玫瑰黄链霉菌Men-myco-93-63 nsdA_(mgh)基因阻断突变株的构建
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摘要
为研究从玫瑰黄链霉菌Men-myco-93-63中克隆到的,与天蓝色链霉菌M 145中的一个重要负调控基因nsd A基因同源的nsdA_(mgh)基因的功能,本文构建了nsd A_(mgh)基因破坏型重组质粒pSRNA2500(pKC1139::1.5 kb nsdA_(mgh)::1.0 kb Km~r),转化ET12567(pUZ8002)获得接合转移供体菌ET12567(pUZ8002,pSRNA2500),通过接合转移将重组质粒导入玫瑰黄链霉菌Men-myco-93-63中。在高温和抗生素双重筛选压力下,筛选得到表型为Am~sKm~r的nsd A_(mgh)基因阻断突变株,通过PCR、Dot bloting和Southern blotting验证了突变株中的nsdA_(mgh)基因已被正确阻断。与出发菌株相比,突变株在摇瓶水平上对棉花黄萎病菌的抑制能力提高了一倍。
Insertional mutagenesis is a widely used method to determine the function(s) of a gene. To study the function(s) of the gene nsdA_(mgh) in Streptomyces roseoflavus, a homologous recombination vector pSRNA2500 was structured in this paper. The recombination donor vector was then transformed into Strempomyces roseoflavus strain Men-myco-93-63 by conjugative transfer. The transformants were subjected to selection under the pressure of high temperature and appropriate antibiotics. As a result, several disrupted mutants of nsdA_(mgh) gene, with a phenotype of Am~sKm~r, were isolated and verified using PCR and Dot-blotting and Southern blotting hybridization methods. Functional analysis showed that the disrupted mutants of nsdA_(mgh) had a two-fold higher inhibition against Verticillium dahlia Kleb than that of the wild strain Men-myco-93-63, which all will provide a new study route for future research about positive and negative regulator in Men-myco-93-63.
Insertional mutagenesis is a widely used method to determine the function(s) of a gene. To study the function(s) of the gene nsdA_(mgh) in Streptomyces roseoflavus, a homologous recombination vector pSRNA2500 was structured in this paper. The recombination donor vector was then transformed into Strempomyces roseoflavus strain Men-myco-93-63 by conjugative transfer. The transformants were subjected to selection under the pressure of high temperature and appropriate antibiotics. As a result, several disrupted mutants of nsdA_(mgh) gene, with a phenotype of Am~sKm~r, were isolated and verified using PCR and Dot-blotting and Southern blotting hybridization methods. Functional analysis showed that the disrupted mutants of nsdA_(mgh) had a two-fold higher inhibition against Verticillium dahlia Kleb than that of the wild strain Men-myco-93-63, which all will provide a new study route for future research about positive and negative regulator in Men-myco-93-63.
引文
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[14]Schauwecker F,Pfennig F,Schro DW.Molecular Cloning of the actinomycinsynthetase gene cluster from Streptomyces chrysomallus and functional heterologous expression of the gene encoding actinomycin synthetase II.J Bacteriol,1998,180(9):2468–2474.
[15]Zhu HJ,Liang YX,Zhou JC,et al.Effects of bkd AB interruption on avermectin Biosynthesis.Chin J Biotech,2004,20(2):269–273(in Chinese).朱浩军,梁运祥,周俊初,等.阿维链霉菌bkd AB的基因中断对阿维菌素合成的影响.生物工程学报,2004,20(2):269–273.
[16]Liu ZY,Guo MJ,Qian JC,et al.Disruption of zwf2gene to improve oxytetraclyline biosynthesis in Streptomyces rimosus M4018.Acta Mocrobiol Sin,2008,48(1):21–25(in Chinese).刘志勇,郭美锦,钱江潮,等.龟裂链霉菌zwf2基因阻断提高土霉素生物合成.微生物学报,2008,48(1):21–25.
[17]Liao YL,Liu ZY,Tang ZY,et al.Effects of zwf2gene knock-in and disruption on oxytetracycline biosynthesis in Streptomyces rimosus M4018.Food Drug,2009,11(1):7–10(in Chinese).廖瑜玲,刘志勇,唐振宇,等.龟裂链霉菌zwf2基因敲入及阻断对土霉素合成的影响.食品与药品,2009,11(1):7–10.
[18]Cui QQ.Construction of genetic engineering Metarhizium anisopliae for Plutella xylostella control[D].Beijing:Chinese Academy of Agricultural Sciences,2013(in Chinese).崔倩倩.防治小菜蛾高效绿僵菌工程菌的构建[D].北京:中国农业科学院,2013.
[19]Gu MZ.Gene knockout of Saccharopolyspora erythraea and function research on SACE_0069[D].Shanghai:East China University of Science and Technology,2013(in Chinese).古明珠.红色糖多孢菌基因敲除和SACE_0069基因功能研究[D].上海:华东理工大学,2013.
[20]Zuo Q,Zhao XQ,Liu HJ,et al.Development of genetically stable recombinant Saccharomyces cerevisiae strains using combinational chromosomal integration.Chin J Biotech,2014,30(4):669-673(in Chinese).左颀,赵心清,刘海军.等.稳定遗传的染色体组合整合酿酒酵母重组菌株的构建.生物工程学报,2014,30(4):669–673.
[21]Liu JL,Zhang XB,Jia SS,et al.Construction of genetic engineering strain of introduction extra nod D into native Rhizobium japonicum.J Nat Sci Heilongjiang Univ,2012,29(1):121–129(in Chinese).刘金玲,张喜波,贾珊珊,等.nod D基因的克隆及其导入大豆根瘤菌工程菌株的构建.黑龙江大学自然科学学报,2012,29(1):121–129.
[2]Takano E,Tao M,Long F,et al.A rare leucine codon in adp A is implicated in the morphological defect of bld A mutants of Streptomyces coelicolor.Mol Microbiol,2006,50:475–486.
[3]Li W,Ying X,Guo Y,et al.Identification of a gene negatively affecting antibiotic production and morphological differentiation in Streptomyces coelicolor A3(2).J Bacteriol,2006,188:8368–8375.
[4]Wang Q.Application of nsd A,a negative regulator of Streptomyces differention in antibiotic breeding[D].Wuhan:Huazhong Agricultural University,2004(in Chinese).王茜.链霉菌分化负调节基因nsd A在抗生素高产育种上的应用[D].武汉:华中农业大学,2004.
[5]Chen F,Xiong W,Min Y,et al.Construction of the conjugal transfer system of Streptomyces cinnamonensis and effect of PCR-mediated nsd A gene disruption on its secondary metabolism.J Agric Biotechnol,2007,15(6):1042–1047(in Chinese).陈芬,熊伟,闵勇,等.肉桂地链霉菌接合转移体系的构建及nsd A基因中断对其次级代谢的影响.农业生物技术学报,2007,15(6):1042–1047.
[6]Liu DQ.Biological control of Streptomyces scabies and other plant pathogens.Minneapolis:The University of Minnesota,1992.
[7]Shen FY,Liu LQ,Wu WG,et al.Cloning and sequence analysis of nsd Amgh gene in Streptomyces roseoflavus Men-myco-93-63.Acta Agric Boreali-Sin,2009,24(5):77–80(in Chinese).沈凤英,刘力强,吴伟刚,等.玫瑰黄链霉菌Men-myco-93-63负调控基因nsd Amgh的克隆及序列分析.华北农学报,2009,24(5):77–80.
[8]Tobias K,Bibb MJ,Mark JB,et al.Practical Streptomyces Genetics.Norwich:The John Innes Foundation,2000.
[9]Sambrook J,Fritsch EF,Maniatis T.Molecular Cloning:A Laboratory Manual.2nd ed.New York:Cold Spring Harbor Laboratory Press,1989.
[10]Chen F.Construction of the conjugal transfer system of Streptomyces cinnamonensis and effect of PCR-mediated nsd A gene disruption on yield of monensin[D].Wuhan:Huazhong Agricultural University,2007(in Chinese).陈芬.肉桂地链霉菌接合转移体系的构建及nsd A基因中断对莫能菌素产量的影响[D].武汉:华中农业大学,2007.
[11]Shen FY,Wu WG,Ji HL,et al.The validation of nsd Amgh gene in Streptomyces roseoflavus Men-myco-93-63.Henan Agric Sci,2010,39(3):59–62(in Chinese).沈凤英,吴伟刚,冀红柳,等.玫瑰黄链霉菌Men-myco-93-63 nsd Amgh基因的验证.河南农业科学,2010,39(3):59–62.
[12]Wang XF,Chen XY,Zhang XM,et al.Molecular characteristics and specific PCR detection of transgenic rice containing Cry1Ab.Hereditas,2012,34(2):208–214(in Chinese).汪小福,陈笑芸,张小明,等.转Cry1Ab基因水稻分子特征及其特异性PCR检测方法.遗传,2012,34(2):208–214.
[13]Shen S,Wang JJ,Tong YM.et al.Cloning and functional analysis of St AC gene in Setosphaeria turcica.Sci Agric Sin,2013,46(5):881–888(in Chinese).申珅,王晶晶,佟亚萌,等.玉米大斑病菌腺苷酸环化酶基因的克隆与功能分析.中国农业科学,2013(5):881–888.
[14]Schauwecker F,Pfennig F,Schro DW.Molecular Cloning of the actinomycinsynthetase gene cluster from Streptomyces chrysomallus and functional heterologous expression of the gene encoding actinomycin synthetase II.J Bacteriol,1998,180(9):2468–2474.
[15]Zhu HJ,Liang YX,Zhou JC,et al.Effects of bkd AB interruption on avermectin Biosynthesis.Chin J Biotech,2004,20(2):269–273(in Chinese).朱浩军,梁运祥,周俊初,等.阿维链霉菌bkd AB的基因中断对阿维菌素合成的影响.生物工程学报,2004,20(2):269–273.
[16]Liu ZY,Guo MJ,Qian JC,et al.Disruption of zwf2gene to improve oxytetraclyline biosynthesis in Streptomyces rimosus M4018.Acta Mocrobiol Sin,2008,48(1):21–25(in Chinese).刘志勇,郭美锦,钱江潮,等.龟裂链霉菌zwf2基因阻断提高土霉素生物合成.微生物学报,2008,48(1):21–25.
[17]Liao YL,Liu ZY,Tang ZY,et al.Effects of zwf2gene knock-in and disruption on oxytetracycline biosynthesis in Streptomyces rimosus M4018.Food Drug,2009,11(1):7–10(in Chinese).廖瑜玲,刘志勇,唐振宇,等.龟裂链霉菌zwf2基因敲入及阻断对土霉素合成的影响.食品与药品,2009,11(1):7–10.
[18]Cui QQ.Construction of genetic engineering Metarhizium anisopliae for Plutella xylostella control[D].Beijing:Chinese Academy of Agricultural Sciences,2013(in Chinese).崔倩倩.防治小菜蛾高效绿僵菌工程菌的构建[D].北京:中国农业科学院,2013.
[19]Gu MZ.Gene knockout of Saccharopolyspora erythraea and function research on SACE_0069[D].Shanghai:East China University of Science and Technology,2013(in Chinese).古明珠.红色糖多孢菌基因敲除和SACE_0069基因功能研究[D].上海:华东理工大学,2013.
[20]Zuo Q,Zhao XQ,Liu HJ,et al.Development of genetically stable recombinant Saccharomyces cerevisiae strains using combinational chromosomal integration.Chin J Biotech,2014,30(4):669-673(in Chinese).左颀,赵心清,刘海军.等.稳定遗传的染色体组合整合酿酒酵母重组菌株的构建.生物工程学报,2014,30(4):669–673.
[21]Liu JL,Zhang XB,Jia SS,et al.Construction of genetic engineering strain of introduction extra nod D into native Rhizobium japonicum.J Nat Sci Heilongjiang Univ,2012,29(1):121–129(in Chinese).刘金玲,张喜波,贾珊珊,等.nod D基因的克隆及其导入大豆根瘤菌工程菌株的构建.黑龙江大学自然科学学报,2012,29(1):121–129.