To construct the vector 35S::CaWRKY41, the full-length open reading frame was cloned into pDONR207 and transferred into the pGWB2 expression vector (Invitrogen, USA). To construct the reporter vector (pCaWRKY41::GUS) for histochemical β-glucuronidase (GUS) analysis, the promoter of CaWRKY41 of ~2 kb in length (pCaWRKY41) was amplified via PCR from pepper genomic DNA and cloned into the pMDC163 vector (Invitrogen). The constructs 35S::CaWRKY41 and pCaWRKY41::GUS were then transformed into Agrobacterium tumefaciens strain GV3101 using the freeze–thaw method. A. tumefaciens-mediated transformation of Arabidopsis was performed using the floral dip method (Clough and Bent, 1998 (link)), and transgenic plants were identified by sowing seeds on ½ MS agar plates containing 50 mg l−1 hygromycin and selecting hygromycin-resistant seedlings.
Pgwb2 expression vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PGWB2 expression vector is a plasmid that can be used for the expression of proteins in eukaryotic cells. It contains a strong promoter and a multiple cloning site for the insertion of the gene of interest.
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Lab products found in correlation
2 protocols using pgwb2 expression vector
1
CaWRKY41 Transcription Factor Characterization
2
Protein Localization and Overexpression of OsEL2 and Related Genes
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To observe protein localization of OsEL2, the sequence of the OsEL2 promoter and full‐length cDNA was amplified by PCR and the amplified fragment was introduced into the pGWB4 expression vector (Nakagawa et al., 2007a ,b ) using the Gateway system (Invitrogen, http://www.invitrogen.com/ ). To generate the OsEL2, OsEL2like and OsNCED1 overexpression plants, the full‐length cDNAs of these genes were amplified and introduced into the pGWB2 expression vector (Nakagawa et al., 2007a ,b ) using the Gateway system (Invitrogen, http://www.invitrogen.com/ ). The primers for vector construction are listed in Table S7 . For the generation of loss‐of‐function mutants, CRISPR‐CAS9 system was used.
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