Cloning of CsGGCT2;1 and generation of the gene
construct
A Complementary DNA (cDNA) library generated from Camelina wild-type
leaves was used to perform PCR with sense (5’
TACGTCGTCGACGTTTAAACATGGTTTTGTGGGTA 3’) and antisense primers (5’
AGATTCGAATCATGATACAAAGACTCTTTGC 3’) designed from CamelinaGGCT2;1 (Csa08g024050.1, www.Camelinadb.ca) gene coding
sequences. The PCR amplification conditions were 94°C for 2 min (1
cycle); 94°C for 45 sec, 55°C for 1 min, 72°C for 45 sec (40 cycles);
final extension at 72°C for 10 min (1 cycle). The resulting 654
nucleotide PCR product was gel purified using Zymoclean Gel DNA Recovery
Kit (Zymo Research, Irvine, CA) and ligated into pGem-T easy cloning
vector (Promega Corporation, Madison, WI). The insert was verified by
sequencing and then subcloned into the binary pCambia Redseed vector
using Sal 1 and Xba 1 sites for overexpression in plants.
The expression of the CsGGCT2;1 gene is driven under an enhanced
35S promoter. The 35S promoter was amplified from the pEarlygate 103
plasmid via PCR with primers (5’ TAGCTGGGGCCCGGCGCGCCGAGATCTCC 3’), (5’
CGTCGACACTAGTTCCTCTCCAAATGAAA 3’) and after sequence verification, it
was subsequently sub-cloned at Apa 1 and Sal 1 sites of
pCambia Redseed vector. The pCambia RedSeed vector contains a DsRed
reporter gene and a hygromycin resistance gene for transgenic seed
selection using a DsRed filter or hygromycin antibiotics. The resulting
plasmid pCambiaRedSeed/35Sp::CsGGCT2;1 (Figure 1) was transformed
into plants for driving constitutive expression of the CsGGCT2;1gene.