炭团木霉中非核糖体多肽基因NRPS1的功能研究

Identification and functional study of NRPS1 in Trichoderma hypoxylon

摘要:

[目的] 木霉属真菌是应用最为广泛和潜力最大的生防真菌，其产生的典型化合物哌珀霉素（peptaibols）类抗生素在生物防治中发挥重要作用。本研究采用基因组挖掘技术（genome mining）发现炭团木霉（Trichoderma hypoxylon）的潜在哌珀霉素生物合成基因簇及对病原菌的防治作用。[方法] 生物信息学分析预测合成哌珀霉素的基因簇，利用Quick-change技术构建基因骨架敲除盒，通过PEG介导的原生质体转化方法获得敲除突变株，通过平板对峙法和菌丝生长毒力实验验证该基因簇对炭团木霉生物活性的影响。[结果] 基因挖掘鉴定一个非核糖体多肽合成酶（nonribosomal peptide synthetases，NRPS）可能合成哌珀霉素类抗生素，命名为NRPS1，对该基因进行部分敲除，成功获得3株NRPS1缺失突变株。对峙实验表明，突变株对寄生曲霉（Aspergillus parasiticus）、尖孢镰刀菌（Fusarium oxysporum）、黑白轮枝菌（Verticillium alboatrum）等9株植物病原真菌的抑制作用与野生株相比显著下降，且突变株的粗提物的抑菌活性明显弱于野生型。[结论] NRPS1是一个潜在的哌珀霉素合成基因，该基因在宿主与病原真菌对抗过程中起关键作用，该研究为炭团木霉哌珀霉素结构解析及生物防治机理研究奠定了基础。

Abstract:

[Objective] Trichoderma is a genus of common filamentous fungi used for biological control, by producing a variety of secondary metabolites that play an important role in biological control. In this study, a putative nonribosomal peptide synthetase (NRPS) NRPS1 was identified in Trichoderma hypoxylon by genome mining approach and the function of this gene's function was studied by using NRPS1 mutant against the pathogens. [Methods] Bioinformatics analysis reveals a putative NRPS containing biosynthetic gene cluster that is potential for the biosynthesis of peptaibol. The deletion cassette containing the hygromycin resistance gene was constructed by Quick-change method. The partial gene was deleted by using PEG mediated transformation in T. hypoxylon. NRPS1's function was identified by the biological activity assay against pathogenic fungi. [Results] We identified a putative peptaibol synthesis gene NRPS1 and obtained three deletion mutants. Deletion of NRPS1 significantly decreased the ability to resistance the pathogenic fungi, such as Aspergillus parasiticus, Fusarium oxysporum, and Verticillium alboatrum. [Conclusion] NRPS1 is a potential peptaibol synthesis gene in T. hypoxylon and plays important roles in the resistance to pathogenic fungi. This work lays the foundation for the elucidation of peptaibol biosynthesis and its application for the biological control in agriculture.