(b) Genotyping of SALK_ (AT1g51690) and SALK_ (At1g17720) to find homozygous single mutant plants.

Size: px

Start display at page:

Download "(b) Genotyping of SALK_ (AT1g51690) and SALK_ (At1g17720) to find homozygous single mutant plants."

Alvin Logan
5 years ago
Views:

1 Supplemental Table 1. Primers used for cloning and RT-PCR. The B-type subunits cdna CDS were amplified using high-fidelity PCR amplification with gene specific primers. The cdna CDS sequences were obtained from TAIR webpage at www. arabidopsis.org. For all proteins fused to YFP by their C-terminus (B-YFP), 5 primers contained gene specific sequences with the start codons, and in 3 primer sequences no stop codons were included to maintain ORFs. In addition, all primers contained appropriate restriction enzymes sequences (indicated in parentheses). For the proteins fused to YFP by their N-terminus (YFP- B), 5 primers contained gene specific sequence with start codons, and 3 primer sequences included stop codons. Primers for cloning into pw EN18, pwen18-cy,and pwen18-ny AT1G5169Sal1L (SalI) 5' TTATGTCGACATGAACGGTGGTGATGAGGTCGTC 3' AT1G5169accRsec (Acc65I) 5' ATGGTACCAGCATAGTACATGTACAAGCTAT 3' At1g1772Xho1Ltre (XhoI) 5' TATACTCGAGATGAACGGTGGTGACGATGCC 3' at1g1772accrsec (Acc65I) 5' ATGGTACCTGCATAGTACATGTACAAGCTGT 3' AT3G2165L-2 (XhoI) 5' ATCTCGAGATGATCAAACAGATATTTGGGAAA 3' At3g2165R-3 (Acc65I) 5' TATAGGTACCCGACCCTGTGGACTCAGAGCT 3' AT3G988L-2 (XhoI) 5' ATCTCGAGATGTTTAAGAAAATCATGAAAGGTG 3' AT3G988R-2 (Acc65I) 5' TATAGGTACCGGAAGTGATCATATGATCTTCTTC 3' AT3G263L-2(KpnI) 5' TTAGGTACCCTTCGCCATTGAAGCAACAATC 3' AT3G263R-2 (XhoI) 5' TACTCGAGATGTTTAAGCAGATACTTGGGAAGC 3' AT1G1346L-2 (XhoI) 5' TACTCGAGATGTGGAAACAGATTCTGAGTAAGC 3' AT1G1346BsrGI-R5 (BsrGI) 5' ATAATGTACACAATGAACTCTTTTGCTTTTGATTAC 3' AT4G1541L-2 (XhoI) 5' ATCTCGAGATGATCAAACAGATATTTGGGAAA 3' AT4G1541R-2 (KpnI) 5' TATAGGTACCACTACCCGAAGTTTTACCGGTTG 3' AT3G262L-2 (XhoI) 5' TACTCGAGATGTGGAAACAGATTCTAAGTAAGCT 3' AT3G262R-2 (XhoI) 5' TTACTCGAGTGAAGCCTTTCGCACTCCGAGT 3' AT5G53LP-2 (XhoI) 5' TTATCTCGAGGTGATGGGTGGTTTGGCTATG 3' AT5G53-RP2 (Acc65I) 5' TATAGGTACCGCCACAAGGTGTGAGTTTCT 3' AT5GO347L-2(XhoI) 5' ATCTCGAGGCCATGTTTAAGAAGATCATGAAAG 3' AT5GO347R-2(KpnI) 5' TATAGGTACCAGAAGTGATCATAGGATCTTCTCTTTCT 3' AT5G1858LP-2 (Acc65I) 5' TATAGGTACCATGTATAGCGGATCTAGCGATG 3' AT5G1858RP-2 (Acc65I) 5' TTATGGTACCCTGAGACTCTTCCTCAGGTGGT 3' AT3G5493L-2 (XhoI) 5' TACTCGAGATGTTCAACAAAATCATAAAACTGG 3' AT3G5493R-2 (KpnI) 5' ATATGGTACCGTTAGCAGCTAGAGAAGCAGCTT 3' AT1G7776Xho1LP 5' TTACTCGAGATGGCGACCTCCGTCGATAACCG 3' AT1G7776Acc65IRP 5' TATAGGTACCGAAGATTAAGAGATCCTCCT 3' AT1G7776Xho1LP-A 5' TTACTCGAGATGGCGACCTCCGTCGATAACCG 3' AT1G7776Acc65IRP-A 5' TATAGGTACCTCTCGGATCTAAAATCGACG 3' AT1G7776Xho1LP-D 5' TTACTCGAGATGTCTCTCGTTGATGCTGAGCTGG 3' AT1G7776Acc65IRP-D 5' TATAGGTACCGGTGATGAGTTCACCGATAC 3' AT1G7776Xho1LP-E 5' TTACTCGAGATGACTGGCTACGACTCTTCCCCT 3' AT1G7776Acc65IRP-E 5' TATAGGTACCGAAGATTAAGAGATCCTCCT 3' AT1G7776Xho1LP-F 5' TTACTCGAGATGGATCCGAGAGATGAATCAACG 3' AT1G7776Acc65I-F 5' TATAGGTACCAGGAAGGACTCGATTGTCTTT 3' AT1G3713Sal1-LP2 5' TTAGTCGACATGGCGGCCTCTGTAGATAATCG 3' AT1G3713Acc65I-RP2 5' TTATGGTACCGAATATCAAGAAATCCTCCTTGATG 3' Primers for cloning into pw EN25 AT1G5169L-3Sal1 (SalI) 5' TTAGTCGACCCATGAACGGTGGTGATGAGGTCGTC 3' AT1G5169L-3Xma1 (XmaI) 5' TACCCGGGTTAAGCATAGTACATGTACAAGCTAT 3' At1g1772L-3SAL1 (SalI) 5' TTAGTCGACAAATGAACGGTGGTGACGATGCC 3' At1g1772R-3SAC1 (SacI) 5' ATAAGAGCTCATGCATAGTACATGTACAAGCTGT 3' RT-PCR Primers AT1G2549LP1 5' GCTTCGTTTGAACTCGATCC 3' AT1G2549RP1 5' CAAGAAGCACCTCATCGTCA 3' âtub4lp 5' TTTCTCAGTGTTTCCTTCTCC 3' âtub4rp 5' CCTCATTGTCCAAACCATAC 3' AT1G1332LP 5' GGTGCTCAGATGAGGGAGAG 3' AT1G1332RP 5' AGCAGCTTCACGGATTGAGT 3' AT3G258LP 5' GCTCTTGGGAAATTGTTGGA 3' AT3G258RP 5' GCGGATTGAGTGAACCTTGT 3' AT1G2549LP 5' TGACGATGAGGTGCTTCTTG 3' AT1G2549RP 5' CAGAGCGGACATGTTGAGAA 3' AT1G5169LP 5' ATGAACGGTGGTGATGAGGTCGTC 3' AT1G5169RP 5' CATGAGTTTGTAATGTTTGAATTGC 3' AT1G1772LP 5' ATGAACGGTGGTGACGATGCC 3' AT1G1772RP 5' TCATGCATAGTACATGTACAAGCTGT 3'

2 Supplemental Figure 1. Selecting Double Mutant At1g5169 x At1g1772. Homozygous single mutants were selected using Salk lines SALK_954 (At1g5169) and SALK_62514 (At1g1772). The single mutants were crosspollinated resulting in heterozygous plants for both of the genes. With due attention to the fact that these two genes are linked genes it is not possible to get 1:16 double mutants in the first progeny after cross pollination. Frequency of obtaining double mutant by crossing over is not so high and we chose to select homozygous knockouts for one gene and heterozygous for the other, and then search for double mutants (after crossing over had taken place) in the second progeny with higher probability (i.e. 25%). By genotyping 4 plants we found two plants that were both null in At1g1772 (Bβ) gene and heterozygous for At1g5169 (Bα) gene. In third progeny after genotyping 24 plants no mutant plant null in both Bα and Bβ could be found. This was confirmed in another cross when testing 7 plants. Apparently the homozygous double mutation would be lethal. (a) Diagram of At1g5169 (Bα) and At1g1772 (Bβ) genes structure [blue boxes exons; thin lines, introns] with the relative position of the two T-DNA insertions (white boxes) corresponding to SALK_954 and SALK_62514 mutant lines. (b) Genotyping of SALK_954 (AT1g5169) and SALK_62514 (At1g1772) to find homozygous single mutant plants. (c) Verification of mutant plants using RT-PCR. (d) Genotyping first progeny after cross pollination to find plants that have insertion in both of the loci. (e) Two plants (1, 2) which are homozygous for mutation in Bβ and heterozygous for mutation in Bα compared with wild type plant.

3 NR activity state, % pp5ox pp5ko WT A NR activity state, % B bsl1 bsl3 WT bsl

4 NR, total amount (EDTA assay) A Tamoxifen WT Mock WT Mock amirna Tamoxifen amirna NR, non-phosphoryalted (Mg assay) B Tamoxifen WT Mock WT Mock amirna Tamoxifen amirna after light-on after light-on