产酶溶杆菌OH11菌株clp基因的克隆及功能分析
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摘要
产酶溶杆菌OH11是从辣椒根际土壤中分离得到的一种新型生防菌,属于黄单胞科,溶杆菌属。该菌富含G+C,有滑行现象,能产生多种胞外酶,对细菌、真菌、线虫等许多植物病原菌都有很好的生防效果,具有广阔的应用前景。本研究以克隆生防相关基因为目的,建立了产酶溶杆菌OH11菌株的转座子随机插入文库,从突变体文库中筛选拮抗活性丧失的突变株,进行基因克隆并对克隆的基因进行功能分析。
利用mariner转座子对产酶溶杆菌OH11进行转座诱变,成功构建了产酶溶杆菌OH11的突变体文库。以油菜菌核病菌为靶标菌株,通过对峙拮抗法,从2000多株突变株中筛选到一株对油菜菌核病菌拮抗活性完全丧失的突变株3F-11。通过亚克隆的方法,鉴定了mariner转座子在染色体上的插入位点为clp基因。通过对clp基因系统发育树的研究,我们发现,OH11与Xanthomonas属菌株的clp序列同源性达到80%～90%,与Stenotrophomonas属菌株的序列同源性达81%,与同属的菌株Lysobacter enzymogenes strain C3序列同源性比较,同源性高达99%。
利用自杀载体pEX18GM,通过同源重组的方法,构建了clp基因的插入失活突变体(Mclp)。Mclp对油菜菌核病菌的拮抗活性与3F-11一致；通过基因互补,构建了Mclp的互补菌株pMclp, pMclp基本恢复了对油菜菌核病菌的拮抗活性。随后,对clp突变株的表型进行了研究,结果表明clp突变株Mclp在很多表型上发生了改变：(1)突变株Mclp丧失或降低了对病原真菌及卵菌的拮抗能力；(2)Mclp在营养缺陷型培养基(MMX)中不能正常生长；(3) Mclp降低了几丁质酶、蛋白酶和纤维素酶等多种胞外酶的产生量；(4) Mclp增强了生物膜的形成能力；(5)clp基因的突变还影响了菌落形态和菌体的沉降速率性等特性。
Lysobacter enzymogenes strain OH 11 is isolated from pepper rhizosphere soil, which is a new biocontrol agent for plant disease. The genus Lysobacter belongs to the family Xanthomonadaceae characterized by a highG+C content, gliding motility, can produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against various plant pathogens, including bacteria, fungi, and nematodes, having a broad application prospects. In this study, aming at cloning biocontrol-related genes, the mutant library of Lysobacter enzymogenes strain OH 11 was constructed. A mutant losing antimicrobial activity was selected from mutant library. The gene was cloned and the function was analysised.
The mutant library of Lysobacter enzymogenes strain OH11 was successfully constructed by mating mariner transposon into strain OH11. One mutant 3F-11, which lost the antifungal activity against Sclerotinia sclerotiorum was selected from the mutant library. The transposon insertion site was identified as clp gene by sub-clone strategy. clp homology analysis of OH 11 indicated that the homology between OH 11 and Xanthomonas strain was 80% to 90%, reached 99% when compared with Lysobacter enzymogenes strain C3.
A clp disruption mutant Mclp, which had the same phenotype as strain OH11, was constructed by a method of homologous recombination. Complement strain pMclp was constructed, which restored the antifungal ability against Sclerotinia sclerotiorum. Then, other phenotypes of Mclp was studied, the results showed that:(ⅰ) clp mutant strain Mclp lost or reduced the antimicrobial activity against various fungal and oomycetous species; (ⅱ) strain Mclp could not grow normally in the auxotrophic media (MMX); (ⅲ) strain Mclp had decreased the production of multiple extracellular hydrolytic enzymes, such as protease, chitinase and cellulase;(ⅳ) The ability of forming biofilm of strain Mclp was enhanced; (ⅴ) Other features-colony morphology and sedimentation rate of strain Mclp was effected.
利用mariner转座子对产酶溶杆菌OH11进行转座诱变,成功构建了产酶溶杆菌OH11的突变体文库。以油菜菌核病菌为靶标菌株,通过对峙拮抗法,从2000多株突变株中筛选到一株对油菜菌核病菌拮抗活性完全丧失的突变株3F-11。通过亚克隆的方法,鉴定了mariner转座子在染色体上的插入位点为clp基因。通过对clp基因系统发育树的研究,我们发现,OH11与Xanthomonas属菌株的clp序列同源性达到80%～90%,与Stenotrophomonas属菌株的序列同源性达81%,与同属的菌株Lysobacter enzymogenes strain C3序列同源性比较,同源性高达99%。
利用自杀载体pEX18GM,通过同源重组的方法,构建了clp基因的插入失活突变体(Mclp)。Mclp对油菜菌核病菌的拮抗活性与3F-11一致；通过基因互补,构建了Mclp的互补菌株pMclp, pMclp基本恢复了对油菜菌核病菌的拮抗活性。随后,对clp突变株的表型进行了研究,结果表明clp突变株Mclp在很多表型上发生了改变：(1)突变株Mclp丧失或降低了对病原真菌及卵菌的拮抗能力；(2)Mclp在营养缺陷型培养基(MMX)中不能正常生长；(3) Mclp降低了几丁质酶、蛋白酶和纤维素酶等多种胞外酶的产生量；(4) Mclp增强了生物膜的形成能力；(5)clp基因的突变还影响了菌落形态和菌体的沉降速率性等特性。
Lysobacter enzymogenes strain OH 11 is isolated from pepper rhizosphere soil, which is a new biocontrol agent for plant disease. The genus Lysobacter belongs to the family Xanthomonadaceae characterized by a highG+C content, gliding motility, can produces multiple extracellular hydrolytic enzymes and displays antimicrobial activity against various plant pathogens, including bacteria, fungi, and nematodes, having a broad application prospects. In this study, aming at cloning biocontrol-related genes, the mutant library of Lysobacter enzymogenes strain OH 11 was constructed. A mutant losing antimicrobial activity was selected from mutant library. The gene was cloned and the function was analysised.
The mutant library of Lysobacter enzymogenes strain OH11 was successfully constructed by mating mariner transposon into strain OH11. One mutant 3F-11, which lost the antifungal activity against Sclerotinia sclerotiorum was selected from the mutant library. The transposon insertion site was identified as clp gene by sub-clone strategy. clp homology analysis of OH 11 indicated that the homology between OH 11 and Xanthomonas strain was 80% to 90%, reached 99% when compared with Lysobacter enzymogenes strain C3.
A clp disruption mutant Mclp, which had the same phenotype as strain OH11, was constructed by a method of homologous recombination. Complement strain pMclp was constructed, which restored the antifungal ability against Sclerotinia sclerotiorum. Then, other phenotypes of Mclp was studied, the results showed that:(ⅰ) clp mutant strain Mclp lost or reduced the antimicrobial activity against various fungal and oomycetous species; (ⅱ) strain Mclp could not grow normally in the auxotrophic media (MMX); (ⅲ) strain Mclp had decreased the production of multiple extracellular hydrolytic enzymes, such as protease, chitinase and cellulase;(ⅳ) The ability of forming biofilm of strain Mclp was enhanced; (ⅴ) Other features-colony morphology and sedimentation rate of strain Mclp was effected.
引文
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