Rosa rugosa, as a deciduous shrub in Rosacea, has many prominent traits such as intense perfume, strong resistance to winter hardiness, drought, disease, pests and salinization, which makes it highly valued in landscaping application and own a great market potential. But monotonous flower color (most flower is rose red, rare is pink and white, lack yellow, orange and other complex color) has seriously restricted its application in gardens for a long time. Studies have shown that the color of plant tissues is mainly determined by anthocyanins, which is water-soluble pigment that belongs to the flavonoid family of compounds and giving red, blue and purple colors in a range of flowers, fruits, foliage, seeds and roots  . Anthocyanin biosynthesis is regulated by structural genes and regulatory genes. Structural genes synthesize anthocyanins by encoding a series of biosynthetic enzymes in the anthocyanin pathway, while regulatory gene is a transcription factor, which can activate or inhibit the temporal and spatial expression of structural genes through the specific DNA-protein and protein-protein interactions, thus regulating the synthesis of anthocyanins. There are three main types of transcription factors involved in the regulation of anthocyanin biosynthesis: MYB, b HLH and WD-40 protein. As the most important transcription factors, MYB TFs can not only regulate anthocyanin biosynthesis but participate in many other life activities such as secondary metabolism, stress resistance, cell differentiation and regulation of cell differentiation and cycle, cell wall formation, lignin precipitation, biosynthesis of glucosinolates, plant germination and development of vegetative reproductive organs, and regulation of transcription factors  -  . Anthocyanin-regulating MYBs have been isolated from many species to date, including Arabidopsis thaliana AtMYB75 or PAP1, AtMYB90 or PAP2, AtMYB113 and AtMYB114  , Solanum lycopersicum ANT1  , Petunia hybrida AN2  , Capsicum annuum A  , Vitis vinifera VvMYB1a  , Zea mays P  , Oryza saliva C1  , Ipomoea batatas IbMYB1  , Antirrhinum majus ROSEA1, ROSEA2 and VENOSA  , Gerbera hybrid GhMYB10  , Picea mariana MBF1  , Garcinia mangostana GmMYB10  , Malus × domestica MdMYB10, MdMYB1/MdMYBA    , and Gentian GtMYB3  . Although structural genes operating in this biosynthetic pathway in Rosa rugosa have been well characterized   , to date there is little research on the transcriptional level in regulating anthocyanin biosynthesis.
Basing on the transcriptome of R. rugosa, we cloned one R2R3-MYB gene associated with anthocyanin biosynthesis, named RrMYB10.1, then analyzed its bioinformatics and constructed phylogenetic tree to predict its functions. The expression level of RrMYB10.1 in different tissues and different flower developments was identified by using qPCR technology. By figuring out how RrMYB10.1 works in anthocyanin biosynthesis, we may provide a certain foundation on moderating the accumulation of anthocyanin and further design the petal color of R. rugosa.
2. Materials and Methods
The experiment was conducted at the flower germplasm resource nursery of Shandong Agricultural University and the Flower Institute of Forestry College.
2.1. Plant Material
We chose healthy individuals with stable floral inheritance of Rosa rugosa ‘Zizhi’ as experiment material, which grown in the field of rose germplasm resource nursery. The petals in different stages of bud development (identified as S1-S5 in Table 1 and Figure 1) and flowering periods (identified according to the method of Chen Chen, the flower opening period is divided into five stages: bud stage (S5), early opening stage (S6), half opening stage (S7), full blooming stage (S8), and late blooming stage (S9)) were harvested in May for qPCR to analyze its expression in petals of different developments. Stages, Sepals, stems, leaves, stamens, pistils and petals in early opening stage were collected for gene spatial expression. All samples were collected and frozen in liquid nitrogen immediately, then stored at −80˚C until used.
2.2.1. RNA Extraction, cDNA Synthesis and Gene Cloning
The extraction of RNA from petals in early opening period was operated according to the instructions of EASYspin plant RNA rapid extraction kit, the integrity of RNA was detected by 1% agarose gel electrophoresis and its concentration and purity were tested by ultraviolet spectrophotometer. The cDNA was synthesized by RNA reverse transcription kit according to the description of abm reverse transcription kit. Based on the transcriptome, one R2R3-MYB gene was selected, the sequence was identified as complete CDS by BLAST. The specific primers for complete ORF amplification (Table 2) were designed by DNAMAN software. The synthesized cDNA was used as template, and the reaction system was as follows: 2 × Gflex PCR Buffer 12.5 µL, the target gene upstream and downstream primers each 1 µL, template cDNA 1 µL, TKS Gflex DNA Polymerase 0.5 µL, sterilization ddH2O supplement to 25 µL; The PCR reaction conditions are as follows: 94˚C for 1 min; 98˚C for 10 s, 55˚C for 15 s, and 68˚C for 30 s for a total of 30 cycles. The PCR products was detected by 1% agarose gel electrophoresis, and the target product was reclaimed according to the description of Hipure Gel Pure DNA Mini Kit(Magen), then ligated with the carrier pMD18-T to transform Escherichia coli DH5α. Positive clones were selected and sequenced at Ruibiotech.
Table 1. Identification and characteristics of different stages in bud development.
Table 2. Primers used to clone and expression analysis of RrMYB10.1 in R. rugose.
Figure 1. Characteristics of petals in different development in bud stage. (a) Diameter of bud in different developments; (b) petal color in n different developments in bud stage.
2.2.2. Bioinformatics Analysis
The ORF finder (NCBI) was used to search for an open reading frame, and the Conserved Domains database (NCBI) was used to analyze the conserved domains. ExPaSy-SOPMA was used to predict protein secondary structure. We used BLASTX (NCBI) to study the homology of the nucleotide sequence and conducted multiple sequence alignment by DNAMAN, and then constructed phylogenetic tree with other anthocyanin-promoting proteins by MEGA5.0 software. The phylogenetic tree was constructed according to the neighbor-joining method, and tested by bootstrap, which was repeated 1000 times.
2.2.3. Expression Analysis of RrMYB10.1 in Different Tissues and Different Flower Developments
The cDNA synthesized from RNA in different tissues and different flower developments was used as template, and it was carried out following the instruction of SYBR®Premix ExTaqTM kit by CFX96TM Real-Time System RT-qPCR instrument. The suitable primers of RrMYB10.1 for RT-PCR were selected by standard curve method. The specific primers and internal reference primers (Actin) are shown in Table 2. The reaction system is: Mix10ul, cDNA 1 µL, 0.4 µL each of upstream and downstream primer, adding sterilizing ddH2O up to 20 µL. The reaction procedure was: predenaturation at 95˚C for 30 s; 95˚C for 30 s, 60˚C for 30 s for a total of 39 cycles. The relative expression level was calculated by the method of 2−ΔΔCT. And the data analysis was graphed through Sigmaplot10.0.
2.2.4. Construction of Expression Vector
Choosing Spe I and Bst EII as restriction enzymes, designing primers with restriction enzyme sites and then cloning the full length. Then the RrMYB10.1 and expression vector pCAMBIA1304 were digested by restriction endonucleases Spe I and Bst EII, and the target fragments were recycled and connected with solution I. Positive clones were selected and sequenced at Ruibiotech. The diagram of pCAMBIA1304 were shown in Figure 2.
3. Results and Analysis
3.1. Cloning and Sequence Analysis of RrMYB10.1
One R2R3-MYB TF, RrMYB10.1 was cloned from the petals in early opening stage of Rosa rugosa ‘Zizhi’. We acquired a clear single target band about 750bp by PCR (Figure 3). Multiple sequence alignment analysis showed that RrMYB10.1 had a full length of 750bp, encoding 249 amino acids. The alignment showed that the conserved ANDV motif (Box A in Figure 4) in R3 domain and conserved KPRPR[S/T]F motif (Box B in Figure 4, which were identified to contribute to anthocyanin biosynthesis, were present in RrMYB10.1 and were modified to GNDV and RPQASKY respectively   . A conserved amino acid signature (EL × 2R × 3L × 6L × 3R) (the locations indicated by the arrows in Figure 4) has been shown to be functionally important for the interaction between MYB and R/B-like bHLH proteins  . The phylogenic tree constructed with anthocyanin-promoting MYBs revealed that RrMYB10.1 showed high similarity with other proteins in Rosacea and sharing the highest identity (98.39%) with RhMYB10. And the MYBs from dicot species cluster into one group, while monocot species (Rice and Maize) grouped into another clade (Figure 5).
3.2. Bioinformatics Analysis of RrMYB10.1 Gene
There are 249 amino acids encoded by RrMYB10.1. The protein’s formula is C1248H1970N374O395S14 with a molecular weight of 28982.48 and a theoretical pI:
Figure 2. Diagram of pCAMBIA1304.
Figure 3. RrMYB10.1 gene cloning. M: Marker A: RrMYB10.1.
5.86. There are 39 negatively charged residues (Asp + Glu), 34 positively charged residues (Arg + Lys), and 176 neutral amino acids. RrMYB10.1 belongs to unstable protein with an instability index of 55.13 and it’s a hydrophilic protein with the total average hydrophobic index at −0.847. The secondary structure of RrMYB10.1 demonstrates that there are 73α-helix, 150 random coil, 14 extended peptide chain, and 12 β-turn. The phosphorylation site prediction results reveals that there are 18 Ser phosphorylation sites, 6 Thr phosphorylation sites, and 5 Tyr phosphorylation sites, so we can infer that it may participate in phosphorylation control.
Figure 4. Multiple alignment of theRrMYB10.1 with other MYB TFs. Box (A) a conserved motif [A/S/G]NDV in the R2R3 domain for dicot anthocyanin-promoting MYBs. Box (B) a C-terminal-conserved motif [R/K] Px [P/A/R] xx [F/Y] for anthocyanin-promoting MYBs  . Arrows indicate specific residues that contribute to a motif implicated in b HLH co-factor interaction  .
3.3. Expression Analysis of RrMYB10.1 in Different Tissues and Different Flower Developments
The RT-PCR results in different tissues showed that the expression level of RrMYB10.1 was significantly high in petals (at the early opening stage ) , while it could be detected in other tissues(stems, leaves, stamens , sepals and pistil) with little difference but was relatively low (Figure 6(a)). When it comes to the flower development, the expression level increased from stage 1 in bud stage, which was relatively low, to the highest transcript level at stage 5 in bud stage, then declined rapidly with the flower opening (Figure 6(b)). Totally, the transcript level of RrMYB10.1 in petals in bud stage was significantly higher than in opening period.
3.4. Construction of Plasmid for Transient Gene Expression Assay
The RrMYB10.1 and expression vector pCAMBIA1304 were digested by restriction endonucleases Spe I and Bst EII, and the target fragments were recycled and
Figure 5. Phylogenetic relationships between RrMYB10.1 and other anthocyanin-related MYBs of rosaceous and other species.
Figure 6. Relative expression level of RrMYB10.1. (a) RrMYB10.1 relative expression level in different development stages; (b) RrMYB10.1 relative expression level in different tissues.
connected with solution I. Enzyme digestion analysis and DNA sequencing showed that the co-expressed protein expressing vectors were constructed successfully. Next, we are going to transfer the recombinant plasmid RrMYB10.1- pCAMBIA1304 to Arabidopsis thaliana to verify its function.
3.5. Construction of Plasmid for Transient Gene Expression Assay
The RrMYB10.1 and expression vector pCAMBIA1304 were digested by restriction endonucleases Spe I and Bst EII, and the target fragments (as shown in Figure 7) were recycled and connected with solution I. Enzyme digestion analysis and DNA sequencing showed that the co-expressed protein expressing vectors were constructed successfully. Next, we are going to transfer the recombinant plasmid RrMYB10.1-pCAMBIA1304 to Arabidopsis thaliana to verify its function.
One MYB TF, RrMYB10.1, was cloned from Rosa rugosa ‘Zizhi’. And the multiple sequence analysis showed that RrMYB10.1 had conservedR2R3 domain, two anthocyanin-promoting motifs and a conserved amino acid signature for the interaction with b HLH proteins. These results showed that RrMYB10.1 belonged to R2R3-MYB subfamily and may interact with b HLH proteins to regulate anthocyanin biosynthesis. Studies have shown that R2R3-MYB plays a key role in regulating anthocyanin biosynthesis in many plants such as Arabidopsis thaliana  , Prunus avium  , Daucus carota  and Muscari spp  . Some R2R3-MYB TFs can regulate specific structural genes individually, however, many reports show that the R2R3-MYB TFs interact with common bHLH and WD-40 factors form a MYB-bHLH-WD40 (MBW) transcriptional activator complex to regulate the anthocyanin biosynthesis. For example, In Arabidopsis thaliana, AtPAP1 (MYB), AtTT8 (b HLH) and AtTTG1(WD-40) form a ternary complex to activate the structural genes AtANS, AtDFR, AtUFGT to regulate the anthocyanin concentration in seed coat  . In Malus domestica, MdMYBA bind to the promoter regions of MDANS  , while MDMYB1 and MDBHLH3 directly bound to the promoter regions of MDUFGT and MDDFR to regulate anthocyanin accumulation  . In Grape hyacinth, the expression of MaMybA alone cannot activate the expression of early genes, but it can activate the expression of late structural genes MaDFR and MaANS. While MaAN2 can’t activate structural genes alone, but can activate MaDFR and MaANS significantly when binding with MabHLH1 or AtTT8  . What’s more, R2R3-MYB TFs also participant in many biological processes, such as cell morphogenesis and response to abiotic and biological stress. The phylogenic tree showed that MYB10.1 clustered according to their taxonomic relationship, for RrMYB10.1 is closely related to the members in Rosacea with the highest homology to RhMYB10 of 98% identify. And the MYBs from dicot species cluster into one group, while monocot species (Rice and Maize) grouped into another clade.
The RT-PCR results showed that RrMYB10.1 exhibited a tissue-specific expression, for its expression level in petals was significantly higher than in other tissues. In bud stage, the expression level of RrMYB10.1 increased with the petal color deepening, and reached the highest level at late bud stage, then it decreased rapidly in initial opening period and continued to decline slightly with flower
Figure 7. Identification of recombinant expression vector by double enzyme. M: Marker; A: RrMYB10.1 by Spe I and Bst EII double enzyme; B: pCAMBIA1304 by Spe I and Bst EII double enzyme; C: pCAMBIA1304 with no enzyme
opening, finally reached the minimum expression in late blooming period. It indicated that RrMYB10.1 was mainly expressed in the petals in bud stage and expressed at a low level in flower opening stage, which was consistent with the anthocyanin accumulation trend in Rosa rugosa. Study has shown that anthocyanin has synthesized completely in late bud stage, and some anthocyanin is degraded with petals opening when exposed to sunlight  . So we may draw a conclusion that RrMYB10.1 plays a key role in the pathway of anthocyanin biosynthesis in Rosa rugosa.
In summary, one R2R3-MYB TF, RrMYB10.1, was cloned and found to play a key role in regulating anthocyanin concentration in Rosa rugosa, thus providing a certain foundation on regulating flower color formation. Finally, it needs further study on the function of RrMYB10.1 and how it interacts with b HLH proteins to co-regulate specific structural genes to control anthocyanin biosynthesis in Rosa rugosa.
*These authors contribute equally.
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