BioVector? NTCC? pBIB-BASTA-GFP植物雙元表達質粒BioVector?載體

The promoter of PzIPT1 was inserted into a binary vector pBIB-BASTA-GUS modified from pBIB vector (Becker, 1990) at the HindIII and SalI sites to make pPzIPT1::GUS. The PzIPT1 promoter was also recombined into the Gateway-compatible pFYTAG binary vector to drive the expression of fused coding regions of histone 2A (HTA6; At5g59870) and enhanced YFP (EYFP; Zhang et al., 2005). The binary vector pBIB-BASTA-GFP (Ge et al., 2011) was modified to a Gateway-compatible destination vector, pBIB-BASTA-GFP-GWR, by inserting the Gateway module at HindIII and XbaI sites for promoter analyses.

Deletion fragments were amplified by PCR from cloned PzIPT1 promoter and transferred into pDONR/Zeo vector by Gateway in vitro DNA recombination for sequencing analysis. Following sequence verification, these truncated promoter fragments were in vitro recombined into pBIB-BASTA-GFP-GWR and pBIB-BASTA-GUS-GWR (Yuan et al., 2007) to create final binary transformation constructs. IPTPromPB2 was used as a reverse primer for all ten 5′-deletions. Forward primers (?761PB1, ?531PB1, ?368PB1, ?255PB1, ?154PB1, ?130PB1, ?105PB1, ?87PB1, ?63PB1, ?39PB1) were designed according to the positions of the deletions in the PzIPT1 promoter. The deletion constructs ?88-95, ?64-87, and ?40-63 were created according to the manual of QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) using primers ?88-95F, ?88-95R, ?64-87F, ?64-87R, ?40-63F, and ?40-63R.

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