A Comparative Transcriptomic with UPLC-Q-Exactive MS Reveals Differences in Gene Expression and Components of Iridoid Biosynthesis in Various Parts of Gentiana macrophylla
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. RNA Extraction and Sequencing
2.3. Transcript Assembly and Unigene Functional Annotation
2.4. Analysis of Differentially Expressed Genes
2.5. Identification of Transcription Factors
2.6. qRT-PCR Validation of Key Genes in the Biosynthesis of Iridoid Compounds
2.7. Analysis of Constituents by UPLC-Q-Exactive MS
2.7.1. Sample Preparation
2.7.2. UPLC-Q-Exactive MS Analysis
2.7.3. UPLC-Q-Exactive MS Data Acquisition and Analysis
2.8. Correlation Analysis between Expression of Key Enzyme Genes and Constituents
2.9. Statistical Analysis
3. Results
3.1. RNA-Seq and De Novo Transcriptome Assembly
3.2. Functional Annotation of Unigenes
3.3. Correlation Analysis of Secondary Metabolism
3.4. Differential Gene Analysis
3.5. Analysis of Transcription Factors
3.6. Validation of Key Enzyme Genes Using qRT-PCR
3.7. Metabolite Analysis of G. macrophylla by UPLC-Q-Exactive MS
3.8. Correlation Analysis between the Expression of Key Enzyme Genes and Contents of Iridoids
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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No. | Pathway | Number of Unigenes | Proportion % | KEGG ID |
---|---|---|---|---|
1 | Phenylpropanoid biosynthesis | 354 | 16.31 | K09753 |
2 | Flavonoid biosynthesis | 129 | 5.94 | K00660 |
3 | Ubiquinone and other terpenoid-quinone biosyntheses | 96 | 4.42 | K12501 |
4 | Terpenoid backbone biosynthesis | 92 | 4.24 | K05906 |
5 | Phenylalanine, tyrosine, and tryptophan biosynthesis | 78 | 3.59 | K00500 |
6 | Tropane, piperidine, and pyridine alkaloid biosynthesis | 65 | 3.00 | K08081 |
7 | Fatty acid biosynthesis | 64 | 2.95 | K00665 |
8 | Stilbenoid, diarylheptanoid, and gingerol biosynthesis | 63 | 2.90 | K13065 |
9 | Sesquiterpenoid and triterpenoid biosynthesis | 62 | 2.86 | K15803 |
10 | Cutin, suberine, and wax biosynthesis | 56 | 2.58 | K15403 |
11 | Isoquinoline alkaloid biosynthesis | 56 | 2.58 | K14454 |
12 | Biosynthesis of unsaturated fatty acids | 50 | 2.30 | K00507 |
13 | Diterpenoid biosynthesis | 48 | 2.21 | K04125 |
14 | Valine, leucine, and isoleucine biosynthesis | 33 | 1.52 | K00826 |
15 | Indole alkaloid biosynthesis | 26 | 1.20 | K21026 |
16 | Zeatin biosynthesis | 21 | 0.97 | K00279 |
17 | Monoterpenoid biosynthesis | 21 | 0.97 | K15095 |
18 | Monobactam biosynthesis | 18 | 0.83 | K12524 |
19 | Flavone and flavonol biosynthesis | 17 | 0.78 | K05280 |
20 | Benzoxazinoid biosynthesis | 16 | 0.74 | K13229 |
21 | Betalain biosynthesis | 15 | 0.69 | K15777 |
22 | Anthocyanin biosynthesis | 12 | 0.55 | K21383 |
23 | Phenazine biosynthesis | 7 | 0.32 | K01657 |
No. | Ion Mode | tR/min | Excimer Ion Peaks | Molecular Formula | Adduct Ions | Ppm | Maior Fragment Ions (m/z) | Compounds | Parts | ||
---|---|---|---|---|---|---|---|---|---|---|---|
Sample | Control | Measured (m/z) | Calculated (m/z) | ||||||||
1 | − | 1.10 | 341.1094 | 341.1089 | C12H22O11 | [M−H]− | 1.38 | 178.0560, 161.0448, 131.0339, 119.0335, 101.0229, 89.0228, 71.0122 | Sucrose [27] | R, S, L, F | |
2 | − | 1.68 | 191.0191 | 191.0197 | C6H8O7 | [M−H]− | −3.30 | 159.3513, 134.9712, 111.0074, 87.0072, 67.0173, 59.0122, 57.0330 | Citric Acid [27] | R, S, L, F | |
3 | − | 6.95 | 583.1870 | 583.1880 | C22H34O15 | [M+HCOO]− | −1.66 | 375.1304, 313.1309, 213.0761, 169.0865, 113.0229, 59.0122 | Loganic acid 11-O-β-glucopyranosyl ester [4] | R, L | |
4 | − | 10.98 | 10.90 | 375.1299 | 375.1297 | C16H24O10 | [M−H]− | 0.61 | 213.0764, 169.0861, 151.0754, 133.0647, 113.0231, 95.0487, 69.0329 | Loganic acid [28] | R, S, L, F |
5 | + | 11.69 | 429.1366 | 429.1367 | C17H26O11 | [M−Na]− | −0.23 | 267.0845, 235.0575, 203.0521, 185.0418, 110.6607, 79.2134 | Morroniside [29] | R, S, F | |
6 | − | 13.78 | 389.1104 | 389.1089 | C16H22O11 | [M−H]− | 3.78 | 319.0093, 2199.2400, 183.0659, 165.0549, 121.0645, 69.0329, 59.0122 | Secologanoside [30] | R, S, L, F | |
7 | − | 14.21 | 14.16 | 419.1201 | 419.1195 | C16H22O10 | [M+HCOO]− | 1.43 | 302.6173, 179.0552, 149.0597, 141.0181, 119.0336, 113.0230, 89.0228, 59.0122 | Swertiamarine [31] | R, S, L, F |
8 | − | 14.71 | 14.60 | 563.1631 | 563.1618 | C22H30O14 | [M+HCOO]− | 2.38 | 221.0665, 193.0498, 179.0552, 161.0445, 131.0337, 101.0229, 89.0228 | 6’-O-β-D-Glucosyl gentiopicroside [31] | R, S, L, F |
9 | + | 15.66 | 521.1862 | 521.1865 | C22H32O14 | [M+H]+ | −0.58 | 251.4391, 197.0808, 179.0703, 151.0751, 127.0389, 111.0801 | Swertiapunimarin [32] | R, S, F | |
10 | − | 16.79 | 16.83 | 401.1093 | 401.1089 | C16H20O9 | [M+HCOO]− | 0.92 | 324.6015, 219.0556, 149.0597, 121.0648, 113.0231, 93.0333, 89.0228 | Gentiopicroside [31] | R, S, L, F |
11 | + | 16.81 | 177.0545 | 177.0546 | C10H8O3 | [M+H]+ | −0.68 | 147.0440, 131.0491, 121.0647, 119.0497, 103.0543, 91.0492, 79.0544 | Erythrocentaurine [33] | R, S, L, F | |
12 | + | 16.81 | 195.0649 | 195.0652 | C10H10O4 | [M+H]+ | −1.49 | 177.0549, 149.0598, 131.0490, 121.0648, 103.0542, 91.0543, 79.0543 | Ferulic acid [34] | R, S, L, F | |
13 | − | 18.10 | 18.23 | 403.1254 | 403.1246 | C16H22O9 | [M+HCOO]− | 2.03 | 312.8494, 205.4160, 151.0749, 125.0229, 89.0229, 81.0329 | Sweroside [31] | R, S, L, F |
14 | − | 20.10 | 593.1524 | 593.1512 | C27H30O15 | [M−H]− | 2.04 | 557.1314, 473.1095, 431.1017, 341.0662, 311.0567, 282.0520 | Saponarin [35] | R, S, L, F | |
15 | − | 26.94 | 431.0991 | 431.0984 | C21H20O10 | [M−H]− | 1.69 | 341.0669, 311.0566, 283.0620, 269.0458, 239.0713, 163.0392, 117.0332 | Isovitexin [35] | R, S, L, F | |
16 | + | 29.26 | 235.0961 | 235.0965 | C13H14O4 | [M+H]+ | −1.66 | 217.0858, 189.0907, 174.0675, 159.0804, 145.0647, 129.0698, 91.0542 | 2-methoxyanofinic acid [34] | R, S, L, F | |
17 | − | 29.27 | 603.1943 | 603.1931 | C25H34O14 | [M+HCOO]− | 2.06 | 323.0986, 263.0774, 189.0912, 161.0961, 119.0336, 101.0229, 89.0228 | Macrophylloside D [31] | R, S, L, F | |
18 | − | 33.09 | 301.0357 | 301.0354 | C15H10O7 | [M−H]− | 1.06 | 193.0136, 151.0024, 149.0233, 107.0124, 83.0121 | Quercetin [30] | R, S, L, F | |
19 | − | 35.35 | 285.0407 | 285.0405 | C15H10O6 | [M−H]− | 0.84 | 257.0463, 192.0056, 159.0079, 151.0025, 108.0200, 83.0122 | Kaempferol [30] | R, S, L, F | |
20 | − | 36.72 | 531.1522 | 531.1508 | C26H28O12 | [M−H]− | 2.64 | 315.0727, 297.0625, 189.0910, 153.0182, 109.0280 | Gentimacroside[3] | R, S, L, F | |
21 | − | 48.00 | 471.3488 | 471.3480 | C30H48O4 | [M−H]− | 1.74 | 471.3486, 439.8552, 218.8751, 101.9868, 79.8463 | Corosolic acid [36] | R, S, L, F | |
22 | − | 51.76 | 455.3539 | 455.3531 | C30H48O3 | [M−H]− | 1.82 | 455.3540, 229.2049, 177.5234, 151.2601, 141.7751 | Oleanic acid [36] | R, S, L, F |
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Kou, Y.; Yi, X.; Li, Z.; Ai, Y.; Ma, S.; Chen, Q. A Comparative Transcriptomic with UPLC-Q-Exactive MS Reveals Differences in Gene Expression and Components of Iridoid Biosynthesis in Various Parts of Gentiana macrophylla. Genes 2022, 13, 2372. https://doi.org/10.3390/genes13122372
Kou Y, Yi X, Li Z, Ai Y, Ma S, Chen Q. A Comparative Transcriptomic with UPLC-Q-Exactive MS Reveals Differences in Gene Expression and Components of Iridoid Biosynthesis in Various Parts of Gentiana macrophylla. Genes. 2022; 13(12):2372. https://doi.org/10.3390/genes13122372
Chicago/Turabian StyleKou, Yuhang, Xiaoying Yi, Zhuo Li, Yun Ai, Siting Ma, and Qianliang Chen. 2022. "A Comparative Transcriptomic with UPLC-Q-Exactive MS Reveals Differences in Gene Expression and Components of Iridoid Biosynthesis in Various Parts of Gentiana macrophylla" Genes 13, no. 12: 2372. https://doi.org/10.3390/genes13122372
APA StyleKou, Y., Yi, X., Li, Z., Ai, Y., Ma, S., & Chen, Q. (2022). A Comparative Transcriptomic with UPLC-Q-Exactive MS Reveals Differences in Gene Expression and Components of Iridoid Biosynthesis in Various Parts of Gentiana macrophylla. Genes, 13(12), 2372. https://doi.org/10.3390/genes13122372