Part:BBa_K4399013
The negative control for anthocyanin biosynthesis
It is used as a negative control for inducible anthocyanin biosynthesis study, which contains a constitutive GFP expression box, an inducible SbMYB75 expression box and an inducible SbDEL expression box. However, as there is not a XVE box, the activator for PLexA35S, so neither SbMYB75 nor SbDEL can be expressed.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal XbaI site found at 696
Illegal XbaI site found at 1579
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal NheI site found at 1244
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal BglII site found at 4113
Illegal BglII site found at 4925
Illegal BamHI site found at 3547
Illegal BamHI site found at 3849
Illegal BamHI site found at 5168
Illegal BamHI site found at 5237 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal XbaI site found at 696
Illegal XbaI site found at 1579
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1342
Illegal EcoRI site found at 5513
Illegal XbaI site found at 696
Illegal XbaI site found at 1579
Illegal PstI site found at 2759
Illegal PstI site found at 4179
Illegal PstI site found at 4264 - 1000COMPATIBLE WITH RFC[1000]
Results
Construction of level-0 vectors
The DNA elements (golden gate compatible, 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.
Construction of level-1 vectors
The level-0 vectors were then used to construct Level-1 vectors: pEC47732: PAtUBI5-GFP-Tmas, pEC47742: PLexA35S-SbMYB75- Thsp18.2, pEC47751: PLexA35S-SbDEL-Tnos, pEC47761: P2×35S-XVE-T35S, according to the protocol:
| volume / μL | |
|---|---|
| level-1 empty vector (200 ng/μL) | 1.0 |
| promoter | 1.5 |
| CDS | 1.5 |
| terminator | 1.5 |
| NEB T4 buffer | 1.5 |
| BSA (10×) | 1.5 |
| T4 ligase | 0.5 |
| BsaI | 0.5 |
| ddH2O | 10.0 |
| the whole volume | 20.0 |
The reaction ware incubated in a thermocyler 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 clines were confirmed by sequencing.
Construction of level-2 vectors
Level-2 vectors were constructed based on level-1 vectors:
| items | volume / μL |
|---|---|
| level-2 empty vector (200 ng/μL) | 1.0 |
| L1-P1 | 1.5 |
| L1-P2 | 1.5 |
| L1-P3 | 1.5 |
| ELE-X | 1.5 |
| NEB T4 buffer | 1.5 |
| BSA (10×) | 1.5 |
| T4 ligase (NEB) | 0.5 |
| BsaI | 0.5 |
| ddH2O | 9.0 |
| the whole volume | 20.0 |
The reaction ware incubated in a thermocyler for: 37℃ 3 min, 16 ℃ 4 min, 32 cycles; 16℃ 15 min; 50℃ 5 min, 80℃ 5 min. 10 ul of the liagations were used to transform to E. coli. Positive clines were confirmed by sequencing and PCR.
Nucleic acid gel electrophoresis detection results showed that the level-2 vector IA showed one band and its molecular weight were greater than 8000 bp (Fig 2). Monoclonal transformation in plates were obtained by E. coli transformation (Fig 3).PCR detection results showed that SbMYB and SbDEL were integrated in monoclone NC (BBa_K4399013) (Fig 4).
Agrobacterium Rhizogenes transformation
The constructed vector NC 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 (Fig 5).
Explant infection and hairy root emerging induction
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 the infection, induced hairy roots emerged from the infection site (Fig 6).
The co-transgenic roots were detected by screening for GFP fluorescence signal.The successful transformants of hairy roots ware marked and transferred on MS plates (without cefotaxime) in dark at 25 °C for further reproduction.
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 no anthocyanin synthesis was induced in the NC hairy roots on the 1st and 5th day, implying that the negative control for inducible anthocyanin biosynthesis was successfully constructed (Fig 7).
| None |