Supplementary Data
For: Plant Cell Reports
Title: Molecular cloning and characterization of an isoflavone 7-O-glucosyltransferase from
Pueraria lobata
Jia Li1†, Zhaobo Li1,2†, Changfu Li1, Junbo Gou1, 2, Yansheng Zhang1*
1
CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan
Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China
2
*
University of Chinese Academy of Sciences, Beijing 100049, China
To whom correspondence should be addressed. E-mail: zhangys@wbgcas.cn.
Table S1. Primers used for expression profile analyses of glycosyltransferases from P.
lobata by qRT-PCR
Genes
Actin
Primer 5' to 3' sequence
(upstream)
(downstream)
PlUGT1
(upstream)
(downstream)
PlUGT2
(upstream)
(downstream)
PlUGT3
PlUGT4
(upstream)
PlUGT8
PlUGT10
PlUGT11
PlUGT12
PlUGT13
(upstream)
(upstream)
(upstream)
PlUGT16
GTGATTTTGGTGGAGGAA
TTAGTAGAAGTACAAACGGAAC
AAGGTGAGGAAGGGAAGA
GGATTCATTGCTCGAAAG
GGGAAAGAAATTAGACAGAAA
TTATTGAGAAAGAGATTAAGC
TACTTGGCCTCTATTTGC
GAGAAGACCCACCATTTT
(upstream)
GGTGATTATGGTGGAGGA
(upstream)
GTCAAATTATCAATCAAC
GGAGCAGAGGCTGAATAG
(downstream)
AAAGCAACAAGCGAAGAC
(upstream)
AGAAGGTTCCAGAAGCGA
(downstream)
TTTGTACCAAAACAGAGT
(upstream)
CGGAGCAGAGGCTGAATA
(downstream)
CGAAAGCAACAAGCGAAG
(upstream)
GAGCCAAAGAATGGAGAC
(downstream)
GGGCCAAGACATGGTTAG
(upstream)
GCAAGTGAGTTGGAGGAA
(upstream)
(downstream)
PlUGT15
GAATAAATTGTGCTTAGT
(downstream)
(downstream)
PlUGT14
GTGATTTTGGTTGAGGAA
CGATTGGGAATAATGGTG
(downstream)
PlUGT9
AGCTTTTGTACAGAAATCACA
(upstream)
(downstream)
PlUGT7
GGGAAAGAAATTAGACAGAAG
CCATTCTCTCAGATCTCG
(downstream)
PlUGT6
CGGTGAGAAGAACAGGGT
(downstream)
(downstream)
PlUGT5
GGGGTGATGGTTGGGATG
(upstream)
CTGTGAAATCGTTGACCC
GCTGGTGAGAAGAGTGATG
CACCGTAAACTGAAGAAGAT
TAGCAATACATCGCACCT
(downstream)
AGACCAACTCTACGCAAG
(upstream)
GGTAAAGGAATGCGTGAA
(downstream)
PlUGT17
(upstream)
(downstream)
PlUGT18
(upstream)
(downstream)
PlUGT19
(upstream)
(downstream)
PlUGT20
(upstream)
(downstream)
PlUGT21
(upstream)
(downstream)
PlUGT22
(upstream)
(downstream)
PlUGT23
(upstream)
(downstream)
PlUGT24
PlUGT25
PlUGT26
PlUGT27
PlUGT28
(upstream)
PlUGT32
PlUGT34
GGAACAAAGGGAAGAAGA
ATGCCACTACCATACCAA
GGTGAATGAGGAAGGGAT
GCATTTATTATGGGCAAACATC
GACTATGGAAAGTTTGGCTTCG
AGCCTCCTTCCTTGACAGCT
GCAACTGCTTGGGAATGA
GCTAAACTGCGTCTTCTT
AAGGTAAAGGAATGCGTGAA
TTGGGATCAACTAACACTCTGT
GGTGGAACTCAACTCTGG
(downstream)
CCTGGATTAGCTGGGTCA
(upstream)
GGGGTGCCAATGGTTA
(downstream)
TTAGAATGAGAAGAGCC
(upstream)
GTGGATTATAGGGTGGGT
(downstream)
GGTACTACTAGCATGGGTTG
(upstream)
TGTGCTGGTGTGCCAATGTT
(upstream)
(upstream)
(upstream)
TTTTAGAAACGCCTCATT
GAAGCAGAATAGGGTGGT
AGAAATCAATGTCAAATC
TAAGAGGGACAAAATAGAG
ACAAAGGAGTAAGTTAAAAG
TGGTTCATAGGAGGGACG
(downstream)
ATGCTGGCTGCATTCTTG
(upstream)
TTTGGTGGATGTATTTGG
GGAACATAACATAAAGGGA
(upstream)
AGGCAGACCAAAGCACTA
(downstream)
TTCCACCCTCAGAAACAG
(upstream)
(downstream)
PlUGT35
TTCCACCATCACTAACAACT
CCTGCCAATGCTTACTTG
(downstream)
PlUGT33
TCAAAGCACAAATGCCAAGC
(upstream)
(downstream)
PlUGT31
CACCTGGACTAACACTCTTG
CTCATTCTTTCACGCATT
(downstream)
PlUGT30
TGTGCCTATGATTTGCTG
(downstream)
(downstream)
PlUGT29
GCAGCAATACATCCCAGA
(upstream)
(downstream)
AGTAGGAGGCTGAAGGTT
TGGAAGGACTCTAAATCG
AAGAAGATGGGTTTGTGAGT
GATACCAATTCCAGTCCA
PlUGT36
PlUGT37
PlUGT38
PlUGT39
PlUGT40
GT04F14
(upstream)
CTTAGGAGTCCCAACAGT
(downstream)
AAGAACTTCCACCTTCAC
(upstream)
AAACGAATGCGGTTGTGC
(downstream)
CCAGTTTTCCTAAGCCAA
(upstream)
AAAATGGCTTGGTGGAAA
(downstream)
AGGCAAAGATCCCTAACA
(upstream)
TGGAGCATTCCGCCTTAC
(downstream)
TCTATGAGCGCCGTGAGA
(upstream)
AAGGATGAAGATGGGTTT
(downstream)
TGTATCATAGCACCGTAGA
(upstream)
AGGTTTGGTTGTGCCTTC
(downstream)
CCACTATGCCATCCTCGT
Table S2. Primers used for 5' RACE of glycosyltransferase genes from P. lobata
Gene
PlUGT1
Primer 5' to 3' sequence
GSP1: GCTAGTGCTCTCTCCAAAC
GSP2: CACTAACCTATTCAATGCCATGATG
GSP: TTGCTTCGTTGGCACTCA
PlUGT13
GSP1: AACCCAACCAAGTCATTC
GSP2: TCCACCATCGCTGAGTGC
GSP: AGCCACCTTCATCTCCTCAA
PlUGT18
GSP1: TGCTTCTCGTCGCACAACCT
GSP: CTTCCAAACATCTGCCATAA
PlUGT22
GSP1: TTGCCCACCTTTAATTGAGT
GSP: TTCCCAAGCAGTTGCTCCAC
Table S3. Primers used to clone the glycosyltransferase genes from P. lobata to
protein expression vector
Genes
Primers 5' to 3' sequence
For pESC-His
PlUGT1
PlUGT13
PlUGT18
PlUGT22
PlUGT35
PlUGT39
PlUGT40
For pET28a
PlUGT1
PlUGT13
(upstream)
(downstream)
(upstream)
(downstream)
(upstream)
(downstream)
(upstream)
(downstream)
(upstream)
(downstream)
(upstream)
(downstream)
(upstream)
(downstream)
CCCGGATCCATGAAGGACTCCATTGTTC
CCCGTCGACCTAGTGCTCTCTCCAAACTTCC
CCCGGATCCATGAAGGGCACCATAGTTCT
CCCCTCGAGTCATTGCATCCACAACCC
CCCAGATCTATGGAGAAGAAAAGCATAACA
CCCGTCGACTTATGAATGAAACAAGTTATTTG
CCCGGATCCATGGGTATCCCTCACTTTCT
CCCGTCGACCTACTTCGCCCAGTTAATAAAC
CCCGGATCCATGCAACAAGAAGAAACAATAG
CCCGTCGACTCAGATACCAATTCCAGTCC
CCCGGATCCATGGAAACAACACCAGGAGC
CCCGTCGACTCAGTGATTCCCATTCACAT
CCCGGATCCATGGCAGAAACAGAAGGAAAAC
CCCCTCGAGTCACTCTTTCAAATGGATTTTTT
(upstream)
(downstream)
(upstream)
(downstream)
CCCCATATGAAGGACTCCATTGTTC
CCCGTCGACCTAGTGCTCTCTCCAAACTTCC
CCCGGATCCATGAAGGGCACCATAGTTCT
CCCCTCGAGTCATTGCATCCACAACCC
Table S4. Accession data of the glucosyltransferases used in the phylogenetic
analyses
GenBank
accession
Number
Plant
Protein name
ADV71364
Pueraria lobata
GT04F14
ADV71362
ADV71365
ADV71369
P. lobata
P. lobata
P. lobata
GT03H14
GT07O02
GT14A05
ABI94020
Medicago truncatula
UGT73C8
ABI94021
M.truncatula
UGT88E1
ABI94022
M.truncatula
UGT88E2
AAW56092
M.truncatula
UGT71G1
ABI94023
ABI94024
M.truncatula
M.truncatula
UGT84F1
UGT85H2
ABI94025
M.truncatula
UGT78G1
ABI94026
M.truncatula
UGT73P1
BAF64416
Glycine max
BAC78438
Glycyrrhiza echinata
BAM29362
BAA83484
AEE86330
EFH43393
AED92377
BAD52003
BAM37964
AFI71901
G. max
Scutellaria baicalensis
Arabidopsis thaliana
A. thaliana
A. thaliana
Dianthus caryophyllus
Nicotiana tabacum
Paeonia lactiflora
GmIF7GT
(UGT88E3)
GeIF7GT
(UGT73F1)
UGT73F2
UBGT
UGT73B1
UGT73B2
UGT78D2
DicGT1
Nt3GT1
F3GT
Function
active with isoflavone, flavone, flavonol
and coumarin
substrates
weak activity toward liquiritigenin
active toward 6,7,4-trihydroxyisoflavone
catalyzed glycosylation of luteolin on the
7-hydroxyl and of quercetin on the 3and 4'-hydroxyls
active with isoflavone, flavanone,
flavone, flavonol and isoliquiritigenin
active with isoflavone, flavone, flavonol,
coumesttrol and isoliquiritigenin
active with isoflavone, flavanone,
flavone, flavonol, coumesttrol and
isoliquiritigenin
active with triterpenoid ， isoflavone,
flavanone, flavone, flavonol, coumesttrol
and isoliquiritigenin
active with flavonol
active with flavonol, isoliquiritigenin and
biochanin A
active
with
isoflavone,
flavanone ,flavone, flavonol, coumesttrol
and isoliquiritigenin
active with liquiritigenin, kaempferol and
quercetin
isoflavone 7-O-glucosyltransferase
isoflavone 7-O-glucosyltransferase
isoflavone 7-O-glucosyltransferase
flavonoid 7-O-glucosyltransferase
flavonoid 7-O-glucosyltransferase
flavonoid 3-O-glucosyltransferase
flavonoid 3-O-glucosyltransferase
flavonoid 3-O-glucosyltransferase
flavonoid 3-O-glucosyltransferase
flavonol 3-O-glucosyltransferase
AEM37036
AFD61601
Brassica rapa subsp. campestris
Hevea brasiliensis
UF3GT1
ANGT1
flavonoid 3-O-glucosyltransferase
anthocyanidin 3-O-glucosyltransferase
BAA89008
ADZ54786
BAD83701
AAM91686
BAA36421
BAA89009
BAC54093
BAA36423
BAD99560
CP002685
Petunia x hybrida
Prunus avium
Iris x hollandica
A. thaliana
Perilla frutescens var. crispa
Petunia x hybrida
Torenia hybrid cultivar
Glandularia x hybrida
Rosa hybrid cultivar
A. thaliana
PGT8
3GT1
Ih3GT
UGT75C1
PF3R4
PH1
5GT
HGT8
RhGT1
UGT73C6
BAE48239
BAE48240
CAQ77160
Antirrhinum majus
Linaria vulgaris
Oryza sativa
Am4'CGT
L4'CGT
OsCGT
anthocyanidin 3-O-glucosyltransferase
anthocyanidin 3-O-glucosyltransferase
anthocyanidin 3-O-glucosyltransferase
anthocyanidin 5-O-glucosyltransferase
anthocyanidin 5-O-glucosyltransferase
anthocyanin 5-O-glucosyltransferase
anthocyanin 5-O-glucosyltransferase
anthocyanin 5-O-glucosyltransferase
anthocyanidin 5, 3-O-glucosyltransferase
flavonol-3-O-glycoside-7-O-glucosyltran
sferase
chalcone 4-O-glucosyltransferase
chalcone 4-O-glucosyltransferase
flavoniod C-glucosyltransferase
Table S5. Sequence analysis of the PlUGT genes with relatively higher transcription in roots
compared to stems and leaves.
Glycosyltransferase
PlUGT1
BLAST homology search
PREDICTED: anthocyanidin 5, 3-O-glucosyltransferase-like
Amino acid
identities
93%
[Glycine max] (NCBI Reference Sequence accession no.
XP_003548142)
PlUGT13
PREDICTED: anthocyanidin 5, 3-O-glucosyltransf erase-like
92%
[Glycine max] (NCBI Reference Sequence accession no.
XP_003533970)
PlUGT14
glycosyltransferase GT01K01 [Pueraria montana var.
100%
PlUGT16
lobata] (GenBank accession no. ADV71360)
glycosyltransferase GT04F14 [Pueraria montana var. lobata]
98%
PlUGT18
(GenBank accession no. ADV71364)
PREDICTED: UDP-glycosyltransferase 74E2-like
93%
[Glycine max] (NCBI Reference Sequence accession no.
XP_003554595)
PlUGT22
PREDICTED: UDP-glycosyltransferase 83A1-like
87%
[Glycine max] (NCBI Reference Sequence accession no.
XP_003552563)
PlUGT23
glycosyltransferase GT04F14 [Pueraria montana var. lobata]
86%
PlUGT35
(GenBank accession no. ADV71364)
glycosyltransferase GT19J14 [Pueraria montana var. lobata]
98%
PlUGT39
(GenBank accession no. ADV71373)
glycosyltransferase GT18P15 [Pueraria montana var. lobata]
97%
PlUGT40
(GenBank accession no. ADV71372)
glycosyltransferase GT21C20 [Pueraria montana var.
99%
lobata] (GenBank accession no. ADV71374)
Fig. S1 Mass spectra of the products detected in the yeast culture extract. a, b and c showed
the mass spectra of the authentic standards daidzin, genistin and ononin respectively; d, e and
f showed the mass spectra of the products that PlUGT1 reacted with daidzein, genistein or
formononetin respectively; g, h and i showed the mass spectra of the products that PlUGT13
reacted with daidzein, genistein or formononetin respectively. The collision energy is 10 V.
The MS/MS data of the each product were consistent with corresponding authentic standards.
Fig. S2 UV spectra of the products detected in the yeast culture extract in methanol. a, b and c
showed the UV spectra of the authentic standards daidzin, genistin and ononin respectively; d,
e and f showed the UV spectra of the products that PlUGT1 reacted with daidzein, genistein
or formononetin respectively; g, h and i showed the UV spectra of the products that PlUGT13
reacted with daidzein, genistein or formononetin respectively. The UV spectrum of the each
product was consistent with corresponding authentic standards.
Fig. S3 HPLC analysis of the activities of PlUGT1 and PlUGT13 toward genistein. PlUGT1
converted nearly all genistein to genistin (Peak 1) and PlUGT13 only converted part of
genistein to genistin (Peak 2). WAT11 (pESC-HIS), the yeast strain carrying the empty vector
pESC-His as a control; WAT11 (pESC-HIS-PlUGT1), the yeast strain expressing PlUGT1;
WAT11 (pESC-HIS-PlUGT13), the yeast strain expressing PlUGT13. For the mobile phase,
solvent A was Milli-Q water and solvent B was HPLC grade methanol. Samples were
separated with 45% B for 60 min. Genistin and its isomer sophoricoside (glycosylated
isoflavone aglycones at the 4'-hydroxy) have different retention times. The retention times of
the products (Peak 1 and Peak 2) were consistent with genistin.
Fig. S4 Purification of the recombinant protein PlUGT1 and PlUGT13 from E. coli Rosetta
(DE3) cells. Lane M, protein molecular mass standard; lane 1, PlUGT1; lane 2, PlUGT13.
Fig. S5 HPLC analysis of the activities of WAT11 (pESC-HIS-PlUGT1, pESC-TRP-IFS) and
WAT11 (pESC-HIS-PlUGT13, pESC-TRP-IFS) toward liquiritigenin. Both WAT11
(pESC-HIS-PlUGT1, pESC-TRP-IFS) and WAT11 (pESC-HIS-PlUGT13, pESC-TRP-IFS)
converted liquiritigenin to daidzin (Peak 1 and Peak 3) and WAT11 (pESC-HIS-PlUGT1,
pESC-TRP-IFS) showed a relatively higher activity. Daidzein (Peak 2 and Peak 4) could be
detected as well. WAT11 (pESC-HIS, pESC-TRP), the yeast strain carrying the empty vector
pESC-His
and
pESC-TRP,
served
as
a
control;
WAT11
(pESC-HIS-PlUGT1,
pESC-TRP-IFS), the yeast strain expressing PlUT1 and IFS; WAT11 (pESC-HIS-PlUGT13,
pESC-TRP-IFS), the yeast strain expressing PlUGT13 and IFS. The retention times and UV
spectra of the products (Peak 1 and Peak 3) were consistent with daidzin.
Fig. S6 Effect of MeJA on daidzin production at different concentrations. The time on the
x-axis refers to the days after the subculture of the cell line (see the experiment section in the
main text file). The homogenous cell line was treated with MeJA on the 4th day after the
subculture. The daidzin content on the 4th day showed the baseline level before induction.
After the 4th day, the effect of MeJA on daidzin production was investigated every two days
till the 18th day.
Fig. S7 The content of puerarin and daidzin in different organs. Error bars represent the
standard errors (s.e.) from three biological replicates. To measure the content of daidzin and
puerarin in different organs (roots, stems, and leaves) of P. lobata, 40 mg of dried plant
materials were extracted with 1 ml ethanol and repeated for three times. The ethanol extracts
were air dried at room temperature and re-dissolved in methanol for HPLC analysis. The
HPLC method of measuring the content of daidzin and puerarin is same as those described
previously (Li et al. 2014). For chemical quantifications, a standard calibration curve was
assembled from a range of concentrations (1–100 µg ml–1) of each chemical standard.
Fig. S8 Structures of the substrates used in in vitro enzyme activity assays.