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VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in chili pepper leaves.

Zhang Z, Li DW, Jin JH, Yin YX, Zhang HX, Chai WG, Gong ZH - Front Plant Sci (2015)

Bottom Line: Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation.The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern.These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes.

View Article: PubMed Central - PubMed

Affiliation: College of Horticulture, Northwest A&F University Yangling, China.

ABSTRACT
The purple coloration of pepper leaves arises from the accumulation of anthocyanin. Three regulatory and 12 structural genes have been characterized for their involvement in the anthocyanin biosynthesis. Examination of the abundance of these genes in leaves showed that the majority of them differed between anthocyanin pigmented line Z1 and non-pigmented line A3. Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation. Moreover, the expression of multiple genes was altered in the silenced leaves. The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern. Most structural genes including CHS, CHI, F3H, F3'5'H, DFR, ANS, UFGT, ANP, and GST were repressed in CaMYB-silenced foliage with the exception of PAL, C4H, and 4CL. These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes. Besides CaMYB silenced leaves rendered more sporulation of Phytophthora capsici Leonian indicating that CaMYB might be involved in the defense response to pathogens.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the silencing vector. LB, left borders of the T-DNA; RB, right borders of the T-DNA; 2 × 35 S, two copies of the cauliflower mosaic virus 35 S promoter; CP, coat protein; RdRp, RNA-dependent RNA polymerase; MP, movement protein; 16K, 16 KDa protein; R, ribozyme; N, nos-terminator; MCS, multiple cloning sites.
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Figure 2: Schematic representation of the silencing vector. LB, left borders of the T-DNA; RB, right borders of the T-DNA; 2 × 35 S, two copies of the cauliflower mosaic virus 35 S promoter; CP, coat protein; RdRp, RNA-dependent RNA polymerase; MP, movement protein; 16K, 16 KDa protein; R, ribozyme; N, nos-terminator; MCS, multiple cloning sites.

Mentions: The pTRV vector and Agrobacterium tumefaciens strain GV3101 were prepared for VIGS (Wang et al., 2013; Ji et al., 2014). We cloned CaMYB gene in purple pepper Z1 and found that the sequence in the coding region of CaMYB is the same as that deposited in the GenBank (AJ608992). Multiple sequence alignment was performed using MEGA6 to reveal the homology among genes involved in anthocyanin biosynthesis from different species. Online software siRNA-scan1 was used to avoid off-target silencing (Xu et al., 2006). A 332-bp fragment harboring a conserved and a non-conserved region of CaMYB was cloned into the pTRV2 vector by One Step Cloning Kit to yield the pTRV2: CaMYB construct. Empty vector (pTRV: 00) was served as a negative control (NC). The pTRV2: PDS, which contains the phytoene desaturase sequence to induce a photo-bleaching phenotype, was used as a positive control (Figure 2). The primers were shown in Table 1. A 10-mL culture of each strain was incubated for 24–36 h at 28°C in LB broth containing 50 mL-1 kanamycin, 50 mL-1 Gentamicin, and 50 mL-1 rifampicin. The primary culture was resuspended into Induction Medium containing 50 mL-1 kanamycin, 20 mg mL-1rifampicin, 50 mL-1 Gentamicin, 200 μM acetosyringone, and was shacked at 28°C for 20–24 h according to Velásquez et al. (2009). The cells were precipitated by centrifugation for 10 min at 3,000 × g and resuspended in the same volume of Agrobacterium infiltration buffer containing 10 mM MgCl2 and 10 mM MES at pH 5.7 till the OD600 reaching 0.5. A. tumefaciens GV3101 containing pTRV1 was mixed with GV3101 containing either pTRV2:00 or pTRV2: CaMYB in a 1:1 ratio. After mixing them, 400 μM acetosyringone was added.


VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in chili pepper leaves.

Zhang Z, Li DW, Jin JH, Yin YX, Zhang HX, Chai WG, Gong ZH - Front Plant Sci (2015)

Schematic representation of the silencing vector. LB, left borders of the T-DNA; RB, right borders of the T-DNA; 2 × 35 S, two copies of the cauliflower mosaic virus 35 S promoter; CP, coat protein; RdRp, RNA-dependent RNA polymerase; MP, movement protein; 16K, 16 KDa protein; R, ribozyme; N, nos-terminator; MCS, multiple cloning sites.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493389&req=5

Figure 2: Schematic representation of the silencing vector. LB, left borders of the T-DNA; RB, right borders of the T-DNA; 2 × 35 S, two copies of the cauliflower mosaic virus 35 S promoter; CP, coat protein; RdRp, RNA-dependent RNA polymerase; MP, movement protein; 16K, 16 KDa protein; R, ribozyme; N, nos-terminator; MCS, multiple cloning sites.
Mentions: The pTRV vector and Agrobacterium tumefaciens strain GV3101 were prepared for VIGS (Wang et al., 2013; Ji et al., 2014). We cloned CaMYB gene in purple pepper Z1 and found that the sequence in the coding region of CaMYB is the same as that deposited in the GenBank (AJ608992). Multiple sequence alignment was performed using MEGA6 to reveal the homology among genes involved in anthocyanin biosynthesis from different species. Online software siRNA-scan1 was used to avoid off-target silencing (Xu et al., 2006). A 332-bp fragment harboring a conserved and a non-conserved region of CaMYB was cloned into the pTRV2 vector by One Step Cloning Kit to yield the pTRV2: CaMYB construct. Empty vector (pTRV: 00) was served as a negative control (NC). The pTRV2: PDS, which contains the phytoene desaturase sequence to induce a photo-bleaching phenotype, was used as a positive control (Figure 2). The primers were shown in Table 1. A 10-mL culture of each strain was incubated for 24–36 h at 28°C in LB broth containing 50 mL-1 kanamycin, 50 mL-1 Gentamicin, and 50 mL-1 rifampicin. The primary culture was resuspended into Induction Medium containing 50 mL-1 kanamycin, 20 mg mL-1rifampicin, 50 mL-1 Gentamicin, 200 μM acetosyringone, and was shacked at 28°C for 20–24 h according to Velásquez et al. (2009). The cells were precipitated by centrifugation for 10 min at 3,000 × g and resuspended in the same volume of Agrobacterium infiltration buffer containing 10 mM MgCl2 and 10 mM MES at pH 5.7 till the OD600 reaching 0.5. A. tumefaciens GV3101 containing pTRV1 was mixed with GV3101 containing either pTRV2:00 or pTRV2: CaMYB in a 1:1 ratio. After mixing them, 400 μM acetosyringone was added.

Bottom Line: Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation.The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern.These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes.

View Article: PubMed Central - PubMed

Affiliation: College of Horticulture, Northwest A&F University Yangling, China.

ABSTRACT
The purple coloration of pepper leaves arises from the accumulation of anthocyanin. Three regulatory and 12 structural genes have been characterized for their involvement in the anthocyanin biosynthesis. Examination of the abundance of these genes in leaves showed that the majority of them differed between anthocyanin pigmented line Z1 and non-pigmented line A3. Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation. Moreover, the expression of multiple genes was altered in the silenced leaves. The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern. Most structural genes including CHS, CHI, F3H, F3'5'H, DFR, ANS, UFGT, ANP, and GST were repressed in CaMYB-silenced foliage with the exception of PAL, C4H, and 4CL. These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes. Besides CaMYB silenced leaves rendered more sporulation of Phytophthora capsici Leonian indicating that CaMYB might be involved in the defense response to pathogens.

No MeSH data available.


Related in: MedlinePlus