植酸酶酵母工程菌的构建

The Construction of a Transgenic Yeast Strain Expression Phytase

从无花果曲霉 (Aspergillusficuum) 3 432 2中用RT PCR方法扩增出一条约 1 4kb的特异性条带 ,DNA序列测定表明 ,目的片段为不含信号肽的植酸酶编码序列 ,全长 1 347bp。无花果曲霉 (Aspergillusficuum) 3 432 2phyA基因序列已在GenBank注册 (注册号为 :AF5 37344)。将该基因克隆到酵母表达载体pYES2中 ,构建成不带信号肽phyA基因的重组表达载体pYPA2。用醋酸锂法将pYPA2转进Ura缺陷型的酿酒酵母 (S cerevisiaeINVSc1 ) ,筛选获得含植酸酶基因的酵母转化子。经半乳糖诱导表达后 ,用磷钼蓝显色 (AMES)法对酵母菌体进行酶活测定 ,测出了明显的植酸酶活性 ,pYPA2胞内植酸酶活性约 1 1 5 5IU mL ,表明无花果曲霉 (Aspergillusficuum) 3 432

A phyA gene was cloned from Aspergillus ficuum 3.4322 by reverse transcription polymerase chain reaction (RT-PCR). The amplified fragment was cloned into pMD18 T-vector and sequenced. Nucleotide sequence analysis of the phyA gene showed that it comprises 1347 bp without the signal peptide sequence. The phyA sequence has been deposited in GenBank (accession number: AF537344). The expression vector pYPA2 was constructed by cloning the phyA gene of without the signal peptide sequence into the yeast expression vector pYES2. The recombinant plasmid was transformed into Saccharomyces cerevisiae INVSc1 by the method of LiAc. Then, the yeast clone containing phyA gene was screened to test the phytase activity by AMES method. The phytase activity (about 11.55IU/mL) was found in pYPA2 endocellular fluid by galactose inducing. The results demonstrated that the phyA gene had been expressed in Saccharomyces cerevisiae.