Cloning and Expression Analyses of C-glycosyltransferase Gene FtUGT143 in Fagopyrum tataricum

doi:10.13560/j.cnki.biotech.bull.1985.2023-0098

Abstract

Abstract:

Tartary buckwheat[Fagopyrum tataricum（L.）Gaertn]is rich in flavonoid C-glycosides, and it has many health effects, such as anti-oxidation, anti-cancer and anti-inflammation. In this study, in order to explore the flavonoid C-glycosyltransferase gene in tartary buckwheat, a glycosyltransferase gene named FtUGT143 was cloned from tartary buckwheat by RT-PCR（reverse transcription-polymerase chain reaction）, and its bioinformatics, molecular docking, gene expression, correlation between gene expression and metabolites content were also analyzed. The results showed that the full-length CDS sequence of FtUGT143 was 678 bp, encoding a protein of 226 amino acids. The codon preference analysis indicated that FtUGT143 had the codon usage preference of dicotyledons. Multiple sequence alignment and phylogenetic tree analysis suggested that FtUGT143 belonged to the carbon glycosyltransferase subfamily of plant glycosyltransferase gene family. The molecular docking analysis implied that FtUGT143 interacted with apigenin and luteolin, which were the substrates of flavonoid C-glycosides（vitexin, isovitexin, orientin and isoorientin）. RT-qPCR results showed that FtUGT143 gene was expressed in all tissues of tartary buckwheat, but it was significantly expressed in the roots, stems and leaves at seedling stage, and its expression in different tissues had a good correlation with the accumulation of four flavonoid glycosides. The research results showed that FtUGT143 is a C-glycosyltransferase which may participate in the biosynthesis of flavonoid C-glycosides, revealing the important role of FtUGT143 in the synthesis mechanism of flavonoid C-glycosides in tartary buckwheat.

Key words: tartary buckwheat, C-glycosyltransferase, flavonoid C-glycosides, gene cloning, molecular docking, FtUGT143, expression analyses

WANG Jia-rui, SUN Pei-yuan, KE Jin, RAN Bin, LI Hong-you. Cloning and Expression Analyses of C-glycosyltransferase Gene FtUGT143 in Fagopyrum tataricum[J]. Biotechnology Bulletin, 2023, 39(8): 204-212.

Figures/Tables 8

Table 1 List of primers'sequences in this study

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Use
FtUGT143-F	ATGGACTGGATGGATGAGATGCTA	基因克隆Gene cloning
FtUGT143-R	CTGTCAAATTTCGACACTGTCAAG	基因克隆Gene cloning
qFtUGT143-F	TAAGGAGATGGCTGAAGCTG	荧光定量Fluorescent quantification
qFtUGT143-R	CACCGAACTTGGATATCCGA	荧光定量Fluorescent quantification
qFtUPL7-F	TTCACGGGCACCATTACTGG	荧光定量内参Fluorescent quantification of internal reference primers
qFtUPL7-R	AGGTGGAAGCTGAAGGAAGC	荧光定量内参Fluorescent quantification of internal reference primers

Fig. 1 Full-length CDS clone of the FtUGT143 gene of buckwheat（Fagopyrum tataricum） M: DNA maker 2000. 1: Amplified fragment of FtUGT143

Fig. 2 Analysis of FtUGT143 codon's RSCU values The legend below the abscissa axis corresponds to the RSCU value of the codon above（reflecting the proportional relationship between the actual occurrence times of the codon and the expected occurrence times）

Fig. 3 Sequence multi-alignment of plant UGT protein amino acids and comparative analysis of PSPG box A: Multiple Alignment of amino acid sequences of plant UGT protein（FtUGT143: Fagopyrum tataricum. OsCGT（XP_015641684.1）: Oryza sativa; ZmCGT（PWZ07394.1）: Zea mays; FeCGTa（A0A0A1HA03.1）: Fagopyrum esculentum; FeCGTb（A0A0A1H7N4.1）: Fagopyrum esculentum; GmUGT708D1（NP_001347241.1）: Glycine max; At5GlcT（NP_193146.1）: Arabidopsis thaliana; FaGT1（AAU09442.1）: Fragaria × ananassa; CaUGT3（BAH80312.1）: Catharanthus roseus）. The red box indicates the PSPG box, a conserved sequence motif found in glycosyltransferases. B: Comparative analysis of PSPG box

Fig. 4 FtUGT143 phylogenetic tree analysis

Table 2 Docking results of FtUGT143 with four flavonoids metabolizing small molecules

配体小分子 Ligand small molecule	结合能 Binding energy/（kJ·mol^-1）
山奈酚 Kaempferol	- 29.706 4
表儿茶素 Epicatechin	- 31.796 4
木犀草素 Luteolin	- 33.890 4
芹菜素 Apigenin	- 32.216 8

Fig. 5 Schematic diagram of FtUGT143 docking with luteolin and apigenin A: Schematic diagram of FtUGT143 and luteolin docking. B: Schematic diagram of FtUGT143 and apigenin docking

Fig. 6 Correlation between the expressions of FtUGT143 in the different tissue sites of buckwheat and the content of four C-flavonoids A: Expression of FtUGT143 in different tissue sites. B: Relative content of four C-flavonoids in different tissue sites. C: Correlation between the expression of FtUGT143 and the content of four C-flavonoids in different tissue sites

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