毕赤酵母FLD1启动子元件的克隆与测序

为了应用毕赤酵母表达某些食用蛋白或同时表达多个异源蛋白，本文以毕赤酵母GS115基因组DNA为模板,采用prime5.9程序设计了一对不等长引物,经多轮直接多聚酶链式反应，扩增获得了大小为597bp目标DNA片段；再经限制性内切酶和双脱氧末端终止法分析,其DNA片段排列顺序与EMBL发表的FLD1启动子的序列完全一致。

Cloning of FLD1 promoter from genomic DNA of yeast Pichia pastoris GS115

The AOX1 promoter is most frequently used for expression of foreign genes in Pichia pastoris, but is unsuitable for production of food products or when more than one foreign gene needs to be cloned on a single vector. P_FLD1 is an attractive alternative to the AOXI promoter as it can be independently induced by either methanol or methylamine. Two primers have been designed using the Primer 5.9 program and according to the P_FLD1 sequences deposited in EMBLunder accession No. AR533312. Genomic DNA extracted from P. pastoris GS115 strain was used as a template and after several PCR cycles the 597 base pairs FLD1 promoter was cloned on a T-vector and then amplified in E.coli DH5α. Through enzyme restriction and fragment sequencing analyses, the cloned PFLD1 was confirmed to have 100% homology with the EMBL sequence.