应用原生质体技术改良秦岭链霉菌的研究
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摘要
秦岭链霉菌(Streptomyces qinlingensis sp.nov.)是西北农林科技大学农药研究所从陕西秦岭山区土壤中分离到的一新种放线菌,其发酵产物对多种植物病原真菌和细菌具有强烈的抑制作用。秦岭链霉菌发酵产物中含有多种抑菌活性成分,其中包括10种链丝菌素类抗生素。为了提高其发酵液的生物活性和有效成分含量,本试验以原生质体为材料对该秦岭链霉菌进行菌株改良,主要研究结果如下:
1.应用原生质体再生并结合物理化学诱变得到了抗菌活性提高较大、稳定性较好的秦岭链霉菌的诱变菌株。生测结果表明:秦岭链霉菌原生质体再生菌株R-72、亚硝基胍诱变菌株NTG-1和热诱变菌株H30-7对枯草芽孢杆菌、蜡状芽孢杆菌、金黄葡萄球菌、铜绿假单胞菌和大肠杆菌等5种病原细菌和小麦赤霉病菌、苹果炭疽病菌、番茄灰霉病菌、棉枯萎病菌和玉米弯孢病菌等5种植物病原真菌的抗菌活性比出发菌株均有显著提高,并且连续培养10代,其遗传性状均比较稳定。其中,菌株H30-7对上述5种植物病原真菌的毒力是出发菌株的3.16、1.39、1.16、1.87和1.34倍。经邻苯二甲醛(OPA)柱前衍生化HPLC分析,再生及诱变菌株R-72、NTG-1和H30-7的链丝菌素F的含量分别为0.42mg/mL、0.66 mg/mL和0.76 mg/mL,但是与出发菌株的链丝菌素F含量(0.82 mg/mL)相比均有所降低。
2.将秦岭链霉菌原生质体与链霉素产生菌Str2的原生质体进行融合,得到一株抗菌活性有较大提高且遗传稳定性良好的菌株F-47,其培养特征与双亲株均有所差异。抗菌活性测定结果表明:菌株F-47对5种病原细菌和5种植物病原真菌的抗菌活性与出发菌株秦岭链霉菌相比均有明显提高。但是HPLC检测结果表明F-47菌株发酵产物的链丝菌素F含量仅为0.19 mg/mL,远远低于出发菌株秦岭链霉菌中的含量。融合菌株的链丝菌素F的含量大幅度下降而抗菌活性却提高,这可能是由于原生质体融合使融合菌株发酵产物中各种链丝菌素含量比例发生了改变,亦或产生了新的高活性的抗菌物质。
Streptomyces qinlingensis sp.nov., an agricultural antibiotics producer, was isolated from a soil sample collected in Qinling mountains, Shaanxi Province, and identified as a new strain. Its fermentation broth exhibited potent antifungal and antibacterial activities. There are many kinds of bioactive compounds including streptothricins antibiotics with antifungal and antibacterial activities. To improve the antimicrobial activities and antibiotics production of S. qinlingensis, protoplast mutagenesis and protoplast fusion were applied in this study. The main results were as follows.
The first method is protoplast regeneration combined with physical and chemical mutagenesis to select high-yielding strains. The results of bioassay showed that the antibacterial activities against Bacillus subtilis, Bacillus cereus, Staphylicoccus aureus, Pseudomonas aeruginosa and Escherichia coli and antifungal activities against plant pathogenic fungi Gibberella zeae, Colletotrichum gloesporioides, Botrytis cinerea, Fusarium oxysporum and Curvularia lunata of the fermentation broth from 3 strains, R-72 from protoplast regeneration, NTG-1 and H30-7 from NTG and heat mutagenesis of protoplast, respectively, were remarkable increased comparing with that of the parent strain, and the heredity characters of those strains were stable in successive ten generations. Of these, the toxicities of H30-7 against 5 species plant pathogenic fungi increased 3.16, 1.39, 1.16, 1.87, and 1.34 times compared to that of the starting strain. However, the results of HPLC detection revealed that the contents of streptothricin F of those 3 strains were 0.42 mg/mL、0.66 mg/mL and 0.76 mg/mL, respectively, which lower than that of parent strain, 0.82 mg/mL.
The second method is the protoplast fusion between S. qinlingensis and Streptomycin producer Str2 because streptothricin and sreptomycin are both belong to aminoglycoside antibiotics. A fusant named F-47, with potent antifungal and antibacterial activities and stable heredity character, was screened from the fusants. Moreover, the culturing characteristics of F-47 have some clear differences from its parental strains. But, the result of HPLC detection showed that the content of streptothricin F dropped a lot from 0.82 mg/mL in the original strain S. qinlingensis to 0.19 mg/mL in the fusant F-47. This is possible because that there may be another antifungal agents are generated or contents of streptothricins are changed in the fermentation broth from fuant F-47 due to the effect of protoplast fusion.
1.应用原生质体再生并结合物理化学诱变得到了抗菌活性提高较大、稳定性较好的秦岭链霉菌的诱变菌株。生测结果表明:秦岭链霉菌原生质体再生菌株R-72、亚硝基胍诱变菌株NTG-1和热诱变菌株H30-7对枯草芽孢杆菌、蜡状芽孢杆菌、金黄葡萄球菌、铜绿假单胞菌和大肠杆菌等5种病原细菌和小麦赤霉病菌、苹果炭疽病菌、番茄灰霉病菌、棉枯萎病菌和玉米弯孢病菌等5种植物病原真菌的抗菌活性比出发菌株均有显著提高,并且连续培养10代,其遗传性状均比较稳定。其中,菌株H30-7对上述5种植物病原真菌的毒力是出发菌株的3.16、1.39、1.16、1.87和1.34倍。经邻苯二甲醛(OPA)柱前衍生化HPLC分析,再生及诱变菌株R-72、NTG-1和H30-7的链丝菌素F的含量分别为0.42mg/mL、0.66 mg/mL和0.76 mg/mL,但是与出发菌株的链丝菌素F含量(0.82 mg/mL)相比均有所降低。
2.将秦岭链霉菌原生质体与链霉素产生菌Str2的原生质体进行融合,得到一株抗菌活性有较大提高且遗传稳定性良好的菌株F-47,其培养特征与双亲株均有所差异。抗菌活性测定结果表明:菌株F-47对5种病原细菌和5种植物病原真菌的抗菌活性与出发菌株秦岭链霉菌相比均有明显提高。但是HPLC检测结果表明F-47菌株发酵产物的链丝菌素F含量仅为0.19 mg/mL,远远低于出发菌株秦岭链霉菌中的含量。融合菌株的链丝菌素F的含量大幅度下降而抗菌活性却提高,这可能是由于原生质体融合使融合菌株发酵产物中各种链丝菌素含量比例发生了改变,亦或产生了新的高活性的抗菌物质。
Streptomyces qinlingensis sp.nov., an agricultural antibiotics producer, was isolated from a soil sample collected in Qinling mountains, Shaanxi Province, and identified as a new strain. Its fermentation broth exhibited potent antifungal and antibacterial activities. There are many kinds of bioactive compounds including streptothricins antibiotics with antifungal and antibacterial activities. To improve the antimicrobial activities and antibiotics production of S. qinlingensis, protoplast mutagenesis and protoplast fusion were applied in this study. The main results were as follows.
The first method is protoplast regeneration combined with physical and chemical mutagenesis to select high-yielding strains. The results of bioassay showed that the antibacterial activities against Bacillus subtilis, Bacillus cereus, Staphylicoccus aureus, Pseudomonas aeruginosa and Escherichia coli and antifungal activities against plant pathogenic fungi Gibberella zeae, Colletotrichum gloesporioides, Botrytis cinerea, Fusarium oxysporum and Curvularia lunata of the fermentation broth from 3 strains, R-72 from protoplast regeneration, NTG-1 and H30-7 from NTG and heat mutagenesis of protoplast, respectively, were remarkable increased comparing with that of the parent strain, and the heredity characters of those strains were stable in successive ten generations. Of these, the toxicities of H30-7 against 5 species plant pathogenic fungi increased 3.16, 1.39, 1.16, 1.87, and 1.34 times compared to that of the starting strain. However, the results of HPLC detection revealed that the contents of streptothricin F of those 3 strains were 0.42 mg/mL、0.66 mg/mL and 0.76 mg/mL, respectively, which lower than that of parent strain, 0.82 mg/mL.
The second method is the protoplast fusion between S. qinlingensis and Streptomycin producer Str2 because streptothricin and sreptomycin are both belong to aminoglycoside antibiotics. A fusant named F-47, with potent antifungal and antibacterial activities and stable heredity character, was screened from the fusants. Moreover, the culturing characteristics of F-47 have some clear differences from its parental strains. But, the result of HPLC detection showed that the content of streptothricin F dropped a lot from 0.82 mg/mL in the original strain S. qinlingensis to 0.19 mg/mL in the fusant F-47. This is possible because that there may be another antifungal agents are generated or contents of streptothricins are changed in the fermentation broth from fuant F-47 due to the effect of protoplast fusion.
引文
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