嗜热真菌Thermomyces langinosus编码丝/苏氨酸蛋白激酶部分cDNA的克隆及序列分析

根据几种丝状真菌保守的氨基酸序列,设计了一组简并性的引物,从嗜热真菌Thermomyceslanginosus中提取总RNA,利用RT-PCR的方法,我们得到了一个1243bp的cDNA的片段。片段回收纯化后连接到pGEM-Teasy载体上,PCR扩增重组质粒证实这个长约1200bp片段已经插到载体上。序列分析发现和裂殖酵母的蛋白激酶dis1非常相似,比较两者3'氨基酸序列发现同源性达28%以上。其推断的氨基酸序列上包含有6个丝/苏氨酸蛋白激酶的保守亚区。这些都表明它编码的基因可能是一个新的丝/苏氨酸蛋白激酶。

Cloning and sequencing of a cDNA coding a putative ser/thr kinase from Thermomyces langinosus

Based on the conserved amino acid sequence of ser/thr protein kinase from several filamentous fungus, degenerate primers was designed and synthesized. Total RNA was isolated from the fungi Thermomyces langinosus. Using RT-PCR,one 1243bp cDNA fragment was obtained, and was cloned into pGEM-T easy vector. PCR amplification indicated that the 1243bp fragment was inserted into the plasmid vector. Sequencing analysis showed that the cloned cDNA of Thermomyces langinosus was similar to that of fission yeast protein kinase disl. Comparison results showed that the homogenity of the 3'-deduced amino acids was above 28%. Six conservative amino acid subdomains which most ser/thr kinases contain can be found in the deduced amino acid sequence. These showed that it might be a novel ser/thr protein kinase.