Zongda Xu

Establishment of Virus-Induced Gene Silencing (VIGS) System in Perennial Rosa Plants under Field Conditions

Virus-induced gene silencing (VIGS) technique, which is developed in recent years, is a rapid identification of plant gene function from reverse genetics. It is a manifestation of post-transcriptional gene silencing mechanism. Compared with the traditional transgenic technology, VIGS is a transient expression system, which can achieve good results in a short time. At present, it is widely used to study the function of plant genes, but most of them are model plants, and the experiments are carried out always in the indoor environment with controlled light and temperature conditions. In this study, we creatively provided a method to establish VIGS system using perennial Rosa plants as experimental materials under field conditions. The recombinant virus vector was constructed with RrGT1 gene as reporter gene and modified TRV-GFP virus as vector, and the perennial R. rugosa “Zizhi” and R. davurica were used as experimental verification materials. According to the growth conditions of Rosa plants, the natural environment in the field and the optimal conditions for the occurrence of VIGS, the technical problems such as the confirmation of the inoculation period, the preparation of the infective fluid, the inoculation technology of the virus vector and the light and temperature conditions of plant materials cultured after inoculation were solved one by one. When the RrGT1 gene was silenced, the Rosa plants showed a pale petal color phenotype. By detection, it was found that the expression of endogenous RrGT1 gene was significantly down-regulated, and the content of all anthocyanins also decreased significantly. Therefore, we believed that the attempt to establish VIGS system in perennial Rosa plants under field conditions was very successful.

Overexpression of the Rosa rugosa RrGT1 Gene Induces Anthocyanin Accumulation in Tobacco

Rosa rugosa has always been an important plant in landscape application, and the improvements and innovations about its flower color are particularly important. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1161 bp, encoding 386 amino acids, designated as RrGT1, were isolated from flowers of R. rugosa “Zizhi” and then functionally characterized. Sequence alignments with the NCBI database show that the RrGT1 protein is a member of the GTB superfamily and has typical conserved amino acid residues called PSPG that are crucial for RrGT1 enzyme activity. RrGT1 transcripts were detected in five flowering stages and seven tissues of R. rugosa “Zizhi” and their expression patterns corresponded with the accumulation of anthocyanins. Additionally, the in vivo function of RrGT1 was investigated via its overexpression in tobacco. Transgenic tobacco plants expressing RrGT1 induced anthocyanin accumulation in flowers, indicating that RrGT1 could encode a functional glycosyltransferase (GT) protein for anthocyanin biosynthesis and could function in other species. Therefore, we speculated that glycosylation of RrGT1 played a crucial role in anthocyanin biosynthesis in R. rugosa.

Cloning and Expression of Anthocyanin Biosynthesis Related Gene RrMYB6 in Rosa rugosa

R2R3-MYB transcription factor plays an important role in plant anthocyanin synthesis. Based on the transcriptional database of Rosa rugosa, one MYB transcription factor related to floral color, RrMYB6, was cloned. By using bioinformatics analysis method, cloning MYB gene and analyzing its function in anthocyanin biosynthesis regulation, we hope to lay a solid foundation for new color variety breeding of R. rugosa. Using the R. rugosa “Zi zhi” as the material, we obtained the total length of cDNA of RrMYB6 by RT-PCR and RACE. By analyzing its bioinformatics, we found that the formula of the protein was C1491H2368N452O470S17, molecular weight was 34690.97 Da, the theoretical pI was 8.74. In addition, it belonged to unstable protein with an unstable index at 50.59, and it was also a hydrophilic protein with the total average hydrophobic index at -0.847. In the secondary structure of RrMYB6 protein, the Alpha helix accounted for 32.35%, random coil was 47.39%, extended strand was 11.11%, and beta turn was 9.15%. The sequence analysis showed that RrMYB6 had a typical R2R3-MYB domain and bHLH binding domain, and it also had an N1, C1, C2 inhibitory motif, belonging to the Sg4 subfamily MYB protein. What’s more, evolutionary analysis indicated that the RrMYB6 protein was closely related with the MYB protein in Rosacea family, while it was far from those in other families. The expression analysis showed that RrMYB6 protein decreased with the color of petals deeping, and its expression was the lowest in the petals while the highest in stamens. According to the above results, it was speculated that RrMYB6 was involved in regulating the anthocyanin synthesis of R. rugosa, which belonged to negative regulatory mechanism.

Anatomical and biochemical analyses reveal the mechanism of double-color formation in Paeonia suffruticosa ‘Shima Nishiki’

Paeonia suffruticosa ‘Shima Nishiki’ is a very precious double-color cultivar because of its distinctive and colorful flowers. However, our understanding of the underlying mechanisms of its double-color formation is limited. The present study investigated the soluble sugar content, cell sap pH value and anatomical structure, anthocyanin composition and content and expression patterns of genes related to anthocyanin biosynthesis in the red and pink petals of the ‘Shima Nishiki’ cultivar. Here, we found that soluble sugar content, cell sap pH and the shape of outer epidermal cells were not the key factors that determine double-color formation. Five different anthocyanins were detected in both the red and pink petals, and the pelargonidin-3,5-di-O-glucoside (Pg3G5G) and pelargonidin-3-O-glucoside (Pg3G) contents in the red petals were significantly higher than those in the pink petals at every developmental stage. In addition, these gene expression patterns suggested that the significant differential expression of the dihydroflavonol 4-reductase gene (PsDFR) gene might play a key role in double-color formation. These results will provide a valuable resource for further studies unraveling the underlying genetic mechanisms of double-color formation in P. suffruticosa ‘Shima Nishiki’.