苏云金芽孢杆菌几丁质酶基因chiA在枯草芽孢杆菌中的分泌表达Secretory Expression of Chitinase Gene chiA from Bacillus thuringiensis in Bacillus subtilis
宋光明;沈微;孙金凤;饶志明;方慧英;诸葛健;诸葛斌;
摘要(Abstract):
[目的]研究来源于苏云金芽孢杆菌的几丁质酶基因chiA在枯草芽孢杆菌中的表达情况。[方法]以苏云金芽孢杆菌染色体DNA为模板,PCR扩增获取几丁质酶基因chiA,将其与枯草芽孢杆菌表达载体pHSG连接,构建重组菌,经IPTG诱导后,检测培养液中的几丁质酶活性。[结果]扩增得到几丁质酶基因chiA大小为2.5 kb。构建的重组菌对底物[4-MU-(GlcNAc)3]显示出一定的水解活性,培养液酶活约为2.8 U/ml,而pHSG空质粒转化子在同样条件下其培养液没有明显酶活。该重组酶的最适pH值为6.5,最适反应温度为50℃,与苏云金芽孢杆菌自身产生的几丁质酶性质一致。[结论]几丁质酶基因chiA能在枯草芽孢杆菌中成功表达,表达产物可成功分泌到细胞外。
关键词(KeyWords): 苏云金芽孢杆菌;几丁质酶基因;枯草芽孢杆菌;分泌表达
基金项目(Foundation): 国家高技术研究发展计划(863计划,2006AA10Z307)资助项目
作者(Author): 宋光明;沈微;孙金凤;饶志明;方慧英;诸葛健;诸葛斌;
Email:
DOI: 10.13989/j.cnki.0517-6611.2008.26.057
参考文献(References):
- [1]GOMES R C,SEMEDO L T,SOARES R M,et al.Chitinolytic activity ofactinomycetes froma cerradosoil and theirpotential inbiocontrol[J].Let-ters in Applied Microbiology,2000,30(2):146-150.
- [2]SOUZARF,GOMESRC,COELHORRR,etal.Purification and charac-terization of an endochitinase produced byColletrichum gloeosporioides[J].FEMS Microbiology Letters,2002,222(1):45-50.
- [3]HAKI GD,RAKSHITS K.Development in industrially important thermo-stable enzymes[J].Bioresource Technology,2003,89(1):17-34.
- [4]CARLINI CR,GROSSI DS,MARIAF.Planttoxic proteins with insectici-da properties[J].Toxicon,2002,40(11):1519-1539.
- [5]FUCHS R L,MCPHERSON S A,DRAHOS D J.Cloning of aSerratiamarcescensgene encoding chitinase[J].Appl Environ Microbiol,1986,51(3):504-509.
- [6]WORTMANAT,SOMERVILLE C C,COLWELLR R.Chitinase determi-nants ofVibrio vulnificus:Gene cloning and applications of a chitinaseprobe[J].Appl Environ Microbiol,1986,52(1):142-145.
- [7]陈三凤,李季伦,裘维蕃.关于抑制植物病原真菌几丁质酶来源及效应的研究强作用黄杆菌的分离和鉴定[J].植物病理学报,1992,22(4):323-327.
- [8]李继红,邵寒霜,郑学勤.烟草Ⅰ类几丁质酶和β-1,3-葡聚糖酶cDNA在大肠杆菌中的表达[J].生物工程学报,1998,14(3):309-313.
- [9]李育阳.基因表达技术[M].北京:科学出版社,2001.
- [10]唐雪明,邵蔚蓝,王正祥,等.地衣芽孢杆菌感受态细胞的诱导形成及其高效电转化方法[J].无锡轻工大学学报,2002,21(5):460-463.
- [11]CUTTING S M,VANDER-HOM P B.Genetic analysis[C]//HAR-WOOD C R,CUTTINGS M.Molecular biological methods for Bacillus.Chichester,UK:Wiley,1990:78-82,144-145.
- [12]BARBOZA-CORONAJ E,J CCONRERAS,etal.Selection of chitinolyticstrain ofBacillus thuringiensis[J].Biotechnol Lett,1999,21:1125-1129.
- [13]ELEAZAR BARBOZA-CORONA J,ELIZABETHN M,ROCIO V R,etal.Cloning,sequencingand expressionofthe chitinase gene chiA74 fromBacillus thuringiensis[J].Applied and Environmental Microbiology,2003,69(2):1023-1029.
- [14]LEWIS P,MARSTON A L.GFP vectors for controlled exprmession anddual labeling of protein fusions inBacillus subtilis[J].Gene,1999,227:101-109.