paper n a recombinant dna plasmid pgadt7 takara  (TaKaRa)

Journal: Nature Communications

Article Title: A quantitative gibberellin signaling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem

doi: 10.1038/s41467-024-48116-4

Figure Lengend Snippet: a Schematic representation of the domain structure of a typical DELLA protein. Conserved histidine (H471), tyrosine (Y472), and tyrosine (Y473) residues were mutated into alanine (A) to obtain the modified RGA m2 protein. The nucleic acids and amino acids mutated in RGA m2 are indicated in red. b Yeast two-hybrid (Y2H) assays in which RGA and RGA m2 were tested pairwise with four known DELLA-interacting partners: JAZ1, TCP14, IDD2 and BZR1. Empty pGBKT7 and pGADT7 vectors were included as negative controls. Photos show the growth of the yeast on control media (GM) and on selective media (SM). c Pairwise Y2H interaction assays between RGA or RGA m2 and the three GA receptors GID1a, GID1b and GID1c. Photos show the growth of the yeast on control media (GM), selective media (SM), and SM media supplemented with 100 μM GA 3 . d Time-course analysis of GA-induced degradation of RGA (upper panel) and RGA m2 protein (lower panel). Immunodetection of RGA-GFP and RGA m2 -GFP protein in 35S::RGA-GFP and 35S::RGA m2 -GFP N. benthamiana agro-infiltrated leaves treated with 100 mM cycloheximide (CHX) and 100 µM GA 3 for the indicated times. Numbers indicate RGA-GFP and RGA m2 -GFP levels relative to actin levels, used as loading control. The experiment was repeated twice with similar results. e Co-immunoprecipitation assays between RGA or RGA m2 and IDD2 or TCP14. Protein extracts from different combinations of N. benthamiana agro-infiltrated leaves with 35S::RGA-GFP , 35 S::RGA m2 -GFP , 35S::IDD2-RFP , and 35S::TCP14-RFP were immunoprecipitated with anti-GFP antibodies. The co-immunoprecipitated protein (IDD2-RFP and TCP14-RFP) was detected by anti-RFP antibodies. The experiment was repeated twice with similar results. f , g Effect of RGA and RGA m2 on BZR1 ( f ) and TCP14 ( g ) transcriptional activities in N. benthamiana agro-infiltrated leaves with a combination of BZR1, TCP14, RGA, and RGA m2 effector constructs and corresponding Luciferase/Renilla reporter constructs, as indicated (top panels). BZR1 and TCP14 have been fused to VP16 transcriptional activator domain in this experiment. Transcriptional activities are represented as the ratio of Luciferase and Renilla (used as internal control) activities, relative to the value obtained for the reporter construct alone that was set to 1. Data are means ± SD of three biological replicates. P -values were calculated in R using a two-tailed Welch t -test. Bottom panels: immunodetection of RGA-GFP, RGA m2 -GFP, HA-VP16-BZR1, and HA-VP16-TCP14 from N. benthamiana agro-infiltrated leaves used for transcriptional activity assays. These experiments were repeated three times with similar results.

Article Snippet: On the other hand, JAZ1, TCP14, IDD2, BZR1, GID1a, GID1b and GID1c cDNAs inserted into pDONR221 were fused to the activation domain GAL4 (AD) after recombination into pGADT7 (Clontech).

Techniques: Modification, Control, Immunodetection, Immunoprecipitation, Construct, Luciferase, Two Tailed Test, Activity Assay

Journal: eLife

Article Title: Ablation of palladin in adult heart causes dilated cardiomyopathy associated with intercalated disc abnormalities

doi: 10.7554/eLife.78629

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pGADT7 AD vector , Takara Bio , Cat# 630442 , .

Techniques: Knock-Out, Transgenic Assay, Recombinant, Plasmid Preparation, Cloning, Sequencing, DC Protein Assay, Transformation Assay, Isolation, Reverse Transcription, Protease Inhibitor, Western Blot, Software

Journal: Horticulture Research

Article Title: VvSUN may act in the auxin pathway to regulate fruit shape in grape

doi: 10.1093/hr/uhac200

Figure Lengend Snippet: Analysis of the VvSUN promoter and location analysis of VvSUN . (A) VvARF6 interfaces with the VvSUN promoter, as shown by a Y1H assay. As bait, Y1H strains containing the promoters of VvSUN were employed in the experiment. pGADT7 was set as the vector for the control. (B) Measurement of GUS expression levels in grapevine leaves was accomplished based on the transient expression of the VvSUN promoter in response to varying dosages of IAA treatment. The 35S promoter acted as the positive control. Outcomes are presented as the means (± standard deviation) of three biological experiments. Lower-case letters ( P ≤ .01) and capital letters ( P ≤ .05) represent a significant difference among different treatments, as determined by Student’s t -test. (C) Histochemical staining of GUS function mediated by the VvSUN promoter under different concentrations of IAA treatment in grapevine leaves. The 35S promoter is the control and P0, P10, P50, and P100 represent the 0, 10, 50, and 100 mg/l IAA treatments, respectively. (D) Subcellular distribution of VvSUN in the leaves of N. benthamiana that had been transiently converted. 35S-GFP was adopted as the control, and expression was monitored utilizing a confocal laser scanning microscope. Chlorophyll channel (left 1), GFP channel (left 2), bright field (right 2), and merged images (right 1) are shown.

Article Snippet: Co-transformation of recombinant plasmid pGADT7-VvARF6 and pAbAi-VvSUN-pro into yeast strain Y1HGold (Clontech) was performed in accordance with the guidelines provided by the manufacturer.

Techniques: Y1H Assay, Plasmid Preparation, Expressing, Positive Control, Standard Deviation, Staining, Laser-Scanning Microscopy

Journal: Horticulture Research

Article Title: VvSUN may act in the auxin pathway to regulate fruit shape in grape

doi: 10.1093/hr/uhac200

Figure Lengend Snippet: The pathway correlation network and proposed model of fruit shape control. (A) Key pathways in the control of fruit shape by WGCNA and KEGG analysis were characterized and identified, then significantly enriched pathways were used to construct a correlation network by Cytoscape. (B) Proposed model of how VvSUN regulates fruit shape. VvSUN is situated in the plasma membrane and exogenous auxin may induce expression of VvARF6 , then VvARF6 activates cis -elements in VvSUN promoters to induce gene expression. The increased expression of VvSUN stimulates endogenous auxin accumulation and polar transport and/or auxin signal transduction process variations. Therefore, we suppose that VvSUN may not only respond to exogenous auxin treatment but also modulate the elongated fruit shape in the plant hormone signal transduction pathway during the early phases of fruit growth.

Article Snippet: Co-transformation of recombinant plasmid pGADT7-VvARF6 and pAbAi-VvSUN-pro into yeast strain Y1HGold (Clontech) was performed in accordance with the guidelines provided by the manufacturer.

Techniques: Construct, Expressing, Transduction