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棘孢木霉(Trichoderma asperellum)几丁质酶基因的克隆与生物信息学分析
引用本文:汤伟,夏伟,李雅华,丛大鹏,咸洪泉.棘孢木霉(Trichoderma asperellum)几丁质酶基因的克隆与生物信息学分析[J].中国生物化学与分子生物学报,2012,28(4):385-392.
作者姓名:汤伟  夏伟  李雅华  丛大鹏  咸洪泉
作者单位:青岛农业大学生命科学学院应用真菌实验室
基金项目:山东省自然科学基金项目(No.ZR2010CM040);青岛农业大学高层人才启动基金项目(No.631108)~~
摘    要: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.

关 键 词:棘孢木霉  几丁质酶  克隆  表达  生物信息学分析  
收稿时间:2011-12-18

Cloning and Bioinformatics Analysis of Chitinase Gene Tachi1 from Trichoderma asperellum
TANG Wei,XIA Wei,LI Ya-Hua,CONG Da-Peng,XIAN Hong-Quan.Cloning and Bioinformatics Analysis of Chitinase Gene Tachi1 from Trichoderma asperellum[J].Chinese Journal of Biochemistry and Molecular Biology,2012,28(4):385-392.
Authors:TANG Wei  XIA Wei  LI Ya-Hua  CONG Da-Peng  XIAN Hong-Quan
Institution:(Applied Microbiology Laboratory,College of Life Sciences,Qingdao Agricultural University,Qingdao 266109,Shandong,China)
Abstract:Chitinases, are thought to be a major factor for resisting plant pests and diseases by Trichoderma, and play an important role in biological control and environmental protection. In order to study bio-control mechanism and obtain the related functional genes of T. asperellum, the full-length DNA and mRNA of (GenBank: GU457410, GU457411) were cloned by using RT-PCR, 3′-RACE and 5′-TAIL-PCR technology, bioinformatics analysis of the gene was performed and also Tachi1 was transformed into Pichia pastoris expression system. Results showed that the full-length DNA of Tachi1 was 1635 bp, including an ORF with 1275 bp, contained three introns, and encoded 424 amino acids. Its product, Tachi1 belonged to Family 18 glycoside hydrolases. The sequence contained a substrate binding site (SIGGW), an active center site (FDGIDXDWE), and a 22- amino acid signal peptide sequence. The molecular weight of mature peptide was about 44 kDa. α-helices, β-sheets and random coils were the major structural elements in secondary structure of Tachi1, while (α/β)8 rounded buckets appeared in tertiary structure. The recombinant plasmid pPIC9K/Tachi1 was constructed and transformed into P. pastoris. The recombinant chitinase Tachi1 was secreted successfully with the yield reaching 9.25 U/mL by the 8-day methanol induction.
Keywords:T  asperellum  chitinase  cloning  expression  bioinformatics analysis  
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