绿色木霉内切几丁质酶cDNA的克隆及其编码蛋白的序列分析

cDNA cloning of the endochitinase from Trichoderma viride and the sequence analysis of its encoding protein

根据已克隆的内切几丁质酶基因序列的同源性比较,设计引物,采用PCR技术从绿色木霉基因组中分离出一个大小为1467bp的特异DNA片段,采用RT-PCR技术从绿色木霉总RNA中分离出大小约1276bp的cDNA片段。序列对比后发现该内切几丁质酶DNA含有三个内含子,大小分别为52bp,69bp,64bp。同源性分析表明其全长cDNA序列和已经报道的内切几丁质酶序列的同源性高达95%以上,预测其编码蛋白的氨基酸序列含424个氨基酸残基,分子量为46kDa,氨基酸序列分析表明该内切几丁质酶164～172位氨基酸是其活性中心,用同源建模法模拟其空间结构模型,为进一步研究其作用机制奠定了良好基础。

DNA of 1467bp and eDNA of 1 276 of the endochitinase from T. viride were amplified by PCR and RT- PCR using the genome and the total RNA as templates, respectively. The DNA sequenee includes three introns with lengths of 52, 69 and 64 bp. The homology analysis shows that the eDNA sequence of the endochitinase from T. viride has a high homology of 95% with other ones. Deduced amino acids have 424 residues and the theoretical moleeular weight is 46kDa. The amino acid sequence analysis shows that its active sites are 164 - 172 residues. The tertiary structure of the mature endochitinase protein was simulated with homology modeling method, which lays a good foundation for further studying its catalytic mechanism.