| 摘要: |
| 【目的】克隆绿色木霉β-葡萄糖苷酶bglⅠ基因，并在粟酒裂殖酵母细胞中进行表达分析。【方法】利用PCR技术，从绿色木霉中克隆纤维素酶β-葡萄糖苷酶bglⅠ基因的DNA序列，再通过XhoⅠ和NotⅠ2个酶切位点与粟酒裂殖酵母表达载体pREP5N连接，构建酵母真核表达载体，利用电转化方法转化粟酒裂殖酵母SP-Q01细胞，并在SP-Q01中进行表达分析，最后采用DNS法测定表达产物的酶学活性。【结果】PCR扩增得到2 380 bp的bglⅠ基因序列，包括完整的CDS序列和信号肽序列，编码序列与已报道的里氏木霉纤维素酶基因序列相似性为100%；对酵母转化子进行诱导培养后，SDS-PAGE电泳检测得到大小为76 ku的目的蛋白条带，在SP-Q01细胞中重组酶活性最高达155 U/mL，最佳培养时间为72 h，产生酶活的最适温度为60 ℃。【结论】成功克隆出了绿色木霉β-葡萄糖苷酶bglⅠ基因的DNA序列，该基因能在粟酒裂殖酵母细胞中成功表达，为绿色木霉β-葡萄糖苷酶bglⅠ基因的深入研究和应用奠定了基础。 |
| 关键词: 绿色木霉 β-葡萄糖苷酶 粟酒裂殖酵母 酶学活性 |
| DOI： |
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| 基金项目:国家自然科学基金面上项目(31171568)；吉林农业大学博士科研启动基金项目（201202） |
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| Expression and functional analysis of a β-glucosidase gene in Schizosaccharomyces pombe |
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FU Yongping1, LI Bo2, WANG Peiwu,etal1
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1.College of Agronomy,Jilin Agricultural University;2.Jilin City Academy of Agricultural Sciences
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| Abstract: |
| 【Objective】The study aimed to clone β-glucosidase (bglⅠ) gene from Trichoderma viride and analyze its expression in Schizosaccharomyces pombe.【Method】The bglⅠ gene was cloned by PCR method,and inserted into the expressing vector pREP5N of Schizosaccharomyces pombe to generate a recombinant plasmid pREP5N-bglⅠ.The recombinant pREP5N-bglⅠ plasmid was transformed into S.pombe SP-Q01 by electroporation method.Using cellubiohydrolase activity test and SDS-PAGE analysis,the bglⅠ was expressed by S.pombe bglⅠ.【Result】Sequence analysis showed that PCR amplification generated a gene sequence with 2 380 bp,including coding sequence and signal peptide sequence.The DNA sequence and the putative amino acid sequence shared 100% identity with the reported sequence.The molecular weight of purified protein product from S.pombe bglⅠ was 76 ku.The cellubiohydrolase activity of the cultivation supernatant of recombinant S.pombe bglⅠ was 155 U/mL.The enzyme activity culminated when being cultured for 72 h,and the optimal enzyme reaction temperature was 60 ℃.【Conclusion】The bglⅠ gene from Trichoderma viride was cloned,and its function expression in eukaryotic cells was analyzed.This could provide a foundation for further research and application of the cellulase bglⅠ gene from Trichoderma viride. |
| Key words: Trichoderma viride bglⅠ gene Schizosaccharomyces pombe cellubiohydrolase activity |
