芽孢杆菌几丁质酶基因的克隆、序列分析及其在伯克氏菌B418中的表达

采用PCR技术扩增到枯草芽孢杆菌几丁质酶基因的编码序列。片段共2227bp,含有一个具有1788个核苷酸的开放阅读框架,起始密码子位于211bp,终止密码子位于1998bp,共编码氨基酸605个。序列比较发现同已发表的枯草芽孢杆菌几丁质酶核苷酸具有99.39%的同源性。将该基因与大肠杆菌-伯克氏菌穿梭质粒pRK415连接,获得重组质粒pRChi113。重组质粒电击转入伯克氏菌B418中,获得工程菌株B418-9、B418-13。与野生菌株B418相比,平板拮抗试验表明,工程菌株B418-9、B418-13对大丽花轮枝孢、立枯丝核菌、麦根腐长蠕孢、禾谷丝核菌抑菌效果明显提高。

Chitinase Gene from Bacillus subtilis: Cloning,Sequencing and Expression in Burkholderia B418

PCR method was adopted for cloning of chitinase encoding genes.The chitinase gene fragment is about 2227bp,and contains an open reading frame 1788 nucleotides starting with the initiation codon ATG at position 211 and ending with the termination codon TAA at position 1998,and deduced amino acid is about 605.DNA sequencing analysis showed that sequence homology between PCR fragment and chitinase gene of B.subtilis was 99.39%.The chitinase gene chi113 was used to transform Burkhoderia B418 by electroporation with plasmid pRK415.Antagonistic activity test in vitro suggest that the transformants remained the ability to produce chitinase.The recombinant strains showed an increased biocontrol efficacy against Verticillum dahliae,Rhizoctonia solani,Helminthosporium sativum,Rhizoctonia cerealis in vitro.