Supp. 9. ItWRKY1 gene cloning and validation of its function
1. ItWRKY1 gene cloning from I. trifida
1.1 ItWRKY1 CDS and all the primers
ItWRKY1 CDS has a length of 1653 (551*3) bp, coding 550 amino acids plus
one stop codon. The following ItWRKY1 CDS sequence was used in the gene cloning
and agrobacterium-mediated transformation of tobacco.
> ItWRKY1 CDS 1531 bp
ATGGCTGCTTCTTCAGGGACAATAGACGCCCCCACAGCTTCTTCATCTTTCTCTTTCTCCACCGCCTCTT
CATTCATGTCCTCCTCCTTCACTGACCTCCTTTCCTCCGACGCCTATTCCGGCGGCTCTGTGAGCAGAG
GGCTGGGTGATCGGATAGCGGAGAGGACGGGGTCGGGTGTGCCCAAGTTTAAGTCTTTGCCGCCGCC
GTCTCTGCCGCTTTCTTCGCCGGCCGTCTCGCCGTCGTCTTACTTCGCTTTTCCTCCTGGGTTGAGCCC
CAGTGAGCTCCTGGATTCCCCTGTTCTTCTATCTTCCTCAAACATTTTGCCGTCTCCGACAACTGGGAC
TTTTCCTGCTCAGACCTTCAACTGGAAGAATGATTCTAACGCATCCCAGGAAGATGTTAAGCAAGAAG
AGAAAGGATACCCAGATTTCTCTTTCCAGACTAACTCTGCTTCAATGACAATGAATTATGAAGATTCTA
AGAGGAAAGATGAGCTCAATTCTCTGCAGAGCCTTCCCCCTGTGACTACTTCAACTCAGATGAGCTCT
CAGAACAATGGTGGGAGCTACTCTGAGTATAATAATCAATGCTGCCCGCCCTCCCAGACGTTGAGGGA
GCAGAGGCGATCTGATGACGGGTACAATTGGAGGAAATACGGGCAGAAACAGGTGAAGGGGAGCGA
AAACCCGAGGAGTTATTACAAGTGCACGCACCCGAATTGCCCCACGAAGAAGAAGGTCGAGAGGGCT
TTGGATGGGCAGATTACTGAGATTGTCTACAAAGGAGCTCACAATCACCCGAAGCCTCAGTCCACTAG
GAGATCGTCGTCCTCCACAGCTTCTTCGGCTTCAACTTTGGCTGCCCAGTCTTATAACGCGCCTGCCAG
TGATGTCCCGGATCAGTCGTATTGGTCTAATGGTAACGGGCAGATGGATTCTGTTGCCACGCCAGAGAA
TTCTTCGATCTCCGTGGGGGATGATGAATTCGAGCAGAGCTCTCAGAAGAGGGAGCCCGTGGGAGAC
GAGTTTGATGAAGACGAACCCGATGCAAAGAGATGGAAAGTGGAAAACGAAAGCGAGGGAGTTTCT
GCACAGGGGAGTAGGACAGTAAGGGAACCGAGAGTTGTAGTTCAAACGACGAGTGATATTGATATTCT
CGACGATGGTTATAGATGGAGAAAATATGGCCAGAAAGTTGTGAAGGGAAATCCCAATCCAAGGAGCT
ATTACAAATGCACGAGCCAAGGCTGCCCGGTGAGGAAACACGTGGAAAGGGCTTCACACGATATCCG
CTCGGTGATAACAACCTACGAAGGGAAACACAACCACGACGTTCCTGCTGCCCGAGGGAGTGGCAGC
CACGGCCTCAACCGGGGCGCCAATCCTAACAACAATGCGGCCATGGCTATGGCGATTAGGCCTTCGAC
GATGTCTCTCCAATCTAACTACCCCATCCCAATCCCGAGCACGAGGCCAATGCAGCAGGGAGAAGGCC
AAGTGCCTTACGAGATGTTGCAGGGACCGGGCGGTTTTGGGTACTCGGGATTTGGGAACCCGATGAAT
GCCTACGCGAACCAAATCCAGGACAACGCGTTCTCGAGGGCCAAGGAGGAGCCCAGAGATGAGTTGT
TCCTGGAGACATTGCTAGCTTGA
Table S1. Primer information of ItWRKY1
ID
WRKY110-F
110 bp
WRKY110-R
WRKY686-F
686 bp
WRKY686-R
WRKY1653-F
1653
WRKY1653-R
WRKY1669-F
1669
WRKY1669-R
ACTIN209-F
209
ACTIN209-R
ACTIN516-F
Sequence(5’-3’)
Length
516
ACTIN516-R
Tm/℃
TTTGGGTACTCGGGATTTGG
58
GTCTCCAGGAACAACTCATCTC
59
TCCTCCTCCTTCACTGACCTCCT
59.9
ATCTGCCCATCCAAAGCCCTCT
59.7
ATGGCTGCTTCTTCAGGGAC
67.9
TCAAGCTAGCAATGTCTCCAGG
63.4
caggtaccATGGCTGCTTCTTCAGGGAC
67.3
aatctagaTCAAGCTAGCAATGTCTCCAGG
63
CCGTGACATCAAGGAGAAG
58
AGGTGGTAGCGTGGATAC
57
ACGGAGCGTGGCTACTCTTTCA
60
AGACTCGTCGTACTCTGCCTTGG
60.1
WRKY1653-F and WRKY1653-R is the first 20 bp from the 5’ end and the last 22 bp from the 3’ end on the 1,653
ItWRKY1 CDS sequence. WRKY1669-F is WRKY1653-F extended to 5’ end with 6 bp KpnI site plus 2 bp for
protection. WRKY1669-R is WRKY1653-R extended to 3’ end with 6 bp XbaI site plus 2 bp for protection. The
PCR product using the primer WRKY1669-F and WRKY1669-R had the length of 1669 bp. WRKY1653-F and
WRKY1653-R were not used in this study.
1.2 Total RNA extraction and cDNA synthesis
1. Total RNA was extracted from each tissue (0.5 g fibrous root, tuberous root, stem
and leaf) using TRIzon Reagent Kit (CWBIO). The electrophoresis (1% agarose gel)
and Nanodrop were used for RNA quality control.
2. The following reaction mixture was incubated at 37℃ for 25 min. Then, 0.5 μL
EDTA was added to this mixture at 65℃ for 10 min.
DNAase I
1 μL
10×Buffer
1 μL
RNA
8 μL
3. The RNA mixture (10.5 μL) with 2 μL Oligod(T)18 was incubated at 80℃ for 10
min and immediately placed on ice for 3-5min. The following reverse-transcribed
reaction mixture (25 μL) was used to synthesize cDNA at 42℃ for 90 min and the
cDNA product was preserved at -20℃.
5×MLV buffer
5 μL
dNTP
4 μL
RNase inhibitor
0.5 μL
M-MLV
1 μL
4 μL
DEPC
1.3 ItWRKY1 cDNA and DNA amplification
1. The cDNA from the previous step was PCR-amplified, using the primers of
WRKY686 and ACTIN516 (Figure 6B in the manuscript). The following PCR
reaction mixture was incubated at 94˚C for 5 minutes, followed by 35 PCR cycles (30
s at 94˚C, 30 s at 60˚C and 30 s at 72˚C for each cycle) and finally incubated at 72˚C
for 7 minutes. The PCR product was validated by electrophoresis (1% agarose gel).
cDNA
2 μL
10×buffer
2.5 μL
dNTP
2.5 μL
Primers
2 μL
TaqE
0.3 μL
ddH2O
15.7 μL
Total
25 μL
2. The tuberous cDNA and DNA were PCR-amplified using the primers of
WRKY1669 (Figure 6C in the manuscript). The following PCR reaction mixture was
incubated at 94˚C for 5 minutes, followed by 35 PCR cycles (30 s at 94˚C, 30 s at
60˚C and 100 s at 72˚C for each cycle) and finally incubated at 72˚C for 7 minutes. 1
μL of PCR product was validated by electrophoresis (1% agarose gel). The remaining
PCR product was used for ItWRKY1 gene cloning (See 1.4).
cDNA
4 μL
10×buffer
5 μL
dNTP
5 μL
Primers
4 μL
TaqE
0.5 μL
ddH2O
31.5 μL
Total
50 μL
1.4 Construction of ItWRKY1 gene cloning plasmid
1. The remaining PCR product from the previous step was performed
electrophoresis (1% agarose gel). The gel band containing ItWRKY1 gene was cut,
collected and purified by the Gel Extraction Kit. The purified product was validated
by electrophoresis (1% agarose gel).
2. The purified product was ligated to the pEASY-T1 vectors. The following
reagents were mixed at room temperature for 15 min.
PCR product
3 μL
pEASY-T1
1 μL
ddH2O
1 μL
3. The product from the previous step was mixed with 50 μL E. coli strain
Trans1-T1 cells by tapping gently and incubated in ice for 30 min.
4. The mixture was heat-shocked at 42℃ for 30 s and immediately placed on ice
for 2 min.
5. The mixture was inoculated into 250 μL liquid LB medium and incubated at 37℃
with shaking at 200 rpm for 1 h.
6. 8 μL IPTG (500 mM) and 40 μL X-gal (20 mg/mL) were mixed and spread on a
plate processed by solid LB medium containing 100 mg/L Kana. The plate was
incubated at 37℃ for 30 min.
7. After IPTG and X-gal were soaked, 200 μL mixture from step 5 was spread on
the plate and incubated overnight.
8. The streak plate method was used to obtain single isolated pure colonies, by
being incubated at 37℃ overnight. The colony PCR of several positive clones was
performed to identify the transformants using the primers of WRKY1669. The
following PCR reaction mixture was incubated at 94˚C for 10 minutes, followed by
35 PCR cycles (30 s at 94˚C, 30 s at 60˚C and 100 s at 72˚C for each cycle) and
finally incubated at 72˚C for 7 minutes. The PCR product (1 μL) was validated by
electrophoresis (1% agarose gel).
10×buffer
2.5 μL
dNTP
2.5 μL
Primers
2 μL
TaqE
0.3 μL
ddH2O
17.7 μL
Total
25 μL
9. The plasmids from positive clones were selected and validated by Sanger
sequencing.
2. Agrobacterium-mediated transformation of tobacco
2.1 Construction of ItWRKY1 gene expression plasmid
1. The positive clones of ItWRKY1 (E. coli strain Trans1-T1) were inoculated into 5
mL liquid LB medium containing 100 mg/L Kana and incubated at 37℃ with
shaking at 200 rpm. The pEASY-T1 plasmids were extracted using Plasmid
MiniPreparation Kit (Generay, Shanghai, CHINA).
2. E. coli strain DH5-α containing pCAMBIA1301 plasmids was inoculated into 5
mL liquid LB medium containing 100 mg/L Kana and incubated at 37℃ with
shaking at 200 rpm. The pCAMBIA1301 plasmids were extracted using Plasmid
MiniPreparation Kit (Generay, Shanghai, CHINA).
3. The pCAMBIA1301 vector and the recombinant pEASY-T1 vector were digested
using two enzyme KpnI and XbaI. The following reagents were mixed gently and
incubated at 37℃ for 3 h.
Multicore buffer
4 μL
BSA
0.4 μL
plasmid
10 μL
KpnI
1 μL
XbaI
1 μL
ddH2O
23.6 μL
Total
40 μL
4. The PCR product (3 μL) from the previous step was validated by electrophoresis
(1% agarose gel). The ItWRKY1 cDNA was collected from the gel and ligated to the
pCAMBIA1301 vectors. The following reagents were mixed gently and incubated at
16℃ overnight.
ItWRKY1 cDNA
3 μL
pCAMBIA1301
3 μL
Buffer
1 μL
T4 ligated enzyme
0.5 μL
ddH2O
2.5 μL
Total
10 μL
5. The ligated product (5 μL) was added into the 50 μL E. coli strain DH5-α and
performed ice bath for 30 min, hot shock at 42℃ for 90 s, and then ice bath for 6 min.
E. coli strain DH5-α was inoculated into 250 into 500 μL liquid LB medium and
incubated at 37℃ with shaking at 110 rpm for 1 h. The product was spread on solid
LB medium containing 100 mg/L Kana and incubated at 37℃ overnight.
6. The streak plate method was used to obtain single isolated pure colonies, which
then PCR-amplified using the primers of WRKY1669. The following PCR reaction
mixture was incubated at 94˚C for 5 minutes, followed by 30 PCR cycles (30 s at
94˚C, 30 s at 60˚C and 100 s at 72˚C for each cycle) and finally incubated at 72˚C for
7 minutes. The PCR product (5 μL) was validated by electrophoresis (1% agarose
gel).
10×buffer
2.5 μL
dNTP
2.5 μL
Primers
1.5 μL
Taq-DNA polymerase
0.25 μL
ddH2O
18.25 μL
Total
25 μL
7. Positive clones were selected and digested by KpnI and XbaI for validation.
2.2 Transferring ItWRKY1 into A. tumefaciens
1. The positive clones of ItWRKY1 (E. coli strain DH5-α) were inoculated into 5
mL liquid LB medium and incubated at 37℃ with shaking 200 rpm overnight. The
pCAMBIA1301 plasmids containing ItWRKY1 were extracted using Plasmid
MiniPreparation Kit (Generay, Shanghai, CHINA).
2. 10μL plasmids were transferred into 100 μL Agrobacterium tumefaciens strain
LBA4404, performed ice bath for 30 min and immediately frozen in liquid nitrogen
for 3 min, then performed ice bath at 37℃ for 5 min. The bacteria solution was
inoculated into the 500 μL YEB medium containing 100 mg/L Rif and incubated at
28℃ with shaking at 150 rpm for 4-5 h. The mixture was centrifuged, concentrated,
added into the solid YEB medium containing 50mg/L Kana and 50mg/L Rif and
incubated at 28℃ in the dark for 48 h.
3. The colony PCR was performed to identify the transformants using the primers of
WRKY1669. The following PCR reaction mixture was incubated at 94˚C for 5
minutes, followed by 35 PCR cycles (30 s at 94˚C, 30 s at 60˚C and 100 s at 72˚C for
each cycle) and finally incubated at 72˚C for 7 minutes. The PCR product (5 μL) was
validated by electrophoresis (1% agarose gel).
10×buffer
2.5 μL
dNTP
2.5 μL
Primers
1.5 μL
Taq-DNA polymerase
0.25 μL
ddH2O
18.25 μL
Total
25 μL
4. Positive clones were inoculated into liquid YEB medium and incubated at 28℃
with shaking at 200 rpm for 48 h. The concentration of bacterial solution was adjusted
to 20% and preserved at -80℃.
2.3 Construction of transgenic tabacco
1. The preserved Agrobacterium tumefaciens from the previous step was inoculated
into solid YEB medium containing antibiotic and cultured using the streak plate
method at 28℃ in the dark for 72 h. Single clones were selected and inoculated into
liquid YEB medium and incubated at 28℃ with shaking at 200 rpm overnight, then
subcultured for 4 h. The concentration of the bacterial solution was adjusted to
OD600=0.6 for transformation.
2. Full developed tobacco leaves were processed by removing the upper half parts
and edges. The remaining parts of leaves were cut into segments (0.5 x 1.0 cm). These
leaf segments were infected by dipping into a suspension of the ItWRKY1
transformed Agrobacterium tumefaciens LBA4404 for 20 minutes.
3. The leaf segments were blotted on sterile paper towels and cultured on MS
medium at temperature 22-25℃ in the dark.
4. After three days of co-cultivation, the leaf segments were washed with sterile
water and transferred to selective medium containing 300 mg/L Timentin (inhibiting A.
tumefacien) to be screened for four weeks. Calli from leaf segments were subcultured
once every two weeks.
5. Seedlings from calli were transferred to rooting medium.
6. Among 10 rooted transgenic tobacco lines, seven of them grew up in the small
flowerpots under standard conditions.
2.4 Validation of the transformation
2.4.1
The genomic DNA was extracted from transgenic and non-transgenic
tobacco lines, when the young seedlings grew up to have the 3-4 leaves, following
such steps as below:
1. CTAB (2%) buffer was heated at 65℃ in water bath.
2. Several leaves were placed in a 1.5 mL tube and ground to homogenate.
3. 700 ul CTAB (2%) buffer was added into the tube with shaking gently.
4. The mixture was placed in water bath at 65℃ with shaking 2-3 times for 40 min.
5. When the temperature of the mixture reached room temperature, 700 μL
chloroform-isopentanol (24:1) extraction was added with vigorously shaking for 2~3
min for completely mixing.
6. The mixture was centrifuged with 12000 rpm for 10 min, the supernatant was
transferred to another tube. Rerun the step 5.
7. The supernatant was gently transferred to a tube containing 600 μL
Isopropylalcohol after centrifugation at 12000 rpm for 1 min. The tube was shaken for
30 s and kept upright at -20℃ for 30 min.
8. The liquid was removed after centrifugation at 12000 rpm for 1 min.
9. DNA was washed twice by 800 μL Ethanol (75%).
10. Residual liquid was removed after centrifugation at 12000 rpm for 30 s in order
to dry the DNA.
11. The dry DNA was dissolved by 50 μL 0.5 × TE buffer and preserved at -20℃.
2.4.2
ItWRKY1 CDS was validation at the DNA level in seven transgenic tobacco
lines by the PCR amplification using the primers of WRKY686 (Figure 7A in the
manuscript). The following PCR reaction mixture was incubated at 94˚C for 5
minutes, followed by 30 PCR cycles (30 s at 94˚C, 30 s at 60˚C and 40 s at 72˚C for
each cycle) and finally incubated at 72˚C for 7 minutes. The PCR product (5 μL) was
validated by electrophoresis (1% agarose gel).
10×buffer
2.5 μL
Template DNA
1 μL
dNTP
2.5 μL
Primers
1.5 μL
Taq-DNA polymerase
0.25 μL
ddH2O
17.25 μL
Total
25 μL
2.4.3
Total RNA was extracted from seven transgenic tobacco lines (Figure 7B in
the manuscript). The expression of ItWRKY1 CDS in the transgenic tobacco lines
was validated by RT-PCR using the primers of WRKY110 (Figure 7D in the
manuscript).
3. Drought stress experiment
To simulate drought stress, we used 20% PEG solution (30 mL) to irrigate seven
transgenic lines and one non-transgenic line every day for 10 days. We also used 30
mL water to irrigate another non-transgenic line as control. The growth of seven
transgenic and two non-transgenic tobacco lines were recorded on the 1st day (not
processed) and from the 2ND to the 11th day (PEG processed).
Figure S1. A shows the growth status of one non-transgenic tobacco line with PEG processed in 11 days. B shows
the growth status of one non-transgenic tobacco line with water processed in 11 days.
Figure S2. This shows the growth status of No. 1 to No. 7 (left to right) transgenic tobacco lines with PEG
processed in 11 days.