甲基营养型芽胞杆菌G-1抗菌肽的分离及抑菌作用研究

doi:10.16409/j.cnki.2095-039x.2021.06.006

摘要/Abstract

摘要： 为探索甲基营养型芽胞杆菌Bacillus methylotrophicus G-1抑制番茄灰霉病菌的作用机制，采用HPLC、LC-MS等对其分泌的抗菌肽进行分离鉴定，通过扫描电镜观测抗菌肽对番茄灰霉病菌菌丝形态影响，借助RNA-seq研究抗菌肽处理番茄灰霉病菌3、5 d后的差异表达基因。研究结果表明：甲基营养型芽胞杆菌G-1产生的抗菌肽主要为伊枯草菌素A。经菌株G-1抗菌肽处理后，番茄灰霉病菌菌丝生长异常，菌体畸形肿胀，内溶物外渗。基因差异表达分析表明，有40个基因在2个时期均差异表达。GO、KEGG富集分析发现，与萜烯合酶、氨基酸跨膜转运相关的15个基因在各时期均上调表达，与细胞表面受体信号通路、氧化还原酶、果胶裂解酶、单加氧酶等有关的25个基因在各时期均下调表达，其中编码琥珀脱氢酶（SDH）的Bcin02g06840基因及编码3-羟基-3-甲基戊二酸辅酶A还原酶（HMGR）的Bcin04g04450基因表达量显著下调。经qRT-PCR验证，8个差异表达基因的表达模式变化与转录组测序分析结果一致。研究表明菌株G-1抗菌肽可能通过抑制番茄灰霉病菌细胞膜组分中SDH、HMGR的合成抑制菌丝及孢子的生长。

关键词: 甲基营养型芽胞杆菌, 番茄灰霉病菌, 抗菌肽, 抑菌作用

Abstract: This study aims to explore the mechanism of inhibitory effect of Bacillus methylotrophicus G-1 on Botrytis cinerea. High-performance liquid chromatography and liquid chromatography-mass spectrometry were used to isolate and identify the antimicrobial peptides produced by strain G-1. Scanning electron microscopy was performed to observe the influence of the antimicrobial peptides on the hyphal morphology of B. cinerea. RNA-seq was used to investigate the differentially expressed genes after antimicrobial peptide treatment for 3 and 5 days. The results showed that the main antimicrobial peptide produced by B. methylotrophicus G-1 was iturin A. After being treated with the antimicrobial peptides of strain G-1, B. cinerea showed abnormal hyphal growth, deformed and swollen thalli, and leakage of internal contents. Analysis of differentially expressed genes indicated that 40 genes were differentially expressed at both time points. The gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that 15 genes related to terpene synthase and amino acid transmembrane transport were up-regulated, and 25 genes related to the cell surface receptor signaling pathway, oxidoreductase, pectin lyase and monooxygenase were down-regulated. Specifically, the expression levels of Bcin02g06840 gene encoding succinate dehydrogenase (SDH) and Bcin04g04450 gene b encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) were significantly down-regulated. The changes in the expression levels of eight differentially expressed genes according to qRT-PCR were consistent with the results of transcriptome sequencing analysis. These results indicated that the antimicrobial peptides of B. methylotrophicus G-1 might inhibit the growth of hyphae and spores by suppressing the synthesis of SDH and HMGR in the cell membrane components of B. cinerea.

Key words: Bacillus methylotrophicus, Botrytis cinerea, antimicrobial peptide, antifungal effect

中图分类号:

S476

张宝俊, 马超, 郭洁心, 孙卓楠, 张晓杰. 甲基营养型芽胞杆菌G-1抗菌肽的分离及抑菌作用研究[J]. 中国生物防治学报, 2021, 37(5): 1024-1034.

ZHANG Baojun, MA Chao, GUO Jiexin, SUN Zhuonan, ZHANG Xiaojie. Inhibition Effect of Antimicrobial Peptide Isolated from Bacillus methylotrophicus G-1 against Botrytis cinerea[J]. Chinese Journal of Biological Control, 2021, 37(5): 1024-1034.

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