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几丁质酶作为木霉菌防治植物病虫害的主要因子,在生物防治和环境保护等领域发挥着重要的作用.为了研究棘孢木霉(Trichoderma asperellum)的生防机制并获得与其相关的功能基因,本研究通过RT-PCR、3’-RACE及5’-TAIL-PCR技术克隆了T.asperellum 1个几丁质酶基因Tachi1,对该基因进行了生物信息学分析,并利用毕赤酵母表达系统进行表达验证.Tachi1的DNA序列长1 635 bp,含有3个内含子,包含1 275 bp的开放阅读框,编码424个氨基酸;Tachi1属于糖基水解酶18家族内切几丁质酶,包含SIGGW底物结合位点和FDGIDXDWE活性中心位点,信号肽长度为22个氨基酸,成熟肽分子量为44 kD,二级结构以α-螺旋、β-折叠和无规则卷曲为结构元件,三级结构为(α/β)8的圆桶形结构.转Tachi1基因酵母工程菌可高效分泌表达几丁质酶Tachi1,甲醇诱导培养8 d几丁质酶酶活可达9.25 U/mL.
Chitinase plays an important role in the prevention and control of plant diseases and insect pests as Trichoderma.In order to study the mechanism of biocontrol of Trichoderma asperellum and obtain the related functional genes, In this study, a chitinase gene Tachi1 of T.asperellum was cloned by RT-PCR, 3’-RACE and 5’-TAIL-PCR. Bioinformatic analysis of this gene was carried out. The Pichia pastoris expression system The Tachi1 DNA was 1635 bp in length and contained 3 introns, including an open reading frame of 1 275 bp, encoding 424 amino acids. Tachi1 belongs to the endo-chitinase of glycosyl hydrolase 18 family and contains SIGGW substrate binding site and FDGIDXDWE active site, the signal peptide is 22 amino acids in length and the mature peptide has a molecular weight of 44 kD. The secondary structure is composed of α-helix, β-sheet and random coil. The tertiary structure (Α / β) 8. The Tachi1 gene yeast engineered bacteria can efficiently express chitinase Tachi1, and the methanol-induced chitinase activity reached 9.25 U / mL for 8 days.