镁硅酸盐纳米粘土介导的深绿木霉HB20111的遗传转化

Magnesium aminoclay-mediated genetic transformation of Trichoderma atroviride HB20111

摘要:

【背景】木霉菌遗传转化方法的烦琐复杂限制了对其基因克隆、基因功能的研究。【目的】建立深绿木霉HB20111便捷、高效的遗传转化体系。【方法】利用镁硅酸盐纳米粘土作为载体吸附含有荧光蛋白基因gfp的丝状真菌表达载体pCAMBIA1303-gpdA-GFP-TrpC-Hygro，形成质 粒-镁硅酸盐纳米粘土复合物，在超声条件下对深绿木霉HB20111分生孢子进行遗传转化。【结果】当分生孢子浓度为10⁶ CFU/mL、分生孢子悬液培养12 h、镁硅酸盐纳米粘土浓度100 mg/L和超声处理30 s (输出功率为100 W/cm²，发射频率为50 kHz)条件下对深绿木霉HB20111的转化效率最高，可以达到124个转化子/μg-DNA。【结论】使用镁硅酸盐纳米粘土作为载体可以实现对深绿木霉HB20111便捷、高效的遗传转化，转化子抗性基因和报告基因可以稳定遗传。

Abstract:

[Background] The complicated genetic transformation methods hinder the gene cloning and gene function characterization of Trichoderma.[Objective]To establish a convenient and efficient genetic transformation system for Trichoderma atroviride HB20111.[Methods] Magnesium aminoclay was used as a carrier to adsorb the filamentous fungal expression vector pCAMBIA1303-gpdA-GFP-TrpC-Hygro containing the fluorescent protein gene gfp to form a plasmid-magnesium aminoclay complex.The conidia of T. atroviride HB20111 were then genetically transformed under ultrasonic conditions.[Results] Under the conditions of the conidial suspension at a concentration of 10⁶ CFU/mL,culture time of 12 h,magnesium aminoclay concentration of 100 mg/L,and ultrasonic treatment for 30 s (the ultrasonic output power was 100 W/cm²,and the emission frequency was 50 kHz),the transformation efficiency of HB20111 was the highest,which reached 124 CFU/μg-DNA.[Conclusion] Using magnesium aminoclay as a carrier can realize convenient and efficient genetic transformation of T. atroviride HB20111,and the resistance gene and reporter gene can be inherited stably in the transformant.