Bosch Et Al. Supporting Information Table 1: Details of Primers Used for VPE Cloning

Bosch et al. Supporting Information Table 1: Details of Primers used for VPE cloning

Cloning of poppy pollen VPE

The following degenerate primers, based on highly conserved nucleotide sequences from plant VPEs, were designed to amplify poppy VPE from pollen:

Forward #1: 5’-TT(C/T) ATG TA(C/T) GAT GA(C/T) AT(C/T) GC-3’

Forward #2: 5’-GG(A/T) GT(C/T) CC(A/T/C) AAG GA(C/T) TA(C/T) AC-3’

Reverse #1: 5’-TC(G/A/T) CCA (A/T)AT T(G/C)C A(A/T)(A/G) ACA TG-3’

Reverse #2: 5’-AGA TC(G/T/C) GCA TC(A/T) C(G/T)(C/T) TG(A/G) TT-3’

Gene-specific primers for 5'-RACE:

5’race1: 5'-AGA TGT GAT CAT TCG GAC CA-3’

5’race2 (nested): 5’-CCT TCC CAC TAC CCC CTT TA-3’

Gene-specific primers for 3'-RACE:

3’race1 : 5’-CCC ACG TAA TGC AGT ATG GA-3’

3’race2 (nested): 5’-GGG TAC CAA TCC AGC AAA TG-3’

Primers to generate full-length cDNA of poppy VPE:

MB40 forward: 5’-ATG GTG AAA TTC TTA TTT TCA GTG-3’

MB41 reverse: 5’-TTA CGC GCT GAA ACC CTG G-3’

RT-PCR of VPEs from the different Papaver tissues

prVPE specific primers giving a 900-bp product:

Forward: 5'-CAG TGA TAA TTC TCT TCT TTC TAT TAT CGG-3'

Reverse: 5'-GAG AAA TAG ATC TTC CTT GCT AAC TTC GAG-3'

GAPD internal control primers:

Forward: 5'-CTT GAA GGG TGG TGC CAA GAA GG-3'

Reverse: 5'-CCT GTT GTC GCC AAC GAA GTC AG-3'

VPE His-tagged fusion construct primers

pVPEp:

Forward: 5’-GCG CTA GCC GGA ATA TTG AAG AAG ATG GTG TG-3’

(NheI site underlined)

Reverse: 5’-GCC TCG AGC GCG CTG AAA CCC TGG TGA GTT-3’

(XhoI site underlined)

pVPE:

Forward: 5’-GCG CTA GCC GGA ATA TTG AAG AAG ATG GTG TG-3’

(NheI site underlined)

Reverse: 5’-GCC TCG AGT TCA AGT ACT TGA GGA CCT TTT TCA AA-3’ (XhoI site underlined)

mVPE:

Forward: 5’-CGG CTA GCG ATG ATG ATT CTG TCG GTG GGA CT-3’

(NheI site underlined)

Reverse: 5’-GCC TCG AGT TCA AGT ACT TGA GGA CCT TTT TCA AA-3’ (XhoI site underlined)