菌株YN145对稻瘟病菌黑色素合成的影响及全基因组分析

Bacillus subtilis YN145: effect on melanin synthesis of Magnaporthe oryzae and genome-wide analysis

摘要:

【背景】枯草芽孢杆菌YN145是一株从湖南省桃江县的健康稻株中分离的细菌，前期研究中该菌对稻瘟病菌拮抗效果显著，在生物防治方面有很大的应用潜力。【目的】深入研究该菌株的生防机制并挖掘次级代谢产物基因资源。【方法】在4株稻瘟病菌生防菌中，选择其胞外抗菌物质抑制稻瘟病菌黑色素合成效果最佳的菌株YN145，采用紫外-可见分光光度计在波长400 nm处测定胞外和菌丝体内黑色素液的吸光度值，采用菌丝生长抑制平板法和分生孢子萌发抑制法测定抑菌活性。采用PacBio第三代测序和Illumina Hiseq第二代测序相结合的技术对菌株YN145进行全基因组测序，并对测序数据进行组装，注释预测基因的功能，分析次级代谢产物合成基因簇。【结果】菌株YN145的胞外抗菌物质能较好地抑制稻瘟病菌黑色素合成、分生孢子萌发和菌丝生长。菌株YN145全基因组大小为4 167 871 bp，GC含量为43.86%，编码序列(coding sequence, CDS)数量为4 294个；共找到85个tRNA、30个rRNA和92个sRNA。同时预测到5个已知的次级代谢产物合成基因簇，分别编码合成bacillaene、bacillibactin、fengycin、subtilosin-A和bacilysin，并且找到了与抑制黑色素相关的查尔酮合酶基因。【结论】在基因组层面上分析菌株YN145具有良好抑菌效果的内在原因并提供大量基础数据，为深入研究枯草芽孢杆菌次级代谢提供参考，对菌株YN145后续相关研究具有重大意义。

Abstract:

[Background] Bacillus subtilis YN145 is a bacterial strain isolated from healthy rice plants in Taojiang County of Hunan Province. Studies have demonstrated that YN145 has significant antagonistic effect against Magnaporthe oryzae and great potential in biocontrol. [Objective] To further study the biocontrol mechanism and screen out the gene clusters for secondary metabolite synthesis of this strain. [Methods] The strain YN145, whose extracellular substances had the best inhibitory effect on the melanin synthesis of M. oryzae, was selected from four biocontrol bacterial strains. The absorbance values of the extracellular and intramycelial melanin liquids of M. oryzae were measured by a UV-visible spectrophotometer at a wavelength of 400 nm. The antifungal activity of YN145 was determined by mycelial growth inhibition assay and conidial germination inhibition assay. PacBio Sequel IIe and Illumina Hiseq platforms were employed to sequence the whole genome of YN145, and the sequencing data were assembled and annotated for prediction of gene functions and analysis of the gene clusters for secondary metabolite synthesis. [Results] The extracellular antimicrobial substances of YN1145 effectively inhibited the melanin synthesis, conidial germination, and mycelial growth of M. oryzae. The full-length genome of YN1145 was 4 167 871 bp, with the GC content of 43.86% and coding sequence (CDS) number of 4 294. A total of 85 tRNAs, 30 rRNAs, and 92 sRNAs were identified in the genome. Meanwhile, five known gene clusters for secondary metabolite synthesis were predicted, which mainly encoded bacillaene, bacillibactin, fengycin, subtilosin-A, and baclysin. [Conclusion] This study analyzed the intrinsic reasons for the good antimicrobial effect of YN145 at the genome level and obtained rich basic data. It provides a reference for further in-depth research on the secondary metabolite synthetic pathway of B. subtilis and has great significance for the subsequent research of YN145.