耐热植酸酶基因phyA的克隆及新型表达载体的构建

Cloning of Heat-Stable PhyA Gene and Construction of its New Expression Vector

目的 克隆烟曲霉耐热植酸酶基因 ,构建分泌性的真核表达质粒 ,以获取耐热的基因工程植酸酶。方法 提取烟曲霉菌的基因组 ,以此为模板PCR扩增成熟肽编码序列 ,双酶切后与包含黑曲霉葡萄糖淀粉酶启动子和基因片断的真核表达载体 pYG 1 .2相连接 ,转化入大肠杆菌DH5α ,从转化菌株中提取质粒进行序列测定和分析。 结果 PCR扩增出 1 32 6bp的植酸酶编码序列 ,插入 pYG 1 .2质粒中 ,构建了重组质粒 ,重复实验结果表明 ,该植酸酶基因序列与已报道的烟曲霉植酸酶序列有 2个碱基的差异 ,编码的蛋白质序列有 99.8%的同源性。结论 成功构建了用于黑曲霉菌真核表达耐热植酸酶的重组质粒。

Objective To construct a fusion expression plasmid containing heat-stable phytase A gene from A.fumigatus for further expression of phytase A protein in A.niger.Methods Genomic DNA of A.fumigatus was isolated and the phytase A coding sequence was amplified by PCR.The amplified fragment was inserted into the expression vector pYG 1.2 in frame with glucoamylase coding sequence using the promoter from A.niger glaA.The DH5αwere transformed and the transformants were confirmed by PCR.The positive products were verified by restriction enzyme digestion and sequencing.Results A DNA fragment of approximately 1.4kb was generated by PCR and cloned into plasmid pYG 1.2.The sequence cloned shares 99.9% homology with reported phytase A gene sequence except for two base difference.Conclusion The recombined plasmid containing heat-stable phytase was constructed successfully and it will be used to express heat-stable phytase A in large quantity in our A.niger expression system.