农杆菌介导的粘帚菌遗传转化

Genetic transformation of the fungus Gliocladium sp.mediated by Agrobacterium tumefaciens

【目的】建立分离自西藏林芝的能够产生一系列具有抗肿瘤活性的二酮哌嗪类化合物(epipolythiodioxopiperazine,ETP)的一株冬虫夏草定殖真菌——粘帚菌(Gliocladium sp.)6.102的遗传转化体系。【方法】利用农杆菌介导的方式建立了粘帚菌的遗传转化体系;用正交试验研究了影响转化效率的因素,包括共培养所用农杆菌与真菌孢子的比例、共培养的时间、pH值和乙酰丁香酮的浓度。【结果】成功建立了农杆菌介导的粘帚菌的遗传转化体系,得到了转化的最佳条件,其转化效率为50-100个转化子/106真菌孢子。通过农杆菌介导的方式分别将潮霉素B磷酸转移酶基因(hph)和绿色荧光蛋白基因(egfp)转入粘帚菌中,实现了表达并且可以稳定存在。【结论】首次建立了农杆菌介导的粘帚菌遗传转化体系,为研究ETP化合物的生物合成及其调控机制奠定了基础。

[ Objective] To construct a transformation system in Gliocladium sp. 6. 102, a Cordyceps-colonizing fungus producing a variety of epipolythiodioxopiperazine （ETP） compounds with drug potentials. [ Methods ] Agrobacterium tumefaciens-mediated transformation （ATMT） was used to transform Gliocladit^m sp. 6. 102. The factors of bacterial cell concentration, co-cultivation time, pH and acetosyringone concentration were optimized. [ Results] A total of 50 -100 transformants per 106 fungal conidia was obtained via the optimal ATMT method. The genes encoding hygromycin B phosphotase and enhanced green fluorescent protein （EGFP） were transferred into Gliocladium sp. by the optimal ATMT method. The marker genes were successfully expressed and stably maintained in the transgenic fungus. [ Conclusion] A transformation system was established for Gliocladium sp. 6. 102 and this system may be useful to identify ETP biosynthetic genes in Gliocladium.