Optimizing the transformation conditions of highly antagonistic Trichoderma harzianum T2-16 and screening the positive transformants with similar biocontrol characteristics to wild-type strain will lay a foundation for determination of colonization dynamics and distribution of T. harzianum T2-16. In this study, the green fluorescence protein (GFP) expression vector with G418 resistance gene, named pKN-sGFP, was constructed by PCR and molecular cloning technology. T. harzianum T2-16 transformant with high fluorescent expression level was obtained by PEG-CaCl2-mediated protoplast transformation, and a positive transformant with similar biocontrol properties to wild-type strain was screened out. T. harzianum T2-16 strain was sensitive to 1000 μg/mL G418 at 20 ℃. Under the above optimized conditions, the stable genetic positive transformant TG2-10 was obtained and compared with the wild-type strain. There was no significant difference in biocontrol characteristics between the two strains. Thus, the transformant TG2-10 could be used to further study the biocontrol mechanism of T. harzianum T2-16 in the future.%优化高效拮抗生防菌哈茨木霉T2-16的转化条件,筛选出与野生型菌株具有相似生防特性的阳性转化子,为生防木霉菌T2-16的定殖动态、分布规律等研究打下基础.通过PCR和分子克隆技术构建具有G418抗性基因的绿色荧光(GFP)表达载体pKN-sGFP,利用PEG-CaCl2介导的原生质体转化法,获得强荧光表达的哈茨木霉T2-16转化子,并将其与野生型菌株的生物特性进行比较,筛选出与野生型菌株具有相似生防特性的阳性转化子.试验结果显示,哈茨木霉T2-16在20℃培养条件下对1000μg/mL G418敏感,在上述优化条件下,转化获得稳定遗传的阳性转化子TG2-10;进一步比较其与野生型菌株的生物特性发现,两者之间无明显差异,可用于下一步哈茨木霉T2-16生防机理的研究.
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