菌株Snef5杀线虫活性物质分离纯化及相关活性基因簇分析
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摘要
本文主要以形态学鉴定为基础并结合分子生物学手段对具有高杀线虫活性新型菌株Snef5进行了分类地位的确定,同时以南方根结线虫和大豆胞囊线虫等4种线虫为靶标测定了菌株Snef5发酵液的生物活性,并针对几种植物的安全性进行了评价,而后采用层析法、色谱光谱法和核磁共振等方法对菌株Snef5代谢产物中活性物质进行分离纯化和结构解析,并利用SSH方法对相关活性基因簇进行分析,首次报道菌株Snef5对线虫具有生物活性并获得如下创新性研究结果：
1.Snef5的鉴定及杀线虫活性测试：以形态学鉴定为基础并结合菌株的ITS序列、β-微管蛋白序列及18S rDNA序列测定结果,绘制系统发育树亲缘关系,鉴定该菌株为真菌肉座菌目,虫草菌科,Simplicillium chinense。Snef5菌株发酵液对南方根结线虫(Meloidogyne incognita)J2、大豆胞囊线虫(Heterodera glycines)J2、水稻干尖线虫(Aphelenchoides besseyi)和小杆线虫(Caenarhabditis sp.)都具有较高的致死作用,而且具有较高的稀释限点和稳定性。
2. Snef5的安全性测试：真菌菌株Snef5高浓度(稀释1×和5×)发酵液对番茄NewL402种子以及黄瓜绿龙七号种子的发芽情况均有一定程度的抑制作用,但对大豆辽豆15种子发芽抑制作用不明显；而菌株Snef5(稀释1×和5×)发酵液对番茄以及大豆种子的生长情况均无抑制作用,植株也未见药害产生,而当处理黄瓜绿龙七号植株时,其表现出对发酵液中某些活性物质较为敏感的现象。菌株Snef5对黑麦草以及小麦不同品种致病性鉴定结果显示,菌株Snef5对黑麦草以及小麦辽10和小蜜穗等2个品种均无致病性。但菌株Snef5可以侵染并寄生柞蚕茧蛹。
3. Snef5的杀线虫活性物质分离纯化及结构解析：本试验通过对Snef5发酵液粗提液的理化性质试验结果显示,该活性物质是一种强极性弱酸性的水溶性抗生素。同时对菌株发酵液稳定性测定结果表明,杀线虫活性物质是一种耐光照、耐高温、耐酸碱,且可长期贮存的稳定性物质。而后对活性物质进行分离纯化,用乙醇对发酵液中活性物质进行萃取,并经离子交换树脂层析、硅胶薄层层析、硅胶柱层析、Sephadex LH-20凝胶柱层析,最后经HPLC进行活性组分的分离纯化,得到具有杀线虫活性的3个组分,后经光谱和NMR进行结构解析,初步判断发酵液中的三个组分为五环糖组分1、2号,及3号葡萄糖。薄层层析氨基酸预试试验结果表明发酵液中存在氨基成分,因此也可推测3个组分中可能为带氨基结构的五环糖类及氨基葡萄糖,有待进一步验证与研究。
4. Snef5突变株的获得：本试验采用紫外照射、硫酸二乙酯及ATMT等3种诱变方法对菌株Snef5进行诱变育种,筛选其强毒菌株与弱毒菌株。共筛选得到8株强毒菌株,3株弱毒菌株。经连续转代培养5代后,筛选毒力稳定菌株其中强毒菌株UV-17与UV-18共2株,弱毒菌株UV-15和T-25共2株。
5.Snef5杀线虫活性相关基因簇的分析：应用抑制消减杂交法SSH构建了真菌Snef5弱毒菌株Snef5-T-25与强毒菌株Snef5-UV-17的cDNA文库。经Blast比对,检测序列同源性结果表明：多数候选基因功能未知,比对结果中具有功能的EST片段仅有10条。有2条序列分别与来源于绿僵菌的糖类化合物代谢途径及氮、磷及硫代谢途径相关；1条序列与来源于樟子松与异担子菌混合EST的反转录rRNA的同源性高；5条序列与来源于绿僵菌的假设蛋白同源性高；1条序列与来源于长枝木霉菌的假设蛋白同源性高；其中还有1条拼接序列比对结果显示差异片段与来源于绿僵菌的博来霉素水解酶(Bleomycin Hydrolase,BH)具有较高的同源性,因此该差异片段的功能有可能与杀线虫活性相关基因簇具有一定的相关性,具有进一步深入的研究价值。
Snef5isolated in this study was identified as S. chinense based on morphological characteristics and molecular phylogenetic analysis. The filtrate of fermentation fluid of Snef5is tested for nematicidal activities against4targeted nematode species and showed a high level of nematicidal activity. The experiments are studied systematically on bioactive compounds and structural analysis of metabolites by chromatography and nuclear magnetic resonance (NMR) methods, and its safety to plants have been evaluated. SSH was used in research on analysis of bioactive compounds gene cluster of Snef5. It is the first time to report that S. chinense can be used as a biological control agent against plant parasitic nematodes. And there are some innovative results as follow:
1. Identification and the test of bioactivity to nematodes of Snef5:The Snef5was further identified as Simplicillium chinense, by morphological characteristics, sequence determination of ITS, β-tubulin gene and18S rDNA, molecular phylogenetic analysis. The filtrate of fermentation fluid of Snef5showed a high level of nematicidal activity to Meloidogyne incognita J2, Heterodera glycines J2, Aphelenchoides besseyi and Caenarhabditis sp. There are some advantage of Snef5that are high dilution limit points and stability.
2. The evaluation of safety:The high concentration filtrate of fermentation fluid (Dilute with1x and5x) of Snef5showed some inhibition to the seeds of tomato L402and cucumber Lvlong7, but it was not obvious to soybean L15. The cucumber Lvlong7plant treated by Snef5indicated some sensitive of growth. By contrast it is safety to the plant of tomato L402, soybean L15, ryegrass and wheat. But Antheraea pernyi pupaes can be infected by Snef5.
3. Separation, purification and structure determined of nematicidal metabolites of fungus strain Snef5: The nematicidal metabolites of Snef5is a strongly polar and faintly acid soluble components. It possessed light, thermal, acid and base stability. The three of pure nematicidal compound are extracted from fermentation fluid, separated and purified by ion exchange chromatography, TLC, silica column chromatography, Sephadex LH-20chromatography, and HPLC. It showed that among the three compound1,2were furanose amino and3was glucose according to them NMR data and amino acids test. And it need be futher research.
4. The successful construction of the mutant:After many UV, DES and ATMT mutagenetic treatments, and the selections from a lot of mutants. We acquired8high toxic strains and3low toxic strains. Finally high stable high toxic strain UV-17, UV-18and low toxic strains UV-15, T-25have been screened after five generation.
5. SSH is used in research on analysis of bioactive compounds gene cluster of Snef5:Forward and reverse SSH libraries were contructed by using RNA from Snef5-UV-17and Snef5-T-25strains. By comparing sequences in the GenBank, there are10function-known EST, such as two EST with function of nitrogen, phosphate, sulfur metabolism from Metarhizium acridum, one EST with function of transcript antisense to ribosomal RNA from Pinus sylvestris and Heterobasidion annosum mixed EST library, five EST with function of hypothetical protein from Metarhizium acridum, one EST with function of hypothetical protein from Trichoderma longibrachiatum and one EST with function of bleomycin hydrolase from Metarhizium acridum. And it need be futher research.
1.Snef5的鉴定及杀线虫活性测试：以形态学鉴定为基础并结合菌株的ITS序列、β-微管蛋白序列及18S rDNA序列测定结果,绘制系统发育树亲缘关系,鉴定该菌株为真菌肉座菌目,虫草菌科,Simplicillium chinense。Snef5菌株发酵液对南方根结线虫(Meloidogyne incognita)J2、大豆胞囊线虫(Heterodera glycines)J2、水稻干尖线虫(Aphelenchoides besseyi)和小杆线虫(Caenarhabditis sp.)都具有较高的致死作用,而且具有较高的稀释限点和稳定性。
2. Snef5的安全性测试：真菌菌株Snef5高浓度(稀释1×和5×)发酵液对番茄NewL402种子以及黄瓜绿龙七号种子的发芽情况均有一定程度的抑制作用,但对大豆辽豆15种子发芽抑制作用不明显；而菌株Snef5(稀释1×和5×)发酵液对番茄以及大豆种子的生长情况均无抑制作用,植株也未见药害产生,而当处理黄瓜绿龙七号植株时,其表现出对发酵液中某些活性物质较为敏感的现象。菌株Snef5对黑麦草以及小麦不同品种致病性鉴定结果显示,菌株Snef5对黑麦草以及小麦辽10和小蜜穗等2个品种均无致病性。但菌株Snef5可以侵染并寄生柞蚕茧蛹。
3. Snef5的杀线虫活性物质分离纯化及结构解析：本试验通过对Snef5发酵液粗提液的理化性质试验结果显示,该活性物质是一种强极性弱酸性的水溶性抗生素。同时对菌株发酵液稳定性测定结果表明,杀线虫活性物质是一种耐光照、耐高温、耐酸碱,且可长期贮存的稳定性物质。而后对活性物质进行分离纯化,用乙醇对发酵液中活性物质进行萃取,并经离子交换树脂层析、硅胶薄层层析、硅胶柱层析、Sephadex LH-20凝胶柱层析,最后经HPLC进行活性组分的分离纯化,得到具有杀线虫活性的3个组分,后经光谱和NMR进行结构解析,初步判断发酵液中的三个组分为五环糖组分1、2号,及3号葡萄糖。薄层层析氨基酸预试试验结果表明发酵液中存在氨基成分,因此也可推测3个组分中可能为带氨基结构的五环糖类及氨基葡萄糖,有待进一步验证与研究。
4. Snef5突变株的获得：本试验采用紫外照射、硫酸二乙酯及ATMT等3种诱变方法对菌株Snef5进行诱变育种,筛选其强毒菌株与弱毒菌株。共筛选得到8株强毒菌株,3株弱毒菌株。经连续转代培养5代后,筛选毒力稳定菌株其中强毒菌株UV-17与UV-18共2株,弱毒菌株UV-15和T-25共2株。
5.Snef5杀线虫活性相关基因簇的分析：应用抑制消减杂交法SSH构建了真菌Snef5弱毒菌株Snef5-T-25与强毒菌株Snef5-UV-17的cDNA文库。经Blast比对,检测序列同源性结果表明：多数候选基因功能未知,比对结果中具有功能的EST片段仅有10条。有2条序列分别与来源于绿僵菌的糖类化合物代谢途径及氮、磷及硫代谢途径相关；1条序列与来源于樟子松与异担子菌混合EST的反转录rRNA的同源性高；5条序列与来源于绿僵菌的假设蛋白同源性高；1条序列与来源于长枝木霉菌的假设蛋白同源性高；其中还有1条拼接序列比对结果显示差异片段与来源于绿僵菌的博来霉素水解酶(Bleomycin Hydrolase,BH)具有较高的同源性,因此该差异片段的功能有可能与杀线虫活性相关基因簇具有一定的相关性,具有进一步深入的研究价值。
Snef5isolated in this study was identified as S. chinense based on morphological characteristics and molecular phylogenetic analysis. The filtrate of fermentation fluid of Snef5is tested for nematicidal activities against4targeted nematode species and showed a high level of nematicidal activity. The experiments are studied systematically on bioactive compounds and structural analysis of metabolites by chromatography and nuclear magnetic resonance (NMR) methods, and its safety to plants have been evaluated. SSH was used in research on analysis of bioactive compounds gene cluster of Snef5. It is the first time to report that S. chinense can be used as a biological control agent against plant parasitic nematodes. And there are some innovative results as follow:
1. Identification and the test of bioactivity to nematodes of Snef5:The Snef5was further identified as Simplicillium chinense, by morphological characteristics, sequence determination of ITS, β-tubulin gene and18S rDNA, molecular phylogenetic analysis. The filtrate of fermentation fluid of Snef5showed a high level of nematicidal activity to Meloidogyne incognita J2, Heterodera glycines J2, Aphelenchoides besseyi and Caenarhabditis sp. There are some advantage of Snef5that are high dilution limit points and stability.
2. The evaluation of safety:The high concentration filtrate of fermentation fluid (Dilute with1x and5x) of Snef5showed some inhibition to the seeds of tomato L402and cucumber Lvlong7, but it was not obvious to soybean L15. The cucumber Lvlong7plant treated by Snef5indicated some sensitive of growth. By contrast it is safety to the plant of tomato L402, soybean L15, ryegrass and wheat. But Antheraea pernyi pupaes can be infected by Snef5.
3. Separation, purification and structure determined of nematicidal metabolites of fungus strain Snef5: The nematicidal metabolites of Snef5is a strongly polar and faintly acid soluble components. It possessed light, thermal, acid and base stability. The three of pure nematicidal compound are extracted from fermentation fluid, separated and purified by ion exchange chromatography, TLC, silica column chromatography, Sephadex LH-20chromatography, and HPLC. It showed that among the three compound1,2were furanose amino and3was glucose according to them NMR data and amino acids test. And it need be futher research.
4. The successful construction of the mutant:After many UV, DES and ATMT mutagenetic treatments, and the selections from a lot of mutants. We acquired8high toxic strains and3low toxic strains. Finally high stable high toxic strain UV-17, UV-18and low toxic strains UV-15, T-25have been screened after five generation.
5. SSH is used in research on analysis of bioactive compounds gene cluster of Snef5:Forward and reverse SSH libraries were contructed by using RNA from Snef5-UV-17and Snef5-T-25strains. By comparing sequences in the GenBank, there are10function-known EST, such as two EST with function of nitrogen, phosphate, sulfur metabolism from Metarhizium acridum, one EST with function of transcript antisense to ribosomal RNA from Pinus sylvestris and Heterobasidion annosum mixed EST library, five EST with function of hypothetical protein from Metarhizium acridum, one EST with function of hypothetical protein from Trichoderma longibrachiatum and one EST with function of bleomycin hydrolase from Metarhizium acridum. And it need be futher research.
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