猕猴桃胶孢炭疽病菌的农杆菌介导遗传转化体系构建及荧光标记菌株鉴定

Construction of Agrobacterium Tumefaciens-mediated Genetic Transformation System of Colletotrichum Gloeosporioides and GFP-tagged Transformants Identification

为了明确炭疽病菌对猕猴桃的侵染机制,以猕猴桃胶孢炭疽病菌菌株及含潮霉素B抗性基因和绿色荧光蛋白(green fluorescent protein,GFP)基因的双元载体pCAMBgfp为材料,成功构建了由农杆菌EHA105介导的遗传转化体系并得到了稳定表达绿色荧光的转化子。研究表明,当潮霉素B质量浓度为160μg/mL时可有效筛选阳性转化子;分子鉴定结果证实GFP表达基因已整合至转化子基因组中;荧光观察下继代培养的转化子菌丝及分生孢子均表现出强烈的绿色荧光信号,说明GFP标记在转化子中遗传表达稳定;与野生型菌株相比,2株转化子的菌落形态特征、菌丝生长速率对猕猴桃果实的致病力无明显差异。该研究成功建立了猕猴桃胶孢炭疽病菌的遗传转化体系并得到了稳定表达的荧光转化子,对进一步解析该菌株与宿主之间的互作机理具有重要意义。

In order to clarify the pathogenic mechanism of Colletotrichum gloeosporioides on kiwifruit,pathogen and the binary vector pCAMBgfp containing hygromycin resistance gene and green fluorescent protein(GFP)gene were used as materials.Through the method of Agrobacterium tumefaciens EHA105 mediated transformation,the genetic transformation system of C.gloeosporioides was successfully constructed,and transformants stably expressing green fluorescence were obtained.The research results indicated that the mass concentration of hygromycin B that effectively screen positive transformants is 160μg/mL.Molecular identification showed that the GFP expression gene had been integrated into the transformant genome.Under fluorescence observation,the hyphae and conidia of the subcultured transformants showed strong green fluorescence signals,indicating that GFP stably inherited in the transformants.Compared with wild-type strain,two transformants were no significant differences in colony morphology,mycelial growth rate,and pathogenicity on kiwifruit.In this study,the genetic transformation system of C.gloeosporioides was established and the transformants stably expressing green fluorescent protein were obtained,which is of great significance for studying the interaction between C.gloeosporioides and the host.