pgbkt7 bait vector  (Addgene inc)

Journal: Plant Biotechnology Journal

Article Title: Elucidating the Genetic Basis of Columnar Upright Architecture in Populus Through CRISPR Disruption of TILLER ANGLE CONTROL1

doi: 10.1111/pbi.70415

Figure Lengend Snippet: PtrLAZY1 transcription is unaffected in TAC1‐CRISPR hybrid poplars, and PtrTAC1 does not interact with PtrLAZY1 at the protein level. (a) Semi‐quantitative RT‐PCR analysis of PtrTAC1‐1 , PtrTAC1‐2 , PtrWEEP and PtrLAZY1 expression in BH, TAC1‐CRISPR lines (#24 and #26) and Lombardy poplar. PtrACTIN2 was used as a loading control. (b) Schematic showing the upper and lower petiole regions used for tissue‐specific transcriptional analysis in (c). The upper and lower parts were separated by the red dashed line. (c) Semi‐quantitative RT‐PCR analysis of PtrTAC1‐1 , PtrLAZY1 and PtrWEEP in the upper and lower petiole tissues of BH and TAC1‐CRISPR lines (#24 and #26) with PtrACTIN2 as a control. (d) Yeast two‐hybrid (Y2H) assay testing protein–protein interaction between PtrTAC1‐1 and PtrLAZY1. Y2H assay was performed by using various combinations of vector constructions (right, see Section ). AtSTM/AD+PtrTALE12ΔC/BD serves as a positive control (Bae et al. ). Yeast was grown on selective media: SD‐W (−Trp), SD‐LW (−Leu, −Trp) and SD‐AHLW (−Ade, –His, −Leu, −Trp). AD and BD represent pGADT7 (activation domain, Leu selection) and pGBKT7 (binding domain, Trp selection) vectors, respectively.

Article Snippet: Briefly, the full‐length PtrTAC1‐1 cDNA was cloned into the pGBKT7 bait vector (Addgene #61703), fused in‐frame with the GAL4 DNA‐binding domain, and selected using tryptophan (Trp) dropout media.

Techniques: CRISPR, Quantitative RT-PCR, Expressing, Control, Y2H Assay, Plasmid Preparation, Positive Control, Activation Assay, Selection, Binding Assay

Journal: Plant Biotechnology Journal

Article Title: Elucidating the Genetic Basis of Columnar Upright Architecture in Populus Through CRISPR Disruption of TILLER ANGLE CONTROL1

doi: 10.1111/pbi.70415

Figure Lengend Snippet: Lombardy poplar has a non‐sense mutation in the PnTAC1‐1 gene. (a) Lombardy poplar ( P. nigra var. italica ) showing its characteristic upright, columnar growth habit in the field. (b) Comparison between Lombardy poplar (left) and the hybrid poplar clone BH ( P. alba × P. glandulosa , clone BH) grown at the Forest Bioresources Department of NIFoS, Republic of Korea. Red arrows indicate representative branch orientations, showing markedly steeper, upright branches in Lombardy poplar relative to BH. (c) Gene structure and sequence alignment of TAC1 homologues. A schematic of the PnTAC1‐1 gene shows a point mutation (T → A) in the third exon, converting a leucine codon (TTA) into a premature stop codon (TAA), likely resulting in a nonfunctional protein. Aligned nucleotide and amino acid sequences of TAC1‐1 from P. trichocarpa ( PtrTAC1‐1 ), P. alba ( PaTAC1‐1 ), P. glandulosa ( PgTAC1‐1 ) and P. nigra var. italica ( PnTAC1‐1 ) highlight the nonsense mutation in red with an asterisk.

Article Snippet: Briefly, the full‐length PtrTAC1‐1 cDNA was cloned into the pGBKT7 bait vector (Addgene #61703), fused in‐frame with the GAL4 DNA‐binding domain, and selected using tryptophan (Trp) dropout media.

Techniques: Mutagenesis, Comparison, Sequencing

Journal: Plant Biotechnology Journal

Article Title: Elucidating the Genetic Basis of Columnar Upright Architecture in Populus Through CRISPR Disruption of TILLER ANGLE CONTROL1

doi: 10.1111/pbi.70415

Figure Lengend Snippet: PtrLAZY1 transcription is unaffected in TAC1‐CRISPR hybrid poplars, and PtrTAC1 does not interact with PtrLAZY1 at the protein level. (a) Semi‐quantitative RT‐PCR analysis of PtrTAC1‐1 , PtrTAC1‐2 , PtrWEEP and PtrLAZY1 expression in BH, TAC1‐CRISPR lines (#24 and #26) and Lombardy poplar. PtrACTIN2 was used as a loading control. (b) Schematic showing the upper and lower petiole regions used for tissue‐specific transcriptional analysis in (c). The upper and lower parts were separated by the red dashed line. (c) Semi‐quantitative RT‐PCR analysis of PtrTAC1‐1 , PtrLAZY1 and PtrWEEP in the upper and lower petiole tissues of BH and TAC1‐CRISPR lines (#24 and #26) with PtrACTIN2 as a control. (d) Yeast two‐hybrid (Y2H) assay testing protein–protein interaction between PtrTAC1‐1 and PtrLAZY1. Y2H assay was performed by using various combinations of vector constructions (right, see Section ). AtSTM/AD+PtrTALE12ΔC/BD serves as a positive control (Bae et al. ). Yeast was grown on selective media: SD‐W (−Trp), SD‐LW (−Leu, −Trp) and SD‐AHLW (−Ade, –His, −Leu, −Trp). AD and BD represent pGADT7 (activation domain, Leu selection) and pGBKT7 (binding domain, Trp selection) vectors, respectively.

Article Snippet: Briefly, the full‐length PtrTAC1‐1 cDNA was cloned into the pGBKT7 bait vector (Addgene #61703), fused in‐frame with the GAL4 DNA‐binding domain, and selected using tryptophan (Trp) dropout media.

Techniques: CRISPR, Quantitative RT-PCR, Expressing, Control, Y2H Assay, Plasmid Preparation, Positive Control, Activation Assay, Selection, Binding Assay