在蜡状芽胞杆菌中AHL内酯酶对ZwA抗软腐病的增强效果
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摘要
双效菌素(Zwittermicin A，简称ZwA)是由蜡状芽胞杆菌(Bacillus cereus)和苏云金芽胞杆菌(Bacillus thuringiensis，简称Bt)产生的一种新型广谱的氨基多元醇类抗生素。该抗生素可抑制多种植物病原菌：对卵菌、藻类原生生物具有很高的抑菌活性，对很多革兰氏阴性与阳性细菌也表现出一定的抑菌活性。
N-酰基高丝氨酸内酯(Acyl-homoserine Lactones，简称AHLs)是广泛存在于革兰氏阴性细菌群体感应系统(Quorum-Sensing System，简称QS系统)中的信号分子，该信号分子可在细胞内外穿梭，参与多种病原菌致病基因的表达调控，当该分子浓度达到一定阈值时，能够启动病原菌致病基因的表达，酰基高丝氨酸内酯酶(AHL内酯酶)通过可以降解AHLs信号分子，减弱相关致病基因的表达从而达到抗病的效果。
本研究结合以上两种物质的不同抗病机理，将来自苏云金芽胞杆菌中的AHL内酯酶基因(aiiA)导入到ZwA产量不同的蜡状芽胞杆菌中，得到相应的重组菌株。本研究从两条途径来提高AHL内酯酶的作用效果：1．采用苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa的启动子来增强aiiA基因在细胞内的表达，构建带有pro3A-aiiA融合基因的重组质粒pBMB686；2．利用苏云金芽胞杆菌S-层蛋白表面展示技术将AHL内酯酶这一胞内蛋白带到胞外并锚定在细胞表面，以与环境中的AHLs分子直接作用，提高其降解活性，构建了同时带有slh-aiiA和pro3A-aiiA融合基因的重组质粒pBMB0624，并且分别将pBMB686和pBMB0624两个质粒转化到蜡状芽胞杆菌当中，比较其抗病效果。
实验表明，aiiA基因(pro3A-aiiA和slh-aiiA)均可在重组菌中正常表达，并且不影响受体菌ZwA的表达和产量；感病实验证明，同时表达ZwA和AHL内酯酶的重组菌对于胡萝卜软腐欧文氏菌感染马铃薯所引起的病害的抗病效果比单独含产ZwA或AHL内酯酶的蜡状芽胞杆菌的抗病效果有明显增强。说明结合ZwA和AHL内酯酶的不同抗病机理来提高抗病效果是可行的。
Zwittermicin A(ZwA) is a broad spectrum, novel antibiotic produced by Bacillusthuringiensis and Bacillus cereus. ZwA shows diverse inhibitory activity against a broadtarget of plant pathogens, including highly activity against algal protists and a wide rangeof plant pathogenic fungi, and moderate activity against diverse Gram-negative bacteriaand certain Gram-postive bacteria.
N-acyl-homoserine Lactones(AHLs), widely-spreaded signal molecules inQuorum-Sensing systems of many Gram-negative bacteria, can contribute to theinducement of the expression of many virulence genes. And Acyl-homoserineLactonase(AHL lactonase) can suppress certain plant diseases by degrading AHLs, whichcan shuttle in and out of cells.
In order to increase disease suppression by combining the two different diseasesuppression mechanisms of ZwA and AHL lactonase, AHL lactonase gene was introducedinto ZwA~+ Bacillus cereus strains. In this study, two ways of enhacing the activity of AHLlactonase were adopted: on one hand, the expression of AHL gene(aiiA) was increased byconstructing a fusion gene pro3A-aiiA including aliA gene and a strong promoter ofinsecticidal crystal protein coding gene cry3Aa of Bacillus thuringiensis, and the fusiongene pro3A-aiiA was placed in pBMB686. On the other hand, in order to degrade AHLsout of the cells directly, AHL lactonase was displayed on the cell surface by constructinga fusion gene slh-aiiA including aiia gene and SLH motif of S-layer protein from Bacillusthruringiensis, and the fusion gene slh-aiiA was carried by pBMB3439. PlasmidspBMB686 and pBMB3439 were transformed into Bacillus cereus separately, and thedisease suppression of the two difference recombinants was compared.
The results indicated that both pro3A-aiiA and slh-aiiA genes could be expressed inrecombinant strains, without affecting the expression level of ZwA. Furthermore, therecombinant stains exhibited stronger ability of AHLs inactivation and higher restraint tothe potato rot disease caused by Erwinia carotovora than their parental stains. Thus, it wasindicated it was feasible to increase disease suppression by combining the biologicalfunctions of ZwA and AHL lactonase.
N-酰基高丝氨酸内酯(Acyl-homoserine Lactones，简称AHLs)是广泛存在于革兰氏阴性细菌群体感应系统(Quorum-Sensing System，简称QS系统)中的信号分子，该信号分子可在细胞内外穿梭，参与多种病原菌致病基因的表达调控，当该分子浓度达到一定阈值时，能够启动病原菌致病基因的表达，酰基高丝氨酸内酯酶(AHL内酯酶)通过可以降解AHLs信号分子，减弱相关致病基因的表达从而达到抗病的效果。
本研究结合以上两种物质的不同抗病机理，将来自苏云金芽胞杆菌中的AHL内酯酶基因(aiiA)导入到ZwA产量不同的蜡状芽胞杆菌中，得到相应的重组菌株。本研究从两条途径来提高AHL内酯酶的作用效果：1．采用苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa的启动子来增强aiiA基因在细胞内的表达，构建带有pro3A-aiiA融合基因的重组质粒pBMB686；2．利用苏云金芽胞杆菌S-层蛋白表面展示技术将AHL内酯酶这一胞内蛋白带到胞外并锚定在细胞表面，以与环境中的AHLs分子直接作用，提高其降解活性，构建了同时带有slh-aiiA和pro3A-aiiA融合基因的重组质粒pBMB0624，并且分别将pBMB686和pBMB0624两个质粒转化到蜡状芽胞杆菌当中，比较其抗病效果。
实验表明，aiiA基因(pro3A-aiiA和slh-aiiA)均可在重组菌中正常表达，并且不影响受体菌ZwA的表达和产量；感病实验证明，同时表达ZwA和AHL内酯酶的重组菌对于胡萝卜软腐欧文氏菌感染马铃薯所引起的病害的抗病效果比单独含产ZwA或AHL内酯酶的蜡状芽胞杆菌的抗病效果有明显增强。说明结合ZwA和AHL内酯酶的不同抗病机理来提高抗病效果是可行的。
Zwittermicin A(ZwA) is a broad spectrum, novel antibiotic produced by Bacillusthuringiensis and Bacillus cereus. ZwA shows diverse inhibitory activity against a broadtarget of plant pathogens, including highly activity against algal protists and a wide rangeof plant pathogenic fungi, and moderate activity against diverse Gram-negative bacteriaand certain Gram-postive bacteria.
N-acyl-homoserine Lactones(AHLs), widely-spreaded signal molecules inQuorum-Sensing systems of many Gram-negative bacteria, can contribute to theinducement of the expression of many virulence genes. And Acyl-homoserineLactonase(AHL lactonase) can suppress certain plant diseases by degrading AHLs, whichcan shuttle in and out of cells.
In order to increase disease suppression by combining the two different diseasesuppression mechanisms of ZwA and AHL lactonase, AHL lactonase gene was introducedinto ZwA~+ Bacillus cereus strains. In this study, two ways of enhacing the activity of AHLlactonase were adopted: on one hand, the expression of AHL gene(aiiA) was increased byconstructing a fusion gene pro3A-aiiA including aliA gene and a strong promoter ofinsecticidal crystal protein coding gene cry3Aa of Bacillus thuringiensis, and the fusiongene pro3A-aiiA was placed in pBMB686. On the other hand, in order to degrade AHLsout of the cells directly, AHL lactonase was displayed on the cell surface by constructinga fusion gene slh-aiiA including aiia gene and SLH motif of S-layer protein from Bacillusthruringiensis, and the fusion gene slh-aiiA was carried by pBMB3439. PlasmidspBMB686 and pBMB3439 were transformed into Bacillus cereus separately, and thedisease suppression of the two difference recombinants was compared.
The results indicated that both pro3A-aiiA and slh-aiiA genes could be expressed inrecombinant strains, without affecting the expression level of ZwA. Furthermore, therecombinant stains exhibited stronger ability of AHLs inactivation and higher restraint tothe potato rot disease caused by Erwinia carotovora than their parental stains. Thus, it wasindicated it was feasible to increase disease suppression by combining the biologicalfunctions of ZwA and AHL lactonase.
引文
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