【作者】 田向荣；

【导师】 赵德明； 高齐瑜；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2013， 博士

【摘要】 冬虫夏草十分珍贵,资源奇缺。为了开发冬虫夏草的替代品,本文开展了冬虫夏草无性型菌株的研究及其人工培植。通过野外调查掌握了冬虫夏草寄主昆虫的生物学特性。冬虫夏草寄主昆虫整个变态周期内呈幼虫、蛹、成虫、卵四种形态；幼虫常年土栖；独住,三龄以上有互咬的习性；植食性且食谱广泛；对温度敏感；各龄虫对虫草菌敏感,病虫害严重。通过人工模拟实验证实,幼虫以休眠状态在冻土层越冬,身体皱缩、发黄。通过病理切片、显微观察发现,虫草僵虫体内菌丝通过毛囊薄弱处成簇穿出体外,通过包裹身体周围的土壤而形成膜皮；有些皮肤毛囊增厚的僵虫没有膜皮,通过子囊孢子的单孢子培养,发现菌落早期形成穿透钉结构。甘肃天祝虫草海拔较低,体型较小,采用PCR及DNA测序的方法,对其分离株的rDNA ITS和5.8S RNA全序列进行分析,结果表明分离株与中国被毛孢相似性为98%以上。冬虫夏草菌生长速度极慢,实验研究发现,通过人工断裂菌丝增加菌丝生长点和浅层培养技术能极大地提高冬虫夏草无性型菌丝的产量,产业化生产的结果也对此结论做出了进一步证实。用结晶紫染色法检测细胞存活率结果表明,冬虫夏草菌丝液可以显著抑制HepG2细胞的生长,并呈浓度-效应关系；用AnnexinV-FITC/PI双染法检测细胞凋亡情况并经流式细胞仪测定,结果表明冬虫夏草菌丝液可诱导HepG2细胞的凋亡,也呈浓度-效应关系。采用DHE荧光探针进行检测,冬虫夏草菌丝液确实可以诱导细胞产生ROS, ROS抑制剂NAC对冬虫夏草诱导细胞凋亡有明显的保护作用；采用蛋白免疫印迹法对HepG2细胞中的蛋白表达进行分析,随着浓度的增加,p-JNK的蛋白表达量明显上升,Bcl-2的表达量逐渐下降；揭示ROS的促凋亡作用可能与激活JNK通路和下调抗凋亡蛋白Bcl-2表达有关,其相关性有待进一步研究。通过人工养殖对比试验表明,琼脂加水溶化凝固后,可代替土壤养殖冬虫夏草寄主幼虫,优势明显。冬虫夏草寄主幼虫染菌试验结果表明,自然情况下,冬虫夏草菌的感染形式为子囊孢子和分生孢子,感染途径为通过外伤和消化道,其中子囊孢子主要依靠外伤感染,分生孢子主要通过消化道感染。更多还原

【Abstract】 Ophiocordyceps sinensis is very precious and its resources are scarce.The phorozoon of ophiocordyceps sinensis and its artificial cultivation were tesed in this study to develop cordyceps substitute. The biological characteristics of host insect includes worm-shaped sclerotium and weed-like stroma of ophiocordyceps sinensis was mastered through field investigation. The whole metamorphosis cycles of host insect of ophiocordyceps sinensis were four morpha included larva, pupa, adult and egg; larvae perch in soil perennially; it lived alone with habit of biting each other after3instar; it was phytophagous with wide recipe and sensitive to temperature; each age’s insect was sensitive to Hirsutella sinensis, plant diseases and insect pests were serious. Artificial simulated experiment confirmed that larvae overwintered in a dormant state with its body shrinked, and its body became yellow. Hypha in body of bombys batryticatus went through in clumps via the weak point of hair follicle, and formed membrane by packing surrounding soil detected by pathological section and microscope; some bombys batryticatus have no membrane due to the skin in hair follies became more solid and thick so that hyphae couldn’t get through.We found the penetration pegs in the early time of colony through the monospore culture of ascospore.Tianzhu ophiocordyceps sinensis in Gansu had lower altitude and smaller size, the complete sequence of isolate rDNA ITS and5.8S RNA was analyzed by the methods of PCR and DNA sequencing, the result showed that the isolates had similar characteristics with Hirsutella sinensis over98%.Ophiocordyceps sinensis grew very slow,.However,the current study indicated that breaking hypha artificially increased growth point and shallow cultivation could vastly improve the production of agamotype hypha. Furthermore, industrialization result confirmed the conclusion.Crystal violet staining assay was used to observe the cell viability, which indicated that HepG2cells could be inhibited obviously by ophiocordyceps sinensis hyphae solution with a dose-dependent manner. Then apoptosis of HepG2cell were assessed by Annexin V-FITC/PI staining and flow cytometry analysis, the results showed that HepG2cells could be induced to apoptosis by ophiocordyceps sinensis hyphae solution, also with a dose-dependent manner. Further conformation detected by fluorescent chemosensors was that ophiocordyceps sinensis hyphae solution could induce HepG2cell to produce ROS, meanwhile ROS inhibitor NAC could significantly prevent the cell from apoptosis mediated by ophiocordyceps sinensis, and subsequently, western-blot was used to analyse the expression of p-JNK. and Bcl-2proteins. With ascending concentrations,, the expression of p-JNK increased, while Bcl-2decreased. It revealed that the pro-apoptosis of ROS may be due to the activation of JNK pathway and down-regulation of Bcl-2expression, and their correlation needed further research.According to the result of artificial cultivation contrast test, agar can replace the soil to cultivate the host larva of ophiocordyceps sinensis and it had an advantage over the soil.The result of the host larva of ophiocordyceps sinensis infection test showed that the infection form of ophiocordyceps sinensis included ascospore and conidium. The routes of infection of ascospore and conidium were trauma and digestive tract,respectively.更多还原