苏云金芽胞杆菌AHL内酯酶功能及其抗魔芋软腐病研究
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摘要
本文主要研究了苏云金芽胞杆菌AHL内酯酶的抗病功能和其本身在苏云金芽胞杆菌中的调控功能。主要研究结果如下：
1 苏云金芽胞杆菌中AHL内酯酶的抗病活性检测
通过对苏云金芽胞杆菌不同菌株的抗病活性进行检测，发现各菌株之间对大白菜软腐病菌SCG1的致病性抑制活性存在较大差异。从抗病活性较高菌株H_(38)和H_(42)中克隆aiiA基因并在大肠杆菌中表达，表达的AHL内酯酶也能较强抑制大白菜软腐病菌SCG1的致病作用。另外，对这种蛋白的潜在防治对象进行调查时发现植物病原细菌中除了黄单胞杆菌中AHL信号较弱外，其余菌株均有较强的AHL信号存在；在革兰氏阳性细菌和真菌中没有检测到AHL信号分子。
2 抗病苏云金芽胞杆菌工程菌的田间应用
利用抗病工程菌株BMB686和BMB3439于2003年、2004年及2005年连续3年对魔芋新区与老区进行灌根防治试验，同时以化学农药作为对照。结果表明：抗病工程菌株在老区的相对防效可达32—40％，在新区的相对防效可达40—45％；化学农药在魔芋软腐病发病盛期的最高相对防效只有50％左右，绝大部分化学农药的相对防效为40％或更低。
3 aiiA基因的改造及转化花魔芋
依据魔芋的密码子偏爱性改造aiiA基因，构建转化花魔芋的双元载体，利用优化的花魔芋组织培养与再生体系转化花魔芋，潮霉素标记的合适筛选压前期为22.5mg／L，后期为37.5mg／L，抗性苗经PCR检测确定该基因已成功转化到花魔芋中。
4 AHL内酯酶在苏云金芽胞杆菌中的调控功能研究
苏云金芽胞杆菌中以下生理现象与AHL内酯酶的表达水平有关：1)在LB培养基中，提高AHL内酯酶的表达量会缩短细菌的代时；当在LB培养基中添加体积比为2％或4％的乙醇时，苏云金芽胞杆菌的代时会随着AHL内酯酶的表达量提高而大大延长。2)在37℃培养时，AHL内酯酶的表达水平与苏云金芽胞杆菌黑色素的产量呈正相关。3)在swarming评价盘中于28℃恒温保湿条件下培养48h后，AHL内酯酶的表达水平与细菌的swarming能力呈负相关。4)AHL内酯酶的表达水
This thesis focused on AHL-lactonase that can block the bacterial quorum sensing by hydrolyzing AHL-lactone and greatly attenuate the disease caused by many bacterial pathogens employing quorum sensing to regulate the expression of virulence genes. The results are summarized as following:
1. Activity of AHL-lactonase against soft rot caused by Erwinia carotovora.
Different Bacillus thuringiensis strains have distinct activity to protect potato slice from soft rot caused by E. carotovora SCG1. B. thuringiensis strains H_(38) and H_(42) with high activity were choiced to clone aiiA gene. The purified AHL-lactonase expressed in E.coli BL21(DE3) greatly attenuated the pathogenicity of E. carotovora SCG1.
To investigate the potential target of AHL-lactonase, the AHL signal was detected from bacteria and fungi. The strong AHL signal was observed from plant pathogens except for Xanthomonas campestris with faint AHL signal. While no AHL signal was found in gram-positive bacteria and any fungus.
2. Ability of B. thuringiensis strains over-expressing AHL-lactonase to protect Amorphophallus konjac from soft rot disease caused by E. carotovora pv. carotovora.
Engineering B. thuringiensis BMB686 and BMB3439 was used to protect A. konjac from soft rot disease in 2003, 2004 and 2005 year. Common germicides and water acted as control. The results showed that the relative control effect of engineering B. thuringiensis reached 40-45% in new A. konjac field and 35-40% in old A. konjac field (cultivated for one year). The best relative control effect of germicides reached 50% and those of so many germicides were lower than 40%.
3. Transform to A. konjac with alterated aiiA gene via Agrobacterium.
Genetic transformation system of Amorphophallus was established via. Agrobacterium. First, the tissue culture conditions of A. konjac were optimized. Second, the aiiA gene was alterated and transformed to A. konjac. The concentration of Hygromycin, the
1 苏云金芽胞杆菌中AHL内酯酶的抗病活性检测
通过对苏云金芽胞杆菌不同菌株的抗病活性进行检测，发现各菌株之间对大白菜软腐病菌SCG1的致病性抑制活性存在较大差异。从抗病活性较高菌株H_(38)和H_(42)中克隆aiiA基因并在大肠杆菌中表达，表达的AHL内酯酶也能较强抑制大白菜软腐病菌SCG1的致病作用。另外，对这种蛋白的潜在防治对象进行调查时发现植物病原细菌中除了黄单胞杆菌中AHL信号较弱外，其余菌株均有较强的AHL信号存在；在革兰氏阳性细菌和真菌中没有检测到AHL信号分子。
2 抗病苏云金芽胞杆菌工程菌的田间应用
利用抗病工程菌株BMB686和BMB3439于2003年、2004年及2005年连续3年对魔芋新区与老区进行灌根防治试验，同时以化学农药作为对照。结果表明：抗病工程菌株在老区的相对防效可达32—40％，在新区的相对防效可达40—45％；化学农药在魔芋软腐病发病盛期的最高相对防效只有50％左右，绝大部分化学农药的相对防效为40％或更低。
3 aiiA基因的改造及转化花魔芋
依据魔芋的密码子偏爱性改造aiiA基因，构建转化花魔芋的双元载体，利用优化的花魔芋组织培养与再生体系转化花魔芋，潮霉素标记的合适筛选压前期为22.5mg／L，后期为37.5mg／L，抗性苗经PCR检测确定该基因已成功转化到花魔芋中。
4 AHL内酯酶在苏云金芽胞杆菌中的调控功能研究
苏云金芽胞杆菌中以下生理现象与AHL内酯酶的表达水平有关：1)在LB培养基中，提高AHL内酯酶的表达量会缩短细菌的代时；当在LB培养基中添加体积比为2％或4％的乙醇时，苏云金芽胞杆菌的代时会随着AHL内酯酶的表达量提高而大大延长。2)在37℃培养时，AHL内酯酶的表达水平与苏云金芽胞杆菌黑色素的产量呈正相关。3)在swarming评价盘中于28℃恒温保湿条件下培养48h后，AHL内酯酶的表达水平与细菌的swarming能力呈负相关。4)AHL内酯酶的表达水
This thesis focused on AHL-lactonase that can block the bacterial quorum sensing by hydrolyzing AHL-lactone and greatly attenuate the disease caused by many bacterial pathogens employing quorum sensing to regulate the expression of virulence genes. The results are summarized as following:
1. Activity of AHL-lactonase against soft rot caused by Erwinia carotovora.
Different Bacillus thuringiensis strains have distinct activity to protect potato slice from soft rot caused by E. carotovora SCG1. B. thuringiensis strains H_(38) and H_(42) with high activity were choiced to clone aiiA gene. The purified AHL-lactonase expressed in E.coli BL21(DE3) greatly attenuated the pathogenicity of E. carotovora SCG1.
To investigate the potential target of AHL-lactonase, the AHL signal was detected from bacteria and fungi. The strong AHL signal was observed from plant pathogens except for Xanthomonas campestris with faint AHL signal. While no AHL signal was found in gram-positive bacteria and any fungus.
2. Ability of B. thuringiensis strains over-expressing AHL-lactonase to protect Amorphophallus konjac from soft rot disease caused by E. carotovora pv. carotovora.
Engineering B. thuringiensis BMB686 and BMB3439 was used to protect A. konjac from soft rot disease in 2003, 2004 and 2005 year. Common germicides and water acted as control. The results showed that the relative control effect of engineering B. thuringiensis reached 40-45% in new A. konjac field and 35-40% in old A. konjac field (cultivated for one year). The best relative control effect of germicides reached 50% and those of so many germicides were lower than 40%.
3. Transform to A. konjac with alterated aiiA gene via Agrobacterium.
Genetic transformation system of Amorphophallus was established via. Agrobacterium. First, the tissue culture conditions of A. konjac were optimized. Second, the aiiA gene was alterated and transformed to A. konjac. The concentration of Hygromycin, the
引文
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