Transient Protein Expression in Nicotiana benthamiana

Full-length cDNAs corresponding to BDH1 and BDH2 were cloned into the vector pDONR201 and verified by sequencing. Corresponding entry clones were used in LR recombination reactions to transfer the cDNA fragments to the gateway-compatible expression vector pGWB605 [33 (link)]. The binary constructs obtained were used to transform Agrobacterium tumefaciens GV3101. Each pGWB605 construct was coexpressed in 6-week-old Nicotiana benthamiana leaves after leaf infiltration with GV3101 strains containing the tested construct plus an antisilencing agrobacterial strain expressing P19. In addition, the agrobacterial strain transformed with 35S::H2B-RFP was used to visualize the nuclei as described previously [11 (link)]. Fluorescence was analyzed 2 d after infiltration using a Nikon D-Eclipse C1 laser scanning confocal microscope.

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Stachula P., Kapela K., Malecka E., Jaronczyk K., Patryn J., Siwirykow N., Bucholc M., Marczak M., Kotlinski M, & Archacki R. (2023). BRM Complex in Arabidopsis Adopts ncBAF-like Composition and Requires BRD Subunits for Assembly and Stability. International Journal of Molecular Sciences, 24(4), 3917.

Publication 2023

D-Eclipse C1 laser scanning confocal microscope

Agrobacterium tumefaciens Cdna Clones Fluorescence Laser scanning microscope Leaf Nicotiana Nuclei Recombination Strain Vector

Corresponding Organization : Institute of Biochemistry and Biophysics, Polish Academy of Sciences

Top 5 similar protocols

Variable analysis

independent variables

Expression of BDH1 and BDH2 cDNAs in pGWB605 vector
Coexpression of pGWB605 constructs with P19 antisilencing agrobacterial strain
Expression of 35S::H2B-RFP in agrobacterial strain

dependent variables

Fluorescence of expressed proteins analyzed using confocal microscopy

control variables

6-week-old Nicotiana benthamiana leaves used for leaf infiltration
Agrobacterium tumefaciens GV3101 strain used for transformation

controls

Positive control: 35S::H2B-RFP expression to visualize nuclei
Negative control: Not explicitly mentioned

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