温度胁迫及生长发育过程中广东虫草热激蛋白CgDnaJ的功能研究

Heat shock protein DnaJ, one chaperon used to re-establish protein conformation and dynamic equilibrium, plays important regulation roles in organism growth and development and the resistance against kinds of stresses. Nevertheless, only one report on DnaJ protein function in basidiomycete Lentinula edodes was only found for macro-fungi, and DnaJ function in macro ascomycetes has been no documented. Cordyceps guangdongensis (= Tolypocladium guangdongense), an economic fungus domesticated and cultivated by our research team, not only possesses kinds of dramatically bioactive function and extensive application prospect, but also is suitable to study growth and development, temperature stresses and bioactive ingredient metabolism in Cordyceps sp., demonstrating that C. guangdongensis is a promising and ideal material used to explore DnaJ function. This project will launch the function analysis of CgDnaJ encoding DnaJ protein in C. guangdongensis. The expression pattern of CgDnaJ protein during different growth and development stages and temperature stress process will be analyzed. Transgenic strains obtained via reverse genetic methods (overexpression and RNA interference) were used to analyze the differences of the growth and development and resistance to temperature stresses compared to the wild type strains, which will reveal the regulation effect of CgDnaJ in growth and development as well as temperature stress process for C. guangdongensis. Meanwhile, comparative metabolome analysis between wild type and transgenic strains will be used to explore the regulation role of CgDnaJ in bioactive ingredient metabolism and the detailed metabolic process in C. guangdongensis. This project will not only provide the theoretical proof of growth and development, resistance to temperature stress and bioactive ingredient metabolism in Cordyceps sp., but also possess the significant reference value on growth and development, stress resistance and compound metabolism of macro fungi.

热激蛋白DnaJ是一种重建蛋白构象和动态平衡的分子伴侣，对生物生长发育和胁迫抗性具有重要的调控作用，但在大型真菌中仅对担子菌的香菇有所报道，对大型子囊菌的研究尚未见报道。广东虫草是由申请人所在团队驯化栽培的经济真菌，不但具有多种显著的活性功效和应用前景，而且特别适合开展生长发育、温度胁迫及活性成分代谢的研究。本项目拟开展广东虫草DnaJ蛋白编码基因CgDnaJ功能研究，明确该基因在广东虫草生长发育和温度胁迫过程中的表达模式，并对该基因进行沉默和超量表达，比较分析野生型和转化子菌株生长发育、温度胁迫抗性的差异，明确该基因在虫草生长发育及温度胁迫过程中的调控作用。并采用代谢组差异分析，探究CgDnaJ调节广东虫草活性成分代谢及具体的代谢途径。本项目将为虫草类真菌的生长发育、温度胁迫抗性和活性成分代谢机理研究提供的理论依据，对大型真菌生长发育、胁迫抗性及成分代谢具有重要的参考意义。

热激蛋白DnaJ是一种重建蛋白构象和动态平衡的分子伴侣，对生物生长发育和胁迫抗性具 有重要的调控作用。本项目开展了广东虫草DnaJ蛋白编码基因CgDnaJ家族基因鉴定分析、不同发育时期多组学分析和候选基因CgDnaJ05进行基因功能分析。根据广东虫草基因组信息，鉴定到22个DnaJ家族基因，对其进行亚细胞定位和启动子响应元件分析，qRT-PCR分析结果显示多个DnaJ家族基因在不同发育时期和温度胁迫条件下差异表达，表明该家族基因在该过程中可能具有重要的调控作用。不同发育时期转录组数据显示，在5个发育时期共有9076个基因被表达，菌丝期基因转录水平表达谱与原基期和子实体期表达谱显著差异；蛋白组数据表明，在菌丝期、原基期和子实体发育中期鉴定到2040个蛋白；基因蛋白表达模式分析，发现多个热激蛋白、转录因子和涉及萜类、麦角甾醇、腺苷和多糖生物合成基因在广东虫草生长发育过程中显著差异表达；靶标代谢组分析显示，色氨酸和色胺含量在原基期达到最高，3-吲哚乙酸含量随着生长发育降低。以CgDnaJ05蛋白J保守结构域240bp的反向互补片段为干扰片段，采用广东虫草Cgactin和Cggpd基因启动子双向启动改干扰片段，构建该基因沉默载体；通过根癌农杆菌Agrobacterium tumefaciens侵染广东虫草菌丝，获得4个CgDnaJ05基因沉默效率为20%-82%的转化子；与野生菌丝相比，4个沉默转化子菌丝生长速度显著下降，且于沉默效率正相关；转化子原基的形成受到显著抑制。本项目将为虫草类真菌的生长发育、温度胁迫抗性及品种选育机理研究提供的理论依据，对大型真菌生长发育和胁迫抗性具有重要的参考意义。