Molecular Cloning and Functional Analysis of Cysteine Protease Inhibitor Gene AtCYS6 in Arabidopsis thaliana

doi:10.7668/hbnxb.20190371

Abstract

Abstract: In order to study the role and function of the cysteine protease inhibitor AtCYS6 gene in plant growth and development,the AtCYS6 gene was cloned from Arabidopsis thaliana. Bioinformatics analysis,subcellular localization analysis,protein induction,expression,purification and functional were carried out to study this gene. The results showed that the isoelectric point of AtCYS6 protein was 5.85,the chemical formula was C₁₁₇₉H₁₈₄₈N₃₂₂O₃₄₈S₆,which contained 3 703 atoms and it was a hydrophilic protein;AtCYS6 had high homology with Capsella rubella and Camelina sativa,the homology was 81.1% and 80.3% respectively,and it had low homology with Oryza sativa, Zea mays and Glycine max,the homology was 55.5%,54.3% and 56.6% respectively,among the amino acid sequences,there was a highly conserved Q-V-G (Gln-Val-Gly) sequence;evolutionary tree analysis showed that AtCYS6 was closely related to Capsella rubella and Camelina sativa,but far from Oryza sativa, Zea mays, Sorghum bicolor and Setaria italic;subcellular localization results showed that it was located in cytoplasm,which was consistent with the predicted results;the fusion protein was induced,expressed and purified to be about 52 ku in size,the molecular weight of AtCYS6 protein was about 26 ku,which accorded with the predicted value;Western Blot analysis showed that the fusion protein was correctly translated and expressed without structural breakage or degradation;enzyme activity analysis showed that AtCYS6 had inhibitory effect on papain. The results of this study provide a basis for further study of the related mechanism of AtCYS6 in plants.

Key words: Arabidopsis thaliana, Cysteine protease inhibitor, Subcellular localization, Prokaryotic expression, Enzyme activity

CLC Number:

Q78
Q943.2

GUO Kunyuan, ZHANG Xinxin. Molecular Cloning and Functional Analysis of Cysteine Protease Inhibitor Gene AtCYS6 in Arabidopsis thaliana[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(4): 8-14. doi: 10.7668/hbnxb.20190371.

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