Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (20): 3507-3519.doi: 10.3864/j.issn.0578-1752.2019.20.002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Functional Characterization of a UDP: Flavonoid Glycosyltransferase Gene UGT73C19 in Glycine max

ShaoKang DI1,QingGang YIN2,YaYing XIA1,2,YongZhen PANG1()   

  1. 1 Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193
    2 Institute of Botany, Chinese Academy of Sciences, Beijing 100093
  • Received:2019-04-25 Accepted:2019-06-12 Online:2019-10-16 Published:2019-10-28
  • Contact: YongZhen PANG E-mail:pangyongzhen@caas.cn

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Abstract:

【Background】 Flavonoids are a group of important plant secondary metabolites accumulate in soybean, which are involved in many physiological activities, including soybean growth, development and stress resistance. Glycosylation catalyzed by UDP-glycosyltransferase is a key step in flavonoid biosynthesis. 【Objective】 The objective of the present study is to investigate the in vitro enzymatic activity and in vivo function of a soybean glycosyltransferase protein encoded by the UGT73C19 gene, the achievement of which will deep our understanding on the mechanism of the flavonoid biosynthesis in soybean. This study will provide gene resource and theoretical basis for the genetic modification in soybean. 【Method】 Flavonoids in the leaves of soybean core germplasm resources were detected by HPLC, and the expression level of UGT genes were detected by qRT-PCR. The coding region of the UGT73C19 gene was cloned from cDNA of soybean leaf (Williams 82). The amino acid sequences of UGT73C19 were searched in the NCBI database, and the software MEGA5 and DNAMAN were used for multiple sequence alignment and the construction of a phylogenetic tree. The recombinant UGT73C19 protein was expressed in E. coli and its enzymatic activity was determined towards various flavonoid aglycones. All the enzymatic products were identified by HPLC-MS. The expression profile of the UGT73C19 gene in soybean was analyzed by qRT-PCR. UGT73C19 was over-expressed in Arabidopsis thaliana by floral dipping method. Flavonoid content and composition were determined in seedlings and seeds in homozygous lines that showed the relatively high UGT73C19 expression level. 【Result】 Flavonoids in the leaves of soybean core germplasm showed significant differences in flavonoid composition and content in different varieties. Soybean core germplasm can be divided into 12 different types according to flavonoid composition. There was a positive correlation between the content of flavonoids and the expression level of UGT73C19 gene in the leaves of soybean core germplasm resources. The coding sequence of UGT73C19 gene was cloned,and the coding region was found to be 1482 bp, encoding a protein of 493 amino acids. The deduced UGT73C19 protein was found to have a conserved PSPG domain at the C-terminal. In vitro enzymatic activity analysis revealed that the recombinant UGT73C19 protein exhibited glycosyltransferase activity toward six flavonoid aglycones (kaempferol, quercetin, myricetin, apigenin, daidzein and genistein), and it showed the highest catalytic efficiency toward quercetin. The glycosylation sites were at the 5 and 7 hydroxy groups of flavonoid substrates, and the glycosylation substrates and sites of the recombinant UGT73C19 protein showed high diversity. It was found that the total flavonoid contents in the seedlings and seeds of the transgenic A. thaliana increased significantly, by 49% to 70% in leaves and 34% to 37% in seeds, in particular quercetin 3-O rhamnose in the seeds. 【Conclusion】 The recombinant UGT73C19 protein can catalyze the glycosylation of a group of flavonoid compounds and over-expression of UGT73C19 gene can increase the content of flavonols in plants like A. thaliana.

Key words: soybean, UDP-glucosyltransferase, flavonoids, flavonols

Fig. 1

Twenty groups of the flavonol profiles in the leaves of the soybean core germplasm resources"

Fig. 2

Correlation analysis between soybean flavonoid contents and transcript level of UGT73C19 gene"

Fig. 3

The sequence alignment of plant UGT proteins Identical residues (100%) are indicated in black background, similar residues with >75% and 50%-75% identity are indicated in dark gray and light gray. The conserved PSPG domain was boxed with dashed lines"

Fig. 4

Phylogenetic analysis of UGT73C19 and other reported UGT proteins"

Fig. 5

The relative transcript levels of UGT73C19 detected by qRT-PCR"

Fig. 6

Analysis of the products from enzymatic activity reaction of the recombinant UGT73C19 protein Representative HPLC chromatograms of reaction products of UGT73C19 with Kaempferol (a); Quercetin(b); Myricetin(c); Apigenin(d); Daidzein (e) and Genistein (f). K: Kaempferol; A: Apigenin; M: Myricetin; D: Daidzein; Q: Quercetin; G: Genistein; K5G: kaempferol 3-O-glucoside; A5G: apigenin 5-O-glucoside; Q5G: quercetin 5-O-glucoside; Q4G: quercetin 4-O-glucoside; Q7G: quercetin 7-O-glucoside; D7G: daidzein 7-O-glucoside; M7G: myricetin 7-O-glucoside; G7G: genistein 7-O-glucoside; they were identified with retention time, UV spectrum, mass spectrum, and references"

Fig. 7

Mass spectra of the reaction product of the recombinant UGT73C19 protein with various substrates"

Table 1

Analysis of the enzymatic kinetics of the recombinant UGT73C19 protein"

底物 Substrates Vmax（nmol·min-1） Km（μmol·L-1） Kcat（s-1） Kcat/Km（s-1?M-1）
Kaempferol 0.113±0.026 186.400±113.700 0.051±0.008 297.622±92.697
Quercetin 0.269±0.116 403.730±189.080 0.120±0.052 302.504±16.544
Myricetin 0.022±0.009 34.690±14.520 0.010±0.004 291.258±9.472
Apigenin 0.014±0.001 76.870±4.430 0.008±0.000 101.608±0.789
Daidzein 0.011±0.001 85.160±6.400 0.006±0.000 70.010±2.522
Genistein 0.008±0.000 42.470±0.490 0.004±0.000 97.918±2.819

Fig. 8

The analyses of flavonoids in 21_day seedlings and seeds of transgenic A. thaliana over-expressing UGT73C19 gene in comparison with the wild type control"

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