苦荞糖基转移酶基因FtUGT143的克隆及表达分析

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

摘要/Abstract

摘要：

苦荞[Fagopyrum tataricum（L.）Gaertn]富含类黄酮C-糖苷，具有抗氧化、抗癌和消炎等多种保健作用。通过RT-PCR（reverse transcription-polymerase chain reaction）技术从苦荞中克隆得到了一个糖基转移酶基因，命名为FtUGT143，对其进行生物信息学、分子对接、基因表达、基因表达量与代谢物含量相关性等分析。结果表明，FtUGT143全长CDS序列为678 bp，编码226个氨基酸。密码子偏好分析结果显示，FtUGT143具有双子叶植物的密码子使用偏好性。多序列比对和进化树分析表明，FtUGT143是植物糖基转移酶基因家族中的C-糖基转移酶亚家族成员。分子对接结果表明，FtUGT143能与合成黄酮C-糖苷（牡荆素、异牡荆素、荭草素和异荭草素）的底物芹菜素和木犀草素相互作用。RT-qPCR结果显示，FtUGT143基因在苦荞的各个组织部位中均有表达，但在芽苗期的根、茎、叶中显著表达，且其在不同组织部位的表达量与4种黄酮C-糖苷积累量具有较好的相关性。研究结果表明FtUGT143是C-糖基转移酶，它可能参与苦荞中黄酮C-糖苷生物合成，对于揭示苦荞中黄酮C-糖苷的合成机制具有重要意义。

关键词: 苦荞, C-糖基转移酶, 黄酮C-糖苷, 基因克隆, 分子对接, FtUGT143, 表达分析

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

王佳蕊, 孙培媛, 柯瑾, 冉彬, 李洪有. 苦荞糖基转移酶基因FtUGT143的克隆及表达分析[J]. 生物技术通报, 2023, 39(8): 204-212.

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.

图/表 8

表1 本研究中所用引物序列

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

图1 苦荞FtUGT143基因全长CDS克隆 M：DNA maker 2000；1：FtUGT143的扩增片段

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

图2 FtUGT143密码子RSCU值分析横坐标下方的图例分别对应上方该密码子的RSCU值（反映密码子实际出现次数与预期出现次数的比例关系）

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）

图3 植物UGT蛋白氨基酸的序列多重比对与PSPG box比对分析 A：植物UGT蛋白氨基酸的序列多重比对（FtUGT143：苦荞；OsCGT（XP_015641684.1）：水稻；ZmCGT（PWZ07394.1）：玉米；FeCGTa（A0A0A1HA03.1）：甜荞；FeCGTb（A0A0A1H7N4.1）：甜荞；GmUGT708D1（NP_001347241.1）：大豆；At5GlcT（NP_193146.1）：拟南芥；FaGT1（AAU09442.1）：草莓；CaUGT3（BAH80312.1）：长春花）；红框标注为糖基转移酶保守序列的PSPG box；B：PSPG box比对分析

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

图4 FtUGT143系统进化树分析

Fig. 4 FtUGT143 phylogenetic tree analysis

表2 FtUGT143与4种类黄酮代谢小分子的对接结果

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

图5 FtUGT143和木犀草素、芹菜素对接示意图 A：FtUGT143和木犀草素对接示意图；B：FtUGT143和芹菜素对接示意图

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

图6 FtUGT143在苦荞不同组织部位中的表达量与4种C-黄酮含量间的相关性 A：FtUGT143在不同组织部位中的表达；B：4种C-黄酮在不同组织部位的相对含量；C：FtUGT143在不同组织部位表达量与4种C-黄酮含量间的相关性

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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