To study the native promoter of xylanase Ⅱ of Trichoderma asperellum, cloning and sequence analysis of xylanase Ⅱ structural gene and its 5' flanking region was performed.On the basis of the genomic conserved sequence of Trichoderma spp., degenerate PCR was designed to amplify the xylanase Ⅱ structural gene and its 5'flanking region of Trichoderma asperellum.The product was cloned into T vector and confirmed by EcoR Ⅰ digestion.Sequence analysis showed that cloned fragment was 1 875 bp long, comprising the 795 bp xylanase Ⅱ structure gene and its 1 080 bp 5'flanking region.The structure gene encoded a polypeptide of 223 amino acids, where the Glyco-hydro-ll superfamily domains were detected.In the 5' flanking region, core promoter region, transcriptional start site, CAAT-Box and TATA-Box were detected.Several typical transcriptional factor binding domain, such as CreI, AreA, XlnR, Acel, AlcR were also found.The 1080 bp 5' flanking region was a typical promoter.The isolation of this native promoter can benefit the development of an efficient Trichoderma asperellum host system for gene expression.%作者克隆及分析了棘孢木霉木聚糖酶Ⅱ结构基因和上游调控区,并获得了内源启动子.根据木霉属木聚糖酶Ⅱ结构基因及上游调控区的保守性,以棘孢木霉基因组DNA为模板,进行简并PCR扩增,产物纯化并克隆至T载体,经酶切鉴定后进行序列分析.扩增获得片段长1875 bp,由795 bp的木聚糖酶Ⅱ结构基因和1 080 bp的上游调控区组成.结构基因编码223个氨基酸,具有糖基水解酶第11家族的典型保守区域.上游调控区具备核心启动子和转录起始点,有CAAT-Box,TATA-Box等启动子特征元件,分析其还有CreI,AreA,XlnR,Acel,AlcR等转录因子结合位点.综上表明,克隆的1 080 bp上游调控区是典型的丝状真菌基因启动子,可作为内源启动子用于构建棘孢木霉高效外源基因表达系统.
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