Original article

IiWRKY34 positively regulates yield, lignan biosynthesis

and stress tolerance in Isatis indigotica
Ying Xiaoa,†, Jingxian Fenga,†, Qing Lib, Yangyun Zhoub, Qitao Buc, Junhui Zhoud,
Hexin Tanc, Yingbo Yange, Lei Zhangc,*, Wansheng Chena,b,*
a
Research and Development Center of Chinese Medicine Resources and Biotechnology,
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, China
b
Department of Pharmacy, Changzheng Hospital, Naval Medical University (Second Military
Medical University), Shanghai 200003, China
c
Department of Pharmaceutical Botany, School of Pharmacy, Naval Medical University
(Second Military Medical University), Shanghai 200433, China
d
National Resource Center for Chinese Meteria Medica, China Academy of Chinese Medical
Sciences, Beijing 100700, China
e
Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, China
Received 6 November 2019; received in revised form 14 December 2019; accepted 24 December
2019
*
Corresponding authors. Tel./fax: +86 21 51322403.
E-mail addresses: chenwansheng@smmu.edu.cn (Wansheng Chen), smmuipb@163.com (Lei
Zhang).
†These authors made equal contributions to this work.
Table of contents
Figure S1 Multiple sequence alignment of the WRKY domain among IiWRKYs.
Figure S2 Phylogenetic and structural analysis of IiWRKY proteins.
Figure S3 Phylogenetic relationship of WRKY in I. indigotica (red) and Arabidopsis (green).
Figure S4 Ploidy analysis of diploid and tetraploid I. indigotica.
Figure S5 Schematic representation of IiWRKY34-OVX and IiWRKY34-RNAi vectors.
Figure S6 Representative PCR analyses for the specific genes of transgenic hairy root lines.
Figure S7 The expression levels of IiWRKY34 in different tissues and under drought, salt, SA,
MeJA and UV-B treatments.
Figure S8 GO annotations of DEGs via pairwise contrasts of IiWRKY34-OVX, IiWRKY34-RNAi
and WT roots.
Figure S9 W-boxes in the promoter region of Ii4CL3.
Table S1 PCR primers and biotin-labelled probes used in the text.
Table S2 Sequences of 64 IiWRKY genes.
Table S3 Sequences of 64 IiWRKY proteins.
Table S4 Relative levels of metabolites among IiWRKY34-OVX, IiWRKY34-RNAi and WT lines
based on GC/MS data.
Table S5 Sequences of lariciresinol biosynthetic genes.
Figure S1 Multiple sequence alignment of the WRKY domain among IiWRKYs.
.
1
Figure S2 Phylogenetic and structural analysis of IiWRKY proteins.

2
Figure S3 Phylogenetic relationship of WRKY in I. indigotica (red) and Arabidopsis (green).

Figure S4 Ploidy analysis of diploid and tetraploid I. indigotica.

3
Figure S5 Schematic representation of IiWRKY34-OVX and IiWRKY34-RNAi vectors.

Figure S6 Representative PCR analyses for the specific genes of transgenic hairy root lines.

4
Figure S7 The expression levels of IiWRKY34 in different tissues and under drought, salt, SA,
MeJA and UV-B treatments.

Figure S8 GO annotations of DEGs via pairwise contrasts of IiWRKY34-OVX, IiWRKY34-RNAi

and WT roots.
5
Figure S9 W-boxes in the promoter region of Ii4CL3.

6
Table S1 PCR primers and biotin-labelled probes used in the text.
Primer name Sequence

IiWRKY33-qRT-F CACAAGAACAACAGAGGAAGAACC

IiWRKY33-qRT-R ACACTTGTAGTAACTCCGAGGGT

IiWRKY34-qRT-F GTTAACAAGAGCAATATGGCCG

IiWRKY34-qRT-R CCCTTGAGACTTTCTCCTTCACAA

IiWRKY48-qRT-F AGCTCCTCTCATGACCCGGTT

IiWRKY48-qRT-R GGGATACGTACACTTGAAG

IiWRKY49-qRT-F GCTCCTCTCATAGTTTGATATCTCC

IiWRKY49-qRT-R AACCGGGTCCTGCTTCT

IiWRKY50-qRT-F CAGCAGCAGCAGCAACAGAA

IiWRKY50-qRT-R TGTTAGCGACGCTACCATCTA

Actin-qRT-F ATCCTCCGTCTTGACCTTGCT

Actin-qRT-R TTTCCCGTTCTGCTGTTGTG

IiWRKY34-F AATGATCAATGGACCAGTACTCATCGTCTT
IiWRKY34-R AAACTAGTTTTCTCGGTTTGATTCTGTTGA

IiWRKY34-sNcoI-aSalI CCATGGGTCGACTTGTGCTCCAAGCTGT

IiWRKY34-sKpnI-aXbaI GGTACCTCTAGAACAGCTGCTGCTAATG

IiWRKY34-ovx-F CCCTTAATGATCAATGGACCAGTAC

IiWRKY34-ovx-R TTGCCCTTAAACTAGTTTTCTCGGTT

JDPDK-1F TTGGATTGATTACAGTTGGGA

JDPDKR GGCGGTAAGGATCTGAGCTA

hpt-F CGATTTGTGTACGCCCGACAGTC

hpt-R CGATGTAGGAGGGCGTGGATATG

rolb-F GCTCTTGCAGTGCTAGATTT

rolb-R GAAGGTGCAAGCTACCTCTC

rolc-F CTCCTGACATCAAACTCGTC

rolc-R TGCTTCGAGTTATGGGTACA

IiWRKY34-pET-F GGCCATGGCTGACCAGTACTCATCGTCTT

IiWRKY34-pET-R CGGAGCTCTTTCTCGGTTTGATTCTGTTGA
Ii4CL3pF TCCGATTTGACCCATAATTAATTTGAAATGTGCGGTTGATTGACTTCCAA

Ii4CL3pR TTGGAAGTCAATCAACCGCACATTTCAAATTAATTATGGGTCAAATCGGA

Ii4CL3mpF TCCGATTTTTTTCATAATTAATTTGAAATGTGCGGTTGATTTTTTTCCAA

Ii4CL3mpR TTGGAAAAAAATCAACCGCACATTTCAAATTAATTATGAAAAAAATCGGA