ZENG Jia-yu,ZHENG Qun,LIAO Shi-qi,et al.Construction of Soybean Peroxidase Gene Random Mutation Library[J].Soybean Science,2011,30(05):731-737.[doi:10.11861/j.issn.1000-9841.2011.05.0731]
大豆过氧化物酶基因随机突变文库的构建
- Title:
- Construction of Soybean Peroxidase Gene Random Mutation Library
- 文章编号:
- 1000-9841（2011）05-0731-07
- Keywords:
- Soybean peroxidase; Error-prone PCR; Gene library; Random mutation; Directed evolution of enzymes
- 分类号:
- S565.1
- 文献标志码:
- A
- 摘要:
- 为提高大豆过氧化物酶（soybean peroxidase，SBP）的活性及研究该酶一级结构与酶活性间的关系，采用连续易错PCR方法对大豆过氧化物酶基因（sbp）进行了2轮随机突变，将第二轮易错PCR产物与质粒载体pPICZα-A分别进行双酶切，经T4DNA连接酶催化连接后，CaCl2法转化重组质粒pPICZα-A-sbp至大肠杆菌DH5α中，成功构建出sbp随机突变文库，库容量约为1.77×106 cfu。
- Abstract:
- In order to improve enzymatic activity of soybean peroxidase(SBP)and study the correlation between its primary structure and enzymatic activity，sequential error-prone PCR method was used on soybean peroxidase genes（sbp） for two times randon mutation.The second round error-prone PCR products and plasmid vectors pPICZα-A were digested by double enzymes.After the PCR products ligated into the vectors by T4DNA ligase，recombinant plasmids pPICZα-A-sbp were transformed into Escherichia coli DH5α through calcium chlorid method.A random mutation library was constructed，and the capacity was about 1.77×106 cfu.
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备注/Memo
基金项目：甘肃省教育厅资助项目(0601-28,0501B-16)；甘肃省自然科学基金资助项目(096RJZA035)；甘肃省高分子材料重点实验室开放课题资助项目(KF-06-01)。