生防菌株DZY6715在不同生长期的代谢差异分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0148

摘要/Abstract

摘要：

特基拉芽孢杆菌DZY6715是对油茶炭疽菌有较好抑制作用的生防菌，为明确该菌株在生长前期和生长后期的差异代谢物质及其调控功能。以菌株DZY6715的生长曲线为基础，先利用平板对峙法测定该菌株在生长前期和生长后期对油茶炭疽菌的抑制活性，然后通过非靶代谢组学技术，对这两个时期的发酵液进行生物信息学分析。结果表明，菌株生长前期的抑菌活性弱于生长后期，差异代谢物共注释到32条通路，前20条富集到183个差异代谢物，其中潜在重要性高的代谢通路有5条，富集的差异代谢物有84个，27种，涉及的代谢物以氨基酸和有机酸为主，并伴有诺氟沙星、N-乙酰-d-氨基葡萄糖、腺苷、苯酚等物质。另外，生长前期氨基酸的总表达量显著高于生长后期，而有机酸及其他差异代谢物在生长后期的总表达量则明显高于生长前期。因此，本文认为氨基酸主要参与细胞结构的构建，同时附有抑菌功能，而有机酸、诺氟沙星、腺苷等更多的是作为抵抗胁迫过程的重要调节剂。

关键词: 特基拉芽孢杆菌, 生长前期, 生长后期, 差异代谢物, 非靶向代谢组学

Abstract:

Bacillus tequilensis DZY6715 is a biocontrol strain that has a good inhibitory effect on Colletotrichum fructicola in Camellia oleifera. In order to clarify the different metabolic substances and their regulatory functions of the strain at the early and late stages of growth, based on the growth curve of strain DZY6715, plate confrontation method was used to determine the inhibitory activity of the strain against C. fructicola in the early and late stages of growth, and then non-targeted metabolomic technology was employed to perform bioinformatics analysis of the fermentation broth in these two stages. The results showed that antifungal activity of strain DZY6715 in the early growth stage was significantly weaker than that in the late growth stage. In the sample DZYB72h_vs_DZYB24h, 32 pathways were annotated, and 183 differential metabolites were enriched in the top 20 pathways. Among them, there were 5 pathways with high potential importance, and 84 differential metabolites of 27 kinds were enriched. The involved differential metabolites were mainly amino acids and organic acids, accompanied by norfloxacin, N-acetyl-d-glucosamine, adenosine, phenol, etc. Furthermore, the total expression of amino acids in the early growth stage was significantly higher than that in the late growth stage, while the total expressions of organic acids and other differential metabolites in the late growth stage were significantly higher than that in the early growth stage. Therefore, this study confirmed that amino acids are mainly involved in the construction of cell structure and have antibacterial function, while organic acids, norfloxacin, adenosine and other substances are more important regulators in the process of resistance to stress.

Key words: Bacillus tequilensis, early growth stage, late growth stage, differential metabolites, non-targeted metabolomics

周嫒婷, 彭睿琦, 王芳, 伍建榕, 马焕成. 生防菌株DZY6715在不同生长期的代谢差异分析[J]. 生物技术通报, 2023, 39(9): 225-235.

ZHOU Ai-ting, PENG Rui-qi, WANG Fang, WU Jian-rong, MA Huan-cheng. Analysis of Metabolic Differences of Biocontrol Strain DZY6715 at Different Growth Stages[J]. Biotechnology Bulletin, 2023, 39(9): 225-235.

图/表 12

图1 菌株DZY6715的生长曲线 N为活菌数

Fig. 1 Growth curve of strain DZY6715 N refers to the number of live bacteria

图2 菌株DZY6715在不同生长期对油茶炭疽菌的抑菌效果

Fig. 2 Antifungal effects of strain DZY6715 against C. fructicola at different growth stages

图3 菌株DZY6715在不同生长期对油茶炭疽菌的抑菌率

Fig. 3 Inhibition rate of strain DZY6715 against C. fructicola at different growth stages

图4 不同生长期的菌株DZY6715处理后对油茶炭疽菌菌丝形态的影响（10×100倍）

Fig. 4 Effects of strain DZY6715 on mycelial morphology of C. fructicola at different growth stages（10×100 times）

图5 QC样本的PCA得分图 A：正离子模式，B：负离子模式，下同

Fig. 5 PCA score chart of the QC samples A: Positive ion mode. B: Negative ion mode. The same below

图6 DZYB72h-vs-DZYB24h的PCA得分图

Fig. 6 PCA score chart of DZYB72h-vs-DZYB24h

图7 DZYB72h-vs-DZYB24h的得分图（A1, B1）和置换检验图（A2, B2）

Fig. 7 PLS-DA score（A1, B1）and permutation test chart（A2, B2）of DZYB72h-vs-DZYB24h

图8 DZYB72h-vs-DZYB24h的差异代谢物火山图

Fig. 8 Differential metabolite volcano plot of DZYB72h-vs-DZYB24h

图9 DZY6715 KEGG富集通路图

Fig. 9 KEGG enrichment pathway map of strain DZY6715

表1 5条重要代谢通路中的差异代谢物

Table 1 Differential metabolites in 5 metabolic pathways

通路ID KEGG ID	通路名称 Map_Name	上调代谢物数量 Up-numb	下调代谢物数量 Down-numb	P值 P-value	重要性 Significance
ko04974	蛋白质消化和吸收Protein digestion and absorption	8	11	1.04582E-14	0.3830
ko00970	氨酰tRNA生物合成Aminoacyl-tRNA biosynthesis	4	10	2.37618E-08	0.2500
ko01230	氨基酸的生物合成Biosynthesis of amino acids	8	11	5.8421E-06	0.2759
ko02010	ABC转运蛋白ABC transporters	5	12	0.001180783	0.2174
ko01210	2-氧羰基酸代谢2-Oxocarboxylic acid metabolism	8	7	0.001180783	0.2174

表2 5条代谢通路的差异代谢物分析

Table 2 Differential metabolite analysis of five metabolic pathways

序号No.	KEGG ID	代谢物名称Metabolic name	类别Category
1	C00062	精氨酸 Arginine	氨基酸Amino acid
2	C00099	β-丙氨酸 Beta-alanine
3	C00025	谷氨酸 Glutamic acid
4	C00148	DL-脯氨酸 DL-proline
5	C00188	DL-苏氨酸 DL-threonine
6	C00078	DL-色氨酸 DL-tryptophan
7	C00082	DL-酪氨酸 DL-tyrosine
8	C00183	DL-缬氨酸 DL-valine
9	C00037	甘氨酸 Glycine
10	C00407	异亮氨酸 Isoleucine
11	C00049	L-天冬氨酸 L-aspartic acid
12	C00123	亮氨酸 Leucine
13	C00047	赖氨酸 Lysine
14	C00079	苯丙氨酸 Phenylalanine
15	C00233	酮亮氨酸 Ketoleucine
16	C00624	N-乙酰-l-谷氨酸 N-acetyl-l-glutamate
17	C00437	N-α-乙酰-l-鸟氨酸 N-.alpha.-acetyl-l-ornithine
18	C00246	丁酸 Butanoic acid	有机酸 Organic acids
19	C00163	丙酸 Propionic acid
20	C00956	2-氨基己二酸 2-aminoadipic acid
21	C08262	异戊酸 Isovaleric acid
22	C01606	邻苯二甲酸 1,2-benzenedicarboxylic acid
23	C01251	高柠檬酸盐 Homocitrate	/
24	C00146	苯酚 Phenol	/
25	C00212	腺苷 Adenosine	/
26	C00140	N-乙酰-d-氨基葡萄糖 N-acetyl-d-glucosamine	/
27	C06687	诺氟沙星 Norfloxacin	/
28	C00378	硫胺素 Thiamine	/

图10 差异代谢物表达量 *表示显著性（P <0.05）

Fig. 10 Expressions of differential metabolites * indicates significance（P <0.05）

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