The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii
Abstract
:1. Introduction
2. Results and Discussions
2.1. Cullins in C. Reinhardtii
2.2. Expression Patterns of CrCUL Genes under Different Nutrition Deficiency Conditions
2.3. RNAi Expression of CrCULs in C. reinhardtii
2.4. Growth Kinetics and Lipid Contents of CrCUL RNAi Lines
2.5. Fatty Acid Composition Analysis
2.6. Subcellular Localization of CrCUL4
3. Materials and Methods
3.1. In Silico Identification and Sequence Analysis of CrCULs
3.2. C. reinhardtii Strain and Growth Conditions
3.3. Gene Expression Analysis of CrCULs
3.4. RNA Interference (RNAi) Vector Construction and Algae Transformation
3.5. Growth and Neutral Lipid Analysis
3.6. Fatty Acid Methyl Ester Profiling
3.7. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
CRLs | Cullin-RING E3 ligases |
CUL | Cullin |
C. reinhardtii | Chlamydomonas reinhardtii |
RNAi | RNA interference |
TAG | Triacylglycerol |
PDAT | Diacylglycerol acyltransferase |
DGAT | Diacylglycerol acyltransferase |
GPAT | Glycerol-3-phosphate acyltransferase |
GPDH | Glycerol-3-Phosphate Dehydrogenases |
Fat | Acyl-ACP thioesterase |
ACCase | Acetyl-CoA carboxylase |
PEPC | Phosphoenolpyruvate carboxylase |
UPP | Ubiquitin proteasome pathway |
E3s | E3 ubiquitin ligases |
LD | Lipid droplets |
SRS | Substrate-recognition subunit |
SKP1 | S-phase kinase-associated protein 1 |
SOCS/BC | Suppressor of cytokine signaling/elongin-BC |
BTB | Broad complex, Tramtrack, Bric-a-brac |
DDB1 | DNA damage-binding protein-1 |
RBX1 | RING Box 1 |
PARC | PARkin-like cytoplasmic protein |
FA | Fatty acid |
TAP | Tris Acetate Phosphate medium |
HSM | Sueoka’s High Salt Medium |
-N | Nitrogen deficiency |
-S | Sulfur deficiency |
-Fe | Iron deficiency |
COP I | Coat Protein Complex I |
WDR | WD40 repeat domain |
IR | Tandem inverted repeat |
GFP | Green fluorescent protein |
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Gene Name | Chromosome Position | Introns | ORF | Deduced Polypeptide | ||||
---|---|---|---|---|---|---|---|---|
Amino Acids | MW (kDa) | pI | GRAVY | Transmembrane Helices | ||||
CrCUL2 | 17:4895502…4903556F | 19 | 2244 | 747 | 87.6 | 6.98 | −0.214 | 3 |
CrCUL3 | 7:1516277…1525175F | 18 | 2211 | 736 | 84.8 | 8.14 | −0.473 | 0 |
CrCUL4 | 12:3957943…3963740F | 14 | 2301 | 766 | 84.8 | 8.49 | −0.576 | 1 |
FA | Maa7-7 | CrCUL2-i35 | CrCUL3-i44 | CrCUL4-i57 |
---|---|---|---|---|
C12:0 | 0.04 ± 0.01 | 0.04 ± 0.00 | 0.03 ± 0.00 | 0.04 ± 0.00 |
C14:0 | 0.04 ± 0.01 | 0.12 ± 0.11 | 0.04 ± 0.00 | 0.05 ± 0.01 |
C16:0 | 12.25 ± 0.65 a | 14.68 ± 0.89 b | 11.28 ± 1.60 a | 16.77 ± 1.47 c |
C16:1 | 3.95 ± 0.11 | 3.45 ± 0.63 | 2.01 ± 0.68 | 4.55 ± 2.21 |
C16:2 | 1.21 ± 0.12 a | 1.15 ± 0.20 b | 2.00 ± 0.03 a | 1.35 ± 0.19 a |
C16:3 | 1.08 ± 0.36 | 0.80 ± 0.06 | 1.29 ± 0.11 | 1.35 ± 0.27 |
C16:4 | 4.68 ± 0.19 | 6.35 ± 2.53 | 7.66 ± 0.85 | 7.37 ± 2.04 |
C18:0 | 1.40 ± 0.12 a | 1.64 ± 0.09 b | 1.27 ± 0.09 a | 1.83 ± 0.15 b |
C18:1n9t | 4.10 ± 0.40 | 4.77 ± 0.51 | 3.12 ± 1.13 | 4.44 ± 2.90 |
C18:1n9c | 5.50 ± 0.54 | 5.88 ± 0.89 | 4.39 ± 0.56 | 6.85 ± 2.37 |
C18:2 | 5.74 ± 0.23 | 6.27 ± 0.52 | 7.45 ± 0.98 | 7.31 ± 1.59 |
C18:3 (5,9,12) | 6.30 ± 0.32 a | 6.47 ± 0.25 a | 5.56 ± 0.74 a | 9.65 ± 0.51 b |
C18:3n3 | 8.20 ± 0.63 a | 9.67 ± 2.35 a | 10.81 ± 1.36 a | 12.64 ± 3.16 b |
C20:0 | 0.42 ± 0.03 a | 0.50 ± 0.10 a | 0.55 ± 0.03 a | 0.63 ± 0.15 b |
C20:1 | 0.26 ± 0.03 a | 0.47 ± 0.31 a | 0.60 ± 0.03 b | 0.44 ± 0.16 a |
Total FA | 55.19 ± 0.92 a | 62.24 ± 2.78 b | 58.04 ± 2.11 a | 75.30 ± 2.00 c |
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Luo, Q.; Zou, X.; Wang, C.; Li, Y.; Hu, Z. The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii. Int. J. Mol. Sci. 2021, 22, 4695. https://doi.org/10.3390/ijms22094695
Luo Q, Zou X, Wang C, Li Y, Hu Z. The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii. International Journal of Molecular Sciences. 2021; 22(9):4695. https://doi.org/10.3390/ijms22094695
Chicago/Turabian StyleLuo, Qiulan, Xianghui Zou, Chaogang Wang, Yajun Li, and Zhangli Hu. 2021. "The Roles of Cullins E3 Ubiquitin Ligases in the Lipid Biosynthesis of the Green Microalgae Chlamydomonas reinhardtii" International Journal of Molecular Sciences 22, no. 9: 4695. https://doi.org/10.3390/ijms22094695