枯草芽孢杆菌BBs-27发酵液性质及脂肽对黄色镰刀菌的抑菌作用

doi:10.13560/j.cnki.biotech.bull.1985.2023-0086

摘要/Abstract

摘要：

枯草芽孢杆菌BBs-27从药用植物石豆兰中分离，对黄色镰刀菌（Fusarium culmorum）具较强抑菌活性。为进一步掌握发酵液中抑菌成分及抑菌机制，对发酵液耐酸碱、紫外照射、酶解等特性进行研究，在此基础上，借助LC-MS技术对发酵液中脂肽类型进行鉴定，并从形态、生理和转录组3个层面上分析抑菌机制。结果表明，发酵液pH=6时抑菌活性最强，pH<4或>10时活性明显降低。超过10 min紫外照射活性显著降低。易被其他3种蛋白酶水解，但对胃蛋白酶不敏感，推测抑菌物是以脂肽类为主且包含少量蛋白质，LC-MS分析证实，抗菌肽主要是伊枯草菌素和丰原素两大类。SEM结果显示，脂肽处理下菌丝卷曲、畸形、异常膨大。脂肽对菌丝体内SOD、POD、CAT及可溶性蛋白、可溶性糖均有显著影响，5项生理指标均与抑菌率极显著相关。以禾谷镰刀菌基因组为参照组装，样品中Q20>96%，Q30>91%，GC含量在52.27%-52.87%，比对率>78%。共筛选出712个DEGs，包括393个上调基因和319个下调基因。Go功能注释分类显示，712个DEGs主要涉及到6个生物学过程、11个细胞组分和12个分子功能。KEEG分析显示，DEGs显著富集在碳水化合物、脂、氨基酸、辅助因子和维生素代谢等途径，筛选出了重要的脂肽响应基因GAO、CHS3、MPG1、FGSG_02016、IPDH、ODC，它们通过干扰细胞壁、细胞膜、氨基酸、抗氧化酶的正常合成来影响菌体生长。该结果为进一步开发和利用安全微生物农药奠定理论基础。

关键词: 脂肽, 芽孢杆菌, 黄色镰刀菌, 扫描电镜, 菌丝形态, 转录组, 发酵液性质, 抑菌率

Abstract:

Bacillus subtilis strain BBs-27 isolated from the medicinal plant Bulbophyllum sp. shows strong antifungal activity against Fusarium culmorum. In order to further understand the composition of fermentation broth and its antifungal mechanism, the properties of fermentation broth were studied by acid-base treatment, ultraviolet irradiation and enzymolysis. On the above basis, the type of lipopeptides（LPs）in the fermentation broth was identified using LC-MS. The antifungal effects of LPs were analyzed from the morphology, physiology and transcriptome levels. The results showed that the antifungal activity of the fermentation broth was the strongest at pH=6, and decreased significantly at pH < 4 or > 10, and declined significantly when UV irradiation in over 10 min, easily hydrolyzed by other three proteases, but insensitive to pepsin. It was speculated that antifugal antifugal substances were mainly lipopeptides, and contained a small amount of protein. LPs LPs were mainly iturin and fengycin by LC-MS analysis. The fungus mycelium treated by LPs was twisted, informed and abnormally swollen under SEM. LPs had a significant impact on the activities of SOD, POD, CAT and the contents of soluble protein and soluble sugar, and all five physiological indicators were highly correlated with antifungal rate. With F. graminis genome as reference, Q20 and Q30 were above 96%, 91% respectively, GC content ranged from 52.27% to 52.87%, comparison rate was above 78%. Total 712 differentially expressed genes（DEGs）were screened, of which 393 were up-regulated and 319 were down-regulated. The Go functional annotation classification showed that 712 DEGs were mainly involved in 6 biological processes, 11 cell components and 12 molecular functions. KEGG enrichment analysis revealed that the DEGs were mainly enriched in the metabolisms of carbohydrate, lipid, amino acid, cofactors and vitamins. Genes including GAO, CHS3, MPG1, FGSG_02016, IPDH and ODC responding to LPs affected the fungus growth by interfering with the normal synthesis of cell wall, cell membrane, amino acids and antioxidant enzymes. These results provide a theoretical basis for further development and utilization of safe microbial pesticides.

Key words: lipopeptides, Bacillus, Fusarium culmorum, scanning electron microscope, mycelium morphology, transcriptome, fermentation property, antifungal rate

苗永美, 苗翠苹, 于庆才. 枯草芽孢杆菌BBs-27发酵液性质及脂肽对黄色镰刀菌的抑菌作用[J]. 生物技术通报, 2023, 39(9): 255-267.

MIAO Yong-mei, MIAO Cui-ping, YU Qing-cai. Properties of Bacillus subtilis Strain BBs-27 Fermentation Broth and the Inhibition of Lipopeptides Against Fusarium culmorum[J]. Biotechnology Bulletin, 2023, 39(9): 255-267.

图/表 13

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指标 Index	抑菌率 Antifungal rate/%	SOD活力 SOD activity/ （U·g^-1）	POD活力 POD activity/ （U·g^-1）	CAT活力 CAT activity/ （U·g^-1）	可溶性蛋白含量 Soluble protein content/ （mg·g^-1）	可溶性糖含量 Soluble sugar content/ （mg·g^-1）
抑菌率Antifungal rate/%	1	-0.927**	-0.952**	-0.998**	-0.985**	0.966**
SOD活力SOD activity/（U·g^-1）	-0.927**	1	0.811*	0.902*	0.857*	0.842*
POD活力POD activity/（U·g^-1）	-0.952**	0.811*	1	0.963**	0.945**	0.892*
CAT活力CAT activity/（U·g^-1）	-0.998**	0.902*	0.963**	1	0.992**	0.971**
可溶性蛋白含量 Soluble protein content /（mg·g^-1）	-0.985**	0.857*	0.945**	0.992**	1	0.991**
可溶性糖含量 Soluble sugar content /（mg·g^-1）	-0.966**	0.842*	0.892*	0.971**	0.991**	1
平均值Mean	-0.966	0.868	0.913	0.965	0.954	0.932

指标 Index	抑菌率 Antifungal rate/%	SOD活力 SOD activity/ （U·g^-1）	POD活力 POD activity/ （U·g^-1）	CAT活力 CAT activity/ （U·g^-1）	可溶性蛋白含量 Soluble protein content/ （mg·g^-1）	可溶性糖含量 Soluble sugar content/ （mg·g^-1）
抑菌率Antifungal rate/%	1	-0.927**	-0.952**	-0.998**	-0.985**	0.966**
SOD活力SOD activity/（U·g^-1）	-0.927**	1	0.811*	0.902*	0.857*	0.842*
POD活力POD activity/（U·g^-1）	-0.952**	0.811*	1	0.963**	0.945**	0.892*
CAT活力CAT activity/（U·g^-1）	-0.998**	0.902*	0.963**	1	0.992**	0.971**
可溶性蛋白含量 Soluble protein content /（mg·g^-1）	-0.985**	0.857*	0.945**	0.992**	1	0.991**
可溶性糖含量 Soluble sugar content /（mg·g^-1）	-0.966**	0.842*	0.892*	0.971**	0.991**	1
平均值Mean	-0.966	0.868	0.913	0.965	0.954	0.932

样品 Sample	原始序列数 Raw reads	质控后序列数 Clean reads	质控后序列比例 Clean reads rate/%	错误率 Error rate/%	Q20/%	Q30/%	GC 含量 GC content/%	覆盖率 Mapped ratios/%
C1	44 046 600	42 603 392	96.72	0.03	97.00	91.69	52.60	78.75
C2	43 260 998	41 992 094	97.07	0.03	96.99	91.62	52.87	79.61
C3	58 845 594	56 937 404	96.76	0.03	98.03	94.15	52.30	79.27
T1	54 973 812	52 895 734	96.22	0.03	97.93	93.83	52.31	78.44
T2	58 068 196	55 878 726	96.23	0.03	97.95	93.99	52.27	78.47
T3	57 570 042	55 363 728	96.17	0.03	97.84	93.66	52.55	79.33
综合Total	316 765 242	305 671 078	96.50

样品 Sample	原始序列数 Raw reads	质控后序列数 Clean reads	质控后序列比例 Clean reads rate/%	错误率 Error rate/%	Q20/%	Q30/%	GC 含量 GC content/%	覆盖率 Mapped ratios/%
C1	44 046 600	42 603 392	96.72	0.03	97.00	91.69	52.60	78.75
C2	43 260 998	41 992 094	97.07	0.03	96.99	91.62	52.87	79.61
C3	58 845 594	56 937 404	96.76	0.03	98.03	94.15	52.30	79.27
T1	54 973 812	52 895 734	96.22	0.03	97.93	93.83	52.31	78.44
T2	58 068 196	55 878 726	96.23	0.03	97.95	93.99	52.27	78.47
T3	57 570 042	55 363 728	96.17	0.03	97.84	93.66	52.55	79.33
综合Total	316 765 242	305 671 078	96.50

二级类别 Second category	通路名称 Pathway name	基因编号 Gene ID	基因名称 Gene name	Log₂FC	P-value	上下调Regulation
碳水化合物代谢Carbohydrate metabolism	半乳糖代谢 Galactose metabolism	FGSG_03569	Galactose oxidase precursor	1.20	3.85×10^-3	Up
		FGSG_11032	Galactose oxidase precursor	2.11	3.43×10^-3	Up
		FGSG_00096	Hypothetical protein	2.57	3.88×10^-8	Up
		FGSG_03288	Hypothetical protein	1.77	8.10×10^-4	Up
		FGSG_08415	Hypothetical protein	1.90	1.13×10^-7	Up
		FGSG_13188	Hypothetical protein	-1.78	2.89×10^-3	Down
	氨基糖和核苷糖代谢途径 Amino sugar and nucleotide sugar metabolism	FGSG_10327	Chitin synthase 3	-1.22	5.28×10^-6	Down
		FGSG_03591	42 kD endochitinase precursor	-1.77	1.93×10^-3	Down
		FGSG_08773	Mannose-1-phosphate guanyltransferase	1.19	3.13×10^-3	Up
		FGSG_04752	Hypothetical protein	6.07	5.10×10^-5	Up
		FGSG_02355	Hypothetical protein	-1.69	1.39×10^-3	Down
		FGSG_04903	Hypothetical protein	1.88	3.10×10^-3	Up
		FGSG_13188	Hypothetical protein	-1.78	2.89×10^-3	Down
		FGSG_13883	Hypothetical protein	-1.05	9.45×10^-4	Down
脂代谢 Lipid metabolism	类角甾醇生物合成Steroid biosynthesis	FGSG_02502	C-5 sterol desaturase	3.39	2.14×10^-10	Up
		FGSG_02346	Delta（14）-sterol reductase	1.10	3.88×10^-3	Up
		FGSG_09830	C-4 methylsterol oxidase	1.46	4.18×10^-3	Up
		FGSG_02016	Hypothetical protein	-1.32	1.08×10^-4	Down
		FGSG_06215	Hypothetical protein	1.38	3.87×10^-3	Up
氨基酸代谢Amino acid metabolism	缬氨酸、亮氨酸和异亮氨酸生物合成 Valine, leucine and isoleucine biosynthesis	FGSG_09589	3-isopropylmalate dehydratase	-1.05	4.61×10^-3	Down
		FGSG_10118	Ketol-acid reductoisomerase	1.17	1.28×10^-3	Up
		FGSG_10119	Hypothetical protein	1.33	1.07×10^-6	Up
		FGSG_04570	Hypothetical protein	-1.52	8.46×10^-5	Down
	谷胱甘肽代谢 Glutathione metabolism	FGSG_05903	Ornithine decarboxylase	-1.36	1.82×10^-7	Down
		FGSG_00172	Hypothetical protein	-1.47	3.62×10^-3	Down
		FGSG_00443	Hypothetical protein	-1.19	2.42×10^-4	Down
		FGSG_12154	Hypothetical protein	1.58	7.48×10^-6	Up
		FGSG_04902	Hypothetical protein	4.34	3.65×10^-4	Up
辅助因子和维生素代谢Metabolism of cofactors and vitamins	泛酸盐和乙酰辅酶A生物合成 Pantothenate and CoA biosynthesis	FGSG_10118	Ketol-acid reductoisomeras	1.17	1.28×10^-3	Up
		FGSG_12108	Hypothetical protein	1.27	3.02×10^-3	Up
		FGSG_04570	Hypothetical protein	1.52	8.46×10^-5	Uup
		FGSG_03176	Hypothetical protein	-1.21	1.17×10^-3	Down