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本研究通过平板透明圈法筛选获得一株具有几丁质酶活性的生防细菌CAB-1,该菌株对番茄灰霉病菌等多种病原真菌表现较强的拮抗活性。通过生理生化、16S rDNA和gyrB基因序列测定,将菌株CAB-1鉴定为萎缩芽胞杆菌(Bacillus atro-phaeus)。对菌株CAB-1全基因组序列进行分析和功能预测,发现该菌株存在2个几丁质酶编码基因chit1和chit2。通过PCR技术从菌株CAB-1中克隆出这两个几丁质酶的编码基因并在大肠杆菌中表达,其原核表达产物均表现几丁质酶活性,其中chit1的原核表达产物能够显著抑制灰霉菌分生孢子的萌发。对其原核表达条件进行优化,发现在30℃下振荡培养24 h,IPTG浓度为0.2～1.0 mmol/L时,其蛋白表达量最高。
In this study, a biocontrol bacterium CAB-1 with chitinase activity was screened by plate transparent circle method, which showed strong antagonistic activity against many pathogenic fungi such as Botrytis cinerea. The strain CAB-1 was identified as Bacillus atro-phaeus by physiological and biochemical, 16S rDNA and gyrB gene sequencing. The full-length CAB-1 genome sequence analysis and functional prediction, the strain found two chitinase gene chit1 and chit2. The two chitinase genes were cloned by PCR from strain CAB-1 and expressed in E. coli. The prokaryotic expression products of chitinase showed chitinase activity, and the prokaryotic expression products of chit1 could significantly inhibit the expression of gray Germination of mold conidia. The prokaryotic expression conditions were optimized and found that when the IPTG concentration was 0.2 ~ 1.0 mmol / L, the highest protein expression was obtained after shake culture at 30 ℃ for 24 h.