DSM–18020菌株expI基因的克隆及生物信息学分析
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摘要
为克隆和研究软腐病菌Dickeya dadantii的信号分子合成酶基因expI,从Dickeya属模式菌DSM–18020的基因组中扩增获得了基因expI。生物信息学分析显示,该基因全长639 bp,与Dickeya dadantii 3937的N–酰基高丝氨酸内酯(N–acyl–homoserine lactone,AHLs)合成酶基因同源性达99%;DSM–18020 expI编码的蛋白质相对分子质量为24 704,理论等电点为5.85,整个肽链中均匀分布疏水氨基酸,且比亲水性氨基酸多,没有信号肽存在;将获得的exp I基因构建到表达载体p ET–28α(+)并转入大肠杆菌BL21(DE3),诱导后的表达产物与理论值一致。
In order to study the signal molecule synthase gene expI of Dickeya dadantii, the gene expI was amplified from the genome of the type strain DSM–18020. Bioinformatics analysis showed that there was a complete open reading frame(ORF) in DSM–18020 expI, which was 639 bp in length and had 99% homology to N–acyl–homoserine lactone(AHLs) synthetase of D. dadantii 3937. The molecular weight of the protein encoded by DSM–18020 expI is 24,704, and the theoretical isoelectric point is 5.85. The hydrophobic amino acids are more abundant than hydrophilic amino acids and distributed evenly throughout the peptide chain, with no signal peptide. expI gene was cloned into the expression vector p ET28α and transformed into Escherichia coli BL21(DE3). The expression product was basically identical with the expectation.
In order to study the signal molecule synthase gene expI of Dickeya dadantii, the gene expI was amplified from the genome of the type strain DSM–18020. Bioinformatics analysis showed that there was a complete open reading frame(ORF) in DSM–18020 expI, which was 639 bp in length and had 99% homology to N–acyl–homoserine lactone(AHLs) synthetase of D. dadantii 3937. The molecular weight of the protein encoded by DSM–18020 expI is 24,704, and the theoretical isoelectric point is 5.85. The hydrophobic amino acids are more abundant than hydrophilic amino acids and distributed evenly throughout the peptide chain, with no signal peptide. expI gene was cloned into the expression vector p ET28α and transformed into Escherichia coli BL21(DE3). The expression product was basically identical with the expectation.
引文
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[16]送顺,金志强.香蕉Ma ERF–1基因的克隆、序列分析及表达载体构建[J].热带作物学报,2017,38(1):76–82.
[17]陈儒钢,文超.辣椒水通道蛋白基因Ca AQP的克隆与序列分析[J].中国农业科学,2010,43(20):4323–4329.
[18]杨成军.甘蓝型油菜CHSA、CHSB基因以及WRKY44同源基因片断的克隆与序列分析[D].重庆:西南农业大学,2004.
[19]王敏敏,赵婕,常亚磊,等.双酶切和无缝克隆方法构建HCVCORE表达载体的比较[J].山西医药杂志,2017,46(3):267–270.
[20]高海有.β–甘露聚糖酶和木聚糖酶基因在大肠杆菌中共表达[J].微生物学通报,2012,39(3):344–352.
[2]黄媛媛,边子睿.胡萝卜软腐欧文氏菌中信号分子合成酶exp I基因的克隆、表达及其分析[J].生物学杂志,2012,29(4):60–64.
[3]ZHOU J N,ZHANG H B,WU J,et al.A novel multidomain polyketide synthase is essential for zeamine antibiotics production and the virulence of Dickeya zeae[J].Molecular Plant Microbe Interactions,2011,24(10):1156–1164.
[4]SLAWIAK M,van BECKHOVEN J R C M,SPEKSNIJDER A G C L,et al.Biochemical and genetical analysis reveal a new clade of biovar 3 Dickeya spp.strains isolated from potato in Europe[J].European Journal of Plant Pathology,2009,125(2):245–261.
[5]TOTH I K,van der WOLF J M,SADDLER G,et al.Dickeya species:an emerging problem for potato production in Europe[J].Plant Pathology,2011,60(3):385–399.
[6]SAMSON R,LEGENDRE J B,CHRISTEN R,et al.Transfer of Pectobecterium chrysanthemi(Burkholder et al)Brenner et al.1973 and Brenneria paradisiaca to the genus Dickeya gen.nov.as Dickeya chrysanthemi comb.nov.and Dickeya paradisiaca comb.nov.and delineation of four novel species,Dickeya dadantii sp.Nov.,Dickeya dianthicola sp.Nov.,Dickeya dieffenbachiae sp.nov.and Dickeya zeae sp.nov[J].International Journal of Systematic and Evolutionary Microbiology,2005,55:1415–1427.
[7]MA B,HIBBING M E,KIM H S,et al.Host range and molecular phylogenies of the soft rot enterobacterial genera Pectobacterium and Dickeya[J].Phytopathology,2007,97(9):1150–1163.
[8]GRENIER A,DUPORT G,PAGE S,et al.The phytopathogen dickeya dadantii(Erwinia chrysanthemi3937)is a pathogen of the pea aphid[J].Applied and Environmental Microbiology,2006,72(3):1956–1965.
[9]罗利龙,娄瑞娟,邱健,等.细菌群体感应及其干扰策略的研究进展[J].生物学杂志,2010,27(6):79–82.
[10]陈峰.微生物的群体感应信号分子[J].上海农业学报,2005,21(1):98–102.
[11]HUSSAIN M B B M,ZHANG H B,XU J L,et al.The acyl–homoserine lactone–type quorum–sensing system modulates cell motility and virulence of Erwinia chrysanthemi pv.zeae[J].Journal of Bacteriology,2008,190(3):1045–1053.
[12]宋水山.N–酰基高丝氨酸内醋介导的细菌与其真核寄主之间的信息交流[J].中国细胞生物学学报,2010,32(2):331–335.
[13]HUSSAIN M B B M,ZHANG H B,XU J L,et al.Integeration of the quorum–sensing system in the regulatary networks controlling virulence factor synthesis in Erwinia chrysanthemi[J].Molecular Microbiology,1998,29(6):1407–1418.
[14]Sambrook J,Russell D W.Molecular Cloning:A Laboratory Mannual[M].3rd.New York:Cold Spring Harbor Laboratory Press,2001.
[15]ZHANG J X,LIN B R,SHEN H F,et al.Genome sequence of the banana pathogen Dickeya zeae strain MS1,which causes bacteria soft rot[J].Genome Announcements,2013,1(3):e00317–13.
[16]送顺,金志强.香蕉Ma ERF–1基因的克隆、序列分析及表达载体构建[J].热带作物学报,2017,38(1):76–82.
[17]陈儒钢,文超.辣椒水通道蛋白基因Ca AQP的克隆与序列分析[J].中国农业科学,2010,43(20):4323–4329.
[18]杨成军.甘蓝型油菜CHSA、CHSB基因以及WRKY44同源基因片断的克隆与序列分析[D].重庆:西南农业大学,2004.
[19]王敏敏,赵婕,常亚磊,等.双酶切和无缝克隆方法构建HCVCORE表达载体的比较[J].山西医药杂志,2017,46(3):267–270.
[20]高海有.β–甘露聚糖酶和木聚糖酶基因在大肠杆菌中共表达[J].微生物学通报,2012,39(3):344–352.