黑曲霉N-2植酸酶phy4基因的克隆及其重组酵母表达载体的构建

根据已发表的植酸酶phyA基因序列设计并合成1对引物，应用PCR技术，以黑曲霉N-2总DNA为模板，扩增出不包含假定信号肽序列的phyA基因，将其克隆到pMD18-T载体中，测定其核苷酸序列，并推导其氨基酸序列。该基因全长为1350bp，与已发表的黑曲霉NRRL3135的phyA基因的同源性为92．4％(不计内含子)，编码1个含449个氨基酸残基的蛋白质，推导的氨基酸序列同源性为95．1％。将该基因与分泌型载体pPIC9K连接，构建了植酸酶基因的重组酵母表达载体pPIC9K／phyA。

Cloning of Aspergillus niger N-2 phytase gene and construction of recombinant plasmid pPIC9K/phyA

The phyA encoding phytase of Aspergillus niger N-2 was amplified by PCR, the amplified fragment was cloned and sequenced. After digested by EcoRI and NotI, phyA fragment was linked with vector pPIC9K and transformed into competent JM109. Positive clones were screened and plasmid pPIC9K/phyA was detected by PCR and restriction digestion. The results show that the coding region comprises 1 350 nucleotides without the intron and putative signal sequence, it encodes a polypeptide of 449 amono acids. Comparision with phytase gene phyA of Aspergillus niger NRRL3135(GenBank Accession No. Z16414), it shows 92.4% homology in nucleotides and 95.1% in amino acids residues. The sequenced phyA fragment was inserted into Pichia secretive expression vector pPIC9K correctly, and the recombinant plasmid pPIC9K/phyA was constructed successfully.