球孢白僵菌亲环蛋白影响毒力及次生代谢的机理研究

To isolate virulence associated genes in entomopathogenic fungi, and understand the roles they play in pathogenesis，will aid in research and development of mycoinsecticides with higher virulence. Cyclophilins, which conserved in all prokaryotes and eukaryotes, are a family of peptidyl prolyl isomerase which capable of binding to immunosuppressant cyclosporine. In both plant and human pathogenic fungi, disruption of cyclophilins can lead to attenuation of virulence. In our previous study, however, we found that both disruption and RNA interference of cyclophilins enhanced the virulence of entomopathogenic fungus Beauveria bassiana. Associated with the down regulation of cyclophilins, the expression of some secondary metabolite synthetic genes, such as NRPS and PKS, were found to be up regulated. These findings suggest that the mechanism of cyclophilins affecting virulence in entomopathogenic fungus, might be different from that in plant and human pathogenic fungi. In the present study, we are going to isolate the affected secondary metabolites and the genes responsible for their biosynthesis, in the aid of expression profiling, biological information, gene disruption, over expression, and secondary metabolite profiling. Then, identify the secondary metabolites targeted, and test their insecticidal activity using bioassay method. After finish of this study, it is possible for us to isolate the cyclophilin regulated secondary metabolites and the genes responsible for their biosynthesis, get some recombinant strains with high virulence, and identify some novel toxins promising for insect control.

对毒力相关基因的发掘研究，进而阐明昆虫病原真菌的致病机理，有助于研制开发高毒力的真菌杀虫剂。亲环蛋白是一类普遍存在于原核与真核生物的、能与免疫抑制剂环孢霉素特异结合的肽酰脯氨酰顺反异构酶。在植物和人类病原真菌中，下调亲环蛋白能够降低菌株的毒力。而申请人的研究发现，下调亲环蛋白后，昆虫病原真菌球孢白僵菌的毒力没有降低反而增高，一些次生代谢物合成基因的表达水平也明显提高，说明昆虫病原真菌亲环蛋白影响毒力的方式可能不同于植物和人类病原真菌。为深究其中的机理，本项目拟通过表达谱差异比较、生物信息学、基因敲除、超量表达、次生代谢谱差异分析等方法，找出受亲环蛋白影响的次生代谢物及其合成相关基因。然后对差异次生代谢物进行纯化鉴定，并通过生物测定验证它们和毒力的关系。本项目的完成，预期能够靶定受亲环蛋白影响的次生代谢物及其合成相关基因，有望获得一些高毒力的基因工程菌株，以及具有潜在杀虫价值的次生代谢产物。

亲环蛋白是一类能够同免疫抑制剂环孢霉素特异结合的肽酰脯氨酰顺反异构酶，这类蛋白广泛存在于各界生物并和许多生物学过程相关。丝状昆虫病原真菌球孢白僵菌含有包括cypA/Cyp1在内的11个亲环蛋白基因。BbCypA重组蛋白具有受CsA抑制的PPI活性，然而敲除BbCypA后并不影响菌株的生长、发育、抗逆及毒力。为明确各亲环蛋白的生物学功能，又分别对它们进行了基因敲除和超量表达。敲除菌株ΔBbCypE和ΔBbCyp6的生长受到抑制，产孢下降，毒力也明显降低；菌株ΔBbCypH和ΔBbCyp9除了产孢明显提高外，其它表型和野生菌株没有明显差异；抑菌实验显示BbCypA和BbCypX是环孢霉素CsA的主要胞内受体；在32℃亚高温胁迫条件下，敲除BbCypA不影响菌株生长，而敲除其它基因均会影响菌株生长，此外，超表达任何一个亲环蛋白都会抑制菌株的生长。通过基因沉默下调整个家族基因后，菌株的生长、发育、产孢、以及对高盐和刚果红氧化胁迫的耐受性不受影响；而对CsA的耐受性及高温胁迫的敏感性增加，同时菌株的毒力也明显下降。酵母双杂交实验显示：BbCyp8和BbCyp9没有作用靶标，推测它们能够独立的完成某些生物学功能；而其余亲环蛋白都有一个或多个作用靶标，且许多靶标蛋白能够和2个以上的亲环蛋白相互作用；这些靶标靶标蛋白可能参与了包括DNA复制、染色质组装、转录调控、核糖体组装/翻译调控、物质代谢、胞内传递及运输等诸多生物过程。综上所述，球孢白僵菌11个亲环蛋白，组成一个多功能蛋白家族，参与了诸多生物学过程，并协同影响了菌株的生长、发育、耐热及毒力；此外，我们还获得了一种同时下调多基因家族的基因沉默策略。