Aspergillus Niger N25 Phya Gene Expression The Fragment Cloning And Ppic9k Of Recombinant Yeast Expression Vector Construction

By using modified method of two-step precipitation, the total DNA of chromosom⁽vas extracted from an Aspergiiius Niger (N25, China strain). A pair of wiv^S primers were designed and synthesized according to the sequence of phyA gene (GenBank Accession No. AF2 18813) registered by Wang Hong-ning. The upstream primer started with signal cleavage site (EcoR I site was designed), and the downstream primer ended at stop codon (Kpn I and Not I were designed). By using a high-fidelity polyerase (Advantage-HF), the DNA of encoding region (1.4kb) was amplified by PCR, inserted into EcoR I and Kpn I sites of pUC 18 vector, and transformed into JM 109 strain. As a result, white colonies were screened on LB plate (Amp and x-gal added). The result of enzyme digestion of vector proved that the recombination vector (named pANP-1) was obtained , and the sequence of interesting DNA showed that it was consistent with total DNA of phyA gene without signal peptide and intron sequence. pANP- 1 vector was digested by EcoR I and Not I , and the DNA of encoding region of phyA gene was taken out, inserted into the multi-cloning site of pPIC9K vector, and transformed into E. coil DH5a strain finally. Positive colonies 搼^<sup> selected by situ-hybridization with photo-biotin labeled phyA gene. The result of enzyme digestion of vector proved that recombinatiⁱ vector was obtained (named pPNP- 1). The successful construction of pPNP- 1 vector is the key foundation of phyA gene expressed in Pichia pastoris.