有机溶剂对野生型枯草芽胞杆菌转化效率的影响

摘要

枯草芽孢杆菌(Bacillus subtilis)作为一种动物益生菌,可以直接添加在动物日粮中,提高动物对营养物质的消化吸收.随着基因技术的快速发展,枯草芽孢杆菌表达系统被广泛的应用于畜牧行业.为提高枯草芽孢杆菌感受态细胞的转化效率,在感受态细胞的制备过程中添加不同种类,不同体积浓度的有机溶剂以此达到优化制备方法和提高转化效率的目的.本实验在野生型枯草芽孢杆菌LN感受态细胞制备过程中,分别添加体积比为1.0％、2.0％、3.0％、4.0％和5.0％的吐温-80、甲醇和丙酮,统计感受态细胞的复活菌数.将目的基因长度为500和2 680 bp的外源质粒pGEM-kpgt、pGEM-kmpgt通过化学转化的方式分别转入制备好的感受态细胞中,以P4326F/P4326R、KgF/KgR为引物,利用PCR验证转化结果,并计算转化效率.结果显示,在野生型枯草芽孢杆菌LN感受态细胞的制备过程中添加4.0％的甲醇,所制备的感受态细胞复活数最多为546±13个/μL,经转化后可获得52±4个转化子/μg,转化效率最高;目的基因片段长度延长至2～3 kb,转化效率为29±2个转化子/μg.通过对感受态制备方法的改进,提高了枯草芽孢杆菌感受态的转化效率,为枯草芽孢杆菌基因工程研究提供参考.%Bacillus subtilis is a probiotics that can be added directly to the diet to improve the uptake of nutrients in animals.With the rapid development of gene technology,the expression system of B.subtilis is widely used in the livestock industry.In order to ameliorate the transformation efficiency of B.subtilis competent cells,different types and concentrations organic solvents were added in the process of competent cells preparation to optimize the transformation efficiency.In this study,1.0％,2.0％,3.0％,4.0％ and 5.0％ of the Tween-80,methanol and acetone were added in preparation of wild-type Bacillus subtilis LN competent cell.The number of viable bacteria was calculated by dilution plate counting method to determine the best addition reagent and its concentration.The plasmid pGEM-kpgt and pGEM-kmpgt with the length of 500 and 2 680 bp was transformed into individual competent cells.The transformation results were determined by PCR using P4326F/P4326R and KgF/KgR as primers and the transformation efficiency was calculated.The results showed that the count of viable competent cell is 546± 13 cell/μL and the highest transformation efficiency is 52±4 transformants/μg DNA when adding 4％ methanol in the preparation process.When the length of the target gene fragment was extended to 2～3 kb,the transformation efficiency was 29 ± 2 transformants/μg.Through the optimization of the preparation method of the competent cell,the transformation efficiency of the competent cell of Bacillus subtilis was improved,which provides a potential tool for genetic engineering of Bacillus subtilis.