Jiang Qiong, Ying Sheng-Hua, Feng Ming-Guang
Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, PR China.
J Microbiol Methods. 2007 Jun;69(3):512-7. doi: 10.1016/j.mimet.2007.03.005. Epub 2007 Mar 24.
The techniques of restriction enzyme-mediated integration (REMI) and electroporation (EP) were applied for the first time to improving the blastospore transformation of fungal biocontrol agent Beauveria bassiana for higher frequency. The blastospores from < or =24 h incubation in glucose-mineral medium after shaking conidia for 48 h in Subouraud dextrose broth were found most competent for integrating 1 microg plasmid DNA vectoring the phosphinothricin (PPT) resistance gene bar in 360 microL reaction system containing 100 U HindIII or XbaI. Such blastospores were also most suitable for EP transformation at the optimized field strength of 10 kV cm(-1). The optimized REMI and EP generated averagely 39 and 53 transformants microg(-1) plasmid DNA whereas polyethylene glycol (PEG) integration yielded only 22. All transformants grew well on Czapek's agar containing 400 microg PPT mL(-1) after three rounds of cultivation on the same agar excluding PPT but their parental strain showed no resistance. The target gene inserted into the genomes of 10 transformants randomly taken from REMI or EP transformation was consistently detected by both PCR and Southern blotting. Compared to the PEG integration, REMI and EP enhanced the frequency of the blastospore transformation by 73 and 137%, respectively.
限制酶介导整合（REMI）和电穿孔（EP）技术首次应用于提高真菌生防菌球孢白僵菌芽生孢子的转化频率。在沙氏葡萄糖肉汤中摇瓶培养分生孢子48小时后，于葡萄糖 - 矿物培养基中培养≤24小时的芽生孢子，在含有100 U HindIII或XbaI的360 μL反应体系中，被发现最适合整合携带膦丝菌素（PPT）抗性基因bar的1 μg质粒DNA。这种芽生孢子在10 kV cm⁻¹的优化场强下也最适合进行电穿孔转化。优化后的REMI和电穿孔平均每微克质粒DNA分别产生39个和53个转化子，而聚乙二醇（PEG）整合仅产生22个。在不含PPT的相同琼脂上进行三轮培养后，所有转化子在含有400 μg PPT mL⁻¹的察氏琼脂上生长良好，但其亲本菌株无抗性。通过PCR和Southern印迹法一致检测到从REMI或电穿孔转化中随机选取的10个转化子基因组中插入了目标基因。与PEG整合相比，REMI和电穿孔分别将芽生孢子转化频率提高了73%和137%。
