无花果曲酶植酸酶基因phyA的克隆、序列分析及表达  

Cloning, Sequence Analysis and Expression of the Phytase phyA Gene from Aspergillus ficuum

作　　者：李桂兰[1] 祝建洪[1] 孙建[1] 吴作为[2] 王隆飞[1] 晏晶[1] 陈功[1] 姜洪州[1] 李明刚[1] 

机构地区：[1]南开大学分子生物学研究所 [2]云南大学云南省工业微生物发酵重点实验室,昆明650091

出　　处：《农业生物技术学报》2003年第5期520-524,共5页Journal of Agricultural Biotechnology

基　　金：国家转基因植物研究和产业化专项(J00-B-003-05)。

摘　　要：用RT-PCR方法从无花果曲酶(Aspergillusficuum 3.4322)中扩增出1条约1.4kb的特异性条带,并将其克隆到pMD18-T载体中。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1347bp,编码448个氨基酸。该基因序列已在GenBank注册(注册号为:AF537344)。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(SaccharomycescerevisiaeINVSc1),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性测定,测出了明显的植酸酶活性,pYPA2胞内植酸酶活性约11.55U/L,表明无花果曲酶phyA基因能在酿酒酵母中表达。A phyA gene was cloned from Aspergillus ficuum 3.4322 by reverse transcription polymerase chain reaction(RT-PCR). The amplified fragment was cloned into the pMD18 T-vector and sequenced. Nucleotide sequence analysis of the phyA gene showed that it comprised 1 347 bp without the signal peptide sequence and encoded a polypeptide of 448 amino acids. The phyA sequence has been deposited in GenBank (accession number: AF537344). Expression vector pYPA2 was constructed by cloning the phyA gene 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 after galactose inducement. Phytase activity was found in pYPA2 (about 11.55U/L) endocellular fluid by galactose inducing. The results demonstrated that the phyA gene had been expressed in Saccharomyces cerevisiae .

关 键 词：无花果曲酶 植酸酶 酿酒酵母 表达 PHYA基因 

分 类 号：Q946.5[生物学—植物学] Q786