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棘孢木霉木聚糖酶Ⅱ结构基因和上游调控区的克隆及分析
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国家自然科学基金项目(31071837);广东省科技计划项目(2008B011000005)


Cloning and Sequence Analysis of Xylanase II Structural Gene and Its 5’ Flanking Region of Trichoderma asperellum
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    摘要:

    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1 875 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,Ace1,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。

    Abstract:

    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-11 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,Ace1,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.

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庞漫宇,郭丽琼,林俊芳.棘孢木霉木聚糖酶Ⅱ结构基因和上游调控区的克隆及分析[J].食品与生物技术学报,2011,30(2):245-249.

PANG Man-yu, GUO Li-qiong, LIN Jun-fang. Cloning and Sequence Analysis of Xylanase II Structural Gene and Its 5’ Flanking Region of Trichoderma asperellum[J]. Journal of Food Science and Biotechnology,2011,30(2):245-249.

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