Cloning and Analysis of RrF 3’ H in Rosa rugosa

Rosa rugosa is an important garden ornamental plant which belongs to the genus Rosa of the family Rosaceae. The current wild and cultivated R. rugosa are mostly purple, pink, a small amount of white, but lack of yellow, orange and so on. Flavonoids 3’-hydroxylase belongs to CYP75B subfamily of cytochrome P450, and is an essential enzyme in anthocyanins synthesis. In this experiment, RrF3’H gene was cloned from the petal of Rosa rugosa ‘Hunchun’ using RT-PCR, and bioinformatics analysis was performed. The RrF3’H gene’s full length of opening reading frame was 1687 bp, encoding 510 amino acids. The formulas of proteins encoded by RrF3’H were S 24 . The derived protein had a molecular weight of 58,506.95 Da. The aliphatic index was 90.94. It belongs to unstable hydrophilic protein. The protein consists of 46.76% α-helix, 31.04% random coil, 7.66% β-corner and 14.54% extended strand. The protein contains 21 Ser phosphorylation sites, 12 Thr phosphorylation sites, and 2 Tyr phosphorylation sites. The protein contained two O-glycosylation sites, located at positions 98 and 263 of the amino acid sequence respectively. The protein has a signal peptide site and a transmembrane structure. In addition, by comparing the expression levels of RrF3’H, we found RrF3’H was positively correlated with the depth of flower color.


Introduction
Rosa rugosa originated in China, and it belongs to the genus Rosa in the family Rosaceae. As an important ornamental garden plant, it has graceful shape, sweet-smelling flowers and many varieties. However, the petal colors of R. rugosa are mostly pink, purple and white, but lack of other colors [1] [2] [3]. This monotone coloration seriously limits the use of Rosa rugosa in gardens. Flavonoid 3'-hydroxylase belongs to the CYP75B subfamily of the P450 family and is essential for the synthesis of anthocyanins. It can catalyze the formation of dihydroquercetin from dihydrokaempferol [4] [5]. Brugliera et al. (1999) [6] cloned the first F3'H gene from Petunia hybrida, and it has been cloned from various plants so far, such as Dendranthema morifolium, Ginkgo biloba, Vitis amurensis, Chimonanthus praecox and Phalaenopsis aphrodite [7] [8] [9] [10]. Yang yuxia et al. (2013) [10] found that the expression level of F3'H in Phalaenopsis aphrodite with red flowers was about 19 times that of Phalaenopsis aphrodite with yellow flower, indicating that F3'H had an important influence on the synthesis of anthocyanin. Petunias with red flowers [11] were obtained by reducing the expression of F3'H gene and over expression of DFR. By inhibiting the expression level of F3'H gene in corydalis viola, it was also found that the content of anthocyanin was significantly reduced and the flower color became lighter [12]. All these studies indicate that the study of F3'H gene is of great significance to the improvement of plant color.
In this study, F3'H gene was cloned from petals of R. rugosa, and analysed its bioinformatics and gene expression in different flowering stages, in order to lay a foundation for future research on pigment formation and color improvement in R. rugosa.

Plant Materials
Petals were collected from R. rugosa 'Hunchun', R. 'Jiaomeisanbian', R. 'Miaoyu' ( Figure 1). From mid-April to the beginning of May 2016, we collected the petals at the soft bud stage, initial opening stage, full opening stage and wilting stage at Shandong Agricultural University Rose Planting Garden, Tai'an City (36˚18'N, 117˚13'E), Shandong Province, China. After picking the petals, we placed them in liquid nitrogen and then stored them at −80˚C for further use.

Total RNA Extraction and cDNA Synthesis
Total RNA was extracted from petals of different stages using an EASY Spin Plant RNA Extraction Kit (Aidlab Biotechnologies Co., Ltd.); then, the RNA concentration, purity and integrity were determined using a NanoDrop 2000c Spectrophotometer (Thermo Scientific, USA) and 1.0% nonvariable agarose gel electrophoresis. First-strand cDNA was synthesized directly from the tested RNA samples, and the reaction was performed according to the method of the 5X All-in-One RT MasterMix reverse transcription kit (abm Inc., USA).

Cloning of F3'H
'Hunchun' cDNA was used as the template. The specific primers were designed using Primer 5 software and based on the gene fragment in the R. rugosa transcriptome sequencing results (Table 1). Specific amplification of the F3'H open reading frame (ORF) was carried out with reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using cDNA as the template. PCR reaction system was as follows: ddH 2 O 9.5 µl, 2 × EasyTaqSuperMix 12.5 µl, target gene upstream primer 1 µl and downstream primer 1 µl, template cDNA 1 µl, 25 µl in total. Reaction conditions as follows:: 94˚C for 5 min; 94˚C for 30 s, 53˚C for 30 s, and 72˚C for 1 min, 35 cycles; and then extension at 72˚C for 10 min. PCR products were detected by 1% agarose gel electrophoresis. According to the instructions of Hipure Gel Pure DNA Mini Kit (Magen), the target strip was recovered, then connected with the pmd18-t vector of TaKaRa, transformed into E. coli DH5a, identified by PCR, then the positive clone was selected for sequencing.

Cloning and Sequence Analysis of RrF3'H Gene
The Using MEGA5 to build system phylogenetic tree of the amino acid sequence of 16 kinds of plants ( Figure 5), including R. rugosa, it can be seen that R. rugosa was closely related to the members belonging to Rosaceae family. The R. rugosa and Prunus persica converged first means R. rugosa has the most close relationship  with Prunus persica. Then converged with Prunus mume and Narcissus tazetta means that R. rugosa also has a very close relationship with P. mume and N. tazetta. In addition, they converged to a large group with Prunus cerasifera and Paeonia lactiflora, but it was relatively distant from other plants.

Bioinformatics Analysis
RrF3'H belongs to the P450 superfamily, with corresponding conservative structure domains. The formulas of proteins encoded by RrF3'H were C 2666 H 4149 N 699 O 734 S 24 .
The derived protein had a molecular weight of 58506.95 Da, a calculated pI of American Journal of Molecular Biology  The secondary structure prediction result demonstrated that the protein consists of 46.76% α-helix, 31.04% random coil, 7.66% β-corner and 14.54% extended strand. The protein contains 21 Ser phosphorylation sites, 12 Thr phosphorylation sites, and 2 Tyr phosphorylation sites. The protein contained two O-glycosylation sites, located at positions 98 and 263 of the amino acid sequence respectively. In addition, the protein has a signal peptide site and a transmembrane structure.

Expression Patterns of RrF3'H in Different Flowering Stages
The expression levels of RrF3'H were compared among the petals of R. 'Hunchun', R. 'Jiaomeisanbian', R. 'Miaoyu' at different stages (soft bud stage, initial opening stage, full opening stage and wilting stage). The expression patterns of the gene are shown in Figure 6. The results showed the expression level of this gene was the highest in the full openning stage, and the lowest in the soft bud stage. It shows a trend of rising first and then falling. In addition, in each opening stage, the expression level of RrF3'H was the highest in 'Hunchun', followed by 'Jiaomeisanbian', and the expression level was the lowest in 'Miaoyu'.

Discussion
Previous research has clearly shown that flower color intensity is largely determined by the amount of accumulated anthocyanins, and the anthocyanin biosynthetic pathway is well known [14] [15] [16]. Flavonoid 3'-hydroxylase (F3'H) is one of the key enzymes in the synthesis of anthocyanins in plants. It can catalyze the formation of dihydroquercetin from dihydrokaempferol, therefore, it plays an important role in the formation of plant color [5] [17]. In addition, in R. rugosa, F3'H competes with DFR and FLS for the common substrate dihydrokaempferol, so F3'H is of great significance for changing the color of R. rugosa. In this study, the RrF3'H gene was successfully cloned from R. rugosa, and analysed   [18]. By comparing the amino acid sequences of RrF3'H and the corresponding proteins in other plants, we found that the amino acid sequences of RrF3'H have higher homology with those of other plants, indicating that F3'H is relatively conservative among different species. The secondary structures were composed of α-helix, random coil, β-corner and extended strand. The α-helix domain can cause the bilayer of phospholipids to bend inward, which can resist the cell membrane damage caused by low temperature and protect the cell structure [19]. RrF3'H has multiple phosphorylation sites, indicating that reversible phosphorylation regulation plays an important role in achieving its functions.
By comparing the expression levels of RrF3'H among the petals at different stages, we found the expression level of this gene was the highest in the full openning stage, and the lowest in the soft bud stage. It shows a trend of rising first and then falling. Therefore, the full opening stage may be the time when anthocyanins are synthesized in large quantities in R. rugosa. By comparing the expression level of RrF3'H gene in the petals of the three varieties, we found that the expression level of RrF3'H was positively correlated with the depth of flower color; the gene expression level will be higher in redder flowers. It showed the highest expression level in 'Hunchun' and the lowest expression level in 'Miaoyu'. Therefore, RrF3'H is indeed related to the formation of flower color in R. rugosa, and the higher the expression of RrF3'H gene, the more the anthocyanin synthesis. In this study, F3'H gene in R. rugosa petals was isolated and analyzed to find out the information of this gene, which provided a theoretical basis for the improvement of R. Rugosa's color in the future.

Conclusion
We successfully cloned the RrF3'H from the R. rugosa, and the protein encoded by this gene is highly similar to that in other plants. In addition, this gene plays an important role in the formation of the color of R. rugosa and is positively correlated with the amount of anthocyanin synthesis.