牡丹根腐病原菌拮抗细菌抑菌活性物质分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0337

摘要/Abstract

摘要：

前期研究发现解淀粉芽胞杆菌（Bacillus amyloliquefaciens）md8和md9对牡丹根腐病原菌具有较好的抑制作用,但其抑菌物质的组成尚不清楚。本文首先明确了2个菌株3种脂肽类物质合成的基因片段,利用酸沉淀和葡聚糖凝胶层析法进行抑菌物质的分离纯化,牛津杯对峙法检测脂肽类粗提物和凝胶层析分离组分的抑菌活性;进一步利用实时荧光定量PCR（RT-qPCR）检测菌株在拮抗根腐病原菌过程中脂肽类物质合成基因相对表达量的变化,基质辅助激光解吸飞行时间质谱（MALDI-TOF-MS）分析抑菌物质的种类。结果表明,2个菌株的脂肽类粗提物和凝胶层析分离组分对根腐病原菌均具有良好的平板抑菌效果,RT-qPCR结果表明2个菌株合成伊枯草菌素（iturin）的基因ituD在对峙培养过程中相对表达量显著增加,芬荠素（fengycin）合成基因fenA也呈现上调表达。MALDI-TOF-MS分析表明2个菌株中具有抑菌作用的分离组分主要为伊枯草菌素类物质Iturin B和Bacillomycins D,结合实时荧光定量PCR结果,推测菌株md8和md9在拮抗根腐病原菌过程中发挥主要生防作用的物质为伊枯草菌素。

关键词: 牡丹, 根腐病原菌, 解淀粉芽胞杆菌, 脂肽类物质合成基因, 抑菌活性物质

Abstract:

Previous studies revealed that two stains Bacillus amyloliquefaciens md8 and md9 have strong antagonism to Fusarium solani causing tree peony root rot;however,the components of antimicrobial substances are not clear yet. First,the biosynthesis gene fragments of 3 lipopeptides were identified. Acid precipitation and the Sephadex gel chromatography column were used to separate and purify the antimicrobial substances. Then Oxford Cup method was adapted to detect the inhibitory activity of the crude lipopeptides and separation components. RT-qPCR was employed to detect the expression variations of lipopetide biosynthesis genes in the strains md8 and md9 during inhibiting F. solani process in the dual culture. And finally MALDI-TOF-MS technique was used to analyze the types of antimicrobial substances. The results showed that the crude lipopeptide extracts and separation components via Sephadex gel chromatography had favorable effect against the pathogen of root rot. The RT-qPCR results indicated that gene ituD of md8 and md9 for iturin biosynthesis was constantly expressed in high amounts throughout the antagonism. The gene fenA for fengycin biosynthesis were up-regulated when md8 and md9 were confronted with F. solani. The MALDI-TOF-MS results further verified that iturin B and bacillomycin D were the main antimicrobial components of md8 and md9 against F. solani. Combined with RT-qPCR results,it was speculated that iturin produced by the strains md8 and md9 played an important role in the inhibitory effect on F. solani.

Key words: Paeonia suffruticosa, Fusarium solani, Bacillus amyloliquefaciens, lipopeptide biosynthesis genes, antimicrobial active metabolites

杨瑞先, 刘萍, 王祖华, 阮宝硕, 汪智达. 牡丹根腐病原菌拮抗细菌抑菌活性物质分析[J]. 生物技术通报, 2022, 38(2): 57-66.

YANG Rui-xian, LIU Ping, WANG Zu-hua, RUAN Bao-shuo, WANG Zhi-da. Analysis of Antimicrobial Active Metabolites from Antagonistic Strains Against Fusarium solani[J]. Biotechnology Bulletin, 2022, 38(2): 57-66.

图/表 11

表1 脂肽类物质合成基因片段引物序列

Table 1 Primer sequence for lipopeptide biosynthesis genes fragments

序号 No.	基因 Gene	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物大小 Product size/ bp	产生的脂肽类物质 Lipopeptide
1	fenA	FenAa	AAGAGATTCAGTAAGTGGCCCATCCAG	1 500	Fengycin
1	fenA	FenAb	CGCCCTTTGGGAAGAGGTGC	1 500	Fengycin
2	srfAA	Srfkn-1	AGCCGTCCTGTCTGACGACG	1 500	Surfactin
2	srfAA	Srfkn-2	TCTGCTGCCATACCGCATAGTC	1 500	Surfactin
3	ituD	ItuD-f ItuD-r	ATGAACAATCTTGCCTTTTTA TTATTTTAAATTCCGCAATT	1 300	Iturin

表2 脂肽类物质合成基因荧光定量PCR引物序列

Table 2 Primer sequences for lipopeptide biosynthesis genes in RT-qPCR

序号 No.	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	基因 Gene
1	F1-F	CAGGTGCCGTTACAGGACAT	fenA
1	F2-R	TCGCAGGCTGAATCGCTTC	fenA
2	S1-F	CGGTCAGCAATACGGAAGTCT	srfAA
2	S2-R	TCAGTTCAGGACGGTTGTAGTAG	srfAA
3	I1-F	ATTTCCTGGACAAGGGTCTCAAT	ituD
3	I2-R	TCGCATCGCTCGCTTCTTC	ituD
4	rpsJ-F	GAAACGGCAAAACGTTCTGG	rpsJ
4	rpsJ-R	GTGTTGGGTTCACAATGTCG	rpsJ

图1 拮抗菌株md8和md9菌落和菌体形态特征（10×100）

Fig.1 Colonies and morphological characteristics of anta-gonistic strain md8 and md9（10×100）

图2 基于16S rDNA序列构建的拮抗细菌md8和md9的系统发育树

Fig.2 Phylogenetic tree of antagonistic strain md8 and md9 based on 16 S rDNA sequences

图3 菌株md8和md9脂肽类物质合成基因片段PCR扩增电泳结果 A：菌株md8脂肽类物质合成基因片段扩增结果;B：菌株md9脂肽类物质合成基因片段扩增结果。M：DL2000 marker

Fig. 3 Electrophoresis of PCR amplified lipopeptide biosy-nthesis gene fragments of strain md8 and md9 A：Amplified results of the lipopeptide biosynthesis gene fragments of md8. B：Amplified results of the lipopeptide biosynthesis gene fragments of md9. M：DL2000 marker

图4 菌株md8和md9对牡丹根腐原菌的平板抑制作用（平板对峙培养6 d） A：菌株md8;B：菌株md9

Fig.4 Inhibitory effect of strain md8 and md9 inhibiting F. solani on the plate（plate confrontation cultured 6 d） A：strain md8;B：strain md9

图5 菌株md8脂肽类物质合成基因在抑制牡丹根腐病原菌时的相对表达量

Fig.5 Relative expressions of the lipopeptide biosynthesis genes of strain md8 inhibiting F. solani on the plate

图6 菌株md9脂肽类物质合成基因在抑制牡丹根腐病原菌时的相对表达量

Fig.6 Relative expressions of the lipopeptide biosynthesis genes of strain md9 inhibiting F. solani on the plate

图7 菌株md8和md9脂肽类粗提物和凝胶层析分离组分对牡丹根腐病原菌的平板抑制作用 A：菌株md8和md9脂肽类粗提物的平板抑制作用;B：菌株md8凝胶层析分离组分的平板抑制作用;C：菌株md9凝胶层析分离组分的平板抑制作用

Fig.7 Inhibition effect of lipopeptide extract and separation components via gel chromatography of strain md8 and md9 inhibiting F. solani A：Inhibition effect of lipopeptide extract from strain md8 and md9. B：Plate inhibition effect of separation components of strain md8 via gel chromatography. C：Inhibition effect of separation components of strain md9 via gel chromatography

图8 菌株md8凝胶层析分离组分A4（A）和A5（B）的MALDI-TOF-MS分析

Fig.8 MALDI-TOF-MS analysis of separation component A4（A）and A5（B）produced by the strain md8

图9 菌株md9凝胶层析组分B2（A）、B3（B）和B4（C）的MALDI-TOF-MS分析

Fig.9 MALDI-TOF-MS analysis of separation component B2（A）,B3（B）and B4（C）produced by the strain md9

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