一株枯草芽胞杆菌的分离鉴定及其杀虫活性的研究

Study on Isolation and Insecticidal Activities of a Bacillus Subtilis Strain

以从土壤中自行分离的219株芽胞杆菌中筛选的一株枯草芽胞杆菌菌株HX08为研究对象,通过菌落形态观察、扫描电镜、激光共聚焦显微镜等进行菌体形态观察以及16S rRNA基因同源性分析等一系列方法鉴定,该株细菌属枯草芽胞杆菌Bacillus subtilis,命名为HX08,并将其16S rRNA基因序列提交上Genebank数据库,获得登陆号KF774302.通过生测发现,HX08菌株在60 h发酵时间段对棉铃虫的致死效果最好.对该菌株发酵液提取的活性蛋白进行初步研究,建立了HPLC技术分离体系,使用H2O做流动相,以0.5 mL/min流速洗脱,检测波长分别为215、254和280 nm,收集第15 mL收集管处即保留时间为31 min的2号峰粗分离物活性较高.切取SDS-PAGE胶上相对应活性峰目的蛋白质条带酶解,利用质谱(LTQ-MS)技术进行了分子特性分析,鉴定发现杀虫活性蛋白成分为草酸脱羧酶.

Abstract A Bacillus subtilis strain has been separated from soil among 219 strains, which is named HX08, by using the methods of bacterial shape and microscope observation, the optical microscope, the laser scanning con- focal microscope and 16S rRNA genetic homology analysis. Then 16S rRNA gene sequence has been submitted to the Genebank database, gained ID KF774302. The insecticide activity of this strain to Helicoverpa armigera under different fermentation period and sample concentration were studied. The results show that the fermentation broth at 60 h of this strain HX08 also has significant insecticide activity. Some elementary research has been implemented to the protein from the fermentation broth, such as cutting the target protein stripe from SDS-PAGE gel and enzymolys- is, and LTQ-MS analysis. It was found that the strain HX08 has insecticidal activity because of the oxalate decar- boxylase. At the same time, the HPLC separation system is built, by using a molecular sieve Surperdex 200, H20 as mobile phase, elution flow with 0.5 mL/min, detection wavelength at 215, 254 and 280 nm. And the coarse separation substance from the second peak at 31 min retention time has higher activity.