RACE法克隆黑曲霉NCU-317中α-L-鼠李糖苷酶基因与生物信息学分析

RACE cloning and bioinformatics analysis of α-L-rhamnosidase from Aspergillus niger NCU-317

实验室前期筛选得到一株能够产α-L-鼠李糖苷酶的黑曲霉NCU-317,但由于其完整产酶序列未知,通过逆转录聚合酶链式反应(RT-PCR)技术和改良cDNA末端快速扩增(RACE)法,以克隆得到的黑曲霉NCU-317 cDNA为模板,成功克隆得到其α-L-鼠李糖苷酶基因全长。克隆得到α-L-鼠李糖苷酶基因共3 018 bp,其中含有2 748 bp的完整开放阅读框,5’非编码区长为216 bp,3’非编码区长为54 bp。经过对序列的生物信息学分析显示:编码蛋白由915个氨基酸组成,预测理论分子量为103.94 kD,等电点为5.59;蛋白质二级结构以无规则卷曲为主;将氨基酸序列输入UniProt比对后发现,黑曲霉NCU-317的产酶序列与黑曲霉A0A254UAG7同源性最高,达到了99.6%。

A strain named Aspergillus niger NCU-317 which is capable of producing α-L-rhamnosidase was screened in our laboratory.Due to the unknown sequence of the intact enzyme,in this study,the cloned Aspergillus niger NCU-317 cDNA was successfully cloned by reverse transcription polymerase chain reaction(RT-PCR) and modified rapid amplification of cDNA ends(RACE).Its α-L-rhamnosidase gene is full length.A total of 3018 bp of the α-L-rhamnosidase gene was cloned,which contained a complete open reading frame of 2748 bp.The 5’ non-coding region was 216 bp long and the 3’ non-coding region was 54 bp long.After bioinformatics analysis of the sequence,the encoded protein consists of 915 amino acids with a predicted theoretical molecular weight of 103.94 kD and an isoelectric point of 5.59.The secondary structure of protein was dominated by random coils;When the amino acid sequence was imported into UniProt,it was found that the sequence of Aspergillus niger NCU-317 had the highest homology(99.6%) with the protein(Aspergillus niger A0 A254 UAG7).