Part:BBa_K5218015

RcC3H

p-coumaric acid 3-hdroxylase (C3H) gene from Rosa chinensis.

Base Pairs：1569 bp

Function：p-coumaric acid 3-hdroxylase that catalyzes the formation of caffeic acid from p-coumaric acid.

Figure 1: C3H plays a role in the Caffeic acid cycle.

figure adopted from Tatiana et al., 2020[1]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 652
  Illegal PstI site found at 938
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 652
  Illegal PstI site found at 938
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 652
  Illegal BamHI site found at 241
  Illegal BamHI site found at 1257
  Illegal XhoI site found at 1077
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 652
  Illegal PstI site found at 938
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 652
  Illegal PstI site found at 938
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 779
  Illegal SapI site found at 34
  Illegal SapI.rc site found at 805
  

Introduction

Cytochrome P450 family has been identified in all kingdoms of life, which can catalyze more than 20 types of reactions, including hydroxylation, epoxidation, cyclization, and so on [1]. One of its members, C3H, catalyzes the formation of caffeic acid from p-coumaric acid. This reaction is associated with lignin biosynthesis, which is vital for plant development [2]. Recently scientists have utilized Fungal Bioluminescence Pathway (FBP) to generate autoluminescent plant [3]. C3H, which produced the starting substance for caffeic acid cycle, is a crucial candidate gene for FBP genetical modification.

The engineering objective of this project is to generate brighter autoluminescent plants using synthetic biology approaches. Team BGI-MammothEdu-South 2024 selected a variety of candidate genes from different species and tested their functions in FBP via eGFP reporter system (pS1300-GFP plasmid). The RcC3H gene loci is LOC112191575, transcript loci is XM_024330936.2 (NCBI). Team BGI-MammothEdu-South 2024 extracted Rosa chinensis petal and leaf total RNA, reverse transcribed into cDNA and cloned the RcC3H CDS with specific primer pairs.

Cloning attempt

Team BGI-MammothEdu-South 2024 tried to extract total RNA from Rosa chinensis of different tissues and stages, unfortunately couldn't get proper RNA samples for reverse transcription. We noticed that the Rosa chinensis precipitated RNA was brown color, which was caused by secondary metabolites. The absorbance curves of both total RNA and cDNA were abnormal, indicating the failure of the extraction and RT. Due to limited time, Team BGI-MammothEdu-South did not work on this gene.

Figure 2: Abnormality in Rosa chinensis total RNA and reverse transcription.

A. Nanodrop-2000 measurement of total RNA extracted from Rosa chinensis.

B. Nanodrop-2000 measurement of cDNA from reverse transcription.

Reference

1.Jung S T, Lauchli R, Arnold F H. (2011) Cytochrome P450: taming a wild type enzyme[J]. Current opinion in biotechnology, 22(6): 809-817.

2.Barros J, Escamilla-Trevino L, Song L, Rao X, Serrani-Yarce JC, Palacios MD, Engle N, Choudhury FK, Tschaplinski TJ, Venables BJ, Mittler R, Dixon RA. 4-Coumarate 3-hydroxylase in the lignin biosynthesis pathway is a cytosolic ascorbate peroxidase. (1994) Nature Communication, 2019 Apr 30;10(1).

3.Mitiouchkina, T., Mishin, A.S., Somermeyer, L.G. et al. Plants with genetically encoded autoluminescence. (2020) Nature Biotechnology, 38, 944–946.