3.1 Cloning and sequence analysis of CsMYB113
The CsMYB113 gene was cloned from the tea plant cultivar ‘Fudingdaba’ by RT-PCR. The complete ORF sequence is 726 bp (Fig. 1B) which encoding for a polypeptide of 241 amino acids. The isoelectric point (pI) and molecular weight (MW) were 9.88 and 27.96 kDa, respectively. The gene sequence BLASTx against the Arabidopsis showed the highest similar with AtMYB113, and then this gene was named as CsMYB113. Sequence analysis via the ScanProsite program revealed that the CsMYB113 protein contained two myb-type HTH (helix-turn-helix) DNA-binding domain profiles at N-terminus. Furthermore, the amino acid sequences similar with CsMYB113 were selected in other species, and multiple sequence alignment was performed. The result suggested that the R2 and R3 domains were highly conserved in these species. Moreover, 13th, 33rd, and 53rd positions of the R2 domain encoded the same tryptophan residues, which were conducive to keep the stability of the ‘helix-turn-helix’ configuration of the MYB protein domain (Fig. 1C and 2B). Phylogenetic analysis indicated CsMYB113 was divided into subgroup 6 (S6) and similar with MYBs involved in anthocyanin biosynthesis regulation, suggesting that CsMYB113 may play important role in the anthocyanin metabolism regulation. CsMYB113 shared the highest amino acid sequence identity with tea plant CsAN1 (62%) and kiwifruit AcMYB110 (57.5%), respectively.
3.2 Relative gene expression analysis of CsMYB113 and the subcellular localization of its protein
To investigate the expression patterns of CsMYB113 gene in various tissues, qRT-PCR analysis was conducted to evaluate mRNA expression levels separated from ten tissues of ‘Fudingdabai’. As shown in Fig. 3A, CsMYB113 transcripts accumulated in all analysed tissues. Overall, the transcript levels of CsMYB113 were the highest in root, and decreased in the first leaf (FL). Second leaf (SL) showed the lowest expression level, which had no significant difference with other tissues, including seed, bud, third leaf (TL), mature leaf (ML), old leaf (OL), stem, and flower.
To determine the subcellular localization of CsMYB113, the full-length ORF was fused in pCAMBIA2300 vector with the GFP reporter gene driven by the 35S CaMV promoter. Then the construct was transformed into Agrobacterium and then infiltrated into tobacco epidermal cells. The results showed that GFP fluorescence in control (pCAMBIA2300-GFP) was ubiquitous distribution throughout the cell, while the GFP signal of CsMYB113-GFP overlaps with the nucleus co-localization fluorescence signal (as shown in pseudo colours green and red, respectively) (Fig. 3B). It is clearly indicated that CsMYB113 is exclusively localized in the nucleus of plant cell, which is similar with the CsAN1 (Sun et al. 2016). These results indicated that CsMYB113 is a transcription activator.
3.3 Overexpression of CsMYB113 increased the anthocyanin contents in transgenic Arabidopsis
To determine the potential role of CsMYB113 in the regulation of anthocyanin biosynthetic pathway, the 35S::CsMYB113 construct was employed to ectopically activated CsMYB113 expression into Arabidopsis (Col-0). There was visible phenotypic difference between Col-0 (wide type) plants and the transgenic lines after growing in a 16/8-h (light/dark) photoperiod under illumination of 10000lux in growth chamber. The overexpression of CsMYB113 resulted in the accumulation of anthocyanins in hypocotyl, veins, stems, seeds and roots (Fig. 4A-F). And the anthocyanin contents in four tissues was significantly increased in transgenic Arabidopsis compared with wild-type (Fig. 4G and H).
Further research showed that in transgenic lines, the anthocyanin contents in leaf, stem, root, and seed increased by 6.7- , 41.7- , 29.0- , and 4.5- fold compared with the wild-type (average of the three lines), respectively. It’s indicated that the relative anthocyanin contents had significant differences in four tissues (Fig. 5A). In order to verify whether the phenomenon is caused by the differential expression of CsMYB113, we detected the expression levels of CsMYB113 gene in four tissues of wild-type (WT) and three overexpressing homozygous lines. The results showed that the successive decreasing order of the expression levels was tender stem, root, leaf, seed (average of the three lines), which had the same trend as the increase folds of anthocyanin contents (Fig. 5A and B). Therefore, we supposed that the differential expression of the CsMYB113 gene in four tissues lead to the differences in the anthocyanin contents. At the same time, combined with the results of CsMYB113 is root-specific expression in C. sinensis, which could further prove that the expression of CsMYB113 gene has obvious organizational differences.
3.4 Overexpression of CsMYB113 increased the expression levels of anthocyanin biosynthetic genes
R2R3-MYB TFs play important roles in activating structural genes involved in the anthocyanin biosynthesis. To further study the regulation of CsMYB113 gene, the expression levels of eight structural genes (AtPAL、AtCHS、AtCHI、AtF3H、AtF3’H、AtDFR、AtLDOX、AtUF3G) were detected between four tissues of wild-type and transgenic lines, respectively. In general, compared with the wild-type, overexpressing of CsMYB113 could strongly increase the expression levels of AtCHI、AtF3H、AtDFR、AtLDOX and, AtUF3G (Fig. 6). In four tissues, the expression level of F3H gene was the most significant up-regulated by 5.5- fold in leaves (Fig. 6A), whereas the expression levels of CHI, F3H, UF3G in stems were increased by 13- , 39-, and 114 fold, respectively (Fig. 6B). F3H, DFR and UF3G genes in roots were up-regulated by 42- , 20- , and 40 fold, respectively (Fig. 6C). The expression of each gene in seeds is less than 4- fold (Fig. 6D). These results indicate that CsMYB113 can promote the expression levels of anthocyanin biosynthetic genes, thereby regulating the synthesis and accumulation of anthocyanin. However, the regulation profile has a certain difference, as CsMYB113 gene mainly up-regulated different structural genes in the four tissues.
3.5 Transient expression of GFP protein in leaves of tea plants
With the development of research, transient transformation system has been established in many plants. It has the characteristics of high efficiency, short cycle and fast realization of gene function verification, and has been widely used in herbs (tobacco, tomato, Arabidopsis, rice) and woody plants (citrus, poplar). In order to investigate whether this system was suitable for the leaves of tea plant, we constructed a transient expression vector pK7WG2D with GFP protein as a reporter. The suspensions bring CsMYB113-GFP plasmid was injected into the leaves of three tea cultivars types (‘Fudingdabai’, ‘YingShuang’ and, ‘Wuniuzao’). After infiltration, we detected the GFP fluorescence 10 days later using the inverted microscope. In the non-transformed control, we did not observed any GFP fluorescent signal in the leaves (Fig. 7A2-C2). However, fluorescent signals were obviously detected in the infiltrated parts of the tea plant leaves (Fig. 7A4-C4). These results indicated that the transient expression system could be applied in leaves of tea plant.
3.6 Transient Overexpression of CsMYB113 Stimulated anthocyanin accumulations in the leaves of tea plant
As we found the transient expression system can be applied in leaves of tea plants. In order to further analysed the function of CsMYB113 gene by using the expression system. We determine the content of anthocyanin and the expression level of CsMYB113 gene in leaves (Figure 8). Leaves transformed with empty vector pK7WG2D (a) and target gene CsMYB113 (b) were collected respectively. Control was set as the non-transformed leaves (ck). The results showed that the whole leaves grew well and were only slightly damaged near the injection hole. Moreover, phenotypic differences were observed among different treatments. Transformed with target gene CsMYB113 (b) appeared slight purple spots in leaves of ‘Fudingdabai’ and ‘Wuniuzao’ (Fig 8A-C). The anthocyanin contents were significantly increased in leaves transformed with CsMYB113 gene (Fig 8D). There was an almost 2-fold increase in leaves of three cultivars (p<0.01).
Meanwhile, qRT-PCR analysis showed that there was a significantly increased in the expression level of CsMYB113 gene in transformed leaves (Fig 8E). Compare with the non-transformed leaves (ck), the expression level is increase almost 4.5 times in ‘Fudingdabai’, and almost 2 times in ‘Wuniuzao’ and ‘Yingshuang’. The expression effect in ‘Fudingdabai’ was better than that in the other two varieties. These results above further evidence that the CsMYB113 gene could transient expression in leaves of tea plant and the existence of CsMYB113 could accelerate the synthesis of anthocyanin in tea leaves to a certain extent.