Comparative Transcriptomics Analysis for Gene Mining and Identification of a Cinnamyl Alcohol Dehydrogenase Involved in Methyleugenol Biosynthesis from Asarum sieboldii Miq.
Abstract
:1. Introduction
2. Results
2.1. De Novo Assembly and Sequence Annotation
2.2. Gene Ontology Classification, COG Classification, and Metabolic Pathway Assignment by KEGG
2.3. Gene Expression Analysis of AsCADs by qRT-PCR
2.4. Identifying Genes Involved in Biosynthesis of Phenylpropanoids Pathway in A. sieboldii
2.5. Cloning and Phylogenetic Analysis of AsCADs
2.6. Cloning and Heterologous Expression of AsCADs
2.7. CAD Activity and Kinetic Parameters of the Recombinant AsCADs
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Illumina Sequencing for Transcriptome Analysis
4.3. De Novo Assembly and Functional Annotation Analysis
4.4. Quantitative Real-Time PCR Validation
4.5. Identifying Genes Related to Biosynthesis of Methyleugenol and Asarinin in A. sieboldii
4.6. Multiple Sequence Alignments and Phylogenetic Analysis of AsCADs
4.7. Cloning of AsCADs
4.8. Heterogeneous Expression of Recombinant AsCADs
4.9. CAD Activity Assay and Determination of Kinetic Parameters
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Not available. |
Public Database | Number of Unigenes Annotated in Asarum sieboldii |
---|---|
Nr | 127,167 |
Swiss-Prot | 76,685 |
COG | 22,386 |
KEGG | 35,326 |
Enzyme Kinetic Parameters | Coniferyl Aldehyde | Sinapyl Aldehyde | P-coumaryl Aldehyde |
---|---|---|---|
Km (µM) | 27.32 ± 6.186 | 35.02 ± 8.043 | 45.52 ± 8.888 |
Vmax (nmol s−1 mg protein−1) | 342 ± 26.29 | 448.5 ± 50.54 | 383.8 ± 31.63 |
Kcat (s−1) | 221.7 | 290.8 | 248.8 |
Kcat/Km (µM−1 s−1) | 8.115 | 8.304 | 5.466 |
▲ 48 | ● 75 | ● 86 | ▲ 94 | ■ 118 | ■ 122 | ● 275 | ● 276 | ● 285 | ● 299 | ● 300 | |
---|---|---|---|---|---|---|---|---|---|---|---|
AaCAD | T | V | V | C | W | D | V | I | P | F | I |
AtCAD5 | T | V | V | C | W | D | V | I | P | F | I |
AsCAD5 | A | Q | I | T | 119T | 123S | 276F | 277P | G | V | T |
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Liu, J.; Xu, C.; Zhang, H.; Liu, F.; Ma, D.; Liu, Z. Comparative Transcriptomics Analysis for Gene Mining and Identification of a Cinnamyl Alcohol Dehydrogenase Involved in Methyleugenol Biosynthesis from Asarum sieboldii Miq. Molecules 2018, 23, 3184. https://doi.org/10.3390/molecules23123184
Liu J, Xu C, Zhang H, Liu F, Ma D, Liu Z. Comparative Transcriptomics Analysis for Gene Mining and Identification of a Cinnamyl Alcohol Dehydrogenase Involved in Methyleugenol Biosynthesis from Asarum sieboldii Miq. Molecules. 2018; 23(12):3184. https://doi.org/10.3390/molecules23123184
Chicago/Turabian StyleLiu, Jinjie, Chong Xu, Honglei Zhang, Fawang Liu, Dongming Ma, and Zhong Liu. 2018. "Comparative Transcriptomics Analysis for Gene Mining and Identification of a Cinnamyl Alcohol Dehydrogenase Involved in Methyleugenol Biosynthesis from Asarum sieboldii Miq." Molecules 23, no. 12: 3184. https://doi.org/10.3390/molecules23123184