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Download Kanr T-DNA Supplemental Figure 1. Transgenic complementation of
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A CAC1A-R CAC1A-KO-5 At5g16390 (CAC1A) JL-202 B T-DNA Kanr CAC1A-KO-5 C CAC1A-X RB CAMBIA-1 35S Bar LB Supplemental Figure 1. Transgenic complementation of the cac1a-1 mutant allele. The figures show schematic representations of the CAC1A alleles that were tracked in the transgenic complementation of the cac1a-1 mutation. Exons are represented by blue boxes, introns are represented by pink boxes, UTRs are represented by white boxes, and intergenic chromosomal regions are represented by gray lines. Locations of primer-sets used for allele-specific PCR assays (see Supplemental Figure 2) are indicated with red arrows. (A) Structure of the native CAC1A allele (At5g16390). The yellow-shaded box represents the genomic region cloned into vector pCABMIA3300 for genetic complementation (see panel C). (B) Structure of the cac1a-1 allele showing the position of the T-DNA insertion, which contains the kanamycin resistance gene (Kanr). (C) Structure of the ectopic CAC1A allele used to transgenically complement the mutant cac1a-1 allele. Black shaded elements represent genetic elements that were part of the T-DNA inserted at this locus: 35S = CaMV 35S promoter; Bar = bar gene from Streptomyces hygroscopicus, conferring resistance to glufosinate; LB = T-DNA left border; RB = T-DNA right border. Supplemental Figure 2. Representative PCR genotyping results for transgenic complementation of the cac1a-1 mutant allele. Heterozygous cac1a-1 mutant plants were transformed with CAC1A genomic fragment (see panel C of supplemental Figure 1). Twelve T2 plants derived from 3 independent transgenic events, were genotyped individually to detect the presence of the endogenous CAC1A (At5g16390) allele (using primers CAC1A-KO-5 and CAC1A-R), the cac1a-1 mutant allele (using primers CAC1A-KO-5 and JL-202), and CAC1A transgenic allele (using primers CAC1A-X and CAMBIA-1). Position of primers are shown in supplemental Figure 1. Supplemental Figure 3. Generation of BCCP1- and BCCP2-specific antibodies. (A) Alignment of BCCP1 and BCCP2 amino acid sequences generated by the BESTFIT algorithm of GCG software package (Genetics Computer Group, Madison, WI). Identical residues are black-shaded and conservative substitutions are grey-shaded. The boxed sequences indicate the region with low amino acid similarity between the two proteins. The BCCP1 and BCCP2 cDNAs coding for this region were PCR amplified and cloned into an expression vector. Using the expressed peptides, BCCP1- and BCCP2-specific antisera were generated. Aliquots of protein extracts prepared from Arabidopsis flowers were subjected to SDS-PAGE and western blot analyses. Identical blots were probed with streptavidin and anti-BCCP1 antisera (B) or with streptavidin and anti-BCCP2 antisera (C). Supplemental Figure 4. The CAC1A- and CAC1B-specific probes used in RNA hybridization experiments. CAC1A and CAC1B sense RNAs were transcribed from pBluescript SK (+/-) and pSPORT 1 clones, respectively, and subjected to agarose electrophoresis. Following northern blot transfer of the RNAs to nitrocellulose membrane, they were subjected to hybridization with DIG-labeled antisense CAC1A RNA (positions 451-1114 of the CAC1A cDNA), and DIG-labeled antisense CAC1B RNA (positions 451-1217 of the CAC1B cDNA). Supplemental Table I. Characterization of T-DNA tagged alleles of BCCP subunit genes Gene Name Gene locus Allele CAC1A At5g16390 cac1a-1 1st intron, 4nt deletion SALK_120571 At5g15530 Characterization T-DNA border 5' 3' Embryo lethal LB LB -181, 1nt deletion No phenotype, normal level BCCP1 LB LB SALK_023225 -288 to -234, 54nt deletion No phenotype, normal level BCCP1 LB RB SALK_050082 1st exon Possible T-DNA translocation a Not found LB SALK_138637 Not found T-DNA not found Not found Not found GT_3_38620 Not found T-DNA not found Not found Not found SALK_025081 N/A NOT CHARACTERIZED N/A N/A GABI_217E08 N/A NOT CHARACTERIZED N/A N/A cac1b-1 (SALK_056228) 3rd intron No phenotype Not found LB cac1b-2 (SALK_070569) 1st exon and intron junction, 64nt deletion No phenotype LB RB (BCCP1) CAC1B Insert Position (BCCP2) a see text for more details. Supplemental Table II. PCR primers used to characterize CAC1A antisense plants and TDNA tagged alleles for htACCase subunit genes Primer Sequence Purpose JL-202 5'-CATTTTATAATAACGCTGCGGACATCTAC-3' T-DNA left border for Wisconsin lines XR-2 5'-TGGGAAAACCTGGCGTTACCCAACTAAT-3' T-DNA right border for Wisconsin lines LB 5'-CGTTCTTTAATAGTGGACTCTTGTTCCAA-3' T-DNA left border for SALK lines RB 5'-GCAATAATGGTTTCTGACGTATGTGCTTA-3' T-DNA right border for SALK lines inv6-2 5'-GCTAAGCACATACGTCAGAAACCATTATT-3' transposon left border primer for GT lines spm31 5'-GCTTGTTGAACCGACACTTTTAACATAAG-3' transposon right border primer for GT lines CAC1A-1 5'-GTTGAGAAAAATCAGTTTGCCTCTCTTTT-3' CAC1A gene 5' primer CAC1A-KO-5 5'-TACACGATTCCTTCCTCGATTAAGATAAG-3' CAC1A gene 5' primer CAC1A-X 5'-TGTTAGGTTTGTTAGTTTGGTGAGGAAGA-3' CAC1A gene 5' primer CAC1A-L 5'-CTTTCCGTTATTCTCCGATTACATCTACC-3' CAC1A gene 5' primer CAC1A-2 5'-CAAAAAGATTCATGTATCCTCAACATCCT-3' CAC1A gene 3' primer CAC1A-3 5'-CTTCAATGAGTTTTTCTCCTTTCCTTCAG-3' CAC1A gene 3' primer CAC1A-R 5'-TATTGTAGTCGAAGTGGAACTGATTCTCG-3' CAC1A gene 3' primer CAC1B-5 5'-GACTAATGGTGGGTATATGAACGGAAAAG-3' CAC1B gene 5' primer used for cac1b-1 CAC1B-3 5'-CATTACAGGAGGCATTGAGTGATAAACTG-3' CAC1B gene 3' primer used for cac1b-1 CAC1B-a 5'-GCGGTTTGGTGAAGTTAGTTAAAAGAGTT-3' CAC1B gene 5' primer used for cac1b-2 CAC1B-b 5'-GCAGGAGAAGAACAGAACAGAGAATTATG-3' CAC1B gene 3' primer used for cac1b-2 pC-F 5'-CATCTTATGCCCAGCAAATGGC-3' CAC1A cDNA 5' primer pC-R 5'-CAGTGACAACGAAGGGGAAACG-3' CAC1A cDNA 3' primer CAMBIA-1 5'-TAACAATTTCACACAGGAAACAGCTATGA-3' pCAMBIA3300 vector primer A-F 5'-CCTCAACCTCAAGCTCCT-3' CAC1A cDNA 5' primer A-R 5'-AACAGTAGGAAGTGACGATT-3' CAC1A cDNA 3' primer B-F 5'-CAGCAAGCTGTACCACCA-3' CAC1B cDNA 5' primer B-R 5'-GAGTGGAGGATGAGACGA-3' CAC1B cDNA 3' primer