Integrated Metabolomic and Transcriptomic Analysis Reveals Differential Mechanism of Flavonoid Biosynthesis in Two Cultivars of Angelica sinensis
Abstract
:1. Introduction
2. Results
2.1. Differenence of Flavonoid and Anthocyanin Contents between the Two Cultivars
2.2. Targeted-Flavonoids Metabolomic Analysis
2.2.1. Identification of Differentially Accumulated Flavonoids (DAFs) in M1 vs. M2
2.2.2. Pathway Enrichment of DAFs
2.3. Isoforms Analysis
2.4. Transcriptomic Analysis between M1 and M2
2.4.1. Global Gene Analysis
2.4.2. Identification of Differentially Expressed Genes (DEGs)
2.4.3. Functional Annotation and Enrichment of DEGs
2.5. DEGs Involved in Flavonoids Biosynthesis
2.6. Network of DAFs Regulated by DEGs
2.7. qRT-PCR Validation of Candidate Genes Involved in Flavonoid Biosynthesis
3. Discussion
4. Materials and Methods
4.1. Plant Material
4.2. Chemicals
4.3. Measurement of Total Flavonoid and Anthocyanin Contents
4.3.1. Measurement of Total Flavonoid Content
4.3.2. Measurement of Anthocyanin Content
4.4. Metabolomic Analysis
4.4.1. Sample Preparation and Extraction
4.4.2. UPLC Analysis
4.4.3. MS/MS Analysis
4.4.4. Metabolites Identification
4.4.5. Differential Metabolites Analysis
4.5. Isoform Sequencing and Transcriptomic Analysis
4.5.1. cDNA Library Construction and Single Molecular Real-Time (SMRT) Sequencing
4.5.2. Isoform Data Processing
4.5.3. Transcriptomic Analysis and DEGs Identification
4.6. qRT-PCR Validation of Genes Involved in Flavonoid Biosynthesis
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
DAFs | differentially accumulated flavonoids |
DEGs | differentially expressed genes |
DR | down-regulated |
FLNC | full-length non-chimeric |
GO | Gene Ontology |
KEGG | Kyoto Encyclopedia of Genes and Genomes |
KOG | euKaryotic orthologous groups of proteins |
M1 | Mingui 1 |
M2 | Mingui 2 |
NCBI | National Center for Biotechnology Information |
OPLS-DA | orthogonal projection to latent structures-discriminant analysis |
PCA | principal component analysis |
REL | relative expression level |
RPKM | Reads Per kb per Million |
TF | transcription factor |
UR | up-regulated |
References
- Upton, R. American Herbal Pharmacopoeia and Therapeutic Compendium: Dang Gui Root-Angelica sinensis (Oliv.); American Herbal Pharmacopoeia: Scotts Valley, CA, USA, 2003; pp. 1–41. [Google Scholar]
- Zhang, H.Y.; Bi, W.G.; Yu, Y.; Liao, W.B. Angelica sinensis (Oliv.) Diels in China: Distribution, cultivation, utilization and variation. Genet. Resour. Crop Evol. 2012, 59, 607–613. [Google Scholar] [CrossRef]
- Li, M.F.; Liu, X.Z.; Wei, J.H.; Zhang, Z.; Chen, S.J.; Liu, Z.H.; ** of an anthocyanin-regulating MYB transcription factor and its expression in red and green pear, Pyrus communis. Plant Physiol. Biochem. 2010, 48, 1020–1026. [Google Scholar] [CrossRef]
- Chen, Y.; Yang, X.; He, K.; Liu, M.; Li, J.; Gao, Z.; Lin, Z.; Zhang, Y.; Wang, X.; Qiu, X.; et al. The MYB transcription factor superfamily of Arabidopsis: Expression analysis and phylogenetic comparison with the rice MYB family. Plant Mol. Biol. 2006, 60, 107–124. [Google Scholar]
- Borevitz, J.O.; ** podophyllotoxin biosynthesis and growth-related transcripts with high elevation in Sinopodophyllum hexandrum. Ind. Crop. Prod. 2018, 124, 510–518. [Google Scholar] [CrossRef]
- Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNAseq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef] [Green Version]
- Robinson, M.D.; McCarthy, D.J.; Smyth, G.K. EdgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26, 139–140. [Google Scholar] [CrossRef] [Green Version]
- Willems, E.; Leyns, L.; Vandesompele, J. Standardization of real-time PCR gene expression data from independent biological replicates. Anal. Biochem. 2008, 379, 127–129. [Google Scholar] [CrossRef]
No. | Compounds Name | Formula | log2FC(M1 vs. M2) |
---|---|---|---|
1 | Quercetin-3-O-(6″-O-arabinosyl) glucoside | C26H28O16 | 0.90 ± 0.12 |
2 | Quercetin-3-O-arabinoside (Guaijaverin) | C20H18O11 | 0.65 ± 0.07 |
3 | Quercetin-3-O-apiosyl(1→2) galactoside | C26H28O16 | 0.65 ± 0.06 |
4 | Quercetin-3-O-sambubioside | C26H28O16 | 0.55 ± 0.01 |
5 | Quercetin-3-O-xyloside (Reynoutrin) | C20H18O11 | 0.35 ± 0.07 |
6 | Quercetin-3-O-rhamnoside (Quercitrin) | C21H20O11 | 0.34 ± 0.06 |
7 | Quercetin-7-O-rutinoside | C27H30O16 | 0.21 ± 0.02 |
8 | Quercetin-4′-O-glucoside (Spiraeoside) | C21H20O12 | −1.02 ± 0.11 |
9 | Quercetin-3-O-galactoside (Hyperin) | C21H20O12 | −0.93 ± 0.06 |
10 | Quercetin-3-O-glucoside (Isoquercitrin) | C21H20O12 | −0.78 ± 0.06 |
11 | Quercetin-7-O-glucoside | C21H20O12 | −0.66 ± 0.14 |
12 | Isorhamnetin-3-O-sophoroside | C28H32O17 | 0.63 ± 0.07 |
13 | Isorhamnetin-3-O-Glucoside | C22H22O12 | 0.38 ± 0.01 |
14 | Rhamnetin-3-O-Glucoside | C22H22O12 | 0.38 ± 0.01 |
15 | Chrysoeriol-5-O-glucoside | C22H22O11 | −2.17 ± 0.21 |
16 | Naringenin-7-O-glucoside (Prunin) | C21H22O10 | 1.15 ± 0.16 |
17 | Hesperetin-5-O-glucoside | C22H24O11 | −0.86 ± 0.11 |
18 | 6-Hydroxykaempferol-7,6-O-Diglucoside | C27H30O17 | 0.47 ± 0.06 |
19 | Kaempferol-4′-O-glucoside | C21H20O11 | −0.39 ± 0.05 |
20 | Isosalipurposide (Phlorizin Chalcone) | C21H22O10 | 1.56 ± 0.24 |
21 | Butin-7-O-glucoside | C21H22O10 | 0.95 ± 0.21 |
22 | Luteolin-7-O-rutinoside | C27H30O15 | 0.37 ± 0.06 |
23 | Pelargonidin-3-O-glucoside-5-O-arabinoside | C26H29O14+ | 18.66 ± 1.22 |
24 | Cyanidin-3-O-glucoside (Kuromanin) | C21H21O11+ | 19.15 ± 1.73 |
25 | Cyanidin-3-O-sambubioside | C26H29O15+ | 8.85 ± 1.09 |
26 | Peonidin-3-O-sambubioside | C27H31O15+ | 6.26 ± 0.85 |
M1 | M2 | |
---|---|---|
Filtered data | ||
Data of reads number (million) | 38.65 ± 1.34 | 38.73 ± 1.90 |
Data of reads number × read length (million) | 5773.93 ± 2.00 | 5784.44 ± 2.83 |
Q20(%) | 97.82 ± 0.03 | 98.09 ± 0.21 |
Q30(%) | 93.39 ± 0.08 | 94.05 ± 0.54 |
Mapped data against full-length isoforms | ||
Data of unique mapped reads (million) | 6.21 ± 0.19 | 6.25 ± 0.29 |
Data of multiple mapped reads (million) | 27.92 ± 0.87 | 28.36 ± 1.35 |
Map** ratio (%) | 88.32 ± 0.31 | 89.37 ± 0.17 |
Exon rate (%) | 100 | 100 |
Gene Name | Protein Name | SwissProt ID | log2FC(M1 vs. M2) |
---|---|---|---|
CHS1 | Chalcone synthase 1 | Q9ZS41 | 8.63 |
CHI3 | Probable chalcone--flavonone isomerase 3 | Q8VZW3 | 1.06 |
F3H | Flavanone 3-dioxygenase | Q7XZQ7 | 1.97 |
DFR | Dihydroflavonol 4-reductase | P51105 | 6.50 |
ANS | Leucoanthocyanidin dioxygenase | P51091 | 7.51 |
CGT | UDP-glycosyltransferase 13 | A0A0M4KE44 | 1.06 |
GT6 | UDP-glucose flavonoid 3-O-glucosyltransferase 6 | Q2V6K0 | 2.25 |
UGT85A8 | UDP-glycosyltransferase 85A8 | Q6VAB3 | 1.31 |
UGT73C6 | UDP-glycosyltransferase 73C6 | Q9ZQ95 | −1.52 |
F3GT1 | Anthocyanidin 3-O-galactosyltransferase F3GT1 | A0A2R6Q8R5 | 1.17 |
3MaT | Malonyl-coenzyme A:anthocyanin 3-O-glucoside-6″-O-malonyltransferase | Q8GSN8 | −1.28 |
P5MaT | Pelargonidin 3-O-(6-caffeoylglucoside) 5-O-(6-O-malonylglucoside) 4‴-malonyltransferase | Q6TXD2 | 1.21 |
CYP71A1 | Cytochrome P450 71A1 | P24465 | 1.19 |
CYP71A9 | Cytochrome P450 71A9 | O81970 | 1.04 |
CYP71D313 | Cytochrome P450 CYP71D313 | H2DH20 | 2.21 |
CYP71B26 | Cytochrome P450 71B26 | Q9LTL0 | 1.77 |
CYP71B36 | Cytochrome P450 71B36 | Q9LIP4 | 1.33 |
CYP72A219 | Cytochrome P450 CYP72A219 | H2DH21 | 1.21 |
CYP736A12 | Cytochrome P450 CYP736A12 | H2DH18 | 1.37 |
CYP76AD1 | Cytochrome P450 76AD1 | I3PFJ5 | 2.59 |
CYP76A2 | Cytochrome P450 76A2 | P37122 | 1.15 |
CYP77A3 | Cytochrome P450 77A3 | O48928 | 1.79 |
CYP71B34 | Cytochrome P450 71B34 | Q9LIP6 | −1.05 |
CYP71B35 | Cytochrome P450 71B35 | Q9LIP5 | −1.35 |
CYP81Q32 | Cytochrome P450 81Q32 | W8JMU7 | −1.12 |
Gene Name | Protein Name | SwissProt ID | log2 FC(M1 vs. M2) |
---|---|---|---|
RL1 | Protein RADIALIS-like 1 | F4JVB8 | 3.76 |
RL6 | Protein RADIALIS-like 6 | Q1A173 | 1.15 |
MYB90 | Transcription factor MYB90 | Q9ZTC3 | 1.19 |
MYB114 | Transcription factor MYB114 | Q9FNV8 | 2.40 |
Gene Name | Primer Sequences (5′ to 3′) | Amplicon Size (bp) |
---|---|---|
ACT | Forward: TGGTATTGTGCTGGATTCTGGT | 109 |
Reverse: TGAGATCACCACCAGCAAGG | ||
Flavonoid biosynthesis (22) | ||
CHS1 | Forward: CATTTCGGGGGCCTAACGAT | 197 |
Reverse: CCCAACCTCCCGAAGATGAC | ||
CHI3 | Forward: CACGGACATTGAGATACACTTCC | 111 |
Reverse: TCTCCAGTTTTTCCCTTCCAGT | ||
F3H | Forward: AGTGAGAAGTTGATGGCGCT | 160 |
Reverse: GTCCCAGTGTCAAGTCAGGT | ||
DFR | Forward: ACAGCACTATCACCGCTCAC | 134 |
Reverse: ATGTATCTTCCCTGCGCTGT | ||
ANS | Forward: GGCCTCAAGTGCCTACAGTT | 169 |
Reverse: TGTCCAGCCACTCTAACACG | ||
CGT | Forward: GCAGCCCGCAAAATCTGTAG | 163 |
Reverse: ACGCAACCCTTCCTTGTCTT | ||
GT6 | Forward: GTGCCACAGGTGACGATTCT | 173 |
Reverse: ACTCCCAGTCCCAACTCCTT | ||
UGT85A8 | Forward: ATGCAGTATCGCCAACTCGT | 111 |
Reverse: GTCTTTCATTCCAGGAGCCCA | ||
UGT73C6 | Forward: GTATGGGCAGTAAGGGCTGG | 110 |
Reverse: GCCCAACCACGGATCAAAAG | ||
F3GT1 | Forward: GCTTTGGAACTGTGGCGATG | 165 |
Reverse: AGGCCACGATTTTTCCGGTT | ||
3MaT | Forward: CTCCGTGACATCTCTGCCTC | 175 |
Reverse: AGCCAACGGAGTGAAGTGTT | ||
P5MaT | Forward: AGGCGAAAAAGGGGTGGAAT | 193 |
Reverse: GCACCAGTCGGTAAACAAGC | ||
CYP71A1 | Forward: GTTTACGTGAGTGCATGGGC | 138 |
Reverse: TGCCCCAAAAGGAACCAACT | ||
CYP71A9 | Forward: CAATGCTTGGGCAACAAACG | 153 |
Reverse: TTTCTGCTTCTCGGATAGGGC | ||
CYP71D313 | Forward: GCTTGGTGAGATCCCTCTGG | 108 |
Reverse: TCACCAAGTACAAGTCCTGGC | ||
CYP71B26 | Forward: TGTTGTGTGGGCCATGACTT | 157 |
Reverse: TCTCATTGCCTCCTTCACCAC | ||
CYP71B36 | Forward: GGGCTGAGAACAGGTCAAGT | 199 |
Reverse: CTTGTATCGGCTCCTGCAAC | ||
CYP72A219 | Forward: TTGCTCGTGTGGACTGTTGT | 186 |
Reverse: TCGTAGAAGCATACCTGCCG | ||
CYP736A12 | Forward: GGAAACCTCCCTCATCGCTC | 167 |
Reverse: GCCTCAAATTCTGGACGGCT | ||
CYP76AD1 | Forward: AATCGGAGCGAAAGGAAGCC | 132 |
Reverse: ACGTTGGTCACCGTTTTGTG | ||
CYP76A2 | Forward: GCAGGTTTCACCGAGAGTGT | 164 |
Reverse: TGTTGCCTCTCCATCACACG | ||
CYP77A3 | Forward: TTAGCAGTGCGGATTTGGCT | 134 |
Reverse: CGGACCGTAGAGTGAGGAGT | ||
MYB transcription factor (4) | ||
RL1 | Forward: TTGAAAAGGCTCTGGCTGTGT | 127 |
Reverse: CTGATGTCTGCCACGAGGATT | ||
RL6 | Forward: GCGTAACTGTGGCTCTACCT | 102 |
Reverse: GCTATGTTATGCCAGCGGTC | ||
MYB114 | Forward: TTCGTAAGGGTGCATGGTGT | 140 |
Reverse: AAGCCACCTCAGTCTACAGC | ||
MYB90 | Forward: AAAGGCACAAGCCTACCCTG | 136 |
Reverse: CTGGGGGCAGTGTCTTCATC |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Zhu, T.; Zhang, M.; Su, H.; Li, M.; Wang, Y.; **, L.; Li, M. Integrated Metabolomic and Transcriptomic Analysis Reveals Differential Mechanism of Flavonoid Biosynthesis in Two Cultivars of Angelica sinensis. Molecules 2022, 27, 306. https://doi.org/10.3390/molecules27010306
Zhu T, Zhang M, Su H, Li M, Wang Y, ** L, Li M. Integrated Metabolomic and Transcriptomic Analysis Reveals Differential Mechanism of Flavonoid Biosynthesis in Two Cultivars of Angelica sinensis. Molecules. 2022; 27(1):306. https://doi.org/10.3390/molecules27010306
Chicago/Turabian StyleZhu, Tiantian, Minghui Zhang, Hongyan Su, Meiling Li, Yuanyuan Wang, Ling **, and Mengfei Li. 2022. "Integrated Metabolomic and Transcriptomic Analysis Reveals Differential Mechanism of Flavonoid Biosynthesis in Two Cultivars of Angelica sinensis" Molecules 27, no. 1: 306. https://doi.org/10.3390/molecules27010306