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.