Fig S1. Alignment of predicted MYB domain protein sequences.
Amino acid sequence alignment was obtained with Clustal W2 and then orderly grouped on the basis of homologies
to the consensus R2R3 MYB DNA-binding domain (2a-MYB R2R3-DBD), the SANT/MYB DNA binding region
(2b-SANT region) and the Arabidopsis calmodulin-interaction motif (2c, AtMYB2 CaMBD) as indicated. Black
shading indicates identical amino acid residues and grey shading are the conserved or semi-conserved substitutions
observed and dashes indicate gaps. The numbers on the right indicate the amino acid position relative from
translation start codon. The names on the left indicate the accession numbers of MYBs found in different plants. In
2a, the boxes and lines above the sequences predict the helix and turn structures in the R2 and R3 regions of MYB
domain. Stars indicate the conserved tryptophan residues; the black arrows represent the unusual amino residues
compared to the consensus amino acid sequence of MYB DNA- binding domains of several plant R2R3-MYB
proteins described by Avila J et.al 1993. Intron positions are indicated by triangles I and II. SO- Saccharum
officinarum hybrid, ZM - Zea mays, SB- Sorghum bicolor, OS- Oryza sativa, OS-cad- Oryza sativa MYB18 protein.
M
1
2
3
11kb
1792bp
1792bp
A.
B.
Fig S2. PCR and restriction digestion confirmation of SoMYB18 gene cloning in pBinAR vector
A. Colony PCR confirmation of pBinAR SoMYB18 clones. M-1.0 kb ladder, Lane 1 and 2 white colonies and 3
water control B. Reconfirmation of gene cloned in pBinAR vector by restricts digestion using KpnI and BamHI
enzymes. M-1.0 kb ladder, Lane 1 and 2-plsmid of +ve clones restricted by KpnI and BamHI, Lane3- KpnI and
BamHI restricted SoMYB18 gene used for ligation, Lane 4- KpnI and BamHI restricted pBinAR vector used for
ligation. M: 1.0 kb marker in each gel.
SoMYB18
pBIN-SoMYB18-Nos
Fig S3. pBinAR Vector and pBinAR::SoMYB18 expressing construct
The pBinAR:: SoMYB18 construct was transformed in Tobacco plants by Agrobacterium mediated transformation
Method. Callus was observed after 4 weeks of incubation, After 8 weeks of subculture, the callus was transferred in
glass bottles, Roots were observed after 3 weeks of transfer on rooting media.
(A)
(B)
(C)
Fig S4. Transformation pBinAR::SoMYB18 construct through agro-transformation and regeneration of
transformed tobacco A. Callus B. Regeneration C. Putative transgenic plant
M
1 2
3
4
5
6
7
8
9 10 11 12
1557bp
Fig S5. PCR screening of putative transgenic tobacco plants harboring SoMYB18 gene
M-1 Kb DNA ladder, Lane 1 to 10 putative transgenic tobacco plantsT0-1,T0-2, T0-3,T0-4, T0-5, T0-6, T0-7, T0-8, T09 and T0-10, 11- positive control pBinAR::SoMYB18 plasmid and 12- negative control g DNA of un-transformed
tobacco plant.
1 2
3
4
5
6
7 8
Fig S6. Southern blot analysis of SoMYB18 overexpressing T0 transgenic tobacco plants
Southern blot analysis of tobacco transgenic plants to determine the copy number of the
SoMYB18 gene integration in tobacco genome. Left to right: lane 1- T0-1, 2- T0-2, 3- T0-3, 4- T04 5- T0-5, 6-Co740 Genomic DNA and 7- pBinAR::SoMYB18 plasmid and 8- Un-transformed
tobacco.
Inheritance of transgenic tobacco in T0- T2 generations:
Two single event southern positive plants were hardened, bagged and self-fertilized (T0-2 and
T0-3). The seeds from self fertilized plants were dried and used for germination on Kanamycin
selection medium (50µg/l) (Fig S-2A). The germination percentage of SoMYB18 plants was
12.66%.
Fig S7. Hardening, bagging and self fertilization of single event southern positive SoMYB18
transformed tobacco plants and baging for self-fertilization.
A
B
C
D
Fig S8. A. germination of transformed seeds on kanamycin (50mg L-1), B. PCR positive Plants
were hardened to soil, C. Maturation of T1 generation SoMYB18 transformed tobacco plants and
D. Bagging of generation SoMYB18 transformed tobacco plants for self-fertilization.
These four SoMYB18 putative transformed and one normal tobacco as control plants were
transferred to soil conditions (Soil:Sand:Cocopeat, in 2:1:1 proportion). These plants were
bagged before floral bud development for self-fertilization. The seed pods were collected and
dried. These dried seeds were used for germination test of T2 generation.
Fig S9. Germination of SoMYB18 overexpressing T2 generation transgenic tobacco plants
on Kanamycin (50 mg L-1)
Table S1: Predicted domains, repeats, motifs and features of SoMYB18
Name of Domain
Begin
End
E-value
Function/Description
2 SANT Domains
13
66
63
114
2.78e-12
1.39e-12
SANT SWI3, ADA2, N-CoR and TFIIIB'' DNAbinding domains
Pfam:
binding
14
61
9.70E-12
Encode nuclear DNA-binding proteins
GIYc
31
118
2.37E+03
GIY-YIG type nucleases (URI domain)
CARD
42
122
5.64E+02
Caspase recruitment domain
Pfam:Myb_DNAbinding
BH4
67
112
9.70E-08
Encode nuclear DNA-binding proteins
100
126
1.23E+02
Bcl-2 homology region 4
Pfam: BH4
100
126
104
133
6.80E+00
1.65E+05
Bcl-2 homology region 4
SAP
ARM
122
165
1.30E+03
Armadillo/beta-catenin-like repeats
CLECT
215
350
2.22E+03
Pfam: DUF303
269
423
6.70E+00
C-type lectin (CTL) or carbohydrate-recognition
domain (CRD)
Function of this domain is unknown
Myb_DNA-
Putative DNA-binding motif predicted to be
involved in chromosomal organisation
Table S2: Biochemical analysis of SoMYB18 transformed tobacco plants under drought, salt and cold stress
Proline
content
MDA
content
Total
chloroph
yll
content
SOD
activity
POD
activity
Tim
e
(Hrs
.)
UT
PEG
TT
1
17.91±4.0
Fold
increas
e/
Decrea
se
Salt
UT
TT
67.45±2.9
62.35±6.6
6
26.21±0.7
12
164.74±8.5
103.64±17.
0
245.69±37.
8
59.47±1.7
24
1
180.58±37.6
0.096±0.00
66.31±3.1
0.448±0.02
6
12
0.351±0.03
0.627±0.06
0.426±0.00
0.467±0.03
24
1
0.503±0.02
0.897±0.00
0.477±0.04
1.510±0.02
-1.34
-1.05
1.6
0.607±0.05
1.508±0.14
6
12
1.125±0.04
1.188±0.05
1.882±0.11
1.778±0.15
1.7
1.5
1.735±0.13
1.567±0.05
24
1
2.145±0.02
58.99±4.42
-1.08
2.2
1.239±0.07
15.96±2.76
6
21.34±2.73
6.6
24.15±3.08
12
24.81±2.97
1.984±0.06
131.58±18.
90
141.61±23.
70
77.12±6.99
3.1
24.40±5.27
24
8.24±1.03
27.8
15.42±2.16
1
-4.31
6
112.119±11.
50
71.089±9.27
140.485±13.
47
46.271±3.60
12
43.914±0.69
229.96±25.
19
26.015±6.6
7
15.765±1.9
5
9.754±0.41
24
42.166±4.13
1
39.786±14.2
6
56.793±5.60
12
41.066±5.71
24
62.216±13.4
-1.08
5.78
60.99±2.5
50.73±3.9
83.40±5.7
289.23±
64.9
516.99±7.4
0.361±0.01
4.66
1.21
73.25±4.9
0.384±0.01
0.767±0.05
0.511±0.02
-4.51
47.248±2.55
-4.50
CAT
activity
24.882±2.1
9
14.246±2.5
3
19.814±3.1
3
14.987±3.4
5
32.126±5.3
9
38.462±4.62
-1.69
21.298±7.65
-2.79
-2.86
12.291±1.10
0
10.848±2.16
-2.74
-1.94
Cold
UT
TT
131.04±
23.5
52.90±2.5
77.05±7.6
40.48±2.3
111.34±15.6
27.159±12.7
8
0.605±0.03
0.396±
0.00
0.381±0.00
1.6334±0.1
2
2.578±0.18
2.872±
0.31
1.950±0.18
108.32±5.6
4
71.56±2.52
69.86±
4.63
144.92±25.
93
11.705±0.8
8
12.535±1.9
6
19.349±
4.59
16.924±2.8
4
29.599±3.3
1
24.215±1.8
9
31.809±
1.67
36.962±5.5
6
3.46
7.05
114.54±4.5
0.245±
0.01
0.362±0.02
0.479±0.11
32.50±2.6
0.352±0.01
-1.06
-1.26
-1.29
-1.59
1.0
0.262±0.02
0.766±
0.03
1.666±0.05
0.987±0.04
0.345±0.00
1.662±0.10
1.5
1.8
1.6
6.8
0.718±0.03
41.494±
2.52
33.737±1.6
7
38.042±1.2
6
43.306±6.3
7
71.676±
11.32
98.115±22.
56
15.885±1.6
2
75.147±2.2
1
48.611±
2.80
89.671±19.
24
74.931±12.
59
32.438±5.6
7
1.790±0.11
164.704±19.
26
102.193±3.7
6
123.344±18.
85
97.099±8.74
3.0
2.9
9.4
-12
-3.69
-2.49
-2.27
1.4
2.0
2.9
1.4
Fold
increas
e/
Decrea
se
-1.7
61.53±11.9
-1.20
9.37
-2.77
-2.72
Fold
increa
se
1.16
0.331±0.01
0.293±0.03
1.954±0.07
1.454±0.08
2.75
-3.52
1.43
-1.09
-1.63
1.31
2.17
1.17
1.47
2.49
4.0
3.0
3.2
2.2
17.789±3.37
-4.03
9.158±0.68
-10.72
5.580±0.49
-2.84
8.194±0.37
-19.17
18.111±1.19
-2.68
22.296±1.69
-4.02
20.951±1.46
-3.58
17.981±0.82
-1.80