of the same order as those within C. sinensis from different geographic regions. This confirmed our previous results that C. sinensis should only have H. sinensis as its asexual stage whatever the geographic region from which the samples were collected. An rDNA ITS clone library was established to obtain further evidence for the interpretation of the fungal community structure from C. sinensis and to confirm the accuracy of the taxonomic identities produced by directly sequencing the rDNA ITS region. The discrimination between intraspecies of C. sinensis might provide additional data for the authentication of medicinal material at the species or intraspecies level.     

西藏冬虫夏草无性型的分子生物学研究

从西藏采集的冬虫夏草子实体中分离出 3个菌株C3、C4、C5,通过培养特征的研究以及采用分子生物学方法 ,以rDNAITS区为分子指标 ,与冬虫夏草 [Cordycepssinensis(Berk .)Sacc.) ]的有性世代及中国被毛孢 (HirsutellasinensisLiu ,Guo,Yu&Zeng)进行比较分析 ,发现C4的培养特征与C3、C5 完全不同 ,其序列与冬虫夏草的相似率为 93 %,与中国被毛孢的相似率为 94%,而C3、C5 与冬虫夏草的相似率很低 (分别为 5 5 %和 6 9%) ,从而证明C4是西藏冬虫夏草的无性型 ,为中国被毛孢。     

古尼虫草无性型的分子鉴别

采用PCR技术,以rDNA的ITS区为分子指标,对古尼虫草Cordycepsgunnii的有性和无性阶段进行比较分析,从分子水平上证明古尼虫草的无性阶段是古尼拟青霉Paecilomycesgunnii。     

利用原生质体紫外诱变技术选育可常温发酵的中国被毛孢新菌株

中国被毛孢(Hirsutella sinensis)是天然冬虫夏草[Cordyceps sinensis(Berk)Sacc.]的真正无性型,其生长温度低(低于20℃),生长缓慢(发酵周期长达20～30d),对人工发酵的技术条件要求很高,因而大大增加了工业化发酵生产的成本和技术难度。为了解决上述问题,实验中从天然冬虫夏草子座中分离到中国被毛孢菌株BS-1,并对其进行了原生质体紫外诱变处理。经过粗筛和精筛,从中选出一株可常温下(25～28℃)发酵、生长迅速(发酵周期4～5 d)、有效成分(甘露醇和腺苷)含量显著高于原始菌株的诱变株BS-5962。通过连续10代PDA斜面继代培养和摇瓶试验,以及连续3批的1t罐的中试试验,检查结果表明所得诱变株性状优异而稳定。可为降低中国被毛孢的液体深层发酵成本和技术难度提供一种有效的解决方案。     

Hirsutella uncinata, a new hyphomycete from Australia

Hirsutella uncinata, collected on follicles of Hakea sp. in New South Wales, Australia, is described as a new anamorph species. The fungus produces stiff, erect, verticillate conidiophores with up to 10 whorls of hooked, phialidic conidiogenous cells that have rough-walled necks. The conidia are subfusiform, flattened on one side and produced in groups of 2-3 in a dense slimy ball at the end of each phialide. Parsimony analysis of aligned internal transcribed spacer rDNA sequences suggests that H. uncinata is closely related to the Cordyceps sinensis complex (Clavicipitaceae, Hypocreales), which includes species with morphologically similar anamorphs.     

确证冬虫夏草无性型的分子生物学证据I.中国被毛孢与冬虫夏草之间的关系

从青海的冬虫夏草子实体上分离出中国被毛孢［Ｃｏｒｄｙｃｅｐｓ　ｓｉｎｅｎｓｉｓ（Ｂｅｒｋ．）Ｓａｃｃ．］,并利用ＲＡＰＤ－ＰＣＲ技术,筛选出８种引物,获得了冬虫夏草和中国被毛抱相应的基因组ＤＮＡ指纹图谱,两者相似率高达９６％,从而表明冬虫夏草的无性型为中国被毛孢。     

虫草对肺部疾病治疗的研究进展

肺纤维化、慢性阻塞性肺病和哮喘是人类主要的呼吸系统疾病,它们均以肺部或呼吸道的慢性炎症为特征,当前的治疗方法尚不理想并伴随着很多的副作用,人们迫切需要一种更安全的替代疗法。冬虫夏草是我国传统的名贵药用真菌,被广泛用来治疗多种肺部疾病。近年来,冬虫夏草对肺部疾病治疗作用的研究取得了较大进展,本文综述了冬虫夏草、其无性型中国被毛孢(Hirsutella sinensis或Ophicordyceps sinensis)和其他相关虫草及其提取物对肺纤维化、慢性阻塞性肺病、哮喘等肺部疾病治疗作用的研究进展,并介绍了其作用机制的研究成果。     

冬虫夏草成熟过程中冬虫夏草菌及其突变基因型在子座和僵虫体中的差异表达

文献报道了从冬虫夏草(Cs)中同时检出蝙蝠蛾拟青霉(Ph)、中华被毛孢(Hs)和冬虫夏草菌(Os)的多个基因型;Cs的成熟伴有化学成分的改变和僵虫体中Hs竞争性菌落形成能力的下降.同时检验了Cs成熟过程中Os突变基因型生物量的变化.应用Southern杂交检验发现:Cs的成熟伴有Ph和2组Os基因型生物量的显著增加.o...