To generate transgenic lines expressing compartment-specific markers, a binary vector pCB301 was modified to accommodate an UBQ10 promoter (Geldner et al., 2009 (link)) and a polyadenylation signal, and used to generate N-terminal fusions of the mTurquoise fluorescent protein (Goedhart et al., 2010 (link)) with the actin-binding domain LifeAct (Goedhart et al., 2010 (link)), the transmembrane domain of N-acetylglucosaminyl transferase I (NAG; Grebe et al., 2003 (link)), or SCAMP2 (Toyooka et al., 2009 (link)). An ER-targeted CFP was described before (Peremyslov et al., 2010 (link)). The marker-expressing 3KOR plants were selected using glufosinate. The transgenic plant lines generated in Columbia or 3KOR genetic background had normal developmental phenotypes under optimal growth conditions. This was also the case for the LifeAct-mTurquoise plants in accord with recent independent work (van der Honing et al., 2011 (link)).
Generation and Characterization of Myosin XI-K Marker Lines
To generate transgenic lines expressing compartment-specific markers, a binary vector pCB301 was modified to accommodate an UBQ10 promoter (Geldner et al., 2009 (link)) and a polyadenylation signal, and used to generate N-terminal fusions of the mTurquoise fluorescent protein (Goedhart et al., 2010 (link)) with the actin-binding domain LifeAct (Goedhart et al., 2010 (link)), the transmembrane domain of N-acetylglucosaminyl transferase I (NAG; Grebe et al., 2003 (link)), or SCAMP2 (Toyooka et al., 2009 (link)). An ER-targeted CFP was described before (Peremyslov et al., 2010 (link)). The marker-expressing 3KOR plants were selected using glufosinate. The transgenic plant lines generated in Columbia or 3KOR genetic background had normal developmental phenotypes under optimal growth conditions. This was also the case for the LifeAct-mTurquoise plants in accord with recent independent work (van der Honing et al., 2011 (link)).
Corresponding Organization : State University of New York
Protocol cited in 7 other protocols
Variable analysis
- Insertion of the YFP ORF into the genomic copy of the myosin XI-K gene (AT5G20490) to generate the XI-K:YFP construct
- Transformation of the 3KO xi-k xi-1 xi-2 plants with the XI-K:YFP construct
- Expression levels of XI-K:YFP in the 3KOR line compared to XI-K expression in Columbia-0 plants
- Phenotypes of plant lines expressing XI-K:YFP
- Genomic copy of the XI-K gene used to generate the XI-K:YFP construct
- Genetic background of the 3KO xi-k xi-1 xi-2 plants used for transformation
- Growth conditions for the transgenic plant lines
- Immunoblotting using a rabbit polyclonal XI-K antibody to detect XI-K:YFP expression
- Immunoblotting using a rabbit polyclonal antibody to ER binding protein (BiP) as a loading control
- Columbia-0 plants as a control for comparing XI-K expression levels
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