米曲霉A-F04的β-果糖基转移酶基因的克隆鉴定及其生物信息学分析

Cloning,identification and bioinformatics analysis of β-fructosyltransferase from Aspergillus oryzae A-F04

以实验室前期筛选出的高产β-果糖基转移酶（β-fructosyltransferase,β-FTase）的米曲霉（Aspergillus oryzae）菌株A-F04出发,提取米曲霉A-F04基因组DNA和RNA,采用PCR和RT-PCR技术克隆β-FTase基因,得到β-FTase基因完整编码序列和内含子序列,利用重组技术构建β-果糖基转移酶质粒基因库,并构建基因的进化树,预测β-FTase分子量、等电点、氨基酸组成等物化性质,预测其N-糖基化位点和信号肽,运用SWISS-MODEL对β-FTase的三级结构预测。结果显示,成功鉴定出菌株A-F04具有β-FTase基因;菌株A-F04的β-FTase基因编码区长度为1630 bp,含有一个52 bp的内含子,位于173 bp与224 bp之间;编码序列的开放阅读框总长为1578 bp;软件分析得知β-FTase为亲水性的稳定蛋白,其分子量为57.4653 ku,理论等电点为4.83,由525个氨基酸组成;可能的N-糖基化位点有6处;信号肽分析软件得知其编码的1~17位置处的氨基酸为信号肽;SWISS-MODEL构建的3个模型质量较高,有利于后期对β-FTase的结构和功能的研究。

Based on the efficient expressing Aspergillus oryzae strain A- F04,the DNA and RNA of strain A- F04 were extracted,PCR and RT- PCR strategies were used to obtain β- FTase sequences,thus,β- FTase gene bank was constructed. Evolutionary tree was built,and physicochemical property including formula weight,isoelectric point and amino acid composition were predicted,the possible glycosylation sites were predicted.SWISS MODEL tool was used to predict β- FTase tertiary structure. Experimental results showed that β- FTase gene was successfully identified in strain A- F04. Strain A- F04 ＇s coding region was 1630 bp,including an intron,between173 bp and 224 bp,CDS were 1578 bp. Bioinformatics analysis program analysis showed that β- FTase was hydrophilic stable protein,its formula weight was 57.4653 ku,isoelectric point was 4.83,composed by 525 amino acids,within 6 possible glycosylation sites. Signal P program showed that the amino acids at 1 to 17 sites were signal peptide.The tertiary structure model constructed by SWISS- MODEL contributes to the β- FTase＇s structure and function exploration.