As natural and edible pigments from plants, anthocyanins give food colors and are used for excellent nutraceuticals for their powerful potent anti-cancer and anti-oxidation bioactivities. However, owing to the shortage of raw material and the instability of source supply, anthocyanin products in the market are few and expensive to meet the consumer demand for anthocyanins. Thus, this project aims to establish an anthocyanin biosynthesis system in vitro and scale it up to mass production to meet the demand.
Anthocyanin biosynthesis is mainly regulated at transcriptional level by MYB and bHLH transcription factors. Ectopic expression of MYB and bHLH genes can significantly increase anthocyanin content in many plants. The estradiol induciable system was used to construct an expression vector for SbMYB75 and SbDEl (a bHLH protein), two anthocyanin regulators from Scutellaria baicalensis, which may realize controllable production of anthocyanin in carrot hairy roots.
Primers were designed to be amplified SbMYB75 and SbDEL based on the sequence of Scutellaria baicalensis genome with the project number PRJNA484052 in GenBank
The XVE-PlexA system was used to construct an inducible anthocyanin biosynthesis system, which includes a 35S constitutive promoter and a GFP expression frame. Constitutive expression of XVE transcription factors can be activated in the presence of β-estrogens. Both SbMYB75 and SbDEL were driven by the 35S promoter, named IA (inducible anthocyanin) vector.
The plasmids of pUC57, pUAP1, pEC47732, pEC 4774, pEC 47751, pEC 47761 and pAGM4723 were used as the expression vectors in this project and plasmid was constructed using the method of golden gate assemblycompatible (Carola E. & Sylvestre M., 2014):
DNA elements, including promoters, CDS of functional genes and terminators were synthesized by Gensript based on their sequences and were sub-cloned into pUC57 or pUAP1.
(1) promoters: PAtUBI5, PLexA35S, P2×35S;
(2) CDS of Genes: GFP, SbMYB75, SbDEL, XVE;
(3) Terminators: Tmas, Thsp18.2, Tnos, T35S
(4) (4)Level-0 vectors: pUC57-PAtUBI5, pUC57- PLexA35S, pUC57- P2×35S, pUC57- GFP, pUC57-SbMYB75, pUC57-SbDEL, pUC57-XVE, pUC57-Tmas, pUAP1-Thsp18.2, pUAP1-Tnos, pUAP1-T35S.
Based on the level-0 vectors, level-1 vectors were sub-cloned into pEC47732, pEC 47742, pEC 47751 or pEC 47761:
(1) pEC47732-PAtUBI5- GFP-Tmas (BBa_K4399008)
(2) pEC47742-PLexA35S-SbMYB75- Thsp18.2 (BBa_K4399009)
(3) pEC47751-PLexA35S-SbDEL-Tnos (BBa_K4399010)
(4) pEC47761-P2×35S -XVE-T35S (BBa_K4399011)
Based on the level-1 vectors, the plasmids pAGM4723 was used to construct the level 2 vectors (i.e., the final vectors) - inducible anthocyanin vector (IA) and negative control vector (NC)：
(5) pAGM4723-PAtUBI5-GFP-Tmas--PLexA35S-SbMYB75-Thsp18.2--PLexA35S-SbDEL-Tnos
(6) pAGM4723-PAtUBI5-GFP-Tmas--PLexA35S-SbMYB75-Thsp18.2--PLexA35S-SbDEL-Tnos-- P2×35S -XVE-T35S
In both vectors, GFP gene was used as a genetic selection marker, and the SbMYB75 and SbDEL genes were used as regulatory genes for anthocyanin production. In IA plasmid, the XVE gene was added as a genetic switch to control the expression of the plasmid. With thee addition of β-estradiol, IA genes were induced to be expressed by the XVE protein-β-estradiol complex and then produce anthocyanins.
After PCR detection, the plant expression vector IA and negative control vector NC were electrotransferred into competent cells of Agrobacterium rhizogenes. Single clones appeared in coated Rifampicin and Kana-resistant solid plates, and then PCR was performed to further verify positive transformation.
Sterilized carrot leaves were infested with verified Agrobacterium rhizogenes and then co-cultured on solid MS medium plates containing acetosyringone. Subsequently transferred to co-culture on solid MS medium containing cephalosporin. After 3-5 weeks, the transgenic hairy roots were detected by GFP fluorescence signal and validated by PCR detection of target genes.
The co-transgenic hairy roots were cultured in liquid M medium for about 30 days and then β-estradiol solution was added to induce anthocyanin synthesis in IA root systems.
After 5 days of incubation, anthocyanins were extracted by maceration with acidified methanol solution. A full wavelength scanner was used to determine the wavelength of the supernatant at 530 nm and 657 nm to calculate the relative anthocyanin content (Frank M et al ., 2005). And then buffer solutions of different pH and different juices were used to determine the color change of anthocyanin under various pH