To construct the vector for the transient expression assay, the ORF of CaREM1.4 or CaRIN4-12 without the termination codon was inserted into the SpeI restriction site of the pGinGFP2 vector with a GFP tag. To construct gene-silencing vectors for VIGS, CaREM1.4 and CaRIN4-12 gene fragments of 200–300 bp were identified by searching for the fragment with the lowest similarity to other pepper gene sequences on the NCBI database, then cloned into the gene-silencing vector pTRV2 (Invitrogen), separately. To construct the vector for the Y2H assay, the CaREM1.4 ORF was constructed into the pGBKT7 and the ORF of CaRIN4-12 was subcloned into pGADT7 with the EcoRI/BamHI sites. To construct the vector for Split-LUC assays, double digestion of nLUC and cLUC plasmids was performed with restriction enzymes BamHI/SalI. The ORF of CaREM1.4 and CaRIN4-12 was constructed into the cLUC and nLUC vector, respectively. To generate constructs for the BIFC assay, CaREM1.4 and CaRIN4-12 were fused to pSPYNE(R)173 and pSPYCE(M) vectors with the BamHI/XhoI sites, respectively [66 (link)]. To construct the vector for the Co-IP assay, the ORF sequence of CaREM1.4 was constructed into the SpeI restriction site of pBI121MCS-mCherry vector. The primer sequences for construction of all vectors are listed in Supplementary Data Table S4 .
Ptrv2
Manufactured by Thermo Fisher Scientific
The PTRV2 is a versatile laboratory equipment designed for general laboratory applications. It provides stable temperature control and consistent performance to support a wide range of experimental procedures. The core function of the PTRV2 is to maintain precise temperature conditions within a designated experimental environment.
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2 protocols using ptrv2
1
Molecular Cloning of Pepper Genes
2
Overexpression and Binding Analysis of CaCBL1 and CaWRKY40
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To analyze CaCBL1 transient overexpression, the ORF of CaCBL1 with or without termination codon was cloned into the entry vector pDONR207 (Invitrogen) by BP reaction and was then cloned into destination vectors such as pEarleyGate103 (Invitrogen) with a GFP protein tag for subcellular localization or pEarleyGate202 (Invitrogen) harboring a Flag protein tag for ChIP assay by LR reaction.
To investigate the possible binding of CaWRKY40 to W-box-2 within the CaCBL1 promoter, the ORF of CaWRKY40 or its mutant (CaWRKY40-m) was cloned into the vector pDONR207 by BP reaction and was then cloned into destination vector pEarleyGate202 for ChIP assay or pDEST15 (harboring a GST protein tag) for EMSA by LR reaction.
To generate constructs for a VIGS assay, a specific DNA fragment of CaCBL1 in its ORF was cloned into the entry vector pDONR207 by BP reaction and was then cloned into destination vector pTRV2 (Invitrogen) by LR reaction. The primers used for vector constructions of CaCBL1 and CaWRKY40 in this study are listed in Supplementary Table S1.
The vectors of CabZIP63 and CaWRKY40 used in this study were previously constructed (Shen et al. 2016a ). The vectors construction followed Gateway Recombination Cloning Technology (Invitrogen Corp.).
To investigate the possible binding of CaWRKY40 to W-box-2 within the CaCBL1 promoter, the ORF of CaWRKY40 or its mutant (CaWRKY40-m) was cloned into the vector pDONR207 by BP reaction and was then cloned into destination vector pEarleyGate202 for ChIP assay or pDEST15 (harboring a GST protein tag) for EMSA by LR reaction.
To generate constructs for a VIGS assay, a specific DNA fragment of CaCBL1 in its ORF was cloned into the entry vector pDONR207 by BP reaction and was then cloned into destination vector pTRV2 (Invitrogen) by LR reaction. The primers used for vector constructions of CaCBL1 and CaWRKY40 in this study are listed in Supplementary Table S1.
The vectors of CabZIP63 and CaWRKY40 used in this study were previously constructed (Shen et al. 2016a ). The vectors construction followed Gateway Recombination Cloning Technology (Invitrogen Corp.).
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