嗜热子囊菌光孢变种cbh1基因的cDNA克隆及在毕赤酵母的高效表达  

cDNA Cloning of the Cellobiohydrolase Gene cbh1 from Thermoascus aurantiacus var. levisporus and Its Expression in Pichia pastoris

作　　者：陈静[1] 李多川[1] 张玉芹[1] 杜欣可[1] 

机构地区：[1]山东农业大学植物保护学院环境生物系,泰安271018

出　　处：《农业生物技术学报》2006年第3期406-411,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2003AA241162);国家自然科学基金项目(No.30270013;30170013)资助。

摘　　要：以嗜热子囊菌光孢变种(Thermoascusaurantiacusvar.levisporus)总RNA为模板,通过RT-PCR克隆出外切纤维二糖水解酶基因cbh1片段,采用RACE方法获得全长cDNA克隆,其全长为1710bp,编码一种由457个氨基酸组成的单肽,推导的氨基酸序列中1～19位为信号肽序列,GenBank的登录号为AY840982。将该片段克隆到毕赤酵母(Pichiapastoris)分泌型表达载体pPIC9K上,获得表达重组质粒pPIC9K/cbh1,转化毕赤酵母GS115,所得重组子经PCR验证后进行诱导表达,筛选出一重组子GSp-15,经144h诱导后,外切纤维二糖水解酶表达量为1.17mg/mL,产酶活力为20.3U/mL。The cellobiohydrolase gene cbhl fragment （GenBank Accession No. AY840982） was amplified by RT-PCR from thermophilic fungus Thennoascus aurantiacus var. levisporus. RACE was used to obtain its full-length cDNA （1710 bp）, encoding 457 amino acids. The first 19 amino acids of the deduced amino acid sequence were presumed to be a signal peptide. The fragment encoding mature cellobiohydrolase was inserted into Pichia pastoris vector pPIC9K to construct recombinant plasmid pPIC9K/cbhl. The pPIC9K/cbhl was then introduced into Pichia pastoris GS115 and 61 transformants were obtained, After confirmed by G418 resis tance and PCR, and induced expression, one clone GSp-15 was selected from the 61 tmnsformants. The expression level of GSp-15 was 1.17 mg/mL after induction for 144 h in methanol, and its activity was 20.3 U/mL with p-NPC as substrate.

关 键 词：嗜热子囊菌光孢变种 外切纤维二糖水解酶 CDNA克隆 RACE 毕赤酵母 

分 类 号：S432.4[农业科学—植物病理学]