根癌农杆菌介导的增强型绿色荧光蛋白基因在淡紫拟青霉中的转化

Transformation of enhanced green fluorescent protein gene in Paecilomyces lilacinus mediated by Agrobacterium tumefaciens

摘要:

为了实现增强型绿色荧光蛋白基因 (egfp) 在生防真菌淡紫拟青霉9410菌株中的转化，借助中间质粒pcDNA3.1(-) 构建nptⅡ-egfp融合基因的表达载体pUPNGT，然后采用根癌农杆菌介导的转化法将egfp基因转化到淡紫拟青霉9410菌株中。PCR检测和Southern blotting分析结果表明，egfp基因以单拷贝形式整合到淡紫拟青霉9410的基因组中。荧光显微镜观察结果显示，转化子在488 nm下能产生绿色荧光。这些结果说明egfp基因已成功转化至淡紫拟青霉9410菌株并获得表达。这些工作可为淡紫拟青霉在不同条件下的防效评价、环境安全评价等提供新的途径和方法。

Abstract:

The main aim of this study was to transform the enhanced green fluorescent protein gene (egfp) into biocontrol fungus Paecilomyces lilacinus strain 9410. We constructed the expression vector pUPNGT of the fusion gene nptII-egfp using pcDNA3.1(-) as a helper plasmid. The egfp gene was then transformed into P. lilacinus strain 9410 via Agrobacterium tumefaciens-mediated transformation. PCR and Southern blotting analysis showed that the egfp gene was integrated into the genomes of the tested transformants and the integration manner was single-copy. The transformants could generate green fluorescence when they were excited by 488 nm blue laser. These results indicated that the egfp gene had been successfully transformed into P. lilacinus 9410 and expressed in the tested transformants. Our work may provide a new approach to assess environmental safety and practical biocontrol efficacy of P. lilacinus under different conditions.