马尔尼菲青霉基因功能研究工具载体pSilent-GFP的构建

目的 建立一种基于载体pSilent-1介导的对马尔尼菲青霉基因进行功能研究的工具。方法 将绿色荧光蛋白（GFP）基因整合于丝状真菌表达质粒pSilent-1中，构建pSilent-GFP载体，将构建成功的pSilent-GFP载体通过电转化方法转化马尔尼菲青霉酵母细胞，经潮霉素阳性筛选及PCR方法鉴定阳性克隆子，并在荧光显微镜下观察GFP在马尔尼菲青霉中的表达。结果 成功构建了pSilent-GFP表达载体，该载体能转化马尔尼菲青霉酵母细胞并在菌体内稳定表达GFP。结论 pSilent-GFP载体在马尔尼菲青霉中稳定表达GFP，pSilent-1载体可应用于马尔尼菲青霉基因的功能研究。

Construction of plasmid vector pSilent-GFP for assessing the gene functions of Penicillium marnefei

Objective To develop a new tool to study the gene function of Penicillium marneffei based on pSilent-1 vector. Methods GFP gene was subcloned into the pSilent-1vector and identified by sequence analysis, then, the reconstructed plasmid pSilent-GFP was transformed into the yeast cells of Penicillium marneffei by electronic transformation. The positive clones were selected by hygromycin and by directed PCR to amplify the GFP gene. The expression of GFP could be detected under inflorescent microscopy. Results It was confirmed that gene of GFP was successfully reconstructed into pSilent-1 plasmid and obtained the pSilent-GFP plamid. The pSilent-GFP plasmid could be transformed into Penicillium marneffei and expressed the GFP in yeast and mycelia phases of Penicillium marneffei. Conclusions The pSilent-GFP could stably express in both phases of, and could be used as a tool to study the gene function of Penicllium marneffei.