以刺芹侧耳菌丝球为受体,潮霉素(Hyg)为筛选标记,应用农杆菌介导法对刺芹侧耳菌丝进行了遗传转化研究。潮霉素敏感性测试结果表明,刺芹侧耳Hyg耐受浓度为50mg/L。农杆菌介导的刺芹侧耳菌丝最佳遗传转化体系为:菌液浓度OD600=0.6–0.7,侵染时间30–35min,共培养时间2d,侵染液和共培养培养基中乙酰丁香酮(AS)浓度为1mg/m L;经潮霉素抗性筛选、PCR鉴定和GUS活性的组织化学分析,表明外源基因GUS已转入到刺芹侧耳菌丝中,并获得表达。本实验成功地建立了稳定的农杆菌介导的刺芹侧耳遗传转化体系。
The mycelial pellets of Pleurotus eryngii were used as receptor for transformation by Agrobacterium-mediated method, and the genetic transformation system of P eryngii was established. Sensitivity test showed that the tolerance concentration for P eryngii against hygromycin was 50mg/L. The optimum genetic transformation system for Agrobacterium-mediated P eryngii mycelia was OD6oo=0.6-0.7 of the bacterial concentration, 30-35min of infection duration, 2 days of the co-cultivation period and lmg/mL of acetosyringone (AS) concentration. The results of PCR identification and the histochemical analysis of 13-glucuronidase (GUS) activities indicated that the exogenous gene gus was transferred into the mycelia of P. eryng/i by hygromycin resistance selection. A stable Agrobacterium-mediated genetic transformation system of P eryngii was constructed successfully