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目的扩增Fonsecaea monophora中控制黑素合成的多聚酮合酶(polyketide synthases,PKS)基因并测序,构建PKS基因敲除载体。方法扩增PKS基因,根据测序结果设计引物,从Fonsecaea monophora基因组DNA扩增PKS基因5’-同源臂和3’-同源臂,从质粒pBHt1中扩增潮霉素B抗性标记基因(hyg),最后将各片段插入载体PDHt/sk中。结果测序得到5 389bp大小的PKS基因序列,构建了PKS基因的敲除载体,用酶切及测序等方法鉴定载体构建成功。结论成功构建Fonsecaea monophora PKS基因敲除载体,为研究PKS基因及黑素的生物学功能奠定了良好的基础。
Objective To amplify and sequence the gene encoding melanin synthesis polyketide synthases (PKS) in Fonsecaea monophora and construct PKS knockout vector. Methods The PKS gene was amplified. According to the sequencing results, primers were designed to amplify the 5’-homologous arm and 3’-homology arm of PKS gene from Fonsecaea monophora genomic DNA. The hygromycin B resistance marker gene was amplified from plasmid pBHt1 hyg), and finally insert each fragment into the vector PDHt / sk. Results The PKS gene sequence of 5 389bp was sequenced and the knockout vector of PKS gene was constructed. The vector was identified by restriction enzyme digestion and sequencing. Conclusion The Fussecaea monophora PKS gene knockout vector was successfully constructed, which laid a good foundation for studying the biological function of PKS gene and melanin.