Table II.
Primers and probes used in PCR experiments
The primers used for DNA cloning may be constituted of two distinct parts. The part of the sequence that appears in uppercase is specific, while the part that appears in lowercase was added to allow the insertion of the PCR product in the appropriate vector (pRSF2 or pDON207) according to the manufacturer's instructions (Novagen and Invitrogen, respectively). The genomic sequences of Trx s1 and Trx s2 were amplified using primers ORFsens and ORFanti, and leaf genomic DNA. The whole coding regions of Trx s1 and Trx s2 were amplified using the same primers, while the parts corresponding only to putative mature forms of the proteins were amplified with primers MATsens and ORFanti, using cDNAs reverse transcribed from root RNAs. After a preliminary denaturation step of 5 min at 94°C, DNA amplification was performed during five cycles (94°C × 1 min; annealing temperature of the specific part of the primers × 1 min; 72°C × 1 min) and 30 cycles (94°C × 1 min; 72°C × 1 min). Then, a final elongation step was realized for 10 min at 72°C. For quantitative RT-PCR, the amplification cycles in all cases consisted in a preliminary step of 5 min at 94°C, followed by 40 cycles of 94°C × 15 s and 60°C × 1 min.
| Primer Name | Sequence |
|---|---|
| Primers used for DNA cloning | |
| S1 ORFsens | gacgacgacaagATGACTACCGTCACAATAAC |
| S1 MATsens | gacgacgacaagATGCAACTCCTCAACGCTGC |
| S1 ORFanti | gaggagaagcccggtTCATAGGTTTTGTTCCATAC |
| S2 ORFsens | gacgacgacaagATGGCCACCGTCACATTA |
| S2 MATsens | gacgacgacaagATGACCGTACAACTCGAATCCT |
| S2 ORFanti | gaggagaagcccggtTCATATTGATAGTTGAATAAG |
| H2 ORFsens | gacgacgacaagATGGCAGCTGAAGAAGGA |
| H2 ORFanti | gaggagaagcccggtTCAAGCAGTAGCAACAGTT |
| AttB1s1sens | ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccATGACTACCGTCACAATA |
| AttB2s1anti | ggggaccactttgtacaagaaagctgggtctAGGTTTTGTTCCATACG |
| AttB1s2sens | ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccATGGCCACCGTCACATT |
| AttB2s2anti | ggggaccactttgtacaagaaagctgggtctATTGATAGTTGAATAAGTTC |
| Primers and probes used for quantitative RT-PCR | |
| S1TaqMsens | CGCTGCCACTCAAGATGCT |
| S1TaqManti | GAGTTAAGGACAAGGGAACCAAAA |
| S1TaqMprobe | FAM-CCAATGGAGTGGCTTATGTTACCGATGAAA-TAMRA |
| S2TaqMsens | CAGTGCCGCCGATGCT |
| S2TaqManti | GGGACGAAGGAACCAAAAGTT |
| S2TaqMprobe | FAM-CCGATGGAGTGGCTCCTGTTACTGATG-TAMRA |
| Qti-ARN18SFor | ACTGCGAAAGCATTTGCCAA |
| Qti-ARN18SRev | CGGCATCGTTTATGGTTGAGAC |