少孢节丛孢菌Aoz1基因的生物信息学分析及原核表达条件的优化
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摘要
为获悉少孢节丛孢菌内蒙古株Aoz1基因的详细信息,以研究捕食线虫性真菌对线虫的作用机制,本试验以捕食线虫性真菌的代表种——少孢节丛孢菌内蒙古株为研究对象,首先扩增其胞外丝氨酸蛋白酶的基因编码序列,并进行生物信息学分析,然后将其构建至原核表达载体pET-32a中,再转化至Transetta(DE3)表达感受态细胞中,进行表达条件的优化以及表达产物的纯化。结果表明,少孢节丛孢菌内蒙古株丝氨酸蛋白酶Aoz1基因与已发表的丝氨酸蛋白酶Aoz1基因的同源性为99%,其蛋白具备螺旋结构;在原核系统中进行表达,其最适IPTG诱导浓度为1.5 mmol/L,最佳诱导时间为8 h,确定了重组蛋白的最佳表达条件,并获得了重组蛋白。对该酶基因的分析,为后续有关蛋白酶和捕食线虫性真菌杀灭寄生性线虫机理方面的研究提供了参考资料。
In order to ascertain the information of the serine protease Aoz1 gene,and to study the nematode-trapping mechanism of the fungi,Arthrobotrys oligospora CHIM was selected and the serine protease Aoz1 gene was amplified and analyzed by bioinformatic software.Then the gene was connected with the prokaryotic expression vector pET32 a,and transformed into Transetta(DE3),followed by optimizing the expression conditions and purifying the expression product.The results showed that the serine protease Aoz1 gene of Arthrobotrys oligospora CHIM was 99% homologous to the published serine protease Aoz1 gene.The protein simulated by the biological software had a helical structure and was expressed successfully in the prokaryotic system.The optimal expression condi-tions of the recombinant protein were determined,such as the optimal IPTG induction concentration was 1.5 mmol/L and the optimal induction time was 8 h,and the recombinant protein was finally obtained.The bioinformatic analysis on serine protease Aoz1 gene,would be provided reference materials for the subsequent research on the protease and nematode-killing mechanism of the fungi.
In order to ascertain the information of the serine protease Aoz1 gene,and to study the nematode-trapping mechanism of the fungi,Arthrobotrys oligospora CHIM was selected and the serine protease Aoz1 gene was amplified and analyzed by bioinformatic software.Then the gene was connected with the prokaryotic expression vector pET32 a,and transformed into Transetta(DE3),followed by optimizing the expression conditions and purifying the expression product.The results showed that the serine protease Aoz1 gene of Arthrobotrys oligospora CHIM was 99% homologous to the published serine protease Aoz1 gene.The protein simulated by the biological software had a helical structure and was expressed successfully in the prokaryotic system.The optimal expression condi-tions of the recombinant protein were determined,such as the optimal IPTG induction concentration was 1.5 mmol/L and the optimal induction time was 8 h,and the recombinant protein was finally obtained.The bioinformatic analysis on serine protease Aoz1 gene,would be provided reference materials for the subsequent research on the protease and nematode-killing mechanism of the fungi.
引文
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[2]杨晓野,吴彩艳,杨莲茹,等.口服少孢节丛孢菌孢子对家畜粪便中线虫幼虫的杀灭研究[J].畜牧兽医学报,2005,36(9):927-930.YANG Xiao-ye,WU Cai-yan,YANG Lian-ru,et al.Study of conidia of Arthrobotrys oligospora administrated orally on killing the nematode larvae in faeces of domestic animals[J].Acta Veterinaria et Zootechnica Sinica,2005,36(9):927-930.(in Chinese)
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[6]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 nemaotodes by Arthrobotrys oligospora[D].Hohhot:Inner Mongolia Agricultural University,2008.(in Chinese)
[7]王瑞,杨莲茹,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)
[8]王瑞,杨晓野,杨莲茹,等.不同碳氮源及其比例和培养基初始pH值对少孢节丛孢菌菌丝生长的影响[J].黑龙江畜牧兽医,2008(9):11-14.WANG Rui,YANG Xiao-ye,YANG Lian-ru,et al.Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio and initial pH of medium on the growth of Arthrobotrys oligospora[J].Heilongjiang Animal Science and Veterinary Medicine,2008(9):11-14.(in Chinese)
[9]王俊伟.少孢节丛孢菌侵染性胞外丝氨酸蛋白酶的基因克隆、表达及其杀线虫活性研究[D].石河子:石河子大学,2013.WANG Jun-wei.Gene cloning,expression and analysis of nematicidal activity of infectious extracellular serine protease of Arthrobotrys oligospora[D].Shihezi:Shihezi University,2013.(in Chinese)
[10]汪世华,王文勇,黄益洲,等.丝氨酸蛋白酶研究进展[J].福建农业学报,2007,22(4):453-456.WANG Shi-hua,WANG Wen-yong,HUANG Yi-zhou,et al.On serine protease[J].Fujian Journal of Agricultural Sciences,2007,22(4):453-456.(in Chinese)
[11]赵海龙,孟庆玲,乔军,等.食线虫真菌侵染性胞外酶研究进展[J].家畜生态学报,2017,38(8):81-88.ZHAO Hai-long,MENG Qing-ling,QIAO Jun,et al.Research progress on infection extracellular enzymes from Nematophagous fungi[J].Acta Ecologiae Animalis Domastici,2017,38(8):81-88.(in Chinese)
[12]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.
[13]杨晓野,杨莲茹,刘珍莲,等.捕食线虫性真菌-少孢节丛孢菌体外杀寄生性线虫幼虫的研究[J].畜牧兽医学报,2004,35(3):334-338.YANG Xiao-ye,YANG Lian-ru,LIU Zhen-lian,et al.Study on the killing role of nematode-trapping fungus-Arthrobotrys oligospora to parasitic nematode larvae in vitro[J].Acta Veterinaria et Zootechnica Sinica,2004,35(3):334-338.(in Chinese)
[14]YANG J,HUANG X,TIAN B,et al.Isolation and characterization of a serine protease from the nematophagous fungus,Lecanicillium psalliotae,displaying nematicidal activity[J].Biotechnology Letters,2005,27(15):1123-1128.
[15]李军燕.Duddingtonia flagrans生长特性和捕食线虫活性物质的研究[D].呼和浩特:内蒙古农业大学,2014.LI Jun-yan.Growth characteristics and nematode-trapping substances of Duddingtonia flagrans[D].Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)
[16]赵海龙,孟庆玲,乔军,等.少孢节丛孢菌胞外丝氨酸蛋白酶P186基因的克隆、序列分析及表达[J].华北农学报,2014,29(4):93-97.ZHAO Hai-long,MENG Qing-ling,QIAO Jun,et al.Cloning and prokaryotic expression of serine protease P186 gene of Arthrobotrys oligospora[J].Acta Agriculturae Boreali-Sinica,2014,29(4):93-97.(in Chinese)
[2]杨晓野,吴彩艳,杨莲茹,等.口服少孢节丛孢菌孢子对家畜粪便中线虫幼虫的杀灭研究[J].畜牧兽医学报,2005,36(9):927-930.YANG Xiao-ye,WU Cai-yan,YANG Lian-ru,et al.Study of conidia of Arthrobotrys oligospora administrated orally on killing the nematode larvae in faeces of domestic animals[J].Acta Veterinaria et Zootechnica Sinica,2005,36(9):927-930.(in Chinese)
[3]BARRON G L.Isolation and maintenance of endoparasitic nematophagous hyphomycetes[J].Canadian Journal Botany,1969,47(12):1899-1902.
[4]TUNLID A,JANSSON S.Proteases and their involvement in the infection and immobilization of nematodes by the nematophagous fungus Arthrobotrys oligospora[J].Applied and Environment Microbiology,1991,57(10):2868-2872.
[5]TUNLID A,ROSEN S,EK B,et al.Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora[J].Microbiology,1994,140(7):1687-1695.
[6]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 nemaotodes by Arthrobotrys oligospora[D].Hohhot:Inner Mongolia Agricultural University,2008.(in Chinese)
[7]王瑞,杨莲茹,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)
[8]王瑞,杨晓野,杨莲茹,等.不同碳氮源及其比例和培养基初始pH值对少孢节丛孢菌菌丝生长的影响[J].黑龙江畜牧兽医,2008(9):11-14.WANG Rui,YANG Xiao-ye,YANG Lian-ru,et al.Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio and initial pH of medium on the growth of Arthrobotrys oligospora[J].Heilongjiang Animal Science and Veterinary Medicine,2008(9):11-14.(in Chinese)
[9]王俊伟.少孢节丛孢菌侵染性胞外丝氨酸蛋白酶的基因克隆、表达及其杀线虫活性研究[D].石河子:石河子大学,2013.WANG Jun-wei.Gene cloning,expression and analysis of nematicidal activity of infectious extracellular serine protease of Arthrobotrys oligospora[D].Shihezi:Shihezi University,2013.(in Chinese)
[10]汪世华,王文勇,黄益洲,等.丝氨酸蛋白酶研究进展[J].福建农业学报,2007,22(4):453-456.WANG Shi-hua,WANG Wen-yong,HUANG Yi-zhou,et al.On serine protease[J].Fujian Journal of Agricultural Sciences,2007,22(4):453-456.(in Chinese)
[11]赵海龙,孟庆玲,乔军,等.食线虫真菌侵染性胞外酶研究进展[J].家畜生态学报,2017,38(8):81-88.ZHAO Hai-long,MENG Qing-ling,QIAO Jun,et al.Research progress on infection extracellular enzymes from Nematophagous fungi[J].Acta Ecologiae Animalis Domastici,2017,38(8):81-88.(in Chinese)
[12]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.
[13]杨晓野,杨莲茹,刘珍莲,等.捕食线虫性真菌-少孢节丛孢菌体外杀寄生性线虫幼虫的研究[J].畜牧兽医学报,2004,35(3):334-338.YANG Xiao-ye,YANG Lian-ru,LIU Zhen-lian,et al.Study on the killing role of nematode-trapping fungus-Arthrobotrys oligospora to parasitic nematode larvae in vitro[J].Acta Veterinaria et Zootechnica Sinica,2004,35(3):334-338.(in Chinese)
[14]YANG J,HUANG X,TIAN B,et al.Isolation and characterization of a serine protease from the nematophagous fungus,Lecanicillium psalliotae,displaying nematicidal activity[J].Biotechnology Letters,2005,27(15):1123-1128.
[15]李军燕.Duddingtonia flagrans生长特性和捕食线虫活性物质的研究[D].呼和浩特:内蒙古农业大学,2014.LI Jun-yan.Growth characteristics and nematode-trapping substances of Duddingtonia flagrans[D].Hohhot:Inner Mongolia Agricultural University,2014.(in Chinese)
[16]赵海龙,孟庆玲,乔军,等.少孢节丛孢菌胞外丝氨酸蛋白酶P186基因的克隆、序列分析及表达[J].华北农学报,2014,29(4):93-97.ZHAO Hai-long,MENG Qing-ling,QIAO Jun,et al.Cloning and prokaryotic expression of serine protease P186 gene of Arthrobotrys oligospora[J].Acta Agriculturae Boreali-Sinica,2014,29(4):93-97.(in Chinese)