Identification and Functional Verification of the Glycosyltransferase Gene Family Involved in Flavonoid Synthesis in Rubus chingii Hu

Abstract

Glycosylation is catalyzed by UDP-glycosyltransferase (UGT) and plays an important role in enriching the diversity of flavonoids. Rubus plants contain a lot of natural flavonoid glycosides, which are important plants with a homology of medicine and food. However, information about the Rubus UGT gene family is very limited. In this study, we carried out genome-wide analysis and identified the 172, 121, 130, 121 UGT genes in R. chingii, R. corchorifolius, R. idaeus, and R. occidentalis, respectively, and divided them into 18 groups. The analysis of the protein motif and gene structure showed that there were structural and functional conservations in the same group, but there were differences among different groups. Gene replication analysis showed that raspberry and dicotyledons had a higher homology. The expansion of the UGTs gene family was mainly driven by tandem replication events, and experienced purified selection during the long evolution of the raspberry. Cis-acting element analysis showed that they were related to plant growth and development, hormone regulation, and stress response. In addition, according to a comprehensive analysis of the co-expression network constructed by transcriptome data and phylogenetic homology, RchUGT169 was identified as a flavonoid glucosyltransferase. Through the transient expression in tobacco, it was verified that RchUGT169 could catalyze the conversion of kaempferol and quercetin to the corresponding flavonoid glycosides. In conclusion, this research enriched the understanding of the diversity of UGTs in Rubus and determined that RcUGT169 can catalyze flavonoids.

Keywords: Rubus chingii; UGTs; flavonoid; functional analysis; phylogeny.

Figure 1

Phylogenetic tree of the UGT gene family. The genealogical tree of UGT proteins from A. thaliana, R. chingii (RchUGTs), R. corchorifolius (RcoUGTs), R. idaeus (RidUGTs), and R. occidentalis (RocUGTs). Various colors and capital letters indicate the different groups of UGT genes.

Figure 2

Phylogenetic tree, conserved protein structure, conserved motif and gene structure analysis of the RchUGTs. (a) The evolution tree was created by proteins sequences of 172 RchUGTs, various colors and capital letters indicate different groups of the RchUGT gene. (b) Conserved protein structure analysis of RchUGTs. (c) Conserved motif position of RchUGTs. (d) Exon-intron structure analysis of RchUGTs.

Figure 3

Collinear analysis of the RchUGT gene family. (a) The collinearity of raspberry genes within species. The circle indicates that the seven chromosomes of raspberry have different markers. The gray and red wired genes show all collinear blocks and fragment doubling events. The outermost layer of the circle represents the gene density corresponding to each chromosome. (b–f) Genetic collinearity between R. chingii and different species, including A. thaliana, S. lycopersicum, P. trichocarpa, Z. mays, and O. sativa. Rectangles of different colors represent chromosomes from different species. The grey and red linker genes show the collinear relationships between all collinear blocks and UGTs, respectively.

Figure 4

The analysis of cis-acting elements of the gene promoter of RchUGTs. The gradient color in the heat map represents the number of cis-acting elements of RchUGTs. The color histogram represents the total number of cis-acting elements in each category.

Figure 5

The expression patterns of RchUGTs in four stages of fruits development and corresponding RT-qPCR analysis. (a) RNA-seq results of RchUGTs in four stages of fruits development, and photos of fruits in four stages. (b) RT-qPCR results of RchUGTs in four stages of fruits development. BG (big green, 21 DPA), GY (green-to-yellow, 42 DPA), YO (yellow-to-orange, 48 DAP), and Re (red, 54 DPA). Different lowercase letters represent significant differences (p < 0.05) between different groups.

Figure 6

Protein interaction network, phylogeny, and correlation analysis of RchUGTs related to the flavonoid. (a) Construction of the RchUGTs protein interaction network based on Arabidopsis thaliana homologous genes. (b) Network interaction analysis of 20 flavonoid RchUGTs genes and related genes. (c) The phylogenetic tree based on 172 RchUGTs and 58 UGTs with flavonoid receptors using the ML method, various colors and capital letters indicate different groups of the RchUGT gene. (d) Correlation analysis heat map between the RchUGTs transcription level and flavonoid biosynthesis-related genes. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

Figure 7

Subcellular localization of the empty vector (35S::YFP, CK) and RchUGT169-YFP fusion protein.

Figure 8

Enzyme activity analysis of the RchUGT169 recombinant protein. (a,d) are the detection of kaempferol and quercetin, respectively; (b,e) are the control groups of enzymatic reaction of kaempferol and quercetin; (c,f) are the experimental groups of enzymatic reaction of kaempferol and quercetin; (g,i) are the MS1 of products (2) and (4); (h,j) are MS2 of products (2) and (4). Products (1), (2), (3), and (4) are kaempferol, kaempferol glucoside, quercetin, and quercetin glucoside, respectively.