Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology
Abstract
:1. Introduction
2. Materials and Methods
2.1. Selection of Target Sequences and sgRNA Design
2.2. Plant Transformation and Selection of Mutant Lines
2.3. CKX Activity Assays
2.4. Quantitative RT-PCR
2.5. Transcriptome Analysis by RNA-seq
2.6. Analysis of Phenotypic traits
2.7. Statistical Analysis
3. Results
3.1. Generation of Targeted Mutations in the HvCKX1 and HvCKX3 Genes in Barley
3.2. CKX Activity in the Spikes and Roots
3.3. Expression of IPT Genes in the ckx1 and ckx3 Mutant Lines
3.4. Transcriptome Analysis of the ckx1 and ckx3 Mutant Lines
3.5. Analysis of Yield-Related Traits in the ckx1 and ckx3 Mutant Lines
4. Discussion
4.1. Cross-Regulation of Cytokinin Gene Family Members
4.2. Knockout of HvCKX1 and HvCKX3 Did Not Enhance Grain Yields but Affected the Root Phenotype
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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sgRNA Construct | Edited Gene | No. Explants | No. Transgenic Events (%) | No. Independent Transgenic Plants | No. PCR-RE-Detected Mutants |
---|---|---|---|---|---|
ckx1-sgRNA | HvCKX1 | 1143 | 137 (12) | 71 | 47 |
ckx3-sgRNA | HvCKX3 | 931 | 76 (8) | 53 | 37 |
Gene | Identifier | Description | C1-22 (ckx1) | C3-21 (ckx3) |
---|---|---|---|---|
Spikes at Seven DAP | Change in Expression (Fold Change) | |||
CKX1 | HORVU3Hr1G019850 | Cytokinin oxidase/dehydrogenase | nd * | downregulation (−8.03) |
CKX2.1 | HORVU3Hr1G027460 | Cytokinin oxidase/dehydrogenase | nd | downregulation (−2.83) |
CKX2.2 | HORVU3Hr1G027430 | Cytokinin oxidase/dehydrogenase | upregulation (4.39) | downregulation (−5.29) |
CKX3 | HORVU1Hr1G042360 | Cytokinin oxidase/dehydrogenase | upregulation (7.50) | nd |
CKX4 | HORVU3Hr1G105360 | Cytokinin oxidase/dehydrogenase | downregulation (−5.00) | downregulation (−5.87) |
CKX5 | HORVU3Hr1G075920 | Cytokinin oxidase/dehydrogenase | downregulation (−4.70) | downregulation (−2.57) |
CKX9 | HORVU1Hr1G057860 | Cytokinin oxidase/dehydrogenase | downregulation (−6.70) | upregulation (4.91) |
IPT1 | HORVU2Hr1G062320 | tRNA isopentenyltransferase | downregulation (−4.16) | downregulation (−5.58) |
IPT2 | HORVU5Hr1G055220 | Adenylate isopentenyltransferase | downregulation (−4.17) | nd |
IPT4 | HORVU1Hr1G011480 | Adenylate isopentenyltransferase | upregulation (5.32) | upregulation (3.60) |
IPT10 | HORVU7Hr1G120070 | tRNA isopentenyltransferase | downregulation (−5.88) | downregulation (−4.81) |
cZOG1 | HORVU2Hr1G096890 | Putative cis-zeatin O-glucosyltransferase (ortholog of TacZOG1) | nd | upregulation (4.23) |
ZOG2 | HORVU2Hr1G004720 | Putative zeatin O-glucosyltransferase (ortholog of TaZOG2) | upregulation (4.70) | upregulation (5.64) |
ZOG3 | HORVU7Hr1G038510 | Putative zeatin O-glucosyltransferase (ortholog of TaZOG3) | upregulation (7.19) | upregulation (2.69) |
ZOG3 | HORVU2Hr1G075920 | Putative zeatin O-glucosyltransferase (ortholog of TaZOG3) | nd | upregulation (3.73) |
LOG | HORVU3Hr1G050770 | Putative cytokinin riboside 5’-monophosphate phosphoribohydrolase (ortholog of LOC_Os01g40630.1) | nd | downregulation (−4.46) |
LOG | HORVU3Hr1G066810 | Putative cytokinin riboside 5’-monophosphate phosphoribohydrolase (ortholog of LOC_Os01g51210.1) | downregulation (−4.00) | nd |
LOG | HORVU1Hr1G079570 | Putative cytokinin riboside 5’-monophosphate phosphoribohydrolase (ortholog of LOC_Os05g46360.1) | downregulation (−6.04) | downregulation (−7.07) |
LOG | HORVU4Hr1G005660 | Putative cytokinin riboside 5’-monophosphate phosphoribohydrolase (ortholog of LOC_Os03g49050.1) | downregulation (−6.23) | nd |
LOG | HORVU1Hr1G094290 | Putative cytokinin riboside 5’-monophosphate phosphoribohydrolase (ortholog of LOC_Os05g51390.1) | downregulation (−7.02) | downregulation (−6.32) |
LOG | HORVU5Hr1G124750 | Putative cytokinin riboside 5’-monophosphate phosphoribohydrolase (ortholog of LOC_Os03g64070.1) | nd | upregulation (6.77) |
GLU | HORVU2Hr1G023590 | Β-glucosidase | downregulation (−8.24) | downregulation (−7.67) |
10-day-old roots | change in expression (fold change) | |||
CKX1 | HORVU3Hr1G019850 | Cytokinin oxidase/dehydrogenase | nd | upregulation (0.53) |
LOG | HORVU2Hr1G089620 | Putative cytokinin riboside 5’-monophosphate phosphoribohydrolase (ortholog of LOC_Os04g43840.1) | nd | downregulation (−3.41) |
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Gasparis, S.; Przyborowski, M.; Kała, M.; Nadolska-Orczyk, A. Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology. Cells 2019, 8, 782. https://doi.org/10.3390/cells8080782
Gasparis S, Przyborowski M, Kała M, Nadolska-Orczyk A. Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology. Cells. 2019; 8(8):782. https://doi.org/10.3390/cells8080782
Chicago/Turabian StyleGasparis, Sebastian, Mateusz Przyborowski, Maciej Kała, and Anna Nadolska-Orczyk. 2019. "Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology" Cells 8, no. 8: 782. https://doi.org/10.3390/cells8080782