新疆虫草寄主昆虫的耐冻机理及新疆虫草菌对其耐冻性的影响研究

Ophiocordyceps gracilis, a traditional herb in Xinjiang, is closely related to Ophiocordyceps sinensis, and has the similar active ingredient and curative properties with Ophiocordyceps sinensis. In our primary experiment, we fond that its host insect (Hepialus altaicola, the endemic species in Xinjiang) can survive after its body fluid freezing, it is proposed that the insect Hepialus altaicola has special freezing tolerant mechanism. But we know little about this mechanism. Meanwhile, it is not clear either Ophiocordyceps gracilis survives as a fungal ice nucleus in Hepialus altaicola, or Ophiocordyceps gracilis induces the expression of Hepialus altaicola’s antifreeze protein gene after infecting Hepialus altaicola with Ophiocordyceps gracilis, during overwintering period. In this project, the main research contents include: (i) based on the ice crystal observation platform constructed by applicant, detecting the ice recrystallization inhibition and ice crystal modification activities in insect haemolymph; (ii) testing respectively the super cooling points, the survival rates of larvae treated under the temperatures below the super cooling point, the thermal hysteresis activity, the variations of water and glycerol contents of larvae (including larvae collected in field and reared under laboratory conditions) in different developmental stages; (iii) observing the insect tissue and the subcellular structure changes before and after freezing; (iv) detecting the existence or nonexistence of ice nucleating agents in insect; (v) and testing the expression levels of antifreeze protein gene and ice nucleation protein gene, the all study mentioned above, is used to elucidate the special freezing tolerant mechanism of Hepialus altaicola; (vi) meanwhile, infecting Hepialus altaicola with Ophiocordyceps gracilis, then, repeating above mentioned study, calculating the effects of Ophiocordyceps gracilis on freezing tolerance in Hepialus altaicola. The result of this research will be helpful in comprehensively understanding the subzero temperature adaptation mechanisms of organisms. It will also be helpful to culture and protect Ophiocordyceps gracilis, in the application.

新疆虫草为新疆传统中草药，和冬虫夏草在亲缘关系、成份含量、功效方面相近。其寄主昆虫——阿尔泰蝠蛾为新疆特有种，能耐受体液结冰而存活。但我们尚不清楚其耐冻机理；并且新疆虫草菌感染寄主昆虫后，在虫体越冬过程中，是充当了真菌冰核剂，还是促进了虫体抗冻蛋白的表达，尚有争议。本项目的主要研究内容为：(i)基于申请人创建的冰晶观测平台，检测昆虫血淋巴的冰晶修饰和抑制重结晶作用；(ii)测定不同发育阶段幼虫的过冷却点、热滞值、耐冻存活率、甘油和水的含量变化；(iii) 观测冷冻前后，虫体组织和亚细胞结构的变化；(iv) 观察寄主昆虫体内有无冰核剂；(v) 检测抗冻蛋白和冰核蛋白的表达情况，通过以上研究来揭示寄主昆虫的耐冻机理；(vi)并将新疆虫草菌感染寄主昆虫，再进行上述测试，研究其对寄主昆虫耐冻性的影响。本研究对于全面认识生物的耐冻机理具有理论意义；在应用上，对新疆虫草的培育和保护具有重要价值。

阿尔泰蝠蛾为新疆虫草的寄主昆虫，是新疆特有种昆虫，能耐受体液结冰而存活。为了研究其耐冻机制，我们开展了以下研究：(i) 测定不同季节幼虫的过冷却点和存活率；(ii) 测定虫体不同组织的过冷却点；(iii) 检测幼虫水、血淋巴、甘油、氨基酸和海藻糖等的含量变化；(iv) 基于申请人创建的冰晶观测平台，检测昆虫血淋巴的冰晶修饰作用，以证实抗冻蛋白的存在,并获取该蛋白；(v) 检测昆虫体内有无冰核蛋白存在；(vi)并检测新疆虫草菌对阿尔泰蝠蛾耐冻性的影响。.研究结果显示：(i) 测完过冷却点后幼虫全部存活；幼虫的过冷却点随温度的变化而改变，两者呈正相关，幼虫的过冷却点冬天最低；(ii) 幼虫的体表过冷却点最高，其次是血淋巴，最低是肠道，也就是说结冰的顺序是从外及内；(iii) 在昆虫的生理生化方面，只有氨基酸含量与昆虫的过冷却点成正相关；(iv) 通过我们的冰晶观测平台，我们发现阿尔泰蝠蛾幼虫血淋巴中有抗冻蛋白，并且通过RNA测序技术得到了该蛋白（71647），发现它可以结合到冰晶表面而限制冰晶生长，从而修饰冰晶形态；(v) 没有发现昆虫体内有冰核蛋白；(vi)没有发现新疆虫草菌具有冰核作用，但发现了新疆虫草菌的抑菌活性。.同时，我们还发现了阿尔泰蝠蛾幼虫的吐水现象，吐水量和其血淋巴量相当，这些发现都有助于阿尔泰蝠蛾耐冻机理的揭示。另外，我们还开发了阿尔泰蝠蛾的饲养方法，发现了其蛹的雌雄二型性，培养成功了新疆虫草菌的子座，等等，这些都为新疆虫草菌的人工培育奠定了基础。.