生物技术通报 ›› 2022, Vol. 38 ›› Issue (2): 57-66.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0337
收稿日期:
2021-03-18
出版日期:
2022-02-26
发布日期:
2022-03-09
作者简介:
杨瑞先,女,博士,副教授,研究方向:植物内生细菌及病害生物防治;E-mail: 基金资助:
YANG Rui-xian(), LIU Ping, WANG Zu-hua, RUAN Bao-shuo, WANG Zhi-da
Received:
2021-03-18
Published:
2022-02-26
Online:
2022-03-09
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。
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.
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 |
FenAb | CGCCCTTTGGGAAGAGGTGC | ||||
2 | srfAA | Srfkn-1 | AGCCGTCCTGTCTGACGACG | 1 500 | Surfactin |
Srfkn-2 | TCTGCTGCCATACCGCATAGTC | ||||
3 | ituD | ItuD-f ItuD-r | ATGAACAATCTTGCCTTTTTA TTATTTTAAATTCCGCAATT | 1 300 | Iturin |
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 |
FenAb | CGCCCTTTGGGAAGAGGTGC | ||||
2 | srfAA | Srfkn-1 | AGCCGTCCTGTCTGACGACG | 1 500 | Surfactin |
Srfkn-2 | TCTGCTGCCATACCGCATAGTC | ||||
3 | ituD | ItuD-f ItuD-r | ATGAACAATCTTGCCTTTTTA TTATTTTAAATTCCGCAATT | 1 300 | Iturin |
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 |
F2-R | TCGCAGGCTGAATCGCTTC | ||
2 | S1-F | CGGTCAGCAATACGGAAGTCT | srfAA |
S2-R | TCAGTTCAGGACGGTTGTAGTAG | ||
3 | I1-F | ATTTCCTGGACAAGGGTCTCAAT | ituD |
I2-R | TCGCATCGCTCGCTTCTTC | ||
4 | rpsJ-F | GAAACGGCAAAACGTTCTGG | rpsJ |
rpsJ-R | GTGTTGGGTTCACAATGTCG |
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 |
F2-R | TCGCAGGCTGAATCGCTTC | ||
2 | S1-F | CGGTCAGCAATACGGAAGTCT | srfAA |
S2-R | TCAGTTCAGGACGGTTGTAGTAG | ||
3 | I1-F | ATTTCCTGGACAAGGGTCTCAAT | ituD |
I2-R | TCGCATCGCTCGCTTCTTC | ||
4 | rpsJ-F | GAAACGGCAAAACGTTCTGG | rpsJ |
rpsJ-R | GTGTTGGGTTCACAATGTCG |
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
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