苏云金芽孢杆菌中脂肽类抗生素Iturins的研究
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摘要
多种芽孢杆菌尤其是枯草芽孢杆菌(Bacillus subtilis)在生长代谢过程中能够产生不同种类的抗菌物质,包括非核糖体合成的脂肽类抗生素(lipopeptide antibiotics)生物表面活性素(surfactin)、丰原素(fengycin)和伊枯草菌素(iturin)等。其中的iturin家族类抗生素在农业、生物防治、医学等方面得到越来越广泛的应用。本研究尝试利用PCR、真菌抑制实验和HPLC等三种方法来鉴别生产iturin的菌株,以期从与枯草芽孢杆菌亲缘关系相近但目前尚未发现产iturin家族类抗生素的苏云金芽孢杆菌中发现生产iturinA等具有抗菌作用的脂肽类抗生素的菌株。
利用GenBank上已登录的ituD序列设计引物,以本实验室保存的苏云金芽孢杆菌、枯草芽孢杆菌、解淀粉芽孢杆菌和蜡质芽孢杆菌等菌株的总DNA为模板进行PCR扩增,获得了9株PCR阳性菌株。对克隆得到的ituD基因进行测序及Blast序列分析,结果表明,所克隆的基因与GenBank上已登录iturin家族脂肽抗生素的基因具有95%以上的同源性。对这9株菌株的的发酵液经过酸沉淀、甲醇抽提等步骤获得脂肽类粗提物,并进行抑制香蕉炭疽病菌(Colletotrichum musae)、黄瓜枯萎病菌( Fusarium oxysporum)、灰葡萄孢霉(Botrytis cinerea)和水稻纹枯病菌(Rhizoctonia solani)等常见植物病原真菌的测定。结果表明,所提取的脂肽类粗提物中,枯草芽孢杆菌EPL8、解淀粉芽孢杆菌TB2对4种真菌菌丝生长均有较强的抑制作用,枯草芽孢杆菌BS1对Colletotrichum musae和Botrytis cinere菌丝生长有抑制作用,而苏云金芽孢杆菌BRC-XQ16则对Colletotrichum musae菌丝生长具有抑制作用。
将对部分真菌菌丝具有抑制作用的这4株菌的脂肽粗提物纯化后经过反相HPLC分析,结果表明,EPL8和TB2脂肽类提取物中,峰值最高的3个组分与iturinA标准样品有相对应的接近的峰,据此推测EPL8和TB2中的脂肽类提取物中可能存在与iturinA结构类似的组分。BS1的脂肽提取物中峰值最高的组分保留时间几乎与iturinA标准样品中的同样峰值最高的保留时间相一致,据此推测该组分很可能就是iturinA标准样品中含量最高的组分。BRC-XQ16中峰值最高的2个组分保留时间与iturinA标准样品中的前2个组分保留时间相近,同样推测,BRC-XQ16中的这2种组分结构也类似iturinA。
根据实验推测,本实验中的EPL8、TB2、BS1和BRC-XQ16共4株菌株可产生iturinA或者与iturinA结构类似的其他iturin家族成员。这一实验结果为这些菌株中产生的脂肽类抗生素的进一步的结构测定奠定了基础,也为拓宽苏云金芽孢杆菌的应用奠定基础。
Many different Bacillus spp., especially Bacillus subtilis strains often produce a variety of antimicrobial cyclic lipopeptides, including the non-ribosomal biosynthesis of surfactins, fengycins, and iturins. The iturins shows a strong antibiotic activity with a broad antifungal spectrum, making it an ideal potential biological control agent with the aim of reducing the use of chemical pesticides in agriculture and medical science. This study used three methods, such as polymerase chain reaction (PCR) , fungal growth inhibition assay and quantitative high-performance liquid chromatography (HPLC) to identify whether a number of Bacillus strains can produce iturin A. In order to find the iturins in the B. thuringiensis which has closer genetic relationship with B.subtilis but had not been found to produce iturins.
This study made use of the Bacillus thuringiensis, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus to examine the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD amplification. Nine of Bacillus strains exhibited positive results. Blast sequence analysis shows that the PCR products are over 95% homologous to genes of iturins operon. Crude lipopeptides were extracted with methanol from the precipitate which were obtained by adding 6 mol/L HC1 to the cell-free culture broth. The assay for potential production of iturin A was carried out by placing crude lipopeptides of the tested Bacillus strains on a PDA agar plate with Colletotrichum musae, Fusarium oxysporum, Botrytis cinerea and Rhizoctonia solani. The fungal growth inhibition assay exhibited that four Bacillus strains-- Bacillus subtilis EPL8, Bacillus subtilis BS1, Bacillus amyloliquefaciens TB2, Bacillus thuringiensis BRC-XQ16 have inhibitory activity against some important plant pathogenic fungi.
Crude lipopeptides of the four antifungal Bacillus species were purified and then run on lichrosphere C18 column in reverse phase HPLC system. With HPLC method , it was determined that the antifungal lipopeptides was composed of many components , so as the iturinA standard, because it has different homologues . Through the HPLC analysis , it shows that three major components of the lipopeptides extracted from EPL8 and TB2 had similar retention time to three components of the iturinA standard. The results indicates that the three major components of the lipopeptides extracted from EPL8 and TB2 maybe the other iturin family members , such as mycosubtilin and bacillomycin D. The major component of the lipopeptides extracted from BS1 has almost the same retention time as the major component of iturinA standard, and it maybe just the major component of the iturinA standard. Two major components of the lipopeptides extracted from BRC-XQ16 had similar retention time to two components of the iturinA standard. As the analysis above, the results indicated that the two major components of the lipopeptides extracted from BRC-XQ16 maybe the other iturin family members , such as mycosubtilin and bacillomycin D.
Based on the above analysis , the four Bacillus strains , Bacillus subtilis EPL8, Bacillus subtilis BS1, Bacillus amyloliquefaciens TB2, Bacillus thuringiensis BRC-XQ16 could product the iturinA or other iturin family members.The result of this study settle the basis for the further study on the determination of structures of the lipopeptides extracted from EPL8, TB2, BS1 and BRC-XQ16. If the speculation concluded from the HPLC were consistent with the result of the determination of structures,and widen the application of the B. thuringiensis.
利用GenBank上已登录的ituD序列设计引物,以本实验室保存的苏云金芽孢杆菌、枯草芽孢杆菌、解淀粉芽孢杆菌和蜡质芽孢杆菌等菌株的总DNA为模板进行PCR扩增,获得了9株PCR阳性菌株。对克隆得到的ituD基因进行测序及Blast序列分析,结果表明,所克隆的基因与GenBank上已登录iturin家族脂肽抗生素的基因具有95%以上的同源性。对这9株菌株的的发酵液经过酸沉淀、甲醇抽提等步骤获得脂肽类粗提物,并进行抑制香蕉炭疽病菌(Colletotrichum musae)、黄瓜枯萎病菌( Fusarium oxysporum)、灰葡萄孢霉(Botrytis cinerea)和水稻纹枯病菌(Rhizoctonia solani)等常见植物病原真菌的测定。结果表明,所提取的脂肽类粗提物中,枯草芽孢杆菌EPL8、解淀粉芽孢杆菌TB2对4种真菌菌丝生长均有较强的抑制作用,枯草芽孢杆菌BS1对Colletotrichum musae和Botrytis cinere菌丝生长有抑制作用,而苏云金芽孢杆菌BRC-XQ16则对Colletotrichum musae菌丝生长具有抑制作用。
将对部分真菌菌丝具有抑制作用的这4株菌的脂肽粗提物纯化后经过反相HPLC分析,结果表明,EPL8和TB2脂肽类提取物中,峰值最高的3个组分与iturinA标准样品有相对应的接近的峰,据此推测EPL8和TB2中的脂肽类提取物中可能存在与iturinA结构类似的组分。BS1的脂肽提取物中峰值最高的组分保留时间几乎与iturinA标准样品中的同样峰值最高的保留时间相一致,据此推测该组分很可能就是iturinA标准样品中含量最高的组分。BRC-XQ16中峰值最高的2个组分保留时间与iturinA标准样品中的前2个组分保留时间相近,同样推测,BRC-XQ16中的这2种组分结构也类似iturinA。
根据实验推测,本实验中的EPL8、TB2、BS1和BRC-XQ16共4株菌株可产生iturinA或者与iturinA结构类似的其他iturin家族成员。这一实验结果为这些菌株中产生的脂肽类抗生素的进一步的结构测定奠定了基础,也为拓宽苏云金芽孢杆菌的应用奠定基础。
Many different Bacillus spp., especially Bacillus subtilis strains often produce a variety of antimicrobial cyclic lipopeptides, including the non-ribosomal biosynthesis of surfactins, fengycins, and iturins. The iturins shows a strong antibiotic activity with a broad antifungal spectrum, making it an ideal potential biological control agent with the aim of reducing the use of chemical pesticides in agriculture and medical science. This study used three methods, such as polymerase chain reaction (PCR) , fungal growth inhibition assay and quantitative high-performance liquid chromatography (HPLC) to identify whether a number of Bacillus strains can produce iturin A. In order to find the iturins in the B. thuringiensis which has closer genetic relationship with B.subtilis but had not been found to produce iturins.
This study made use of the Bacillus thuringiensis, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus cereus to examine the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD amplification. Nine of Bacillus strains exhibited positive results. Blast sequence analysis shows that the PCR products are over 95% homologous to genes of iturins operon. Crude lipopeptides were extracted with methanol from the precipitate which were obtained by adding 6 mol/L HC1 to the cell-free culture broth. The assay for potential production of iturin A was carried out by placing crude lipopeptides of the tested Bacillus strains on a PDA agar plate with Colletotrichum musae, Fusarium oxysporum, Botrytis cinerea and Rhizoctonia solani. The fungal growth inhibition assay exhibited that four Bacillus strains-- Bacillus subtilis EPL8, Bacillus subtilis BS1, Bacillus amyloliquefaciens TB2, Bacillus thuringiensis BRC-XQ16 have inhibitory activity against some important plant pathogenic fungi.
Crude lipopeptides of the four antifungal Bacillus species were purified and then run on lichrosphere C18 column in reverse phase HPLC system. With HPLC method , it was determined that the antifungal lipopeptides was composed of many components , so as the iturinA standard, because it has different homologues . Through the HPLC analysis , it shows that three major components of the lipopeptides extracted from EPL8 and TB2 had similar retention time to three components of the iturinA standard. The results indicates that the three major components of the lipopeptides extracted from EPL8 and TB2 maybe the other iturin family members , such as mycosubtilin and bacillomycin D. The major component of the lipopeptides extracted from BS1 has almost the same retention time as the major component of iturinA standard, and it maybe just the major component of the iturinA standard. Two major components of the lipopeptides extracted from BRC-XQ16 had similar retention time to two components of the iturinA standard. As the analysis above, the results indicated that the two major components of the lipopeptides extracted from BRC-XQ16 maybe the other iturin family members , such as mycosubtilin and bacillomycin D.
Based on the above analysis , the four Bacillus strains , Bacillus subtilis EPL8, Bacillus subtilis BS1, Bacillus amyloliquefaciens TB2, Bacillus thuringiensis BRC-XQ16 could product the iturinA or other iturin family members.The result of this study settle the basis for the further study on the determination of structures of the lipopeptides extracted from EPL8, TB2, BS1 and BRC-XQ16. If the speculation concluded from the HPLC were consistent with the result of the determination of structures,and widen the application of the B. thuringiensis.
引文
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