表达Harpin蛋白的枯草芽孢杆菌生防菌的构建
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摘要
生物防治是植物病害综合治理体系中的一个重要组成部分,植物同病原菌互作的进化过程中形成的过敏反应是自然界中植物抵抗病原菌侵染的最有效方式。Harpin是一类植物病原细菌分泌的蛋白质,能诱导非寄主植物产生过敏反应,这种防卫反应的产生,可以抵抗多种多样的病原物对植物的第二次侵染,类似于人或动物的免疫系统。枯草芽孢杆菌本身无毒,对多种植物病原菌具有拮抗作用,是一种天然的植病生防菌。利用Harpin的诱导抗病性和生防菌的拮抗作用开发新型的生物农药具有重要的理论意义和应用价值。本实验将利用枯草芽孢杆菌来表达Harpin蛋白,期望获得一种既能诱导植物抗性又能与植物病原菌竞争、拮抗的多功能生防菌。
为了能使Harpin蛋白分泌到培养基中,与植物细胞直接作用,诱导植物的防卫反应。首先,我们以枯草芽孢杆菌168总DNA为模板扩增组成型强启动子P43和分泌效率较高的nprB信号肽,通过重组PCR连接两片段,克隆至大肠杆菌和芽孢杆菌的穿梭载体pUBC19,构建组成型表达分泌载体pUBC-PS。
其次,以新鲜制备的胡萝卜软腐欧文氏杆菌(Erwinia carotovora subsp. carotovora)Se9总DNA为模板,设计引物扩增hrpNECC9基因,PCR产物克隆于表达载体pUBC-PS,获得重组质粒pUBC-PSh2,将重组质粒转入大肠杆菌DH5α,在含有100μg/mL氨苄青霉素的LB平板上筛选转化子-DH5a(pUBC-PSh2)。小量提取pUBC-PSh2质粒,采用碱金属转化法转入缺失8种蛋白酶基因的枯草杆菌WB800,在含有卡那霉素20μg/mL的LB平板上筛选工程菌,对其酶切、PCR验证结果表明：生防工程菌WB800(pUBC-PSh2)构建成功。
SDS-PAGE分析表明：工程菌培养到72 h时,表达产物Harpin蛋白被分泌到细胞培养液中,表达量约占菌体蛋白总量的15%。由此证明,工程菌中的hrpNECC9基因在枯草杆菌P43启动子和nprB信号肽元件的带动下,不需诱导,实现了表达,并且,表达产物被分泌到胞外。
生物活性检测和诱导植物抗病性分析结果表明：工程菌能够诱导烟草叶片产生过敏反应,并能提高番茄对早疫病菌的抗性。
Biological control is an important component in plant diseases management system. Hypersensitive response is the most effective way in resistance pathogen infection. Harpin encoded by hrpN gene in E. carotovora subsp can induce hypersensitive reaction on non-host plants, once the establishments of systemic acquired resistance (SAR), plants exhibit a broad-spectrum of disease resistance against pathogen attack, similar to humans or animals immune system.
Bacillus.subtilis is a natural plant bacterium with non-toxic, antagonism effect for plant pathogen. It would be feasible to explore biological control pesticides base on characteristics of Bacillus.subtilis and induction resistance of Harpin. This experiment will be used to express Harpin protein in Bacillus subtilis, in order to obtain multifunctional biocontrol bacteria that can induce plant resistance and compete with plant pathogen.
In this research, firstly, by PCR using B.Subtilis168 genomic DNA as template, the P43 promoter (constitutive-expression) and the nprB signal peptide were amplified and connected with E.coli-B.subtilis shutter vector pUBC19, then formed expression vector pUBC-PS.
Secondly,hrpNEcc9 from Erwinia carotovor (Se9) was cloned into vector pUBC-PS to obtain expression and secretion Harpin plasmid pUBC-PSh2, which was transformed into B. subtilis WB800 by alkali metals method. Next, the recombinant strain was cultured at 37℃, shaking 200 rpm for 72h. Then, the target protein was obviously detected in culture supernatant fluid by SDS-PAGE. And the protein expression quantity was about 15% of cell protein. At the same, Cell supernatant was injected into tobacco leaf which induced hypersensitive response. The induction of plant disease resistance analysis showed that:the engineering bacteria can improve resistance for Alternaria solani.
为了能使Harpin蛋白分泌到培养基中,与植物细胞直接作用,诱导植物的防卫反应。首先,我们以枯草芽孢杆菌168总DNA为模板扩增组成型强启动子P43和分泌效率较高的nprB信号肽,通过重组PCR连接两片段,克隆至大肠杆菌和芽孢杆菌的穿梭载体pUBC19,构建组成型表达分泌载体pUBC-PS。
其次,以新鲜制备的胡萝卜软腐欧文氏杆菌(Erwinia carotovora subsp. carotovora)Se9总DNA为模板,设计引物扩增hrpNECC9基因,PCR产物克隆于表达载体pUBC-PS,获得重组质粒pUBC-PSh2,将重组质粒转入大肠杆菌DH5α,在含有100μg/mL氨苄青霉素的LB平板上筛选转化子-DH5a(pUBC-PSh2)。小量提取pUBC-PSh2质粒,采用碱金属转化法转入缺失8种蛋白酶基因的枯草杆菌WB800,在含有卡那霉素20μg/mL的LB平板上筛选工程菌,对其酶切、PCR验证结果表明：生防工程菌WB800(pUBC-PSh2)构建成功。
SDS-PAGE分析表明：工程菌培养到72 h时,表达产物Harpin蛋白被分泌到细胞培养液中,表达量约占菌体蛋白总量的15%。由此证明,工程菌中的hrpNECC9基因在枯草杆菌P43启动子和nprB信号肽元件的带动下,不需诱导,实现了表达,并且,表达产物被分泌到胞外。
生物活性检测和诱导植物抗病性分析结果表明：工程菌能够诱导烟草叶片产生过敏反应,并能提高番茄对早疫病菌的抗性。
Biological control is an important component in plant diseases management system. Hypersensitive response is the most effective way in resistance pathogen infection. Harpin encoded by hrpN gene in E. carotovora subsp can induce hypersensitive reaction on non-host plants, once the establishments of systemic acquired resistance (SAR), plants exhibit a broad-spectrum of disease resistance against pathogen attack, similar to humans or animals immune system.
Bacillus.subtilis is a natural plant bacterium with non-toxic, antagonism effect for plant pathogen. It would be feasible to explore biological control pesticides base on characteristics of Bacillus.subtilis and induction resistance of Harpin. This experiment will be used to express Harpin protein in Bacillus subtilis, in order to obtain multifunctional biocontrol bacteria that can induce plant resistance and compete with plant pathogen.
In this research, firstly, by PCR using B.Subtilis168 genomic DNA as template, the P43 promoter (constitutive-expression) and the nprB signal peptide were amplified and connected with E.coli-B.subtilis shutter vector pUBC19, then formed expression vector pUBC-PS.
Secondly,hrpNEcc9 from Erwinia carotovor (Se9) was cloned into vector pUBC-PS to obtain expression and secretion Harpin plasmid pUBC-PSh2, which was transformed into B. subtilis WB800 by alkali metals method. Next, the recombinant strain was cultured at 37℃, shaking 200 rpm for 72h. Then, the target protein was obviously detected in culture supernatant fluid by SDS-PAGE. And the protein expression quantity was about 15% of cell protein. At the same, Cell supernatant was injected into tobacco leaf which induced hypersensitive response. The induction of plant disease resistance analysis showed that:the engineering bacteria can improve resistance for Alternaria solani.
引文
1.杜华,王玲,等.防治植物病害的生物农药研究开发进展[J].河南农业科学,2000,(9)：39-42
2.于淑池,张利平,等.拮抗细菌作为生物防治手段研究进展[J].河北农业科学,2004,8(1)：62-65
3.程亮,游春平,等.拮抗细菌的研究进展[J].江西农业大学学报,2003,25(5)：732-737
4.贺字典,高玉峰,等.生防菌在植物病害防治中的研究进展[J].河北职业技术示范学院学报,2003,17(2)：56-58
5.王万能,全学军,等.关于植物病害生物防治[J].重庆工学院学报,2004,18(2)：50-52
6. Monica L E, Elizabeth A D J, William E B J et al. Viability and stability of biological control agents on cotton and snap beanseeds [J]. Pest Management. Science,2001, 57(8):695-706
7.张学君,凌宏通,等.生物农药麦丰宁对小麦纹枯病菌的抑制作用[J].植物病理学报,1994,24(4)：361-366
8.郭利,涂洋祥,等.新型微生物农药百抗防治烟草青枯病试验[J].《现代农业科技》,2008,24
9. L. R. Cavaglieri, A. Passone, M. G. Correlation between screening procedures to Select root endophytes for biological control of Fusarium verticillioides in Zea [J]. Biologicalcontrol,2004,31:259-267
10.赵立平等.产harpin的成团泛菌工程菌的构建[J].植物病理学报,1999,29(2)：142
11. Hammond-kosack KE, Jones JDG. Resistance gene-dependent plant defense responses[J]. Plant Cell,1996,8:1378-1391
12. Willis D K, Rich J J, Hrabak E M. Hrp genes of phytopathogenic bacteria [J]. Molecular Plant Microbe Interact,1991,4:132-138
13.胡向阳.激发子诱导植物防卫反应过程中的信号分子[D].上海,中科院上海 生命科学研究院植物生理生态研究所,2003
14.徐文联,王余生.植物诱导性抗病因子[A].董汉松.植物诱导抗病性原理和研究[C].北京：科学出版社,1995,97-105
15. Lindgren P B et al. Gene cluster of Pseudomonas syringae pv. "Phaseolicola" controls pathogenicity of bean plants and hypersensitivity on nonhost plants [J]. Bacteriol,1986,168:512-522
16.Yog.植物与病原物互作相关基因及产物[J].Trends in Life Sciences News,2003, 1:11-12
17. Wengelnik K, Rossier O, Bonas U J. Bacteriol,1999,181:6828-6831
18. Collmer A, Badel JL et al. Pseudomonas syringae Hrp type III secretion system and effector proteins [J]. Proc.Ntal.Acad.Sci.USA.2000,97:8770-8777
19.王金生.分子植物病理学,北京：中国农业出版社,1999
20.汤承,李士丹.Harpin蛋白的研究进展[J].草业与畜牧,2006,(10)：1-4
21. Wei Z M, RON J L et al. Harpin, elicitor of the HR produced by the plant pathogen Erwinia amylovora [J]. Science,1992,25(7):85-88
22.白艳.胡萝卜软腐欧式杆菌Harpin的基因克隆与表达.硕士学位论文,山西大学,2007
23. Barny M A, Rona J P, et al. Harpin, a hypersensi tive response elicitor from Erwinia amylovora, regulatesion channel activities in Arabidopsis thaliana suspension cells [J]. FEBS Lett,2001,497(2-3):82-84
24. Tampakaki A P, Panopoulos N J. Elicitation of hypersensitive cell death by extracellularly targeted HrpZPsph produced in planta [J]. Molecular Plant Microbe Interactions,2000,13:1366-1374
25.闻伟刚,王金生.水稻白叶枯病菌harpin基因的克隆与表达[J].植物病理学报,2001,31(4)：295-300
26.赵梅勤,王磊,张兵,等.植物抗病激活蛋白HarpinXooc防治水稻病害的研究[J].中国生物防治.2006,22(4)：283-289
27.李平.水稻黄单胞菌无毒基因avrXa3的克隆与鉴定以及过敏反应激发子Hrf蛋白质遗传多样性和功能域的研究.博士学位论文,南京农业大学,2002
28.王健,赵立平.细菌Ⅲ型分泌系统[J].生命的化学,2001,21(2)：147-149
29. Mukherjee A, Cui Y, Ma W, et al. RpoS (Sigma-S) Controls Expression of rsmA, a Global Regulator of Secondary Metabolites, Harpin, and Extracellular Proteins in Erwinia carotovora[J]. Journal of bacteriology,1998,180:3629-3634
30.赵立平,梁元存,刘爱新,等.表达harpin基因的大肠杆菌DH5(pCPP430)诱导植物抗病性的研究[J].高技术通讯,1997,7(9)：1
31.邵敏,王金生.转hrfAXoo基因水稻对白叶枯病的抗性[J].南京农业大学学报,2004,27(4)：36-40
32. Stroud R M, Walter P. Signal sequence recognition and protein targeting [J]. Current opinion in structural biology,1999,9:754-759
33.李育阳.基因表达技术[M].第一版.北京：科学出版社,2001
34. Ehrlich SD. Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilus. Proc.SciUSA,1997,74:1680-1682
35.皱立扣,王红宁,潘欣.枯草芽抱杆菌整合载体研究进展[J].生物技术通讯,2003,14：525-527
36. Breitling R, Serokin AU, Behnke D. Thermo-inducible gene expression in Bacillus subtilis mediated by the 1857-encoded repressor of bacteriophageλ[J]. Gene,1990, 93:35
37.彭清忠,张惟材,朱厚咄.枯草杆菌表达系统的研究进展[J].生物技术通讯,2001,12：220-225
38.刘白玲,何先祺.枯草杆菌的分子生物学研究[J].成都科技大学学报,1993,72(5)：34-41
39. Sarvas M. Gene expression in recombinant bacillus [J]. bioprocess technol,1995,22: 53
40. Sau-Ching Wu and Sui-Lam Wong. Engineering of a Bacillus snbtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin [J]. Applied and Environmental Microbiology.2002,68(3):1102-1108
41.吕正兵,张方,等.一种适合芽孢杆菌质粒DNA提取的改良碱裂解法[J].安徽师范大学学学报,2002,25(1)：54-55
42. Yoshito Sadaie et al. Formation of Competent Bacillus subtilis cells [J]. Jouranl of Bacteriology,1982,2(153):813-821
43.杨晟,黄艳红,黄晓冬,等.巨大芽孢杆菌青霉素G酰化酶基因在枯草杆菌中的高效表达[J].生物化学与生物物理学报,1999,31(5)：601-603
44. Ngaarajna, V. AlbesrtonH, et al. Secretion of streptavidin from Bacillus subtilis [J]. Appl Enviorn Microbiol,1993,59:3894-3898
45.张惠展.基因工程[M].第1版.上海：华东理工大学出版社,2005
46. Ye R, Kim J H, Kim B G,et al. High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis [J]. Biotechnol Bioeng,1999,62:87-96
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48.赵士敏,等.保护地番茄早疫病研究初探[J].天津农业科学,1996,2(4)：13-15
2.于淑池,张利平,等.拮抗细菌作为生物防治手段研究进展[J].河北农业科学,2004,8(1)：62-65
3.程亮,游春平,等.拮抗细菌的研究进展[J].江西农业大学学报,2003,25(5)：732-737
4.贺字典,高玉峰,等.生防菌在植物病害防治中的研究进展[J].河北职业技术示范学院学报,2003,17(2)：56-58
5.王万能,全学军,等.关于植物病害生物防治[J].重庆工学院学报,2004,18(2)：50-52
6. Monica L E, Elizabeth A D J, William E B J et al. Viability and stability of biological control agents on cotton and snap beanseeds [J]. Pest Management. Science,2001, 57(8):695-706
7.张学君,凌宏通,等.生物农药麦丰宁对小麦纹枯病菌的抑制作用[J].植物病理学报,1994,24(4)：361-366
8.郭利,涂洋祥,等.新型微生物农药百抗防治烟草青枯病试验[J].《现代农业科技》,2008,24
9. L. R. Cavaglieri, A. Passone, M. G. Correlation between screening procedures to Select root endophytes for biological control of Fusarium verticillioides in Zea [J]. Biologicalcontrol,2004,31:259-267
10.赵立平等.产harpin的成团泛菌工程菌的构建[J].植物病理学报,1999,29(2)：142
11. Hammond-kosack KE, Jones JDG. Resistance gene-dependent plant defense responses[J]. Plant Cell,1996,8:1378-1391
12. Willis D K, Rich J J, Hrabak E M. Hrp genes of phytopathogenic bacteria [J]. Molecular Plant Microbe Interact,1991,4:132-138
13.胡向阳.激发子诱导植物防卫反应过程中的信号分子[D].上海,中科院上海 生命科学研究院植物生理生态研究所,2003
14.徐文联,王余生.植物诱导性抗病因子[A].董汉松.植物诱导抗病性原理和研究[C].北京：科学出版社,1995,97-105
15. Lindgren P B et al. Gene cluster of Pseudomonas syringae pv. "Phaseolicola" controls pathogenicity of bean plants and hypersensitivity on nonhost plants [J]. Bacteriol,1986,168:512-522
16.Yog.植物与病原物互作相关基因及产物[J].Trends in Life Sciences News,2003, 1:11-12
17. Wengelnik K, Rossier O, Bonas U J. Bacteriol,1999,181:6828-6831
18. Collmer A, Badel JL et al. Pseudomonas syringae Hrp type III secretion system and effector proteins [J]. Proc.Ntal.Acad.Sci.USA.2000,97:8770-8777
19.王金生.分子植物病理学,北京：中国农业出版社,1999
20.汤承,李士丹.Harpin蛋白的研究进展[J].草业与畜牧,2006,(10)：1-4
21. Wei Z M, RON J L et al. Harpin, elicitor of the HR produced by the plant pathogen Erwinia amylovora [J]. Science,1992,25(7):85-88
22.白艳.胡萝卜软腐欧式杆菌Harpin的基因克隆与表达.硕士学位论文,山西大学,2007
23. Barny M A, Rona J P, et al. Harpin, a hypersensi tive response elicitor from Erwinia amylovora, regulatesion channel activities in Arabidopsis thaliana suspension cells [J]. FEBS Lett,2001,497(2-3):82-84
24. Tampakaki A P, Panopoulos N J. Elicitation of hypersensitive cell death by extracellularly targeted HrpZPsph produced in planta [J]. Molecular Plant Microbe Interactions,2000,13:1366-1374
25.闻伟刚,王金生.水稻白叶枯病菌harpin基因的克隆与表达[J].植物病理学报,2001,31(4)：295-300
26.赵梅勤,王磊,张兵,等.植物抗病激活蛋白HarpinXooc防治水稻病害的研究[J].中国生物防治.2006,22(4)：283-289
27.李平.水稻黄单胞菌无毒基因avrXa3的克隆与鉴定以及过敏反应激发子Hrf蛋白质遗传多样性和功能域的研究.博士学位论文,南京农业大学,2002
28.王健,赵立平.细菌Ⅲ型分泌系统[J].生命的化学,2001,21(2)：147-149
29. Mukherjee A, Cui Y, Ma W, et al. RpoS (Sigma-S) Controls Expression of rsmA, a Global Regulator of Secondary Metabolites, Harpin, and Extracellular Proteins in Erwinia carotovora[J]. Journal of bacteriology,1998,180:3629-3634
30.赵立平,梁元存,刘爱新,等.表达harpin基因的大肠杆菌DH5(pCPP430)诱导植物抗病性的研究[J].高技术通讯,1997,7(9)：1
31.邵敏,王金生.转hrfAXoo基因水稻对白叶枯病的抗性[J].南京农业大学学报,2004,27(4)：36-40
32. Stroud R M, Walter P. Signal sequence recognition and protein targeting [J]. Current opinion in structural biology,1999,9:754-759
33.李育阳.基因表达技术[M].第一版.北京：科学出版社,2001
34. Ehrlich SD. Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilus. Proc.SciUSA,1997,74:1680-1682
35.皱立扣,王红宁,潘欣.枯草芽抱杆菌整合载体研究进展[J].生物技术通讯,2003,14：525-527
36. Breitling R, Serokin AU, Behnke D. Thermo-inducible gene expression in Bacillus subtilis mediated by the 1857-encoded repressor of bacteriophageλ[J]. Gene,1990, 93:35
37.彭清忠,张惟材,朱厚咄.枯草杆菌表达系统的研究进展[J].生物技术通讯,2001,12：220-225
38.刘白玲,何先祺.枯草杆菌的分子生物学研究[J].成都科技大学学报,1993,72(5)：34-41
39. Sarvas M. Gene expression in recombinant bacillus [J]. bioprocess technol,1995,22: 53
40. Sau-Ching Wu and Sui-Lam Wong. Engineering of a Bacillus snbtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin [J]. Applied and Environmental Microbiology.2002,68(3):1102-1108
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