Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana
Abstract
:1. Introduction
2. Results and Discussion
2.1. Molecular Cloning of the AcCP2 Gene
2.2. Structure Characteristics of the AcCP2 Protein
2.3. Expression Patterns of AcCP2 During Pineapple-Fruit Development
2.4. Expression of His-AcCP2 Recombinant Protein
2.5. Catalytic Characteristics of His-AcCP2 Recombinant Protein Using Synthetic Substrates
Proteinase | Substrates | Km (μM) | kcat (S−1) | kcat/Km (Mm−1·S−1) |
---|---|---|---|---|
AcCP2 a | Z-Arg-Arg-NH-Mec | 72.3 ± 6.4 | 0.004 ± 0.001 | 0.005 |
Bz-Phe-Val-Arg-NH-Mec | 3.2 ± 1.1 | 15.4 ± 4.8 | 4812.5 | |
Commercial bromelain b | Z-Arg-Arg-NH-Mec | 82.5 ± 10.2 | 0.003 ± 0.001 | 0.003 |
Bz-Phe-Val-Arg-NH-Mec | 4.6 ± 1.0 | 14.12 ± 5.2 | 3065 |
2.6. Disease Resistance Analysis in Transgenic Arabidopsis Plants
2.7. Discussion
3. Experimental
3.1. Plant Materials
3.2. Isolation of AcCP2 Gene
3.3. Extraction and Purification of Plant Proteins
3.4. RNA Blot Analysis
3.5. Expression and Purification of pPIC9K-AcCP2 in P. pastoris
3.6. Antibodies and Western Blots
3.7. Enzymatic Activity of Recombinant Proteinase
3.8. Measurement of Sugar Contents and Invertase Activity in Pineapple Fruits
3.9. Agrobacterium-Mediated Floral Dip Transformation of Arabidopsis
3.10. Treatment of Arabidopsis Plants
3.11. Bioinformatic Analysis
4. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Wang, W.; Zhang, L.; Guo, N.; Zhang, X.; Zhang, C.; Sun, G.; Xie, J. Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana. Molecules 2014, 19, 2374-2389. https://doi.org/10.3390/molecules19022374
Wang W, Zhang L, Guo N, Zhang X, Zhang C, Sun G, Xie J. Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana. Molecules. 2014; 19(2):2374-2389. https://doi.org/10.3390/molecules19022374
Chicago/Turabian StyleWang, Wei, Lu Zhang, Ning Guo, Xiumei Zhang, Chen Zhang, Guangming Sun, and Jianghui Xie. 2014. "Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana" Molecules 19, no. 2: 2374-2389. https://doi.org/10.3390/molecules19022374
APA StyleWang, W., Zhang, L., Guo, N., Zhang, X., Zhang, C., Sun, G., & Xie, J. (2014). Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana. Molecules, 19(2), 2374-2389. https://doi.org/10.3390/molecules19022374