地衣芽胞杆菌HS10原生质电转化体系的研究

Studies on the electroporation system for Bacillus licheniformis HS10 protoplast

地衣芽胞杆菌Bacillus licheniformis HS10及其粗蛋白对多种病原真菌具有防治效果,为挖掘其生防功能基因,本研究通过对原生质体的制备、再生培养基、渗透压稳定剂、电击参数等试验条件进行优化,建立了地衣芽胞杆菌HS10原生质体电转化法。结果表明最佳转化条件为:转接培养10h达到对数生长后期的菌体,浓缩4倍后,利用终浓度1mg/mL的溶菌酶,于37℃、150r/min酶解20min,酶解效率达到99.34%,1×SMM洗涤3次,调整菌浓度至1.0×108 cfu/mL,并通过1.6kV、5ms的电击后,迅速在SMMP溶液中100r/min、37℃恢复6~10h,涂布于含Kan和Erm的再生培养基DM3平板。将该方法用于突变体库构建的质粒pMarA(8 253bp)和基因敲除质粒pMADΔCP(13 043bp)的大片段质粒转化,分别获得了37cfu/μg DNA和10cfu/μg DNA阳性转化子。该遗传转化体系为地衣芽胞杆菌HS10中相关基因功能的研究提供了技术支撑。

Bacillus licheniformis HS10 and its crude protein have good effects on various pathogenic fungi.In order to identify the functional genes,genetic manipulation is needed.Though the optimization of the protoplast preparation,regeneration medium,osmotic stabilizer,shock conditions and other experimental conditions,the electroporation method of protoplast was set up in this paper.The experimental results showed that the optimum reaction conditions were as followed：The cells at logarithmic growth period after transferred for 10 h were concentrated by 4 times,and then subjected to enzymolysis for 20 min with a final concentration of 1 mg/mL lysozyme at 37℃ and 150 r/min.Enzymolysis rate reached 99.34%.The products of enzymolysis were washed for three times with1× SMM and adjusting the concentration to 1.0×10^8 cfu/mL.After electroporation at 1.6 kV and5 ms,with 0.5 mol/L sucrose as osmotic stabilizer,the cells were quickly restored for 6-10 h in SMMP solution,then coated on DM3 resistant plate.In the large-fragment transformation using the plasmids pMarA（8 253 bp）and pMADΔCP（13 043 bp）,we obtained 37 cfu/μg DNA and 10 cfu/μg DNA positive transformants.This study established a genetic transformation system for the genetic manipulation of Bacillus licheniformis HS10.