Zhong and Yang, 2011. Characterization of Grape Gibberellin Insensitive1 Mutant Alleles in Transgenic
Arabidopsis. Transgenic Research
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Supplementary Material
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Material and Methods
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Cloning of VvGAI1 cDNA, creation of site-directed mutations of VvGAI1 (VvGAI1*) and
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construction of 35S::VvGAI1* binary vectors.
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A full-length VvGAI1 cDNA fragment was amplified by RT-PCR from the total mRNA derived
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from young leaf tissue of V. vinifera cv. Pinot Noir. The RT-PCR primers (forward 5’-
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GGATCCatgaagagggagtatcatcatcc -3’, beginning of the ORF, + BamHI, reverse 5’-
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GAATTCttcttttattaccaaactatatacttatatatat-3’, end of the cDNA, + EcoRI) were designed from a
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published VvGAI1 cDNA sequence (Boss & Thomas, 2002). Phusion high-fidelity DNA
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polymerase (New England Biolabs, Ipswich, MA) was used in the RT-PCR reactions. The
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VvGAI1 PCR products were cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA).
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Plasmid DNA was extracted using the QIAprep spin miniprep kit (Qiagen, Valencia, CA). The
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constructs were sequenced to verify the authenticity of the VvGAI1 sequences and also their
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insertion orientations. Construct with correct sequence and orientation (Topo-VvGAI1) was used
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in the subsequent cloning steps. Primers for creating point mutations (GAI1L38H, GAI1L38A,
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GAI1L37H, GAI1G42E and GAI1Q333R), internal deletions (GAI1∆37-51, GAI1∆35-39
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GAI1∆75-86, and GAI1E58stop,∆59-60,E61M , also noted as GAI1Rht-B1b), N-terminal truncations
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(GAI1∆1-93 and GAI1∆1-60,E61M , also noted as GAI1Rht-B1b’) are listed in S Table 2. Mutations
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were created using the PCR-based Phusion site-directed mutagenesis kit (New England Biolabs,
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Ipswich, MA). The entire coding regions of mutated VvGAI1 in the constructs, referred as Topo-
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VvGAI1*, were sequenced to ensure that the desired mutations were created and no unwanted
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mutations were introduced. Then the VvGAI1* fragments containing desired mutations were
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released from the Topo-VvGAI1* vectors and placed into the gateway cassette of the pGWB502Ω
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binary vector (Nakagawa et al., 2007) , which has double 35S promoters and a Ω translational
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enhancer, to generate constitutive expression vectors 35S::VvGAI1*. Gateway LR clonase II
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enzyme mix (Invitrogen) was used for vector construction following the manufacturer’s protocol.
, GAI1∆53-56,
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Cloning of VvGAI1 genomic DNA, creation of site-directed mutations of VvGAI1
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(VvGAI1*) and construction of pVv::VvGAI1* binary vectors. A 5.1 kb- long fragment of
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grape GAI1 genomic sequences containing 2171bp putative promoter, 1773bp coding and
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1157bp 3’ regions presumably containing the GAI1 terminator (tVv) was amplified from V.
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Zhong and Yang, 2011. Characterization of Grape Gibberellin Insensitive1 Mutant Alleles in Transgenic
Arabidopsis. Transgenic Research
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vinifera cv. Pinot Noir genomic DNA using Phusion high-fidelity DNA polymerase (Fig. 1). The
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forward primer (5’-ATGGCGCGCCGTCGACtcgtcatgcatactctttcgtc-3’ +AscI+SalI) and reverse
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primer (5’-ATGGCGCGCCGGATCCcctccatacatctatccctctgtt-3’ +AscI+BamHI) were located
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2171bp upstream of the ATG start codon and 1157bp after the TGA stop codon of the VvGAI1,
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respectively. The primers were designed on the basis of the V. vinifera sequence assembly
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(chr1:4901800, 4911799) (http://www.genoscope.cns.fr/cgi-bin/blast_server/projet_ML/blast.pl).
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Analysis of the cloned GAI genomic DNA fragment confirmed that grapevine GAI1 does not
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have introns in its coding region, as what has been observed in other species. The 5.1kb-long
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VvGAI1 genomic fragment was then cloned into a pCR8/GW/TOPO vector (referred as Topo-
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VvGGAI1 below). The authenticity of the entire insertion fragment was validated through
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sequencing with the sequencing primers located on both GAI1 fragment and border sequences of
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the cloning vector. The GAI1 fragment contains single restriction sites of StuI (AGG↓CCT at
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aa47) and AgeI (A↓CCGGT at aa453) in the coding region of the VvGAI1 gene. Since there was
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no StuI or AgeI restriction site on the pCR8/GW/TOPO vector and the cDNA and genomic
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VvGAI1 sequences are the same (no introns in GAI), the same DNA fragments carrying the
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mutations of GAI1Q333R, GAI1∆53-56, GAI1∆75-86 and GAI1E58stop,∆59-60,E61M between the positions
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of aa47 and aa453 from the Topo-VvGAI1* vectors described earlier were subcloned into Topo-
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GGAI1 by StuI/AgeI (NEB) double digestion to generate various mutant versions of Topo-
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VvGGAI1, referred as Topo-VvGGAI1* . For creating mutations (GAI1L38H, GAI1L38A,
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GAI1L37H, GAI1G42E, GAI1∆37-51, GAI1∆35-39, GAI1∆1-93 and GAI1∆1-60,E61M) located outside the
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StuI/AgeI restriction fragment, the Topo-VvGGAI1* was created by site-directed mutagenesis
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using the Phusion site-directed mutagenesis kit. The entire VvGAI1 genomic sequence in the
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Topo-VvGGAI1* was verified as described for Topo-VvGGAI1.
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The VvGAI1* fragments from Topo-VvGGAI1* were released by AscI (NEB) digestion
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and purified using the QIAquick gel extraction kit (Qiagen). The released VvGAI1* fragments
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were then cloned into the AscI cloning site, (whose 5’ phosphate was removed by Antarctic
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phosphatase (NEB)), of pGWB502HS vector to form pVv::VvGAI1* binary vectors. The
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pGWB502HS vector was created by releasing the gateway cassette (including the 35S promoter)
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from the pGWB502Ω vector with HindIII/SacI digestion (NEB), filling-in by T4 DNA
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polymerase (NEB) followed by self-ligation using T4 DNA ligase (NEB).
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Zhong and Yang, 2011. Characterization of Grape Gibberellin Insensitive1 Mutant Alleles in Transgenic
Arabidopsis. Transgenic Research
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Construction of pAt::VvGAI1* binary vectors. A 2-kb long GAI promoter region of
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Arabidopsis (pAt) was amplified by genomic PCR using Phusion high fidelity DNA polymerase
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with the primers 5’-TGCTCTAGAtattacttctttagaaaaaataatgtttggac-3’ plus XbaI restriction site
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and 5’-CGCGGATCCggttggttttttttcagagatgg-3’ plus BamHI restriction site. Similarly, a 2-kb
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long GAI terminator region of Arabidopsis (tAt) was amplified with the primers 5’-
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CCGGAATTCatggtggctcaatgaattgatc-3’ plus EcoRI restriction site and 5’-
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ACATGTCGACcgtggcggaagtaccgct-3’ plus SalI restriction site. The 2kb pAt fragment was
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digested with XbaI/BamHI and ligated into a pGreenII 0179 binary vector digested with
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XbaI/BamHI (http://www.pgreen.ac.uk/JIT/JIT_fr.htm) to generate the pGreenII 0179-pAt
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vector.
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The 2kb tAt fragment was treated by EcoRI/SalI and ligated into pGreenII 0179-pAt
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digested with EcoRI/BamHI to generate the pGreenII 0179-At binary vector. Then the grape
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GAI1 coding sequence (VvGAI*) was amplified with the primers 5’-
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CGCGGATCCatgaagagggagtatcatcatcc plus BamHI restriction site and 5’-
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GGCGAATTCtcagttggaggcaggtgtgg plus EcoRI restriction site from the corresponding Topo-
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VvGAI1* plasmid. The PCR products were digested by BamHI/EcoRI and further cloned into
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pGreenII 0179-At binary vector treated EcoRI/BamHI to create the final pAt::VvGAI1* binary
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constructs. The pAt::VvGAI1* constructs were sequenced to ensure no extra mutations were
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introduced in the VvGAI1 coding region by the PCR reactions.
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All restriction enzymes were purchased from New England Biolabs unless specified. The
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primers used in VvGAI1 mutation creation and cloning, GAI promoter and terminator cloning,
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and quantitative PCR are listed in S Table 2.
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References cited in the Supplementary Material and Methods
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Boss, P.K., and Thomas, M.R. (2002). Association of dwarfism and floral induction with a grape
'green revolution' mutation. Nature 416, 847-850.
Nakagawa, T., Suzuki, T., Murata, S., Nakamura, S., Hino, T., Maeo, K., Tabata, R., Kawai, T.,
Tanaka, K., Niwa, Y., et al. (2007). Improved Gateway binary vectors: high-performance vectors
for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 71,
2095-2100.
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Zhong and Yang, 2011. Characterization of Grape Gibberellin Insensitive1 Mutant Alleles in Transgenic
Arabidopsis. Transgenic Research
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S Fig. 1 Illustration of the traits and trait measurements investigated in this study. Detailed
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descriptions of traits and trait measurements were provided in the Material and Methods. Data
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for plant height (PH), number of rosette leaves (NR) and number of lateral
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branches/inflorescences (NLI) were collected from whole plants as shown in the left panel.
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Measurements of the number of nodes (NN), total length of internodes (TLI), internode length
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proximal to the primary inflorescence (ILPI), inflorescence length (IL), number of pods (NP),
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length of first 10 pod nodes (10PL) and the average length between pods on primary
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inflorescence (IL/NP) were taken on primary shoots as shown in the right panel
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Zhong and Yang, 2011. Characterization of Grape Gibberellin Insensitive1 Mutant Alleles in Transgenic Arabidopsis. Transgenic Research
S Table 1 Types of VvGAI1 mutations, promoters and transgenic plants evaluated in this study
Grape VvGAI1
(wild type)
Grape Vvgai1
Art1
Art2
Barley
Sln1d
Brassica napa
Brrga1-d
Arabidopsis
Atgai
Art3
Maize
D8-1
Maize
D8-2023
Maize
D8-Mp1
Wheat
Rht-B1b
Wheat
Rht-B1b' (Art4)
∆1-60, E61M
L38H
L37H
L38A
G42E
Q333R
∆35-51
∆35-39
∆53-56
∆75-86
∆1-93
E58st,
∆59-60,
E61M
T1
T1
T1
Not evaluated
Not evaluated
T1
T2
T1
T1
T1
T1
T1
Not evaluated
pVv
Not evaluated
T1
T1
T1/ T2
T1/T2
T1
T2
T1
T2
T1
T1
T1
Not evaluated
pAt
T1
T1/T2
T1
T2
T1
T2
Not evaluated
T1
Not evaluated
T1
T1/T2
T1
T1
Mutation
designationa
35S
b
a) Mutation designations follow the formats of the following examples. L38H: amino acid L at the position 38 of the VvGAI1 protein sequence was substituted by the amino acid H,
∆35-39: amino acids from positions 35 to 39 in the GAI protein sequence were deleted, E58st: amino acid E at position 58 was converted into a stop codon
b) 35S, pVv, and pAt designate the 35S promoter from cauliflower mosaic virus, the VvGAI1 promoter from V. vinifera (grape) and GAI promoter from Arabidopsis, respectively;
T1: plants derived from seeds harvested from T0 transgenic plants, T2: plants derived from self-pollinated seeds harvested from T1 transgenic plants
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Zhong and Yang, 2011. Characterization of Grape Gibberellin Insensitive1 Mutant Alleles in Transgenic Arabidopsis. Transgenic Research
S Table 2 Primers used in VvGAI1 mutation creation and cloning, Arabidopsis GAI promoter and terminator cloning,
and quantitative RT-PCR
Type of primer
Forward sequence
Reverse sequence
L38H
gcatggatgagcttcAcgctgttttgggc
Cggcgtcttgctgggg
L38A
gcatggatgagcttGCcgctgttttgggc
Cggcgtcttgctgggg
L37H
ggcatggatgagcAtctcgctgttttgg
Ggcgtcttgctgggggtc
G42E
gcatggatgagcttctcgctgttttggAAtacaacgtcaaggcc
Cggcgtcttgctgggg
Q333R
ctgcgctgatgcGagccctagccctc
gccactgcatcccttgtttc
∆35-51
gaggtcgctcagaagcttg
Catgccggcgtcttgc
∆35-39
gttttgggctacaacgtcaag
Catgccggcgtcttgc
∆53-56
cttgaacagcttgaggaagttattg
cCcagccatgtcggaggc
∆75-86
Ctgtctaactggcttggaagc
Atgagagaggccatcctcct
∆1-93(genomic)
Atgctctccgagttcaaccc
GGATCCAAGGGCGAATTC
∆1-93 (cDNA)
Atgctctccgagttcaaccc
AGTGTGAGGGGGGGTTGATG
E58stop,∆59-60
E61M
ATGgaagttattgttaatgctcaggagg
ttAaagcttctgagcgacctcag
∆1-60, E61M
ATGgaagttattgttaatgctcaggagg
AGTGTGAGGGGGGGTTGATG
Creation of
mutants
Cloning
AtGAI promoter
tgcTCTAGAtattacttctttagaaaaaataatgtttggac(XbaI)
CGCggatccggttggttttttttcagagatgg (BamHI)
AtGAI terminator
ccgGAATTCatggtggctcaatgaattgatc
acatGTCGACcgtggcggaagtaccgct (SalI)
VvGAI1 ORF
cgcGGATCCatgaagagggagtatcatcatcc (BamHI)
ggcGAATTCtcagttggaggcaggtgtgg (EcoRI)
VvGAI1 ORF+3’UTR
GGATCCatgaagagggagtatcatcatcc (BamHI)
GAATTCttcttttattaccaaactatatacttatatatat (EcoRI)
VvGAI1 genomic
atGGCGCGCCGTCGACtcgtcatgcatactctttcgtc
(AscI, SalI)
atGGCGCGCCGGATCCcctccatacatctatccctctgtt(AscI,BamHI)
Actin2(At3g18780)
cgttgcaccacctgaaag
Tgtgaacgattcctggacct
VvGAI1
aagcacttgtcaagcaaatcg
Tctggagaatatcggagaacg
(EcoRI)
Q-RT-PCR
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