黑曲霉NRRL3135 bgl基因的克隆及序列分析

Cloning and Sequence Analysis of β-glucosidase Gene from Aspergillus niger strain NRRL3135

利用RT-PCR技术从黑曲霉菌株NRRL3135中扩增了β-葡萄糖苷酶(BGL)基因的全长cDNA序列,被命名为bgl3135(GenBank登录号:FN430671)。对该基因编码的BGL进行了同源性分析并预测了其三维构象。试验结果表明,bgl3135的全长cDNA为2598bp,含有一个2583bp的开放阅读框(ORF),编码一个长度为860个氨基酸残基的蛋白质。同源性分析表明,该基因编码的蛋白与来自黑曲霉As3.350和CBS513.88的BGL(GenBank登录号分别为DQ655704和XM_001398779)的相似性最高,其氨基酸序列同源性分别达99%。参照已发表的β-葡萄糖苷酶(BGL)的氨基酸保守序列,判断本研究所克隆的bgl基因属于糖苷水解酶基因家族3的成员。三维结构预测表明,该BGL的立体构象中存在20个α-螺旋和15个β-折叠,对构成和维持酶的底物识别和催化活性位点可能起着重要作用。

A full-length cDNA of the β-glucosidase（BGL） gone from the AspergiUus niger strain NRRL3135 was cloned by RTPCR, and designated as bg13135 （GenBank accession number： FN430671 ）. The homology of the cDNA-predicted BGL with the other β-glucosidases was analyzed and the tertiary structure of the BGL was further predicted. The results revealed that the fuU-length cDNA （2 598 bp） of bg13135 has an open reading frame （ORF） of 2583 nucleotides encoding a protein 0f860 amino acids, BLAST similarity searches in GenBank databases with command blastx revealed the deduced protein is most similar to the BGL sequences from the A. niger strains As3. 350 and CBS 513.88, with amino acid identity of 99% , respectively. Based on the conserved amino acid residues in the predicted BGL, this bgl gene should be classified into the glycoside hydrolase gene family 3, Tertiary structure prediction demonstrated that there were 20 a-helixes and 15 β-sheets which might play important roles in construction and retaining the substrate-recognition domain and catalysis domain in the enzyme. The successful cloning of the bgl gene laied a foundation for the heterogenuous expression and construction of gone engineering bacteria in the near future.