内生真菌交织枝顶孢NRPS-ACR06587基因簇合成的生防活性多肽鉴定及其调控机制

Soil-borne diseases such as root-knot nematode and fusarium wilt are the important biological disasters, which restrict seriously the sustainable development of vegetable production facilities. Previously, we have got the biological synthetase gene knockout mutant of NRPS-ACR06587 cluster of Acremonium implicatum, which was mainly responsible for the synthesis of biocontrol active peptides to soil-borne disease. In the proposed project, we plan to conduct the assays by analyzing the cluster NRPS-ACR06587 biological function, purifying the bioactive peptides, exploring the relationship of biosynthesis efficiency, and the synthetase gene fusion promoter (P06587:lacZ) and transcription factor fusion promoter (P06584:lacZ), to reveal the biosynthetic pathway and molecular regulatory system of biocontrol active peptides controlled by NRPS-ACR06587 gene clusters. The research would explain the series objects from the genome to bioactive peptides and its molecular structures. In addition, the results will provide a theoretical basis and technical platform on safe control technology to soil-borne disease.

根结线虫和枯萎病等土传病害是制约我国设施蔬菜生产可持续发展的主要生物灾害。本项目拟在前期获得主效生防活性多肽NRPS-ACR06587基因簇中生物合成酶基因敲除突变体、证明其决定生防活性产物合成作用的基础上，通过NRPS-ACR06587基因簇功能分析，生防肽类物质分离纯化与鉴定，以及生物合成基因启动子融合GUS（P06587:lacZ）和簇内转录因子启动子融合GUS(P06584：lacZ)靶标合成基因簇表达及产物合成等关键技术，揭示生防活性多肽NRPS-ACR06587基因簇的功能及其调控机制，实现从基因组到未知结构生防活性产物的种类及其分子结构解析，为土传病害绿色防控技术的创制提供理论依据和技术平台。

交织枝顶孢TR菌株（Acremonium implicatum）是从受南方根结线虫（Meloidogyne spp.）侵染的番茄根茎中分离得到的一株内生真菌，其发酵的次级代谢产物对南方根结线虫有显著杀死活性。本研究通过对植物内生交织枝顶孢TR菌株的研究，大麦培养基发酵，乙酸乙酯提取物中分离出一种类衍生自二酮哌嗪的代谢产物，对枯萎病具抗菌活性。并完成了该菌株的全基因组和转录组测序，利用antiSMASH预测分析了其肽类产物合成相关的次生代谢基因簇。获得了簇内转录因子ACR-08905和ACR-08909超表达转化子，显著提高了基因簇内相关合成基因的表达量。分别在构巢曲酶A773和LO8030菌株中实现了对NRPS主合成基因ACR06587（更名为ACR-08903）的异源表达，确定了ACR-08903的活性物质。为分离交织枝顶孢次生代谢活性物质奠定了基础。已发表SCI论文3篇（待发表SCI论文1篇），培养硕士研究生3人。