Part:BBa_K5223012
Phaselic Acid Synthesis System in Nicotiana benthamiana
Hydroxycinnamoyl transferase (HCT) is a crucial member of the plant acyltransferase family. HCT has been extensively applied in developing bioenergy, crop improvement, and anti-inflammatory drug production, playing a significant role in the synthesis and post-modification of plant secondary metabolites. Studies have shown that an HCT-M enzyme from red clover exhibits a high affinity for malic acid, and introducing this gene into Nicotiana benthamiana provides the essential activity for the synthesis of caffeoyl malic acid. We optimized the codons for E. coli to enhance the gene's expression efficiency in this bacterial system.
Biology and Usage
| Phaselic Acid Synthesis System in Nicotiana benthamiana | |
| Function | Synthesize caffeoyl shikimic acid |
| Use in | Nicotiana benthamiana |
| Backbone | pZYB11-pEAQ-HT-DEST1 |
| Derived from | Trifolium pratense |
Design and Properties:
Figure 1: Schematic Diagram of Gene Circuit
Figure 2: LC-MS/MS Results of Caffeoyl Malic Acid
The results indicated that the introduced gene effectively enabled tobacco plants to produce caffeoyl malic acid, with higher levels than the wild-type. This suggests that introducing HCT-M can increase the accumulation of caffeoyl malic acid in Nicotiana benthamiana and demonstrates that knocking out HQT enhances the synthesis of caffeoyl malic acid.
Experimental Approach:
Plasmid Construction:
1. Ligate HCT to pDONR207.
2. Transform the HCT-pDONR207 into E. coli TOP 10 and plate the bacterial culture on a gentamycin-resistant medium at 37°C overnight.
3. Perform bacterial screening and plasmid extraction.
4. Ligate HCT to pEAQ-DEST1.
5. Transform the HCT-pEAQ-DEST1 into E. coli TOP 10 and plate the culture on a kanamycin-resistant medium at 37°C overnight.
6. Perform bacterial screening and plasmid extraction.
7. Transform the positive plasmid into Agrobacterium strain GV3101.
8. Perform bacterial screening and store the positive clones.
Agrobacterium-Mediated Transformation:
1. Perform streak plating of the frozen bacterial solution for bacterial screening.
2. If the screening is positive, take 10 µL of the culture and inoculate it in 50 mL LB+Kan, shaking for 48 hours at OD600 = 0.6. Collect cells at 4°C, 5000 g for 10 minutes, discard the medium, resuspend in 15 mL ddH2O, and wash twice. Finally, adjust the resuspension solution to OD600 = 0.6, incubate at 28°C for 2-3 hours in the dark, and inject the solution into tobacco leaves.
References
[1] Michael L. Sullivan, Robert Zarnowski, Red Clover HCT2, a Hydroxycinnamoyl-Coenzyme A: Malate Hydroxycinnamoyl Transferase, Plays a Crucial Role in Biosynthesis of Phaselic Acid and Other Hydroxycinnamoyl-Malate Esters in Vivo, Plant Physiology, Volume 155, Issue 3, March 2011, Pages 1060–1067, https://doi.org/10.1104/pp.110.166793Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1720
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1444
Illegal BsaI.rc site found at 1723
Illegal SapI.rc site found at 89
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