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Table 2 Oligonucleotide primers used in this study

From: Rapid, high efficiency virus-mediated mutant complementation and gene silencing in Antirrhinum

Purpose

Primer name

Sequence (5′-3′)

Notes

VIGS

AscI-PDS-F

TGGCGCGCCCAATGAGCCTTACCGTGCAT

Adds Asc I sites to AmPDS fragment

AscI-PDS-R

TGGCGCGCCGTAGTGCTTCAGAGGATCTTACAA

PDS-R

GTAGTGCTTCAGAGGATCTTAC

First-round AmPDS amplification

AscI-DIV-R

AGGCGCGCCTACCCCATTCTAAGGTAAAAGG

Adds Asc I site to Div fragment

PDS-DIVfusion-F

gtaagatcctctgaagcactacCATATTTTTCCAGCTCAAGCTGG

Adds PDS-R to Div fragment

AscI-Hutr-F

AGGCGCGCCATAATACAAGTTGAGCAACAGCG

Adds Asc I site to H 3′-UTR fragment

PDS-Hutrfusion-R

gtaagatcctctgaagcactacACAGAGTGATACGCCTCGAT

Adds PDS-R to H 3′-UTR fragment

H expression

AscI-Hairy-F

AGGCGCGCCATGCAGTACGACGCAGAACC

Adds Asc I site 5′ to the H ORF

PDS-Hairyfusion-R

gtaagatcctctgaagcactacTTAGAGCCAAAGAGCACCAGC

Adds PDS-R 3′ to the Hairy ORF

AscI-SynH-F

AGGCGCGCCAACAATGCAATATGATGCTGAGCCT

Adds Asc I upstream of SynH ORF

FLAG-SynHfusion-R

CTACTTGTCGTCATCGTCTTTGTAGTCCAACCACAATGCTCCTGCT

Replaces H stop with FLAG-tag

FLAG-PDSfusion-R

GACTACAAAGACGATGACGACAAGTAGgtagtgcttcagaggatcttac

Adds FLAG to AmPDS fragment

RT-PCR

AmPDS-qPCR-F

TCTTTGTAATGGACGGCAAG

RT-PCR for AmPDS

AmPDS-qPCR-R

ACTTGCCAAACTCTTCCCTG

TRV-CP-F

TGGGTTACTAGCGGCACTGA

RT-PCR for TRV2 RNA

TRV-CP-R

GCTCGTCTCTTGAACGCTGA

AmUbi-qPCR-F

CCGAACCATCAGACAAACAAAC

RT-PCR for AmUBI control

AmUbi-qPCR-R

TACCCTGGCCGACTACAATA

AmACT-F

CTTGGCCGTCTCCATTTCTT

RT-PCR for AmACT control

AmACT-R

TCCTCACAGAGCGTGGATATAG

H-qPCR-F2

ACCATCCACAACACTCTAATC

RT-PCR for H

H-qPCR-R

TGAATATCACCACCGGATTCTC

Div-F

GGGTAGTGGTCATGGATTCG

RT-PCR for Div

Div-R

GGAAAGGAAGTTTGTGAATGGAG

  1. The Asc I restriction site, introduced to allow cloning into pTRV2sgP, is underlined. For overlap amplification of AmPDS fused to H or AmDIV sequences, AmPDS was first amplified with AscI-PDS-F and PDS-R and the H or Div sequence with one primer carrying an Asc I site and a reverse primer containing the complement of the PDS-R sequence at its 5′ end (lower case). Products were then fused by overlap PCR. To express SynH with a C-terminal FLAG-tag, the FLAG-encoding sequence (italics) was added in place of the SynH stop coding by amplification with FLAG-SynHfusion-R. To fuse the SynH-FLAG sequence to the AmPDS reporter, the AmPDS fragment was amplified with FLAG-PDSfusion-R, carring the FLAG-encoding sequence at its 5′ end