Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants
Abstract
:1. Introduction
2. Results
2.1. Identification and Analysis of Cinnamoyl-CoA Reductase (CCR) Genes in M. sativa Genome
2.2. Gene Structure, Evolution, and Conserved Motif Analysis of MsCCRs/MsCCR-like Genes
2.3. The Cis-Acting Regulatory Elements in the Promoter of MsCCRs
2.4. The Expression Patterns of MsCCR/MsCCR-like Genes and Lignin Content Analysis during Different Growth Stage
2.5. The Expression Patterns of MsCCRs/MsCCR-likes under Abiotic Stresses and Hormone Treatment
2.6. Cloning, Heterologous Expression, and Enzymatic Assay of MsCCR1 and MsCCR-like1 Gene
2.7. Three-Dimensional Structure Analysis of CCRs in M. sativa and Other Plants
3. Discussion
4. Materials and Methods
4.1. Plant Growth and Treatments
4.2. MsCCR Identification and Classification
4.3. Motif Analysis and Gene Structure Visualization
4.4. Multiple Sequence Alignment and Phylogenic Analysis Evolutionary Analysis
4.5. Cis-regulatory Element Analysis and 3D Structure Prediction
4.6. Collinearity Analysis and Chromosome Localization
4.7. RNA Isolation and RT-qPCR Analysis
4.8. Lignin Content and Staining Analysis
4.9. Cloning, Expression, and Purification of Recombinant MsCCR
4.10. CCR Activity and Enzyme Kinetic Parameter Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Symbols | Feruloyl-CoA | p-Coumaroyl-CoA | Caffeoyl-CoA | Sinapoyl-CoA | ||||
---|---|---|---|---|---|---|---|---|
Km | Kcat/Km | Km | Kcat/Km | Km | Kcat/Km | Km | Kcat/Km | |
MsCCR1 | 45.20 ± 11.42 ab | 2.09 ± 0.18 B | 24.22 ± 4.51 bc | 2.58 ± 0.11 B | ND | ND | 10.57 ± 1.25 c | 5.72 ± 0.36 A |
MsCCR-like1 | ND | ND | 44.14 ± 7.12 ab | 1.09 ± 0.19 C | 48.16 ± 1.75 a | 1.09 ± 0.11 C | ND | ND |
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Cui, W.; Zhuang, Z.; Jiang, P.; Pan, J.; Zhao, G.; Xu, S.; Shen, W. Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants. Int. J. Mol. Sci. 2022, 23, 7762. https://doi.org/10.3390/ijms23147762
Cui W, Zhuang Z, Jiang P, Pan J, Zhao G, Xu S, Shen W. Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants. International Journal of Molecular Sciences. 2022; 23(14):7762. https://doi.org/10.3390/ijms23147762
Chicago/Turabian StyleCui, Weiti, Zihan Zhuang, Peihao Jiang, Jincheng Pan, Gan Zhao, Sheng Xu, and Wenbiao Shen. 2022. "Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants" International Journal of Molecular Sciences 23, no. 14: 7762. https://doi.org/10.3390/ijms23147762