Plasmids for Protein Expression in Yeast

5.4.3.1 pBD-/At1g28480

The isolated At1g28480 cDNA clone was excised from pGAD10/At1g28480 as 550 bp BglII fragment and ligated into pBD opened with the same enzymes. The At1g28480 cDNA was under the control of Met25 promoter.

5.4.3.2 pBD-/At1g50570 P

The isolated At1g50570 partial cDNA clone was excised from pGAD10/At1g50570 as 950 bp

BamH1 fragment and ligated into pBD opened with BglII. The At1g50570 cDNA was under the control of Met25 promoter.

5.4.3.3 pBL-/TGA1

The TGA1 full-length coding sequence was amplified using PCR from an Arabidopsis cDNA library with primers designed with artificial restriction sites. The PCR product was cloned into pGEM-T vector following the manufacturer’s instructions. An 1100 bp BglII excised fragment from the pGEM- T/TGA1 was ligated into pBL opened with the same enzymes. The TGA1 coding sequence was under the control of Met25 promoter.

5.4.3.4 pBL-/TGA2.2

Two-step cloning procedure was employed in order to clone the pBL-/TGA2.2. A pGBT9/TGA2.2 plasmid was digested with NcoI and BamHI. The digestion produces two fragments: a 800 bp, a NcoI fragment that begins from the TGA2.2 start codon, and a 200 bp that represents a NcoI and BamHI fragment or the rest of TGA2.2 coding sequence. The 200 bp NcoI and BamHI fragment was ligated first into pBL vector opened with NcoI and BglII and the cloning product was termed pBL/200bpTGA2.2. The 800 bp NcoI fragment was inserted into pBL/200bpTGA2.2 opened with the same enzyme. The TGA2.2 coding sequence was under the control of Met25 promoter.

5.4.3.5 pBL-/TGA2.2-VP16

Two-step cloning procedure was employed in order to clone the pBL-/TGA2.2-VP16. A pSK2.2VP16 (Lenk, 2001) plasmid was digested with NcoI and SpeI. The digestion produces two fragments: a 800 bp, a NcoI fragment that begins from the TGA2.2 start codon and a 650 bp that represents a NcoI and BamHI fragment or the rest of TGA2.2 and the VP16 coding sequences. The 650 bp NcoI and SpeI fragment was ligated first into the pBL vector opened with NcoI and SpeI and the cloning product was termed pBL/650bpTGA2.2-VP16. The 800 bp NcoI fragment was inserted into pBL/650bpTGA2.2-VP16 opened with the same enzyme. The TGA2.2-VP16 coding sequence was under the control of Met25 promoter.

5.4.3.6 pGAD424/At1g28480 and pGBT9/At1g28480

The isolated At1g28480 cDNA insert was excised from pGAD10/At1g28480 as 550 bp BglII fragment and ligated into pGAD424 or pGBT9 opened with BamHI. The At1g28480 cDNA was under the control of ADH1 promoter and fused in-frame to the GAL4AD or GAL4BD.

5.4.3.7 pGAD424/At1g50570 F and pGBT9/At1g50570 F

The At1g50570 full-length coding sequence was amplified using PCR from an Arabidopsis cDNA

library with primers designed with artificial restriction sites. The PCR product was cloned into pGEM-T vector following the manufacturer’s instructions. An 1100 bp BglII excised fragment from the pGEM- T/At1g50570 was ligated into pGAD424 opened with BamHI. The At1g50570 coding sequence was under the control of ADH1 promoter and fused in-frame to the GAL4AD or GAL4BD.

5.4.3.8 pGAD424/At5g55530 F and pGBT9/At5g55530 F

The At5g55530 full-length coding sequence was amplified using PCR from an Arabidopsis cDNA

library with primers designed with artificial restriction sites. The PCR product was cloned into pGEM-T vector following the manufacturer’s instructions. A 1450 bp BglII excised fragment from the pGEM- T/At5g55530 was ligated into pGAD424 or pGBT9 opened with BamHI. The At5g55530 coding sequence was under the control of ADH1 promoter and fused in-frame to the GAL4AD or GAL4BD.

5.4.3.9 pGAD424/GDM and pGBT9/GDM

The GDM full-length coding sequence (An At1g28480 cysteine double mutant generated by PCR site directed mutagenesis) was amplified using overlap extension PCR method with primers designed with artificial restriction sites and base pair mismatching sequence. The PCR product was cloned into pGEM-T vector following the manufacturer’s instructions. A 450 bp BglII excised fragment from the pGEM-T/GDM was ligated into pGAD424 or pGBT9 opened with BamHI. The GDM coding sequence was under the control of ADH1 promoter and fused in-frame to the GAL4AD.

5.4.3.10 pGAD424/GSM

The GSM full-length coding sequence (An At1g28480 cysteine single mutant generated by PCR site directed mutagenesis) was amplified using overlap extension PCR method with primers designed with artificial restriction sites and base pair mismatching sequence. The PCR product was cloned into pGEM-T vector following the manufacturer’s instructions. A 450 bp BglII excised fragment from the pGEM-T/GSM was ligated into pGAD424 opened with BamHI. The GSM coding sequence was under the control of ADH1 promoter and fused in-frame to the GAL4AD.

5.4.3.11 pBDAt1g28480/Met25::TGA1

The DNA sequence of the Met25::HA-NLS-TGA1::PGK expression cassette was cut out from the pBL/TGA2.2 plasmid as StuI fragment (~1950 bp) and ligated into pGBT9/At1g28480 opened with PvuII.

5.4.3.12 pGBDAt1g28480/Met25::TGA2.2

The DNA sequence of the Met25::TGA2.2::PGK expression cassette was cut out from the pBL/TGA2.2 plasmid as StuI fragment (~1950 bp) and ligated into pGBT9/At1g28480 opened with PvuII.

5.4.3.13 pGBDGDM/Met25::TGA2.2

The DNA sequence of the Met25::TGA2.2::PGK expression cassette was cut out from the pBL/TGA2.2 plasmid as StuI fragment (~1950 bp) and ligated into pGBT9/GDM opened with PvuII.

5.4.3.14 pGBT9/At1g50570 P

The isolated At1g50570 cDNA insert was excised from pGAD10/At1g50570 as 950 bp BglII fragment and ligated into pGBT9 opened with BamHI. The At1g50570 cDNA insert was under the control of ADH1 promoter and fused in-frame to the GAL4BD.

5.4.3.15 pGBT9/At5g20500 and pGBT9/At5g40370

The At5g20500 and At5g40370 full-length coding sequences were amplified using PCR from an Arabidopsis cDNA library with primers designed with artificial restriction sites. The PCR product was cloned into pGEM-T vector following the manufacturer’s instructions. A ~400 bp and 500 bp EcoRI excised fragments from the pGEM-T/At5g20500 pGEM-T/At5g40370 were ligated into pGBT9 opened with EcoRI, respectively. The At5g20500 and At5g40370 coding sequences were under the control of

ADH1 promoter and fused in-frame to the GAL4BD.

5.4.3.16 pGBT9/TGA2.2 C-term

The TGA2.2 C-terminus coding sequence was amplified using PCR method with primers designed with artificial restriction sites. The PCR product was cloned into pGEM-T vector following the manufacturer’s instructions. A 735 bp EcoRI and BglII excised fragment from the pGEM-T/TGA2.2 C- term was ligated into pGBT9 opened with EcoRI and BamHI. The TGA2.2 C-terminus coding sequence was under the control of ADH1 promoter and fused in-frame to the GAL4BD.

5.4.3.17 pGBT9/TGA2.2Cys181Ser

The TGA2.2Cys181Ser full-length coding sequence (A TGA2.2 cysteine single mutant generated by PCR site directed mutagenesis) was amplified using overlap extension PCR method with primers designed with artificial restriction sites and base pair mismatching sequence. The PCR product was cloned into pGEM-T vector following the manufacturer’s instructions. A 1000 bp EcoRI and BglII excised fragment from the pGEM-T/TGA2.2Cys181Ser _{was ligated into pGBT9 opened with EcoRI and BamHI. The}

TGA2.2Cys181Ser coding sequence was under the control of ADH1 promoter and fused in-frame to the

5.4.3.18 pLEU-/Met25::TGA2.2

The DNA sequence of the Met25::TGA2.2::PGK expression cassette was cut out from the pBL/TGA2.2 plasmid as StuI fragment (1950 bp) and ligated into pGAD424 opened with PvuII.