| 引用本文： |
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王利英,刘一,杨登峰,黄日波.绿色木霉葡聚糖内切酶cDNA基因的克隆及其在酿酒酵母中的表达[J].广西科学,2007,14(3):315-319. [点击复制]
- WANG Li-ying,LIU Yi,YANG Deng-feng,HUANG Ri-bo.Cloning of the Endoglucanase cDNA Genes from Trichoderma viride and Their Expression in Saccharomyces cerevisiae[J].Guangxi Sciences,2007,14(3):315-319. [点击复制]
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| 摘要: |
| 用L-山梨糖诱导绿色木霉(Trichoderma viride)AS3.3711纤维素酶基因的转录,提取其总RNA反转录获得cDNA。PCR扩增葡聚糖内切酶Ⅲ(EGⅢ)和葡聚糖内切酶(EG)的cDNA基因,将其克隆到酵母载体中,构建产生纤维素酶的酿酒酵母工程菌。重组菌株能够识别EGⅠ和EGⅢ自身携带的信号肽而将表达产物分泌到胞外,故可采用刚果红平板染色法筛选具有羧甲基纤维素酶(CMCase)活性的重组转化子。重组菌株表达的EGⅠ酶活在诱导70h时达到最高(为0.08U/ml),表达的EGⅢ酶活在诱导60h时达到最高(为0.03U/ml)。 |
| 关键词: 绿色木霉 葡聚糖内切酶 酿酒酵母 |
| DOI： |
| 投稿时间：2007-05-31 |
| 基金项目:国家自然科学基金项目(20666002);广西科技攻关项目(桂科攻0537012)联合资助 |
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| Cloning of the Endoglucanase cDNA Genes from Trichoderma viride and Their Expression in Saccharomyces cerevisiae |
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WANG Li-ying1, LIU Yi1, YANG Deng-feng2, HUANG Ri-bo1,2
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| (1.College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530004, China;2.Guangxi Academy of Sciences. Nanning, Guangxi, 530007, China) |
| Abstract: |
| The cellulase genes from Trichoderma viride AS3.3711 were induced with L-sorbose.Its total RNA was extracted.The cDNA was obtained by reverse transcription.The cDNA genes encoding endoglucanases Ⅰ and Ⅲ were isolated by PCR, and then they were cloned into a yeast vector to construct the cellulases-producing Saccharomyces cerevisiae engineering strains. Recombinant yeast strains can secret EGsⅠ and Ⅲ by the guide of the signal peptide themselves.So the expressions of EGⅠ and EGⅢ can be screened by activity plate assays with Congo Red method respectively.The endoglucanase activity was assayed with CMC-Na as a substrate.The results showed that the enzyme activities of EGⅠ and EGⅢ culminated at 0.08 U/ml and 0.03 U/ml respectively when they were induced for 70h and 60h respectively. |
| Key words: Trichoderma viride endoglucanase Saccharomyces cerevisiae |
