厚垣普可尼亚菌胞外蛋白酶的诱导及发酵液生化性质的研究
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摘要
为探讨不同培养基对厚垣普可尼亚菌胞外蛋白质的诱导效果及相应发酵液的生化性质,以更好地利用该菌对家畜寄生性线虫进行生物防治,在本实验室前期研究的基础上,设计使用3种常用真菌液体培养基对厚垣普可尼亚菌进行发酵培养,并检测发酵液的蛋白质质量浓度、蛋白酶活性,磷酸酶活性及杀虫效力等,筛选出适合该菌液体培养的最佳培养基。结果表明,在3组培养基中,LMZ培养基组的蛋白质种类最丰富,蛋白质质量浓度(2 320 mg/L±10.00 mg/L)、蛋白酶活性(2.95 U/m L±0.23 U/m L)与磷酸酶活性[1.546 U/100 m L±0.035 U/100 m L(酸性磷酸酶)和0.516 U/100 m L±0.069 U/100 m L(碱性磷酸酶)]都较高,对线虫的杀虫率也是3种培养基中最高的,可达60%,与其他各组间差异极显著(P<0.01)。因此从生物防治的角度,在试验的3种培养基中,以LMZ液体培养基为最佳选择。这一发现可为今后对厚垣普可尼亚菌液体发酵培养、杀虫物质的产生以及作用机制进行深入探究提供重要基础资料。
In order to study the effects of the culture media on the extracellular protein production and partial biochemical properties of the fermentation broth of Pochonia chlamydosporia,three types of media,LMZ,CMA liquid medium(0.4 g/L) and potato dextrose liquid medium,were used to produce the fermentation broth,then the protein mass concentration,enzymatic activity,phosphatase activity and their nematocidal activity of the broths were determined.The results showed that,among the tested media,the biggest protein concentration(2 320 mg/L±10.00 mg/L) was detected in the LMZ broth.The enzymatic activity and phosphatase activity were 2.95 U/m L±0.23 U/m L and 1.546 U/100 m L±0.035 U/100 m L(acid phosphatase activity) and 0.516 U/100 m L±0.069 U/100 m L(alkaline phosphatase activity) in the broth,respectively.Besides,it also had the strongest ability of killing nematodes,with fatality of60%.The difference between every two groups was very significant.In conclusion,the LMZ was the best media among the tested media.The results above provided important information for culturing with liquid media,production of extracellular materials,and biochemical mechanism of the fungus to kill eggs and nematodes of parasites.
In order to study the effects of the culture media on the extracellular protein production and partial biochemical properties of the fermentation broth of Pochonia chlamydosporia,three types of media,LMZ,CMA liquid medium(0.4 g/L) and potato dextrose liquid medium,were used to produce the fermentation broth,then the protein mass concentration,enzymatic activity,phosphatase activity and their nematocidal activity of the broths were determined.The results showed that,among the tested media,the biggest protein concentration(2 320 mg/L±10.00 mg/L) was detected in the LMZ broth.The enzymatic activity and phosphatase activity were 2.95 U/m L±0.23 U/m L and 1.546 U/100 m L±0.035 U/100 m L(acid phosphatase activity) and 0.516 U/100 m L±0.069 U/100 m L(alkaline phosphatase activity) in the broth,respectively.Besides,it also had the strongest ability of killing nematodes,with fatality of60%.The difference between every two groups was very significant.In conclusion,the LMZ was the best media among the tested media.The results above provided important information for culturing with liquid media,production of extracellular materials,and biochemical mechanism of the fungus to kill eggs and nematodes of parasites.
引文
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[9]王瑞,杨连茹,杨晓野,等.少孢节丛孢菌胞外蛋白酶的诱导与生化性质[J].中国兽医科学,2010,40(5):446-451.WANG Rui,YANG Lian-ru,YANG Xiao-ye,et al.Induction and biochemical characteristics of extracellular protease from Arthrobotrys oligospora[J].Chinese Veterinary Science,2010,40(5):446-451.(in Chinese)
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[14]YANG J,TIAN B,LIANG L,et al.Extracellular enzymes and the pathogenesis of nematophagous fungi[J].Applied Microbiology&Biotechnology,2007,75(1):21-31.
[15]MEYER W J,WIEBE M G.Enzyme production by the nematode-trapping fungus,Duddingtonia flagrans[J].Biotechnology Letter,2003,25(10):791-795.
[2]张颖,李国红,张克勤.食线虫真菌资源研究概况[J].菌物学报,2011,30:836-845.ZHANG Ying,LI Guo-hong,ZHANG Ke-qin.A review on the research of nematophagous fungal species[J].Mycosystema,2011,30:836-845.(in Chinese)
[3]PARAUD C,CHARTIER C.Biological control of infective larvae of a gastro-intestinal nematode(Teladorsagia circumcincta)and a small lungworm(Muellerius capillaris)by Duddingtonia flagrans,in goat faeces[J].Parasitology Research,2003,89(2):102-106.
[4]TERRILL T H,LARSEN M,SAMPLES O,et al.Capability of the nematode-trapping fungus Duddingtonia flagrans,to reduce infective larvae of gastrointestinal nematodes in goat feces in the southeastern United States:dose titration and dose time interval studies[J].Veterinary Parasitology,2004,120(4):285-296.
[5]王瑞.少孢节丛孢菌(Arthrobotrys oligospora)捕食线虫生化机制研究[D].呼和浩特:内蒙古农业大学,2008.WANG Rui.Studies of biochemical mechanism of trapping and killing nematodes by Arthrobotrys oligospora[D].Hohhot:Inner Mongolia Agricultural University,2008.(in Chinese)
[6]MANZANILLAL魷PEZ R H,ESTEVES I,FINETTISIALER M M,et al.Pochonia chlamydosporia:Advances and challenges to improve its performance as a biological control agent of sedentary endo-parasitic nematodes[J].Journal of Nematology,2013,45(1):1-7.
[7]王瑞,张博伦,赵晓慧,等.捕食线虫性真菌——Duddingtonia flagrans胞外蛋白质的产生及其杀虫作用研究[J].中国兽医科学,2014,44(4):387-393.WANG Rui,ZHANG Bo-lun,ZHAO Xiao-hui,et al.Extracellular protein production and nematocidal or insecticidal activity of the fermentation broth from Duddingtonia flagrans[J].Chinese Veterinary Science,2014,44(4):387-393.(in Chinese)
[8]王瑞,孙晓燕,马亚囡,等.捕食性真菌——Duddingtonia flagrans胞外蛋白质的生化性质研究[J].中国兽医科学,2015,45(8):776-780.WANG Rui,SUN Xiao-yan,MA Ya-nan,et al.Purification and biochemical properties studies of extracellular proteins from Duddingtonia flagrans[J].Chinese Veterinary Science,2015,45(8):776-780.(in Chinese)
[9]王瑞,杨连茹,杨晓野,等.少孢节丛孢菌胞外蛋白酶的诱导与生化性质[J].中国兽医科学,2010,40(5):446-451.WANG Rui,YANG Lian-ru,YANG Xiao-ye,et al.Induction and biochemical characteristics of extracellular protease from Arthrobotrys oligospora[J].Chinese Veterinary Science,2010,40(5):446-451.(in Chinese)
[10]RUBNER A.Revision of predacious hyphomycetes in the Dactylella-Monacrosporium complex[J].Studies in Mycology,1996,39:1-34.
[11]STADLER M,MAYER A,ANKE H,et al.Fatty acids and other compounds with nematicidal activity from cultures of Basidiomycetes[J].Planta Medica,1994,60(2):128-132.
[12]BRAGA F R,ARAUJO J V,SILVA A R,et al.Predatory activity of the nematophagous fungus Duddingtonia flagrans on horse cyathostomin infective larvae[J].Tropical Animal Health&Production,2010,42(6):1161-1165.
[13]MARTON C O,HIRSCH P R,KERRY B R.Infection of plantparasitic nematodes by nematophagous fungi—A review of the application of molecular biology to understand infection processes and to improve biological control[J].Nematology,2004,6(2):161-170.
[14]YANG J,TIAN B,LIANG L,et al.Extracellular enzymes and the pathogenesis of nematophagous fungi[J].Applied Microbiology&Biotechnology,2007,75(1):21-31.
[15]MEYER W J,WIEBE M G.Enzyme production by the nematode-trapping fungus,Duddingtonia flagrans[J].Biotechnology Letter,2003,25(10):791-795.