吡咯伯克霍尔德氏菌JK-SH007产嗜铁素的相关基因克隆及条件优化
|
摘要
为了明确吡咯伯克霍尔德氏菌Burkholderia Pyrrocinia JK-SH007是否产嗜铁素,揭示其生防机制,本文通过CAS检测该菌株产嗜铁素的能力,并对其嗜铁素合成相关基因cepR进行克隆及序列分析,同时采用单因素试验及响应面法对菌株JK-SH007产嗜铁素条件进行优化。结果表明,菌株JK-SH007具有明显的产嗜铁素能力,其嗜铁素合成相关基因cepR大小为720 bp,与B. pyrrocinia(EU034001)的亲缘关系最近,同源性为99%,两者序列相似性为97%,13个位点出现SNP;另外菌株JK-SH007cepR基因编码的氨基酸序列有3个氨基酸发生了替换,两者一致性为97%。该菌产嗜铁素最优培养时间是15 h、p H 8、转速200r/min、最佳碳源和氮源分别是甘油和氯化铵;PB试验筛选出影响该菌株产嗜铁素的关键性因素分别是p H、加碳量、加氮量;CCD试验结果显示,p H和加氮量的交互作用较明显;最终采用Design-Expert软件分析出菌株JK-SH007产嗜铁素最佳方案为碳源加入量15.00 g/L、氮源加入量10.50 g/L、温度30℃、pH7.36,优化后菌株产嗜铁素能力由18.59提升到37.86,响应面优化产嗜铁素条件的效果是显著的,嗜铁素产量得到明显提高。
Siderophore plays an important role in the plant growth promotion and plant disease control. The effective biocontrol strain Burkholderia pyrrocinia JK-SH007 was isolated from the poplar stem. In order to determine whether JK-SH007 strain can produce siderophore and reveal its biocontrol mechanism, the ability of the strain to produce siderophore was detected by CAS detection, the cep R gene of JK-SH007 was cloned and the sequence was analyzed. On the basis of single factor experiment, response surface method(RSM) was used to optimize the siderophore production conditions of JK-SH007 strain. The results showed that JK-SH007 strain has the capacity to produce siderophore, and the cepR gene size of JK-SH007 was 720 bp. The strain was most closely related to B. pyrrocinia(EU034001), with 99% homology, 97% the sequence similarity was and SNP at 13 sites.Compared with the cepR gene of B. pyrrocinia(EU034001), three amino acids encoded by cep R gene of JK-SH007 were found to be replaced and the consistency was 97%. The experiment of siderophore production showed that the optimal culture time for siderophore producing was 15 h, the pH value of the medium was 8, and the rotating speed was 200 r/min. The optimal carbon and nitrogen sources were glycerin and ammonium chloride.According to PB(Plackett-Burman) experiment, the key factors influencing siderophore producing were p H,carbon and nitrogen. CCD(short for Central Composite Design) experiment showed that the interaction between pH and nitrogen addition was obvious. The optimal solution of siderophore producing for JK-SH007 by Design-Expert was as follows: carbon source at 15.00 g/L, nitrogen source at 10.50 g/L, the culture temperature at30 ℃ and the medium starting pH value of 7.36. After optimization, the siderophore production capacity of JK-SH007 increased from 18.59 to 37.86. The culture conditions obtained by RSM were effective and reliable,and the yield of siderophore improved significantly.
Siderophore plays an important role in the plant growth promotion and plant disease control. The effective biocontrol strain Burkholderia pyrrocinia JK-SH007 was isolated from the poplar stem. In order to determine whether JK-SH007 strain can produce siderophore and reveal its biocontrol mechanism, the ability of the strain to produce siderophore was detected by CAS detection, the cep R gene of JK-SH007 was cloned and the sequence was analyzed. On the basis of single factor experiment, response surface method(RSM) was used to optimize the siderophore production conditions of JK-SH007 strain. The results showed that JK-SH007 strain has the capacity to produce siderophore, and the cepR gene size of JK-SH007 was 720 bp. The strain was most closely related to B. pyrrocinia(EU034001), with 99% homology, 97% the sequence similarity was and SNP at 13 sites.Compared with the cepR gene of B. pyrrocinia(EU034001), three amino acids encoded by cep R gene of JK-SH007 were found to be replaced and the consistency was 97%. The experiment of siderophore production showed that the optimal culture time for siderophore producing was 15 h, the pH value of the medium was 8, and the rotating speed was 200 r/min. The optimal carbon and nitrogen sources were glycerin and ammonium chloride.According to PB(Plackett-Burman) experiment, the key factors influencing siderophore producing were p H,carbon and nitrogen. CCD(short for Central Composite Design) experiment showed that the interaction between pH and nitrogen addition was obvious. The optimal solution of siderophore producing for JK-SH007 by Design-Expert was as follows: carbon source at 15.00 g/L, nitrogen source at 10.50 g/L, the culture temperature at30 ℃ and the medium starting pH value of 7.36. After optimization, the siderophore production capacity of JK-SH007 increased from 18.59 to 37.86. The culture conditions obtained by RSM were effective and reliable,and the yield of siderophore improved significantly.
引文
[1]Tan W Z,Verma V k,Jeong K J,et al.Molecular characterization of vulnibactin biosynthesis in Vibrio vulnificus indicates the existence of an alternative siderophore[J].Frontiers in Microbiology,2014,5(1):1-11.
[2]Kim S M,Park J H,Lee H S,et al.LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment,and its expression is induced by host cells[J].Infection and Immunity,2013,81(10):3721-3730.
[3]Ledala N,Pearson S L,Wilkinson B J,et al.Molecular characterization of the fur protein of Listeria monocytogenes[J].Microbiology,2007,153(4):1103-1111.
[4]Guerinot M L.Microbial iron transport[J].Annual Review of Microbiology,1994,48:743-772.
[5]Saha M,Sarkar S,Sarkar B,et al.Microbial siderophores and their potential applications:a review[J].Environmental Science and Pollution Research,2016,23(5):3984-3999.
[6]Sullivan T S,Ramkissoon S,Garrison V H,et al.Siderophore production of African dust microorganisms over Trinidad and Tobago[J].Aerobiologia,2012,28(3):391-401.
[7]Gamalero E,Glick B R.Bacteria in Agrobiology:Plant Nutrient Managemen[M].Berlin:Springer-Verlag Berlin Heidelberg,2011,17-46.
[8]Rungin S,Indananda C,Suttiviriya P,et al.Plant growth enhancing effects by a siderophore-producing endophytic streptomycete isolated from a Thai jasmine rice plant(Oryza sativa L.cv.KDML105)[J].Antonie Van Leeuwenhoek,2012,102(3):463-472.
[9]Yadav S,Kaushik R,Saxena A K,et al.Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil[J].Journal of Basic Microbiology,2011,51(1):98.
[10]李俊峰.有益菌菌株铁载体的研究和铁吸收调节蛋白(Fur)基因的克隆和表达[D].青岛:中海洋大学,2005.
[11]Parra-Cota F I,Pena-Cabriales J J,de los Santos-Villalobos S,et al.Burkholderia ambifaria and B.caribensis promote growth and increase yield in grain amaranth(Amaranthus cruentus and A.hypochondriacus)by improving plant nitrogen uptake[J].PLo S ONE,2014,9(2):e88094.
[12]Singh R K,Malik N,Singh S.Improved nutrient use efficiency increases plant growth of rice with the use of IAA-overproducing strains of endophytic Burkholderia cepacia strain RRE25[J].Microbial Ecology,2013,66(2):375-384.
[13]Lewenza S,Sokol P A.Regulation of ornibactin biosynthesis and N-acyl-L-homoserine lactone production by CepR in Burkholderia cepacia[J].Bacteriology,2001,183(7):2212-2218.
[14]Pappas K M,Weingart C L,Winans S C.Chemical communication in proteobacteria:biochemical and structural studies of signal synthases and receptors required for intercellular signaling[J].Molecular Microbiology,2004,53(3):755-769.
[15]Huber B,Feldmann F,Kothe M,et al.Identification of a novel virulence factor in Burkholderia cenocepacia H111 required for efficient slow killing of Caenorhabditis elegans[J].Infection and Immunity,2004,72(12):7220-7230.
[16]Weingart C L,White C E,Liu S,et al.Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro[J].Molecular Microbiology,2005,57(2):452-467.
[17]任嘉红,吴小芹,刘辉,等.吡咯伯克霍尔德氏菌JK-SH007抗菌蛋白的分离纯化[J].微生物学通报,2010,37(6):872-880.
[18]任嘉红,李浩,刘辉,等.吡咯伯克霍尔德氏菌JK-SH007对杨树根际微生物数量及功能多样性的影响[J].林业科学,2016,52(5):126-133.
[19]任嘉红,班虎栋,叶建仁,等.吡咯伯克霍尔徳氏菌JK-SH007的发酵条件及其对杨树溃疡病的防治效果[J].中国生物防治,2010,26(3):300-306.
[20]陈佳亮.烟草根际促生菌Sm-1的分离及其外泌铁载体的纯化与鉴定[D].长沙:湖南农业大学,2017.
[21]杨常娥,鲁艳莉,倪捍成,等.创伤弧菌产铁载体菌株的筛选及其诱导条件的响应面优化[J].食品工业科技,2017,38(3):159-165.
[22]Schwyn B,Neilands J B.Universal chemical assay for the detection and determination of siderophorest[J].Analytical Biochemistry,1987,160(1):47-56.
[23]Machuca A,Milagres A M F.Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus[J].Letters in Applied Microbiology,2003,36(3):177-181.
[24]梁建根,郝中娜,王连平,等.嗜铁素功能研究概述[J].中国农学通,2011,27(5):284-287.
[25]Arora N K,Kang S C,Maheshwari D K.Isolation of siderophore-producing strains of Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that causes charcoal rot of groundnut[J].Current Science,2001,81(6):673-677.
[26]Ben J D,Jan W M,Peter A H M,et al.Siderophore-mediated competition for iron and induced resistance in the suppression of Fusarium wilt of carnation by fluorescent Pseudomonas spp.[J].Netherlands Journal of Plant Pathology,1993,99:277-289.
[27]Ran L,Xiang M,Zhou B,et al.Siderophores are the main determinants of fluorescent Pseudomonas in suppression of grey mould in Eucalyptus urophylla[J].Acta Phytopathologica Sinica,2005,35(1):6-12.
[28]Lewenza S,Conway B,Greenberg E P,et al.Quorum sensing in Burkholderia cepacia identification of the luxRI homologs cepRI[J].Bacteriology,1999,181(3):748-756.
[29]Lutter E,Lewenza S,Dennis J J,et al.Distribution of quorum-sensing genes in the Burkholderia cepacia complex[J].Infection and Immunity,2001,69(7):4661-4666.
[30]杨芩,余贤美,贺春萍,等.洋葱伯克霍尔德氏菌CAS19嗜铁素合成相关基因cepR的克隆及生物信息分析[J].基因组学与应用生物学,2010,29(2):245-251.
[31]杨芩,余贤美,贺春萍,等.洋葱伯克霍尔德氏菌CAS19产嗜铁素条件的研究[J].热带作物学报,2010,31(2):265-269.
[32]Parke J L,Gurian-Scherman D.Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains[J].Annual Review of Phytopathology,2001,39:225-258.
[2]Kim S M,Park J H,Lee H S,et al.LuxR homologue SmcR is essential for Vibrio vulnificus pathogenesis and biofilm detachment,and its expression is induced by host cells[J].Infection and Immunity,2013,81(10):3721-3730.
[3]Ledala N,Pearson S L,Wilkinson B J,et al.Molecular characterization of the fur protein of Listeria monocytogenes[J].Microbiology,2007,153(4):1103-1111.
[4]Guerinot M L.Microbial iron transport[J].Annual Review of Microbiology,1994,48:743-772.
[5]Saha M,Sarkar S,Sarkar B,et al.Microbial siderophores and their potential applications:a review[J].Environmental Science and Pollution Research,2016,23(5):3984-3999.
[6]Sullivan T S,Ramkissoon S,Garrison V H,et al.Siderophore production of African dust microorganisms over Trinidad and Tobago[J].Aerobiologia,2012,28(3):391-401.
[7]Gamalero E,Glick B R.Bacteria in Agrobiology:Plant Nutrient Managemen[M].Berlin:Springer-Verlag Berlin Heidelberg,2011,17-46.
[8]Rungin S,Indananda C,Suttiviriya P,et al.Plant growth enhancing effects by a siderophore-producing endophytic streptomycete isolated from a Thai jasmine rice plant(Oryza sativa L.cv.KDML105)[J].Antonie Van Leeuwenhoek,2012,102(3):463-472.
[9]Yadav S,Kaushik R,Saxena A K,et al.Diversity and phylogeny of plant growth-promoting bacilli from moderately acidic soil[J].Journal of Basic Microbiology,2011,51(1):98.
[10]李俊峰.有益菌菌株铁载体的研究和铁吸收调节蛋白(Fur)基因的克隆和表达[D].青岛:中海洋大学,2005.
[11]Parra-Cota F I,Pena-Cabriales J J,de los Santos-Villalobos S,et al.Burkholderia ambifaria and B.caribensis promote growth and increase yield in grain amaranth(Amaranthus cruentus and A.hypochondriacus)by improving plant nitrogen uptake[J].PLo S ONE,2014,9(2):e88094.
[12]Singh R K,Malik N,Singh S.Improved nutrient use efficiency increases plant growth of rice with the use of IAA-overproducing strains of endophytic Burkholderia cepacia strain RRE25[J].Microbial Ecology,2013,66(2):375-384.
[13]Lewenza S,Sokol P A.Regulation of ornibactin biosynthesis and N-acyl-L-homoserine lactone production by CepR in Burkholderia cepacia[J].Bacteriology,2001,183(7):2212-2218.
[14]Pappas K M,Weingart C L,Winans S C.Chemical communication in proteobacteria:biochemical and structural studies of signal synthases and receptors required for intercellular signaling[J].Molecular Microbiology,2004,53(3):755-769.
[15]Huber B,Feldmann F,Kothe M,et al.Identification of a novel virulence factor in Burkholderia cenocepacia H111 required for efficient slow killing of Caenorhabditis elegans[J].Infection and Immunity,2004,72(12):7220-7230.
[16]Weingart C L,White C E,Liu S,et al.Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro[J].Molecular Microbiology,2005,57(2):452-467.
[17]任嘉红,吴小芹,刘辉,等.吡咯伯克霍尔德氏菌JK-SH007抗菌蛋白的分离纯化[J].微生物学通报,2010,37(6):872-880.
[18]任嘉红,李浩,刘辉,等.吡咯伯克霍尔德氏菌JK-SH007对杨树根际微生物数量及功能多样性的影响[J].林业科学,2016,52(5):126-133.
[19]任嘉红,班虎栋,叶建仁,等.吡咯伯克霍尔徳氏菌JK-SH007的发酵条件及其对杨树溃疡病的防治效果[J].中国生物防治,2010,26(3):300-306.
[20]陈佳亮.烟草根际促生菌Sm-1的分离及其外泌铁载体的纯化与鉴定[D].长沙:湖南农业大学,2017.
[21]杨常娥,鲁艳莉,倪捍成,等.创伤弧菌产铁载体菌株的筛选及其诱导条件的响应面优化[J].食品工业科技,2017,38(3):159-165.
[22]Schwyn B,Neilands J B.Universal chemical assay for the detection and determination of siderophorest[J].Analytical Biochemistry,1987,160(1):47-56.
[23]Machuca A,Milagres A M F.Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus[J].Letters in Applied Microbiology,2003,36(3):177-181.
[24]梁建根,郝中娜,王连平,等.嗜铁素功能研究概述[J].中国农学通,2011,27(5):284-287.
[25]Arora N K,Kang S C,Maheshwari D K.Isolation of siderophore-producing strains of Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that causes charcoal rot of groundnut[J].Current Science,2001,81(6):673-677.
[26]Ben J D,Jan W M,Peter A H M,et al.Siderophore-mediated competition for iron and induced resistance in the suppression of Fusarium wilt of carnation by fluorescent Pseudomonas spp.[J].Netherlands Journal of Plant Pathology,1993,99:277-289.
[27]Ran L,Xiang M,Zhou B,et al.Siderophores are the main determinants of fluorescent Pseudomonas in suppression of grey mould in Eucalyptus urophylla[J].Acta Phytopathologica Sinica,2005,35(1):6-12.
[28]Lewenza S,Conway B,Greenberg E P,et al.Quorum sensing in Burkholderia cepacia identification of the luxRI homologs cepRI[J].Bacteriology,1999,181(3):748-756.
[29]Lutter E,Lewenza S,Dennis J J,et al.Distribution of quorum-sensing genes in the Burkholderia cepacia complex[J].Infection and Immunity,2001,69(7):4661-4666.
[30]杨芩,余贤美,贺春萍,等.洋葱伯克霍尔德氏菌CAS19嗜铁素合成相关基因cepR的克隆及生物信息分析[J].基因组学与应用生物学,2010,29(2):245-251.
[31]杨芩,余贤美,贺春萍,等.洋葱伯克霍尔德氏菌CAS19产嗜铁素条件的研究[J].热带作物学报,2010,31(2):265-269.
[32]Parke J L,Gurian-Scherman D.Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains[J].Annual Review of Phytopathology,2001,39:225-258.