PEG介导枸杞内生真菌NQ8GⅡ4遗传转化及转化子评价

Establishment of Genetic Transformation System of Lycium barbarum Endophytic Fungus Fusarium nematophilum NQ8GⅡ4 Using PEG-mediated Method and Transformants Evaluation

通过聚乙二醇(PEG)介导原生质体转化法将绿色荧光蛋白(green fluorescent protein,GFP)表达载体转入枸杞内生轮状镰刀菌(Fusarium nematophilum)NQ8GⅡ4菌株中,通过生物学测定和PCR检测,获得与野生型NQ8GⅡ4菌株无明显差异的转化子57株,转化效率(单位质量的DNA转化子数)为2 850株·mg^(-1)。通过遗传稳定性、表型、荧光性、拮抗性和致病性对比,获得1株与野生型NQ8GⅡ4菌株无差异的转化子。该转化体系的建立为枸杞内生真菌在宿主植物中的侵染定殖规律和生防作用研究奠定基础。

In this study,the expression vector of green fluorescent protein was transferred into the strain NQ8GⅡ4 by PEG-mediated protoplast transformation. Transformants of no difference with the wild strain were obtained by biological assay and PCR analysis. The cell walls of young hypha of NQ8GⅡ4 strain was lyzed with enzymes to generate protoplasts,which were used to insert the exogenous DNA randomly under PEG8000 mediated transformation. Fifty-seven transformants of NQ8GⅡ4 that showed hygromycin B resistance and fluorescence signal were obtained. The transformation frequency of NQ8GⅡ4 was 2 850 transformants per mg DNA. The transformants were compared with wild type strain for mitotic stability,phenotype,fluorescence,antagonistic activity and pathogenicity and a transformant with similar properties to wild-type strain was screened out. The results have established foundation for futher study on the endogenesis and biocontrol mechanism of strain NQ8GⅡ4.