中文摘要：

目的：克隆及分析棘孢木霉木聚糖酶Ⅰ结构基因和上游调控区,以获得内源启动子。方法：根据木霉属木聚糖酶Ⅰ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。结果：扩增获得1.2 kb的片段,酶切鉴定及序列分析表明,该片段长1 265 bp,由753 bp的木聚糖酶Ⅰ结构基因和512 bp的上游调控区组成。结构基因编码230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、XlnR、Ace1、AreA等多个转录因子结合位点。结论：克隆的512 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。

英文摘要：

Objective： To study the native promoter of xylanase Ⅰ of Trichoderma asperellum,cloning and sequence analysis of xylanase I structural gene and its 5′ flanking region was performed.Method： On the basis of the genomic conserved sequence of Trichoderma spp.,degenerate PCR was designed to amplify the xylanaseⅠstructural gene and its 5′flanking region of Trichoderma asperellum.The product was cloned into T vector and confirmed by EcoRⅠ digestion.Sequence analysis was then carried out.Result： An 1.2 kb fragment was amplified.Sequence analysis showed that this fragment was 1 265bp long,comprising the 753 bp xylanase Ⅰ structure gene and its 512 bp 5′flanking region.The structure gene encoded a polypeptide of 230 amino acids,where the Glyco-hydro-11 superfamily domain were detected.In the 5′ flanking region,core promoter region,transcriptional start site,CAAT-Box and TATA-Box were detected.Several typical transcripional factor binding domain,such as CreⅠ,XlnR,Ace1,AreA were also found.Conclusion： The 512 bp 5′ flanking region was an typical promoter.The isolation of this native promoter can benefit the development of an efficient Trichoderma asperellum host system for gene expression.