福建农业学报2025，Vol.40Issue(1)：10-17,8.
木薯MePOD10基因克隆及原核表达分析
Cloning and Prokaryotic Expression of Cassava MePOD10
摘要
Abstract
[Objective]The key gene associated with Class III peroxidase(POD)in stress responses of animals and plants,MePOD10from cassava was cloned for bioinformatics,prokaryotic expression,and protease kinetics analyses.[Methods]The coding sequence(CDS)of MePOD10 of South China 124 cassava cultivar(SC124)was amplified and subjected to bioinformatics analysis.A MePOD10-pET28a fusion expression vector was constructed and transformed into BL21 competent cells for protein induction.The protein was confirmed on its expression by SDS-PAGE and western blot and purified for enzymatic activity determination and kinetic analysis.[Results]The length of MePOD10 CDS was 981 bp encoded 326 amino acids with a molecular weight of 35 069.60 Da and a theoretical isoelectric point of 6.59.The unstable and hydrophobic MePOD10 contained a conserved domain of plant POD sharing the highest amino acid sequence similarity of 93.25%with that of rubber trees.Induced by 1 mmol·L-1 isopropyl-β-D-thiogalactoside(IPTG)at 37℃for 6 h,the protein expressions in the supernatant and precipitate were examined.The purified supernatant showed greater enzymatic activity than control.When guaiacol was used as a substrate,the catalytic activity of MePOD10 increased rapidly with increasing substrate concentration to reach a peak.[Conclusion]MePOD10 was determined to contain conserved POD domain.MePOD10-pET28a could be correctly expressed under the induction of 1 mmol·L-1 IPTG at 37℃for 6 h,and the purified fusion protein exhibited a significant catalytic activity.关键词
木薯/过氧化物酶/原核表达/酶活性Key words
Cassava/peroxidase/prokaryotic expression/enzyme activity分类
农业科学引用本文复制引用
何雪强,吴姝,林慧靖,陈奥,王苗,史钊辉,闫语..木薯MePOD10基因克隆及原核表达分析[J].福建农业学报,2025,40(1):10-17,8.基金项目
海南省自然科学基金高层次人才项目(323RC410) （323RC410）
海南省重点研发项目(ZDYF2024XDNY237) （ZDYF2024XDNY237）
海南大学科研启动基金项目[KYQD(ZR)-22108] （ZR）
