鞘脂合成调控因子Orm1在球孢白僵菌中的功能鉴定及其致病机理研究

As structural component of cell membranes, sphingolipid was widely found in human, plants and microorganisms. Orosomucoid (Orm) is the regulator of sphingolipid biosysthesis, which plays important roles in human health and plant stress tolerance, while the functions of Orm are unclear in pathogenic microbes. This study is sought to find conceivable strategy for enhancing the fungal virulence by elucidating the functions and possible mechanisms of Orm1 in filamentous entomopathogen Beauveria bassiana. First, the qRT-PCR and fluorescent protein fusion technology were used to clarify the expression pattern and subcellular localization of BbOrm1. Second, the biological functions of BbOrm1 were elucidated by summarizing the changes of all strains on sphingolipid systhesis, growth, stress tolerance and virulence. Third, the virulence associated factors, such as cell wall struction, endoenzyme activities, and cell cycle, were characterized to probe the possible mechanisms of BbOrm1 in the process of fungal pathogenicity. The work will add the new knowledge to what are known about the virulence of entomopathogen. Such theory and technique would be helpful for fungal pathogenic research and genetic improvement.

鞘脂是细胞膜结构的组成部分，普遍存在于人体、植物和微生物中。作为鞘脂合成通路的调控因子，血清类黏蛋白(orosomucoid, Orm)在人类健康和植物抗逆中发挥着重要作用，而病原微生物中Orm的功能尚不清楚。本课题拟通过分析防治虫害的病原真菌球孢白僵菌BbOrm1的生物学功能及其调控病原真菌致病力的机制，来探求增强生防真菌侵染能力的新途径。首先，利用qRT-PCR、融合荧光蛋白技术，研究BbOrm1的表达规律及亚细胞定位；其次，采用基因敲除和超表达等技术，下调或上调BbOrm1的表达，分析突变菌株鞘脂合成、生长发育、抗逆能力以及侵染毒力等性状的变化，确定BbOrm1的生物学功能；第三，通过比较菌株细胞壁结构、胞内酶类活性、细胞周期等致病相关因素的变化，探索BbOrm1调控病原真菌致病力的可能机制。本项目的实施将有助于发掘生防真菌新的毒力因子，为研究真菌致病机理和生防菌剂改良提供新的思路。

球孢白僵菌Orm1参与了病原真菌致病力的调节，本课题通过基因敲除和回补技术，结合运用生物化学、细胞生物学等方法，对BbOrm1参与调控病原真菌致病力的分子机制进行了初步研究。首先利用qRT-PCR、融合荧光蛋白技术，确定了BbOrm1的表达规律及亚细胞定位；其次通过生物学功能分析，发现Orm1缺失影响球孢白僵菌无性发育、削弱其对氧化、高渗、胞壁干扰剂、杀菌剂药物、高温、紫外线的抵抗力；第三，探讨了Orm1缺失后球孢白僵菌致病相关因素的变化，结果表明Orm1缺失，菌株细胞壁厚度变薄，分生孢子疏水性降低，胞内抗氧化酶类活性减弱，推测由此引起致病力的下降。综合上述结果，本研究为认识病原微生物Orm1的功能提供了线索，为探索其致病机理奠定了基础。项目资助发表SCI论文3篇，待发表1篇，培养硕士研究生3名，其中2名已获得硕士学位，1名在读。项目投入经费22.0万元，支出16.8034万元，剩余经费5.1966万元，将用于本项目研究的后续支出。