以黑曲霉(Aspergillus niger)A3的基因组DNA为模板，根据已报道的xynB基因序列设计简并引物，在合适的PCR反应条件下有效地扩增出了xynB的结构基因及其5’调控区序列片段，并克隆到pBS-T载体上，序列测定表明，该目的片段全长1611 bp。通过序列分析：结构基因部分长744 bp，其中含有一个66 bp的内含子和18个氨基酸的信号肽序列；xynB基因的cDNA序列大小为678 bp，编码225个氨基酸，与GenBank上检索的木聚糖酶基因的核苷酸序列同源性最高达98%，氨基酸序列同源性达99%。在翻译起始点上游92 bp和118 bp处找到转录起始位点和类“TATA”box的核心启动子区特征元件以及“CAAT”box，表明所克隆的调控区序列具有真核生物启动子特点，这为构建木聚糖酶高效表达载体，用于工业育种奠定了基础。
In this paper,the cloning of a β-xylanase structural gene and its 5′flanking regions gene from Aspergillus niger was studied.On the base of nucleic acid DNA sequence of β-xylanase gene,a pair of degenerate primers was designed.Using the genomic DNA of Aspergillus niger as template,a 1.6 kb fragment was amplified by PCR and cloned into the vector pBS-T.Sequence analysis showed that the structural gene of β-xylanase is 744 bp length including a 66 bp intron and a 678 bp encoding region,which encoded a polypeptide of 225 amino acids and included the signal sequence of 18 amino acids.The homology analysis of the xynB cDNA sequence shares 98% in base sequence with the β-xylanase gene in GeneBank and the amino acid sequence shares 99%.Upstream of the tranlation start site,there were classic core element of Eukaryotic Gene Promoter,such as transcription start site,TATA box and CAAT box elements.The work was beneficial in construction of homologus expression for xylanase efficient expression vector.
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