2. Engineering Success

2.1 Gene Cloning

In the beginning, total RNA was isolated from the root of Scutellaria baicalensis using Trizol reagent following the manufacturer’s instructions. The gel electrophoresis results showed that the isolated total RNA had a high enough quality to be used for further molecular investigation. Next, Single-strand cDNAs were synthesized from 5 ug of total RNA and reversely transcribed. The cDNA was used as templates for PCR amplification, SbMYB75-F and SbMYB75-R, SbDEL-F and SbDEL-R, CaMV35S-F and CAMV35S-R were used as PCR primers. The PCR results showed that SbMYB75, SbDEL and CaMV35S were amplified, subcloned and sequenced.

2.2 Vector construction

The plasmids of pUC57, pUAP1 and pEC47732 were used as the expression vectors in this project. Plasmid constructions were performed by using the method of golden gate assembly (Carola E. & Sylvestre M., 2014).

2.2.1 Level-0 Vector construction

The DNA elements (golden gate assembly, promoters: PAtUBI5, PLexA35S, P2×35S; CDS of Genes: GFP, SbMYB75, SbDEL, XVE; Terminators: Tmas, Thsp18.2, Tnos, T35S) were synthesized by Gensript based on their sequences and were sub-cloned into pUC57. The results showed that 11 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) were constructed.

2.2.2 Level-1 Vector construction

Based on the level-0 vectors, the reaction system was shown in Table 1. A PCR reaction was carried out in a thermocycler for 32 cycles of amplification (37℃ for 3 min, 16 ℃ for 4 min) and 32 cycles of amplification (16℃ 15 min；50℃ 5 min，80℃ 5 min). 10 μl of the liagations were used to transform E. coli. Positive clones were confirmed by sequencing.

The results showed that the four level-1 vectors pEC47732: PAtUBI5- GFP-Tmas (BBa_K4399008), pEC47742: PLexA35S-SbMYB75- Thsp18.2 (BBa_K4399009), pEC47751: PLexA35S-SbDEL-Tnos (BBa_K4399010), pEC47761: P2×35S -XVE-T35S (BBa_K4399011) were constructed (Figure 1).

2.2.3 Level-2 Vector construction

Based on the level-1 vectors, according to the reaction system shown in Table 2, a PCR reaction ware incubated in a thermocycler for: 37℃ 3 min, 16 ℃ 4 min, 32 cycles; 16℃ 15 min; 50℃ 5 min, 80℃ 5 min. 10 μl of the liagations were used to transform E. coli. Positive clones were confirmed by sequencing and PCR.

For the level 2 vectors, the plasmids pAGM4723 was used and the following vectors IA and NC were constructed (Figure 2)

(1) Vector IA: pAGM4723-PAtUBI5-GFP-Tmas--PLexA35S-SbMYB75-Thsp18.2--PLexA35S-SbDEL-Tnos-- P2×35S -XVE-T35S (BBa_K4399012)

(2) Vector NC: pAGM4723-PAtUBI5-GFP-Tmas--PLexA35S-SbMYB75-Thsp18.2--PLexA35S-SbDEL-Tnos (BBa_K4399013)

Nucleic acid gel electrophoresis detection results showed that the level-2 vector IA and NC showed one band and its molecular weight were greater than 8000 bp (Figure 3). Monoclonal transformation in plates were obtained by E. coli transformation (Figure 4). PCR detection results showed that SbMYB and SbDEL were integrated in monoclones BBa_K4399012 and BBa_K4399013 (Figure 5).

A. Representative PCR analysis for the presence of the SbMYB gene in BBa_K4399012 and BBa_K4399013.

Line 1-4, PCR results of 4 single colonies of BBa_K4399012 using SbMYB primers;

Line 5-8, PCR results of 4 single colonies of BBa_K4399013 using SbMYB primers;

Line 9, positive control;

Line 10, negative control;

Line 11, marker;

B. Representative PCR analysis for the presence of the SbDEL gene in BBa_K4399012 and BBa_K4399013.

Line 11-14, PCR results of 4 single colonies of BBa_K4399012 using SbDEL primers;

Line 15-18, PCR results of 4 single colonies of BBa_K4399013 using SbDEL primers;

Line 19, positive control;

Line 20, negative control;

Line 21, marker.

2.3 Agrobacterium Rhizogenes transformation

The constructed vectors IA and IC were successfully transferred into Agrobacterium Rhizogenes A4 by electroporation. The positive transformants containing both SbMYB75 and SbDEL were selected and cultured on solid TY medium plates containing antibiotics of spectinomycin and kanamycin (Figure 6).

2.4 Explant infection

Carrot leaves from 4-week-old tissue culture plantlets were used as transformation explants. Carrot leaves were infected by A. Rhizogenes strains containing target genes and co-cultured on the solid MS medium plates containing 50 mM acetosyringone (AS) for 48-72 h at 25°C in the dark and then transferred to MS solid medium containing 400 mg/L cephalexin (cef). 3-5 weeks later after A. Rhizogenes infection, induced hairy roots emerged from the infection site (Figure 6). Hairy roots were derived and cultured in hormone-free half strength MS solid medium. During the culture, some hairy roots turned brown and grown quickly.

The co-transgenic roots were detected by screening for GFP fluorescence signal. With the help of a fluorescence microscope, GFP expression was observed in transgenic hairy roots, successful transformants of hairy roots ware marked and transferred on MS plates (without cefotaxime) in dark at 25 °C for further reproduction.

2.5 Hairy root culture and Induction of anthocyanin synthesis

The Carrot hairy roots were cultured in MS liquid medium with 200 mg-L-1 cefotaxime for about 30 days, then 2 μM β-estradiol solution was added in liquid M medium. The results showed that anthocyanin synthesis was successfully induced in the IA hairy roots on the 1st and 5th day, but not induced in the NC hairy roots (Figure 8), implying that the inducible anthocyanin biosynthesis system was successfully constructed.

2.6 Content analysis of anthocyanins

Anthocyanins were extracted with acidified 80% methanol solution. The absorbance of cyanidin 3-O-rutinoside (C3R) at 530 nm and 657 nm were measured in the concentration range of 0.001 - 0.05 mg / mL for the anthocyanin solution (Table 3). The relative content of anthocyanins was calculated with the following formula (Frank M. at al, 2005) (Table 4). The results showed that different carrot hairy root lines produce different anthocyanin contents, the mean relative anthocyanin content in IA hairy root line 16-10 and 16-5 was 28.6525 relative unites of anthocyanins / g dry weight (RUA/g DW) and 33.6254 RUA/g DW, respectively and that in NC line 17-3 was only 1.7044 RUA/g DW.