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"

[1] HO H M, CHEN R Y, LEUNG L K, CHAN F L, HUANG Y, CHEN Z Y . Difference in flavonoid and isoflavone profile between soybean and soy leaf. Biomedicine & Pharmacotherapy, 2002,56:289-295.
[2] DUEÑAS M, HERNÁNDEZ T, ROBREDO S, LAMPARSKI G, ESTRELLA I, MUÑOZ R . Bioactive phenolic compounds of soybean (Glycine max cv. Merit): Modifications by different microbiological fermentations. Polish Journal of Food & Nutrition Sciences, 2012,62(4):241-250.
[3] SUBRAMANIAN S, STACEY G, YU O . Distinct, crucial roles of flavonoids during legume nodulation. Trends in Plant Science, 2007,12(7):282-285.
[4] 庄炳昌, 岳德荣, 王玉民, 李建平, 谢雪菊, 徐豹 . 大豆不同品种次生代谢产物及相关酶类含量与抗食心虫的关系. 中国油料, 1992(3):20-22.
ZHUANG B C, YUE D R, WANG Y M, LI J P, XIE X J, XU B . The correlation of content of flavonoid, total phenolate and enzyme in soybean with resistance level to L. glycinivorella. Oil Crops of China, 1992(3):20-22. (in Chinese)
[5] LIU C W, MURRAY J D . The role of flavonoids in nodulation host-range specificity: An update. Plants, 2016,5(3):33.
[6] DIXON R A, STEELE C L . Flavonoids and isoflavonoids - a gold mine for metabolic engineering. Trends in Plant Science, 1999,4(10):394-400.
[7] MAGGIONI D, BIFFI L, NICOLINI G, GARAVELLO W . Flavonoids in oral cancer prevention and therapy. European Journal of Cancer Prevention, 2015,24(6):517-528.
[8] HOSTETLER G L, RALSTON R A, SCHWARTZ S J . Flavones: Food sources, bioavailability,metabolism,bioactivity. Advances in Nutrition, 2017,15; 8(3):423-435.
[9] 杨硕, 徐元庆, 邢媛媛, 史彬林 . 植物源黄酮类化合物对动物免疫和抗氧化功能影响的研究进展. 动物营养学报, 2019,31(7):2958-2964.
YANG S, XU Y Q, XING Y Y, SHI B L . Research advances on effects of plant-based flavonoids on immune and antioxidative functions in animals. Chinese Journal of Animal Nutrition, 2019, , 31(7):2958-2964. (in Chinese)
[10] JONES P, VOGT T . Glycosyltransferases in secondary plant metabolism: Tranquilizers and stimulant controllers. Planta, 2001,213:164-174.
[11] GACHON C M, LANGLOIS-MEURINNE M, SAINDRENAN P . Plant secondary metabolism glycosyltransferases: The emerging functional analysis. Trends in Plant Science, 2005,10:542-549.
[12] CAPUTI L, MALNOY M, GOREMYKIN V, NIKIFOROVA S, MARTENS S . A genome-wide phylogenetic reconstruction of family 1 UDP-glycosyltransferases revealed the expansion of the family during the adaptation of plants to life on land. The Plant Journal, 2012,69:1030-1042
[13] YONEKURA-SAKAKIBARA K, HANADA K . An evolutionary view of functional diversity in family 1 glycosyltransferases. The Plant Journal, 2011,66(1):182-193.
[14] KOVINICH N, SALEEM A, ARNASON J T, MIKI B . Functional characterization of a UDP-glucose: Flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.). Phytochemistry, 2010,71(11/12):1253-1263.
[15] NOGUCHI A, SAITO A, HOMMA Y, NAKAO M, SASAKI N, NISHINO T, TAKAHASHI S, NAKAYAMA T . A UDP-glucose: Isoflavone 7-O-glucosyltransferase from the roots of soybean (Glycine max) seedlings. Purification, gene cloning, phylogenetics, and an implication for an alternative strategy of enzyme catalysis. Journal of Biological Chemistry, 2007,282(32):23581-23590.
[16] FUNAKI A, WAKI T, NOGUCHI A, KAWAI Y, YAMASHITA S, TAKAHASHI1 S AND NAKAYAMA T . Identification of a highly specific isoflavone 7-O-glucosyltransferase in the soybean (Glycine max (L.) Merr.). Plant and Cell Physiology, 2015,56(8):1512-1520.
[17] YIN Q, SHEN G, DI S, FAN C, CHANG Z, PANG Y . Genome-wide identification and functional characterization of UDP-glucosyltransferase genes involved in flavonoid biosynthesis in Glycine max. Plant and Cell Physiology, 2017,58(9):1558-1572.
[18] KIM E H, KIM S L, KIM S H, CHUNG I M . Comparison of isoflavones and anthocyanins in soybean [Glycine max (L.) Merrill] seeds of different planting dates. Journal of Agricultural and Food Chemistry, 2012,60(41):10196-10202.
[19] MAMOON REHMANA H, AMJAD NAWAZA M, BAO L, HUSSAIN SHAHB Z, LEE J M, AHMAD M Q, CHUNG G, YANG S H . Genome-wide analysis of Family-1 UDP-glycosyltransferases in soybean confirms their abundance and varied expression during seed development. Journal of Plant Physiology, 2016,206:87-97.
[20] RODAS F R, DI S, MURAI Y, IWASHINA T, SUGAWARA S, MORI T, NAKABAYASHI R, YONEKURA-SAKAKIBARA K, SAITO K, TAKAHASHI R . Cloning and characterization of soybean gene Fg1 encoding flavonol 3-O-glucoside/galactoside (1→6) glucosyltransferase. Plant Molecular Biology, 2016,92(4/5):445-456.
[21] DI S, YAN F, RODAS F R, RODRIGUEZ T O, MURAI Y, IWASHINA T, SUGAWARA S, MORI T, NAKABAYASHI R, YONEKURA-SAKAKIBARA K, SAITO K, TAKAHASHI R . Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1→2) glucosyltransferase. BMC Plant Biology, 2015,15:126.
[22] RODAS F R, RODRIGUEZ T O, MURAI Y, IWASHINA T, SUGAWARA S, SUZUKI M, NAKABAYASHI R, YONEKURA- SAKAKIBARA K, SAITO K, KITAJIMA J, TODA K, TAKAHASHI R . Linkage mapping, molecular cloning and functional analysis of soybean gene Fg2 encoding flavonol 3-O-glucoside (1→6) rhamnosyl transferase. Plant Molecular Biology, 2014,84(3):287-00.
[23] VOGT T, GRIMM R, STRACK D . Cloning and expression of a cDNA encoding betanidin 5-O-glucosyltransferase, a betanidin- and flavonoid-specific enzyme with high homology to inducible glucosyltransferases from the Solanaceae. The Plant Journal, 1999,19(5):509-519.
[24] HORVATH D M, CHUA N H . Identification of an immediate- early salicylic acid-inducible tobacco gene and characterization of induction by other compounds. Plant Molecular Biology, 1996,31(5):1061-1072.
[25] HIROTANI M, KURODA R, SUZUKI H, YOSHIKAWA T . Cloning and expression of UDP-glucose: Flavonoid 7-O-glucosyltransferase from hairy root cultures of Scutellaria baicalensis. Planta, 2000,210(6):1006-1013.
[26] YONEKURA-SAKAKIBARA K, TOHGE T, NIIDA R, SAITO K . Identification of a flavonol 7-O-rhamnosyltransferase gene determining flavonoid pattern in Arabidopsis by transcriptome co-expression analysis and reverse genetics. Journal of Biological Chemistry, 2007,282(20):14932-14941.
[27] SU X, SHEN G, DI S, DIXON R A, PANG Y . Characterization of UGT716A1 as a multi-substrate UDP: Flavonoid glucosyltransferase gene in Ginkgo biloba. Frontiers in Plant Science, 2018,8:e2085.
[28] YIN Q, SHEN G, CHANG Z, TANG Y, GAO H, PANG Y . Involvement of three putative glucosyltransferases from the UGT72 family in flavonol glucoside/rhamnoside biosynthesis in Lotus japonicus seeds. Journal of Experimental Botany, 2017,68(3):597-612.
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