1
Supplementary Material II. Primers for PCR Amplifications and
Construction of Fusion Plasmids.
Primers for PCR Amplifications
Sequence from 5’ to 3’
(Restriction-Enzyme Site: Underlined)
Reference
F*
R*
F
R
F
R
F
F
GGAAGCTTGAGGTGGGTATCAGGTTATCA (HindIII)
GTTTCATGACCAGAAACACGAACTC (Blunt End)
GGTCTAGAGTACGACGTCTGTATATATCA (XbaI)
GTAACCGACCCAAGAACATGAACAC (Blunt End)
GGTCTAGAGCAGGCCCATTTACATCCT (XbaI)
TGCTGGCTTCTCCATTGTTTCAGT (Blunt End)
GGTCTAGATCTCTGAAGTGATTATT (XbaI)
GGTCTAGAATGGCTAGCATGACTGGTGGACAGCAAATG
GGTCTGCAGATGGATCTTGGAGTTCGT (XbaI; Italic, T7epitope tag)
AtGRL1
AtGRL2,
cMyc-AtGRL2
R
F
AtGRL2
R
GGGAGCTCTCACAGAGAAGGAGCAGTA
GGGGATCCATGGAGCAAAAGCTCATTTCTGAAGAGGACT
TGTCTAGAATGGATATTGGTGTTCAT (BamHI; XbaI;
Italic, cMyc-epitope tag)
GGGAGCTCCAGTTTTCAGGTTGTGTAATGAAA (SacI)
AtGRL1
Promoter::GUS
Fusion
Constructs
AtGRL2
AtGRL3
Overexpression
Constucts
AtGRL1
T7-AtGRL1
GCTGGGGTACATTGGAGAACACCATTAGA
F
CAAGCGGATCTTTTCCATCACCGTTCTTA
R
GTAAGCCTCGTTCTTCTCCCTCTGTCTCT
AtGRL2
F
GTAATCGCCATTGTGTCTGTTGTTCTCTG
R
TTCTCCCCGCATCTGTCTTGTCTCACTGT
AtGRL3
F
TTCTCCTTTAGTTTTCGCTTGCAGCATCA
R
*
F and R indicate forward and reverse primers, respectively.
AtGRL1
Genotyping of
Mutants
Construction of Fusion Plasmids
Promoter::GUS fusion constructs. Primers employed to amplify DNA fragments are listed in the Table
above. The corresponding PCR products were digested and ligated to HindIII/SmaI-digested pBI101.1
(Clontech) to create an AtGRL1::GUS construct, or to XbaI/SmaI-pBI101.1 for AtGRL2::GUS and
AtGRL3::GUS constructs.
Overexpression plasmids. DNA fragments that correspond to the full-length of AtGRL1and AtGRL2
were amplified by using each primer set with or without epitope-tag sequences, digested with XbaI/SacI,
and ligated to pCAMBIA1300MCS1 (Sanderfoot et al., 2001). The PCR products of cMyc-AtGRL2 were
digestd with BamHI instead of XbaI, and ligated to BglII/SacI-pCANBIA1300MCS1.