摘要
从黑曲霉N25(A.niger China strain)中提取出染色体DNA,根据已经测定出的植酸酶phyA基因全序列设计了一对引物,采用高保真度的聚合酶Advangage-HF扩增到了去除信号肽和内含子后约1.4kb片段,对该片段进行了克隆及序列测定。将该序列与植酸酶phyA基因全序列进行了比较。以此片段构建成功了pPIC9K-phyA载体(命名为pPNP-1),并转化毕赤巴斯德酵母。经G418抗性筛选、酶活性测定、Southern印迹和Western印迹,获得了高效表达的转化子PP-NP-1(23869.4u/ml)、PP-NP-2(20533.0u/ml)、PP-NP-3(35646.7u/ml),其酶活性分别是出发菌株的酶活(513.4u/ml)的46.46倍、39.99倍和69.46倍,且转化子具有很好的遗传稳定性。
The DNA of chromosome was extracted from an Aspergillus Niger (N25, China strain). A pair of primers were designed and synthesized according to the sequence of phyA gene (GenBank Accession No. AF218813). Using a high-fidelity polyerase (Advantage-HF), an expression fragment (1.4kb), which does not contain signal peptide and intron sequence in the sequence of phyA gene, was amplified by PCR, cloned, sequenced and compared with the full sequence of phyA gene (GenBank Accession No. AF218813). The pPIC9K-phyA vector was constructed (named pPNP-1) and transformed into Pichia Pastoris. Three transformants named PP-NP-1, PP-NP-2, PP-NP-3 were selected by G418 resistance, enzyme activity test, Southern blot, and Western blot. Their enzyme activity of phytase is 23869.4u/ml, 20533.0 u/ml, 35646.7 u/ml, 46.69 times, 39.99 times and 69.43 times compared with the phytase activity of the original strain (513.4u/ml) respectively. The genetic stability of transformant is quite good.
出处
《菌物系统》
CAS
CSCD
北大核心
2001年第4期486-493,共8页
Mycosystema
基金
获国家科技部"九五"国家重点科技攻关计划资助
专题编号99-009-02-02
