CCA1 overexpression construct: subclone TOC1::CCA1 into binary vector pEarlygate303 (obtained from the Arabidopsis Biological Resource Center, ABRC, #CD694).
- Amplify the TOC1(At5g61380.1) promoter fragment from A. thaliana Columbia genomic DNA and using the primer pair (restriction sites lower case) 5’-GGgaattcCGTGTCTTACGGTGGATGAAGTTGA-3’ (EcoRI) and 5’-GGggatccGTTTTGTCAATCAATGGTCAAATTATGAGACGCG-3’ (BamHI) and a full-length CCA1 cDNA fragment using the primer pair: 5’-GCGGCCggatccATGGAGACAAATTCGTCTGGAG-3’ (BamHI) and 5’-GGCCGCtctagaTCATGTGGAAGCTTGAGTTTC-3’ (XbaI).
- Clone the TOC1 promoter fragment and CCA1 cDNA into pBlueScript and validate the insert by sequencing.
- For conventional cloning procedures, use pCAMBIA-based Gateway-compatible binary vectors system; Subclone the validated fragment into pEarlygGate303(CD694) using the primers 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTACGTGTCTTACGGTGGATGAAGTTGA -3’ and 5-GGGGACCACTTTGTACAAGAAAGCTGGGTCTGTGGAAGCTTGAGTTTCCAACCG-3’.
Transform construct (ProTOC1::CCA1) into A. thaliana (Columbia) plants using floral dipping method3.
cca1(RNAi) construct: Subclone TOC1: :cca1(RNAi) into binary vector pFGC5941 (CD3-447).
- Clone inverted CCA1 cDNA fragments into pFGC5941 binary vector by a two-step cloning process using two pairs of restriction enzyme sites in the primer pairs. After subcloning the inverted repeat fragment, replace the original promoter with the TOC1 promoter (see above).
- Amplify partial CCA1 fragments (~250-bp) using A. thaliana (Columbia) cDNA and primer pair (restriction sites lower case): F-RNAi CCA1 XbaI AscI 5’-GCGGCCtctagaggcgcgccTCTGGAAAACGGTAATGAGCAAGGA-3’ and R-RNAi CCA1 BamHI SwaI 5’-GGCCGCcctaggtaaatttaCACCACTAGAATCGGGAGGCCAAA-3’ (note: each primer introduces an ”inner” restriction enzyme site, AscI or SwaI, and an “outer” restriction enzyme site, BamHI or XbaI that are located next to each other).
- Clone the CCA1 PCR products from step 6 into pFGC5941 in a sense orientation using the “inner” restriction enzymes, AscI and SwaI and validate the insert by sequencing.
- Digest the same PCR products from step 6 with the “outer” restriction enzymes, BamHI and XbaI, and clone the digested CCA1 fragment in an anti-sense orientation into the plasmid containing the sense fragment in step 7.
- Amplify the TOC1 promoter fragment (ProTOC1) using the primer pair: F-EcoRI-ProTOC1 5'-GGGAATTCCGTGTCTTACGGTGGATGAAGTTGA-3' and R-ProTOC1-NcoI 5'-GCGGCCCCATGGGTTTTGTCAATCAATGGTCAAATTATGAGACGCG-3'.
- Digest the TOC1 promoter fragment from step 9 with EcoRI and NcoI and clone it into the plasmid from step 8 to yield the pFGC5941-ProTOC1::cca1(RNAi) construct (note: this replaces the 35S promoter in pFGC5941 with the TOC1 promoter as described in the website: "http://www.chromdb.org/rnai/vector_info.html":http://www.chromdb.org/rnai/vector_info.html); Validate the insert by sequencing.