Overexpression of Acyl-ACP Thioesterases, CpFatB4 and CpFatB5, Induce Distinct Gene Expression Reprogramming in Developing Seeds of Brassica napus
Abstract
:1. Introduction
2. Results
2.1. Seed FA Profiles (mol%) and 100-Seed Weights Showed
Different Patterns Depending on the Transgene Expressed
2.2. Transcriptome Data Summary
2.3. CpFatB4 and CpFatB5 Were Differentially Expressed during Seed Development
2.4. CpFatB4 Expression under the Napin Promoter Affected Napin Promoter Activity
2.5. CpFatB4 Expression Resulted in an Increase in the Overall FatB/FatA Ratio, but a Clear Decrease in Endogenous FatB/FatA Ratio
2.6. Depending on Developmental Stages or Genotypes, the Expressed Genes Showed Overlapping yet Distinct Expression Patterns
2.7. Among the Top 20 DEGs, Similarities Were Found in DEGs by Developmental Stages, but No Common Gene Was Identified in DEGs by Genotypes
2.8. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) Analyses of DEGs Showed Gene Enrichment in Some Categories, Such as “Fatty Acid Biosynthetic Process” and “Glycolysis”, in CpFatB4 at 45 DAF
2.9. Lipid Metabolism DEGs with the Strongest Expression Changes Were Different between CpFatB4 and CpFatB5
2.10. Plastidial FA Synthesis Pathway Was Activated by CpFatB4 Overexpression, but TAG Synthesis Was Not Strongly Affected
2.11. RNA-Seq Results Were Validated by RT-qPCR
3. Discussion
3.1. Whole-Pod Transcriptomes in Transgenic B. napus Showed Similar Developmental Gene Expression Changes to Those of the Control
3.2. The Transcriptome Analyses Provided Comprehensive Gene Expression Changes Caused by 16:0 or 10:0/12:0 FA Accumulation in Seeds of B. napus
3.3. DEGs Detected in CpFatB5 Suggest the Modulation of Organellar Gene Expression Responding to Medium-Chain FA Accumulation
4. Materials and Methods
4.1. Plant Materials
4.2. FA Analysis
4.3. RNA Samples for RNA-Seq and Analysis of DEGs
4.4. Illumina Sequencing, Data Processing, Reads Mapping, and Gene Annotation
4.5. DEG Analysis and Gene Annotation
4.6. GO and KEGG Analysis
4.7. Real-Time Quantitative PCR
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
FA | Fatty acid |
ACP | Acyl carrier protein |
FatB | Fatty acyl-ACP thioesterase B |
DAF | Days after flowering |
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Mol % | Youngsan | CpFatB4 | CpFatB5 |
---|---|---|---|
10:0 | 0.0 | 0.1 | 2.1 ± 0.3 |
12:0 | 0.0 | 0.1 | 1.0 ± 0.1 |
14:0 | 0.2 | 1.0 ± 0.1 | 0.6 |
16:0 | 5.3 ± 0.1 | 22.7 ± 1.7 | 9.0 ± 0.2 |
18:0 | 2.3 ± 0.1 | 3.4 ± 0.1 | 2.5 ± 0.1 |
18:1 | 67.8 ± 0.7 | 49.9±1.8 | 61.8 ± 0.7 |
18:2 | 16.6 ± 0.6 | 15.4 ± 0.7 | 15.7 ± 0.6 |
18:3 | 5.3 ± 0.4 | 4.7 ± 0.2 | 4.9 ± 0.2 |
20:0 | 0.8 ± 0.0 | 1.2 ± 0.0 | 0.8 ± 0.1 |
20:1 | 1.3 ± 0.0 | 1.0 ± 0.0 | 1.2 ± 0.0 |
22:1 | 0.3 ± 0.0 | 0.5 ± 0.0 | 0.4 ± 0.0 |
Percentage of saturated fatty acids | 8.7 | 28.4 | 16.0 |
100-seed weight (mg) | 227.4 ± 9.9 | 250.1 ± 9.3 | 216.1 ± 8.9 |
Type and Name of Comparisons | DEG Numbers | Up-Regulated DEG Numbers | Down-Regulated DEG Numbers | ||||
---|---|---|---|---|---|---|---|
Total | Lipid Metabolism Genes (%) | Total | Lipid Metabolism Genes (%) | Total | Lipid Metabolism Genes (%) | ||
Stage | C1_C2 | 2550 | 250 (9.8) | 1688 | 159 (9.4) | 862 | 91 (10.6) |
C2_C3 | 4642 | 267 (5.8) | 2504 | 100 (4.0) | 2138 | 167 (7.8) | |
41_42 | 2486 | 244 (9.8) | 1829 | 172 (9.4) | 657 | 72 (11.0) | |
42_43 | 2445 | 207 (8.5) | 1028 | 72 (7.0) | 1417 | 135 (9.5) | |
51_52 | 2575 | 216 (8.4) | 1692 | 141 (8.3) | 883 | 75 (8.5) | |
52_53 | 4813 | 264 (5.5) | 1753 | 64 (3.7) | 3060 | 200 (6.5) | |
Line | C1_41 | 366 | 10 (2.7) | 174 | 0 (0) | 192 | 10 (5.2) |
C2_42 | 45 | 0 (0) | 24 | 0 (0) | 21 | 0 (0) | |
C3_43 | 1847 | 105 (5.7) | 676 | 83 (12.3) | 1171 | 22 (1.9) | |
C1_51 | 289 | 6 (2.1) | 155 | 0 (0) | 134 | 6 (4.5) | |
C2_52 | 112 | 6 (5.4) | 12 | 1 (8.3) | 100 | 5 (5.0) | |
C3_53 | 1365 | 48 (3.5) | 324 | 3 (0.9) | 1041 | 45 (4.3) |
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Nam, J.-W.; Yeon, J.; Jeong, J.; Cho, E.; Kim, H.B.; Hur, Y.; Lee, K.-R.; Yi, H. Overexpression of Acyl-ACP Thioesterases, CpFatB4 and CpFatB5, Induce Distinct Gene Expression Reprogramming in Developing Seeds of Brassica napus. Int. J. Mol. Sci. 2019, 20, 3334. https://doi.org/10.3390/ijms20133334
Nam J-W, Yeon J, Jeong J, Cho E, Kim HB, Hur Y, Lee K-R, Yi H. Overexpression of Acyl-ACP Thioesterases, CpFatB4 and CpFatB5, Induce Distinct Gene Expression Reprogramming in Developing Seeds of Brassica napus. International Journal of Molecular Sciences. 2019; 20(13):3334. https://doi.org/10.3390/ijms20133334
Chicago/Turabian StyleNam, Jeong-Won, Jinouk Yeon, Jiseong Jeong, Eunyoung Cho, Ho Bang Kim, Yoonkang Hur, Kyeong-Ryeol Lee, and Hankuil Yi. 2019. "Overexpression of Acyl-ACP Thioesterases, CpFatB4 and CpFatB5, Induce Distinct Gene Expression Reprogramming in Developing Seeds of Brassica napus" International Journal of Molecular Sciences 20, no. 13: 3334. https://doi.org/10.3390/ijms20133334