哈茨木霉SOD基因的克隆及功能研究
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摘要
哈茨木霉(Trichoderma harzianum)是一种优秀的生防真菌,在植物病害,尤其是土壤传染病害的生物防治中有重要的应用价值。哈茨木霉主要通过重寄生作用、溶菌作用、抗生素作用、蛋白酶作用以及对空间和营养的竞争机制达到防治植物病原菌的目的。现已在生产上已被广泛应用于植物病害的生物防治,但由于在各种恶劣的生存条件下哈茨木霉的抑菌功能受到了极大限制和破坏,限制了其优秀生防潜力的进一步发挥。因而,有必要提高哈茨木霉的抗逆性,以保证其生防功能顺利实现。
本文利用二元表达载体pBI121,pCAMBIA1301和克隆载体pUC18,成功构建了用于根癌农杆菌介导的丝状真菌遗传转化表达载体pCA-GSOD,可应用于各种丝状真菌的遗传转化。该载体含有超氧化物岐化酶(SOD)基因,以潮霉素抗性基因为选择标记,目的基因的启动子为CaMV35S,含T-DNA区段。pCA-GSOD表达载体的构建,为提高SOD基因在哈茨木霉中的表达,进而增强哈茨木霉的抗逆性奠定了基础。
本实验应用根癌农杆菌介导的遗传转化系统从根本上解决了传统真菌转化方法所面临的操作繁杂、转化效率低、遗传不稳定等问题。试验中对影响哈茨木霉转化的因素进行优化,获得了该系统转化木霉的最佳条件。经过潮霉素抗性选择培养,Southern杂交及传代分析得到稳定遗传的转化子。
采用与植物病原菌室内拮抗实验,检测了木霉菌各转化子的抑菌活性。结果表明通过根癌农杆菌介导的转化系统所获得转化子保持了木霉菌的抑菌活性,对三种植物病原菌都表现出良好的拮抗作用。在拮抗机制上也同木霉菌原菌一样,主要表现为竞争和重寄生作用。这充分表明,本文的转基因操作没有影响木霉菌的抑菌能力。对木霉原菌和转化子进行高温和盐胁迫实验,结果表明转化子在逆境下仍能正常存活,且在与植物病原菌拮抗实验中仍具备较好的拮抗作用;而木霉原菌在逆境下不能正常存活,在与植物病原菌拮抗实验中丧失其抑菌功能。
SOD基因在哈茨木霉中的表达,提高了哈茨木霉对高温和高盐的抗逆性,保证了哈茨木霉能够在高温和高盐的条件下发挥其优秀的生防功能。从而,为哈茨木霉的抑菌功能在更广泛的领域中应用奠定了基础。
Trichoderma. Harzianum was an antimicrobial strain. It had important applied value in the biocontrol of plant diseases, especially diseases infected through soil. T. harzianum biologically controlled the plant pathogenic fungi by mycoparasitism, bactericidal efficacy, antibiotic effect, protease effect and the competitive mechanism with space and nutrition. It was widely used in biocontrol of plant disease now. But because of various adverse suvivial conditions, the antimicrobial efficacy of T. harzianum was restricted and badly damaged, so it was necessary to enhance the adversity resistance of T. harzianum in order to realize its function of biocontrol.
Express vector pCA-GSOD for filamentous fungi transformation mediated by Agrobacterium tumefaciens was successfully constructed by using binary vector of pBI121,pCAMBIA1301 and clone vector of pUC18. Including the SOD gene, hygromycin resistance gene as selected marker and CaMV35S promoter with section of T-DNA.the vector pCA-GSOD could be used for genetic transformation of mycelial fungus. The construction of vector pCA-GSO, suggests a stratergy for the enhancement of SOD expression in T. harzianum and improvement of T. harzianum’environmental resistance.
In this work, genetic transformation system mediated by A. tumefaciens was used to transformate T. harzianum and may solve the problem of traditional fungus transformation, complex operation, low transformation frequency and genetic unstability of transformants faced by traditional fungus transformation system. The best conditions of this system to transformate T. harzianum were got by analysis every transformation factors. Positive transformant was harvested by the selected method, southern blot analysis and passage culturing.
Antifungal activities of the transformants of T. harzianum was tested by means of dual culture with three kinds of plant pathogens, and the results showed that there was no obvious difference in inhibition activity to these pathogens between original strain of T. harzianum and its transformants obtained by A. tumefaciens. The main antifungal mechanisms of the transformants were competition and parasitism, which were the same to that of wild strain. These results indicated that our transgenic operation didn’t decrease its antifungal ability. Then stress experiment of wild T. harzianum and its transformants was tested. The result indicated that transformants still suvived. Moreover, dual cultured with plant pathogens, the transformants alse suvived well; however, wild T. harzianum could not tolerate the stresses and lack the inhibition activity to these pathogens.
This expression of SOD gene in T. harzianum enhanced the resistance of T. harzianum, ensured its excellent biocontriol function in various adverse conditions so as to widen the application area of T. harzianum.
本文利用二元表达载体pBI121,pCAMBIA1301和克隆载体pUC18,成功构建了用于根癌农杆菌介导的丝状真菌遗传转化表达载体pCA-GSOD,可应用于各种丝状真菌的遗传转化。该载体含有超氧化物岐化酶(SOD)基因,以潮霉素抗性基因为选择标记,目的基因的启动子为CaMV35S,含T-DNA区段。pCA-GSOD表达载体的构建,为提高SOD基因在哈茨木霉中的表达,进而增强哈茨木霉的抗逆性奠定了基础。
本实验应用根癌农杆菌介导的遗传转化系统从根本上解决了传统真菌转化方法所面临的操作繁杂、转化效率低、遗传不稳定等问题。试验中对影响哈茨木霉转化的因素进行优化,获得了该系统转化木霉的最佳条件。经过潮霉素抗性选择培养,Southern杂交及传代分析得到稳定遗传的转化子。
采用与植物病原菌室内拮抗实验,检测了木霉菌各转化子的抑菌活性。结果表明通过根癌农杆菌介导的转化系统所获得转化子保持了木霉菌的抑菌活性,对三种植物病原菌都表现出良好的拮抗作用。在拮抗机制上也同木霉菌原菌一样,主要表现为竞争和重寄生作用。这充分表明,本文的转基因操作没有影响木霉菌的抑菌能力。对木霉原菌和转化子进行高温和盐胁迫实验,结果表明转化子在逆境下仍能正常存活,且在与植物病原菌拮抗实验中仍具备较好的拮抗作用;而木霉原菌在逆境下不能正常存活,在与植物病原菌拮抗实验中丧失其抑菌功能。
SOD基因在哈茨木霉中的表达,提高了哈茨木霉对高温和高盐的抗逆性,保证了哈茨木霉能够在高温和高盐的条件下发挥其优秀的生防功能。从而,为哈茨木霉的抑菌功能在更广泛的领域中应用奠定了基础。
Trichoderma. Harzianum was an antimicrobial strain. It had important applied value in the biocontrol of plant diseases, especially diseases infected through soil. T. harzianum biologically controlled the plant pathogenic fungi by mycoparasitism, bactericidal efficacy, antibiotic effect, protease effect and the competitive mechanism with space and nutrition. It was widely used in biocontrol of plant disease now. But because of various adverse suvivial conditions, the antimicrobial efficacy of T. harzianum was restricted and badly damaged, so it was necessary to enhance the adversity resistance of T. harzianum in order to realize its function of biocontrol.
Express vector pCA-GSOD for filamentous fungi transformation mediated by Agrobacterium tumefaciens was successfully constructed by using binary vector of pBI121,pCAMBIA1301 and clone vector of pUC18. Including the SOD gene, hygromycin resistance gene as selected marker and CaMV35S promoter with section of T-DNA.the vector pCA-GSOD could be used for genetic transformation of mycelial fungus. The construction of vector pCA-GSO, suggests a stratergy for the enhancement of SOD expression in T. harzianum and improvement of T. harzianum’environmental resistance.
In this work, genetic transformation system mediated by A. tumefaciens was used to transformate T. harzianum and may solve the problem of traditional fungus transformation, complex operation, low transformation frequency and genetic unstability of transformants faced by traditional fungus transformation system. The best conditions of this system to transformate T. harzianum were got by analysis every transformation factors. Positive transformant was harvested by the selected method, southern blot analysis and passage culturing.
Antifungal activities of the transformants of T. harzianum was tested by means of dual culture with three kinds of plant pathogens, and the results showed that there was no obvious difference in inhibition activity to these pathogens between original strain of T. harzianum and its transformants obtained by A. tumefaciens. The main antifungal mechanisms of the transformants were competition and parasitism, which were the same to that of wild strain. These results indicated that our transgenic operation didn’t decrease its antifungal ability. Then stress experiment of wild T. harzianum and its transformants was tested. The result indicated that transformants still suvived. Moreover, dual cultured with plant pathogens, the transformants alse suvived well; however, wild T. harzianum could not tolerate the stresses and lack the inhibition activity to these pathogens.
This expression of SOD gene in T. harzianum enhanced the resistance of T. harzianum, ensured its excellent biocontriol function in various adverse conditions so as to widen the application area of T. harzianum.
引文
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3王芊.木霉菌在生物防治上的应用及拮抗机制.黑龙江农业科学. 2001,1:41-43.
4刘丕钢,杨谦.哈茨木霉cDNA文库构建及表达序列标签分析.哈尔滨工业大学学报. 2005,11(2):1503-1504.
5李荣森.我国为生物防治研究与微生物农药产业化的进展.中国病毒学. 2000, 15:1-15
6 Y. Hiei, T. Komari, T. Kubo.Transformation of rice mediateacterium tumefacieos. Plant Mol Bio1.2002, 35:205-218.
7土关林,方宏巧.植物基因工程原理与技术.北京:科学出版.2005,473- 474.
8李卫,郭光沁.根瘤农杆菌介导遗传转化研究的若干新进展.科学通报,2005,45:18-67.
9杨合同,唐文华.木霉菌与植物病害的生物防治.山东科学.2005,9(1):1-2.
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