解淀粉芽胞杆菌WH1抗真菌机制的研究
摘要
研究表明,多种芽胞杆菌都可以产生环状脂肽类物质来抑制植物病原真菌的生长。本研究从水稻根部分离出一株有较广抑菌谱、能产生脂肽的解淀粉芽胞杆菌WH1,并通过两步萃取、凝胶过滤层析等方法从WH1发酵液中纯化到了其产生的脂肽。经薄层层析、红外图谱、电喷雾质谱等分析后发现其是一种surfactin类的脂肽,将其命名为WH1fungin。本研究还对WH1fungin的抑菌机制进行了研究。
在抑菌试验中发现用100μg/ml的WH1fungin处理真菌能使真菌坏死,同时也检测到了核浓缩、DNA断裂、活性氧(ROS)积累、磷酯酰丝氨酸(PS)外翻、caspase活性上升等凋亡特征。
研究表明ROS在真菌凋亡中起到非常重要的作用,实验中用脯氨酸、抗坏血酸或还原型谷胱甘肽等活性氧清除剂处理后检测到活性氧积累减少,但磷脂酰丝氨酸外翻并不呈相应的减少,表明WH1fungin诱导的细胞凋亡不完全依赖于活性氧积累。
实验中发现经WH1fungin处理后真菌的葡聚糖合酶活性有明显下降。而葡聚糖合酶活性的降低会导致胼胝质合成量的减少,使新形成细胞没有足够的胼胝质来合成细胞壁,而细胞壁不完整的细胞更加容易发生细胞凋亡。
实验中还利用亲和层析分离到了与WH1fungin相互作用蛋白,经肽指纹图谱分析其为线粒体F-ATPase,在后续的检测中也验证了经WH1fungin处理后的真菌细胞的ATPase活性明显下降。在用FITC标记的WH1fungin处理细胞后能检测到其结合到线粒体膜上,验证了WH1fungin能作用于线粒体。寡霉素是线粒体FoF1-ATPase的抑制剂,WH1fungin处理的真菌细胞在加入寡霉素后凋亡现象更为显著,进一步验证WH1fungin能通过作用于线粒体ATPase来发挥促真菌凋亡的作用。
综上所述,WH1产生的脂肽在低浓度时可通过诱导凋亡的方式表现出抗真菌活性,这种凋亡可能是通过抑制葡聚糖合酶活性及作用于线粒体ATPase完成的。在自然生态中芽胞杆菌产生的脂肽并不足以导致细胞膜的穿孔,但却可诱导凋亡的发生,由此推断,WH1fungin诱导的细胞凋亡可能在解淀粉芽胞杆菌对真菌的拮抗作用中起重要的作用。
Some species of Bacillus could produce lipopeptides for controlling plant pathogenic fungi. In this study, we isolated a strain of B. amyloliquefaciens, called as WH1, from the rice root. WH1 could produce lipopeptide, WHlfungin, to inhibit several plant pathogenic fungi. WH1fungin could be isolated from the culture of WH1 by extraction and gel filtration, and characterized as an anti-fungal lipopeptide (surfactin) by thin layer chromatography, infrared spectrum, Tricine-SDS-PAGE and mass spectrum. We also studied on the anti-fungal mechanism of it.
After treated by WH1fungin, it was found that WH1fungin could directly kill some fungal cells by eliciting pores on membrane, while WHlfungin also could induce apoptosis. Classic apoptotic markers such as nucleus condensation, ROS accumulation, PS externalization, DNA strand breaks and high caspase-like activity were detectable.
Because ROS is a key factor for fungal apoptosis, further investigation was done for elucidating the role of ROS in apoptotic fungal cells. When fungal cells were treated by WHlfungin, antioxidants such as ascorbic acid, proline and reduced glutathione (GSH) were used for cleaning ROS respectively. It was found that ascorbic acid had no effect on the ROS, while proline and GSH could significantly inhibit ROS accumulation in WHlfungin-treated fungal cells. Unfortunately, clean of ROS by proline and GSH couldn't inhibit the relevant PS externalization in fungal cells.
It was reported that several cyclic lipopeptides could inhibit the activity of fungal glucane synthase, resulting in a synthesis decrease of callose. New generated fungal cells without enough callose for forming cell wall were easily found in apoptosis. In this study we found the activity of fungal glucane synthase was obviously decreased, and correspondent apoptotic markers could be detected in WHlfungin-treated fungal cells.
In order to detect the targeted proteins of WH1fungin, we used WH1fungin conjugated Sepharose 4B to isolate proteins in fungal cells. A purified protein was collected after isolation by this affinity chromatography, and was identified as a mitochondrial F-ATPase by peptide mass fingerprinting. After treated by FITC labeled WH1fungin, it was showed that WH1fungin could bind to the mitochondrial memebrane of fungal cells. After treated by WH1fungin, ATPase activity was significantly lower than control groups. It's known that oligomycin is an inhibitor of mitochondrial FoF1-ATPase of fungal cells. In this study more ROS and PS externalization was observed in fungal cells when oligomycin was used for treating fungal cells together with WHlfungin. All of these data verified that WH1fungin could interact with ATPase on the mitochondrial membrane
As a conclusion, low level of WH1fungin produced by WH1 could suppress the fungal glucane synthase, inhibit the mitochondrial ATPase, and then induce apoptosis in fungal cells. Normally Bacillus couldn't produce enough lipopeptide to elicit pores on fungal membrane in the natural environment, so we deduced that induction of apoptosis might be the common anti-fungal mechanism of Bacillus in the natural habitat.
在抑菌试验中发现用100μg/ml的WH1fungin处理真菌能使真菌坏死,同时也检测到了核浓缩、DNA断裂、活性氧(ROS)积累、磷酯酰丝氨酸(PS)外翻、caspase活性上升等凋亡特征。
研究表明ROS在真菌凋亡中起到非常重要的作用,实验中用脯氨酸、抗坏血酸或还原型谷胱甘肽等活性氧清除剂处理后检测到活性氧积累减少,但磷脂酰丝氨酸外翻并不呈相应的减少,表明WH1fungin诱导的细胞凋亡不完全依赖于活性氧积累。
实验中发现经WH1fungin处理后真菌的葡聚糖合酶活性有明显下降。而葡聚糖合酶活性的降低会导致胼胝质合成量的减少,使新形成细胞没有足够的胼胝质来合成细胞壁,而细胞壁不完整的细胞更加容易发生细胞凋亡。
实验中还利用亲和层析分离到了与WH1fungin相互作用蛋白,经肽指纹图谱分析其为线粒体F-ATPase,在后续的检测中也验证了经WH1fungin处理后的真菌细胞的ATPase活性明显下降。在用FITC标记的WH1fungin处理细胞后能检测到其结合到线粒体膜上,验证了WH1fungin能作用于线粒体。寡霉素是线粒体FoF1-ATPase的抑制剂,WH1fungin处理的真菌细胞在加入寡霉素后凋亡现象更为显著,进一步验证WH1fungin能通过作用于线粒体ATPase来发挥促真菌凋亡的作用。
综上所述,WH1产生的脂肽在低浓度时可通过诱导凋亡的方式表现出抗真菌活性,这种凋亡可能是通过抑制葡聚糖合酶活性及作用于线粒体ATPase完成的。在自然生态中芽胞杆菌产生的脂肽并不足以导致细胞膜的穿孔,但却可诱导凋亡的发生,由此推断,WH1fungin诱导的细胞凋亡可能在解淀粉芽胞杆菌对真菌的拮抗作用中起重要的作用。
Some species of Bacillus could produce lipopeptides for controlling plant pathogenic fungi. In this study, we isolated a strain of B. amyloliquefaciens, called as WH1, from the rice root. WH1 could produce lipopeptide, WHlfungin, to inhibit several plant pathogenic fungi. WH1fungin could be isolated from the culture of WH1 by extraction and gel filtration, and characterized as an anti-fungal lipopeptide (surfactin) by thin layer chromatography, infrared spectrum, Tricine-SDS-PAGE and mass spectrum. We also studied on the anti-fungal mechanism of it.
After treated by WH1fungin, it was found that WH1fungin could directly kill some fungal cells by eliciting pores on membrane, while WHlfungin also could induce apoptosis. Classic apoptotic markers such as nucleus condensation, ROS accumulation, PS externalization, DNA strand breaks and high caspase-like activity were detectable.
Because ROS is a key factor for fungal apoptosis, further investigation was done for elucidating the role of ROS in apoptotic fungal cells. When fungal cells were treated by WHlfungin, antioxidants such as ascorbic acid, proline and reduced glutathione (GSH) were used for cleaning ROS respectively. It was found that ascorbic acid had no effect on the ROS, while proline and GSH could significantly inhibit ROS accumulation in WHlfungin-treated fungal cells. Unfortunately, clean of ROS by proline and GSH couldn't inhibit the relevant PS externalization in fungal cells.
It was reported that several cyclic lipopeptides could inhibit the activity of fungal glucane synthase, resulting in a synthesis decrease of callose. New generated fungal cells without enough callose for forming cell wall were easily found in apoptosis. In this study we found the activity of fungal glucane synthase was obviously decreased, and correspondent apoptotic markers could be detected in WHlfungin-treated fungal cells.
In order to detect the targeted proteins of WH1fungin, we used WH1fungin conjugated Sepharose 4B to isolate proteins in fungal cells. A purified protein was collected after isolation by this affinity chromatography, and was identified as a mitochondrial F-ATPase by peptide mass fingerprinting. After treated by FITC labeled WH1fungin, it was showed that WH1fungin could bind to the mitochondrial memebrane of fungal cells. After treated by WH1fungin, ATPase activity was significantly lower than control groups. It's known that oligomycin is an inhibitor of mitochondrial FoF1-ATPase of fungal cells. In this study more ROS and PS externalization was observed in fungal cells when oligomycin was used for treating fungal cells together with WHlfungin. All of these data verified that WH1fungin could interact with ATPase on the mitochondrial membrane
As a conclusion, low level of WH1fungin produced by WH1 could suppress the fungal glucane synthase, inhibit the mitochondrial ATPase, and then induce apoptosis in fungal cells. Normally Bacillus couldn't produce enough lipopeptide to elicit pores on fungal membrane in the natural environment, so we deduced that induction of apoptosis might be the common anti-fungal mechanism of Bacillus in the natural habitat.
引文
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2.陈亮,浦冠勤.化学防治与生物防治在害虫综合防治中的作用.中国蚕业,2008,29(4)：84—86,90
3.陈中义,张杰,黄大防.植物病害生防芽胞杆菌抗菌机制与遗传改良研究.植物病理学报,2003,33(2)：97—103
4.崔艳红,黄现青.生物表面活性剂—表面活性素.生物技术,2006,16(5)：91—94
5.管珺,胡永红,杨文革等.蜡样芽胞杆菌防治植物病虫害的研究进展.现代农药,2007,6(4)：7—10
6.侯红漫,靳艳,金美芳等.环脂肽类生物表面活性剂结构、功能及生物合成.微生物学通报,2006,33(5)：122—128
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