绿色木霉组蛋白去乙酰化酶编码基因TvRpd3在木霉生物防治作用中的功能分析

Role of Trichoderma viride histone deacetylase gene TvRpd3 in the biocontrol of Trichoderma

摘要:

【目的】 本文借助基因编辑技术在具有生物防治潜力的绿色木霉(Trichoderma viride)中敲除组蛋白去乙酰化酶编码基因TvRpd3,来研究TvRpd3基因及其编码蛋白在提高木霉病原菌拮抗能力中的作用。【方法】 利用融合PCR和同源重组策略构建了TvRpd3基因缺失的突变菌株,通过对峙培养、表型观察、免疫组化检测、代谢组学分析等系统比较TvRpd3基因敲除前后菌株的组蛋白乙酰化修饰水平、次级代谢产物合成、病原菌拮抗能力以及田间防治效果等。【结果】 与野生型菌株相比,缺失TvRpd3基因的木霉工程菌(∆TvRpd3)对多种病原菌表现出了更强的对峙抑制效果,其所产的发酵液对小麦白粉病、烟草黑胫病和番茄枯萎病的防治效果分别提高了62.27%、57.45%和70.71%。同时,敲除TvRpd3基因也显著改变了木霉工程菌所产次级代谢产物的种类和产量,抗生性物质的产量大幅提高。【结论】 绿色木霉TvRpd3基因及其介导的组蛋白乙酰化修饰在提高绿色木霉生物防治中起着重要作用。

Abstract:

[Objective] This paper aimed to analyze the role of TvRpd3 encoding histone deacetylase in the biocontrol of Trichoderma viride Tv-1511 by constructing the engineered strain with TvRpd3 deleted (∆TvRpd3). [Methods] Fusion PCR technique and homologous recombination method were employed to delete TvRpd3 in T. viride Tv-1511. Confrontation culture, phenotype observation, immunohistochemistry detection, and metabonomics analysis were carried out to compare the histone acetylation level, secondary metabolite synthesis, antagonism to pathogens, and field biocontrol effect between the engineered and the wild-type strains. [Results] Compared with the wild type, ∆TvRpd3 exhibited enhanced inhibitory effects on pathogens. The biocontrol effects of ∆TvRpd3 fermentation broth on wheat powdery mildew, tobacco black shank, and tomato Fusarium wilt increased by 62.27%, 57.45%, and 70.71%, respectively. Meanwhile, the knockout of TvRpd3 significantly changed the species and yields of secondary metabolites produced by Trichoderma. Specifically, the yields of several vital antibiotic substances including trichomycin, sorbicillinoids, and monocillin I produced by ∆TvRpd3 increased compared with those of the wild type. [Conclusion] TvRpd3 mediates histone deacetylation to play a role in the biocontrol of T. viride.