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首页> 外文期刊>African Journal of Biotechnology >Polyethylene glycol (PEG)-mediated transformation of the fused egfp-hph gene into Pleurotus ostreatus
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Polyethylene glycol (PEG)-mediated transformation of the fused egfp-hph gene into Pleurotus ostreatus

机译：聚乙二醇（PEG）介导的融合的egfp-hph基因转化为平菇

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A transformation system for the basidiomycete?Pleurotus ostreatus?was established using PEG-mediated transformation. The homologous glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter and terminator regions of?P. ostreatus?were amplified and cloned onto the restriction site of the pUC19 vector. Enhanced green fluorescent protein-encoding gene (egfp) and a selection marker, the hygromycin phosphotransferase gene (hph), were then cloned and an expression vector, pUEGFP-hph, was constructed. Protoplasts of?P. ostreatus?were prepared and used as the recipients in transformation procedures. An improved transformation method was established in the present study and about 100 to 200 resistant colonies per microgram DNA per 107?viable protoplasts were obtained. After subculturing, putative transformants were screened by PCR and verified by Southern blot and RT-PCR analyses showing the correct insertion into the genomic DNA and transcription of the transformed DNA. Moreover, the quantitative PCR and green fluorescence observation were carried out, thus demonstrating expression of the?egfp?gene driven by the homologous?gpd?promoter. This was the first report of transformation of?P. ostreatus?using homologous?gpd?promoter by PEG-mediated methods. These data will be helpful in future investigations using PEG-mediated transformation for functional characterisation of genes in the edible fungi basidiomycete?P. ostreatus.
机译：利用PEG介导的转化建立了担子菌？平菇的转化体系。同源的3-P甘油醛-磷酸脱氢酶基因（gpd）启动子和终止子区域。扩增了平菇，并将其克隆到pUC19载体的限制性酶切位点上。然后克隆了增强的绿色荧光蛋白编码基因（egfp）和选择标记潮霉素磷酸转移酶基因（hph），并构建了表达载体pUEGFP-hph。 P的原生质体。准备了平菇，并将其用作转化程序中的接收者。在本研究中建立了一种改进的转化方法，每107活的原生质体每微克DNA可获得约100至200个抗性菌落。继代培养后，通过PCR筛选推定的转化体，并通过Southern印迹和RT-PCR分析验证，显示正确插入基因组DNA和转化DNA的转录。此外，进行了定量PCR和绿色荧光观察，从而证明了由同源βgpdβ启动子驱动的egfpβ基因的表达。这是对P的首次报道。用同源的gpd启动子通过PEG介导的方法制备平菇。这些数据将有助于今后使用PEG介导的转化对食用菌担子菌P中的基因进行功能表征的研究。平菇

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