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Supplementary figure legends
Figure S1 Homology of GhSusA1 to other sucrose synthases
The amino acid sequence of GhSusA1 was compared with the amino acid
sequences of two cotton sucrose synthases and 17 SuSys from other plants.
Identical amino acids were indicated by black shading, and similar amino
acids were indicated by gray shading. “DxD” motif was marked with black
triangles. Conserved feature sites were boxed and phosphorylate sites were
marked with “P”. (Gh, Gossypium hirsutum; Cit, Citrus unshiu; Ps, Pisum
sativum; Pv, Phaseolus vulgaris; St, Solanum tuberosum; Sl, Solanum
lycopersicum; At, Arabidopsis thaliana; Bv, Beta vulgaris; Vit, Vitis vinifera;
Pin, Pinus taeda; Os, Oryza sativa; Zm, Zea mays; Ta, Triticum aestivum; Bo,
Bambusa oldhamii.)
Figure S2 Comparison of deduced amino acid sequences of plant sucrose
synthase. A phylogenetic dendrogram was generated by the multi alignment
using UPGMA based on identity. The branch lengths indicate the percentage
of sequence homology. The deduced amino acid sequences of plants come
from the following sources: Citrus SuS1 (BAA88905), Cotton GhSus1
1
(AAD28641),
Poplar
(ABW76422),
Pea
(AAC28107),
Kidney
bean
(AAN76498), Arabidopsis SuS4 (NP_566865), Coffee (ABI17891), Potato
SuS4 (CAD61188), Potato SuS2 (AAO34668), Tomato (CAA09593), Cotton
GhSus2 (CAT04586), Bamboo (AAV64256), Maize SuS1 (NP_001105323),
Wheat
SuS2
(CAA03935),
Barley
SuS2
(CAA75793), Wheat
SuS1
(CAA04543), Rice SuS2 (ABL74568), Maize Sh1 (ACG43170), Sorghum 1
(ACM69042), Sugarcane SuS2 (AAF85966), Arabidopsis T2H3.8 (AAC28175),
Arabidopsis SuS3 (NP_192137), Pear SuS1 (BAB20799), Citrus SuSA
(BAA88904.1), Cotton GhSusA1 (HQ702185), Maize SuS2 (NP_001105194),
Rice Os03g0340500 (NP_001050064), Sugar beet SuS1 (ABR87939), Potato
1 (AAO67719), Arabidopsis SuS2 (NP_199730), Grape (CAO44821), Pine
(ABR15470). The black full trigles noted sucrose synthases in cotton.
Figure S3 Purification of GhSusA1 recombination protein in E. coli and the
enzyme activity assay.
(a) SDS-PAGE gel of recombinant GhSusA1. The strains of E. coli carried a
plasmid pET-30a(+)-GhSusA1 with the gene encoding Sus from cotton placed
under the control of the IPTG-inducible promoter. Lane M, protein maker; Lane
1, extracts of un-induced cells; lane 2, IPTG-induced cells; Lane 3,
recombinant GhSusA1 purified from the IPTG-induced E. coli culture.
(b) The activities of crude protein and purified protein expressed in E. coli
2
induced by addition of IPTG.
Figure S4 Genomic evolution and molecular mapping of GhSusA1.
(a) Southern blotting analysis of GhSusA1 in G. hirsutum TM-1 genome. Lane
1 to 4, total DNA digested with EcoRI, HindIII, XbaI, BamHI, respectively.
(b) Molecular mapping of GhSusA1 in tetraploid cotton. Genetic map was
constructed using a BC1 population obtained from the interspecific cross: G.
hirsutum L. acc. TM-1 × G. barbadense L. cv. Hai7124, and based on
previous report from Guo et al. at 2007. Positions of GhSusA1 are underlined.
Distance between loci is given in centiMorgans.
Figure S5 Structures of the GhSusA1 gene in four cotton species. A and D
represent G. herbaceum L. var. africanum and G. raimondii Ulbr, respectively.
TM-1-At and TM-1-Dt denote A and D subgenome, respectively, of G. hirsutum
L. cv. TM-1. 7124-At and 7124-Dt represent A and D subgenome, respectively,
of G. barbadense L. cv. 7124. Note orthologs having the same structures
among four different cotton species.
Figure S6 Agrobacterium-mediated transformation and plant regeneration of G.
hirsutum W0 using hypocotyls and leaves as explants.
(a) – (d) Callus induction of hypocotyl and leaf segments after infected by
3
Agrobacterium.
(e) – (f) Calluses excised from hypocotyl and leaf segments showing growth on
media.
(g) – (h) Callus multiplication; i: Embryogenic calli generated and proliferation.
(i) – (j) Embryogenic lines showing differentiation of somatic embryos.
(k) – (n) Plant regeneration.
(o) Transgenic plants in greenhouse.
(p) T1 transgenic plants in field.
Figure S7 Construction and molecular identification of the sense GhSusA1
plant expression vector.
(a) Structure of the GhSusA1 sense expression vector driven by the 35S
promoter.
(b) Southern bolt analysis of some sense-GhSusA1 transgenic lines. Genomic
DNA was digested with EcoRI and hybridized with a 0.75-kb fragment of the
NPTII. Lane P: plasmid of 35S-SA; Lane C: non-transformed W0 plant; lane
2-15: different transgenic plants.
Figure S8 Expression of GhSus1 in GhSusA1 transgenic lines and wild type
plants.
Expression level of GhSus1 in fibers of W0, Line 9 and Line 45 during fiber
4
elongation time 10, 15 and 20 DPA. Values are mean ± SD of three biological
replicates (n = 3). Primers used for specific amplified of GhSus1 were Forward:
5’-TGTAGGTGGTGATAGGCGAAAG-3’
and
Reverse:
5’-TCCTCTGCCAATGGAACTGA-3’. No significant differences at P ≤ 0.05
according to randomization one-way ANOVA test.
5
Figure S1
P
P
6
7
8
9
Figure S2
Dicot
Citrus SuS1
Cotton GhSUS1
Poplar
Pea
Kidney bean
Arabidopsis SuS4
Coffee
Potato SuS4
Potato SuS2
Tomato
Ⅰ
Cotton GhSUS2
0.05
10
Monocot
Bamboo
Maize SuS1
Wheat SuS2
Barley SuS2
Wheat SuS1
Rice SuS2
Maize Sh1
Sorghum 1
Sugarcane SuS2
Arabidopsis T2H3.8
Arabidopsis SuS3
Pear SuS1
Citrus SuSA
Cotton GhSUSA1
Maize SuS2
Rice Os03g0340500
Sugar beet SuS1
Potato 1
Arabidopsis SuS2
Grape
Pine
Ⅱ
Figure S3
(a)
(b)
-1
SuSy Activity(nmol Fru.•mgProtein•min )
KDa
97.2
92.1
1.2
1
-1
66.4
1.4
44.3
29.0
M
1
2
3
0.8
0.6
0.4
0.2
0
Crude
11
Purified
Figure S4
(a)
2027 bp
2322 bp
21226 bp
1
2
3
4
(b)
A8(Chr.08)
0.0
0.1
0.3
0.4
0.8
1.0
1.5
1.8
2.5
2.8
3.3
3.9
4.5
NAU3207-275
NAU2914-125
NAU3632-470
NAU4045-350
NAU3199-200
NAU3769-350
GhSusA1A-108
em5DC1-300
NAU520-205
GW55-250
BNL1303-2000
NAU3058-250
JESPR90-260
D8(Chr.24)
0.0
0.3
0.6
0.7
1.0
1.2
1.4
1.7
1.9
2.3
3.2
NAU2914-225
NAU1531-240
TMA5-180
em1DC1-205
BNL3084-165
BNL2655-150
BNL2499-250
GhSusA1D-272
EM1OD26-500
BNL2568-180
NAU5335-180
NAU2292-440
12
Figure S5
13
Figure S6
a
b
c
d
e
f
g
h
i
l
j
k
m
n
p
o
14
Figure S7
(a)
(a)
P-NOS NPT Ⅱ TT-NOS
NOS
(a)
P-35S
GhSUSA1
GUS
T- NOS
35S -SA
35S -SA
RB
P-NOS NPT Ⅱ TT-NOS
NOS
P-35S
RB
GhSUSA1
GUS
T- NOS
LB
LB
(b)
(b)
(b)
P
21226bp
21226bp
131315159 96-16-117172121 1111 2828 24
P4 46 616 16
24 27
27 27-1
27-1 C
C
5148bp
5148bp
4268bp
4268bp
3530bp
3530bp
2027bp
2027bp
15
Relative Expression
Figure S8
2.0
1.8
1.6
1.4
1.2
1.0
.8
.6
.4
.2
0.0
w0
9
45
15d
10d
20d
DPA
16
Table S1 Primers for TAIL-PCR
Round
s
1
2
3
Type
s
GSP1
5’ TAIL primers (5’-3’)
3’ TAIL primers (5’-3’)
GATACAGCCCTGGAAGAGT
ACGGCACTGCATGGCTCA
GSP2
TCTTGGTATGTGCTGGTGA
CATGGCAAGGCTGGATCG
GSP3
TGTAAGCCAACAAAGATGC
GCTGAGGGAATTAGCAAACC
DP
CCNGGNGTNTGGGARTA
(A/T)CAGNTG(A/T)TNGTNCTG
GSP1
ATGGATGAAGAGCGGTTGG
GGCAAGGCTGGATCGAGTTA
GSP2
ATGGTTGTCCTCCCTACGC
GSP3
TTGCTCCACGCTGAGTTCAT
CCTCGTTGTTGTTGCTGGTTA
C
CCCAAACAAATCGAGCACGCA
AP
GTCGGAGTGG
NGTNGCNGA(A/G)GA(C/T)ATG
GSP1
GSP2
TTTAAAGACGCAGCATCGAGG
T
ACTGCCTCATGAATCACGGAC
GSP3
G
GGGGAAATGACCTGTGCAGAT
AP
CACGGACCGA
Note: GSP is gene specific primer and AP is the arbitrary primer.
17