Pentr5 topo ta cloning kit

For p5E entry clones, all PCR products were amplified using Roche Expand High Fidelity PCR kit (Sigma, 11732641001), which contains a 3′-adenine overhang-depositing Taq. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, 28104) or the QIAquick Gel Extraction Kit (Qiagen, 28706) using deionised H₂O elution as the last step. PCR products were then TOPO-cloned into MultiSite Gateway entry vectors with the pENTR5′-TOPO TA Cloning Kit (Invitrogen, K59120): 4 μl of purified PCR product was immediately combined with 1 μl pENTR5′-TOPO TA and 1 μl of salt solution, and incubated at room temperature for a minimum of 2 h to overnight to ligate. 3 µl of the TOPO reactions were then transformed with One Shot TOP10 Chemically Competent E. coli (Invitrogen, C404010) and plated on Kanamycin-infused agar plates. Half reactions can also be used. Primer sequences for pE5′ enhancer/promoter fragments are as follows: for pCK079 p5E Mm BMP4ECR2, oBuS014 Mm BMP4 ECR2 fw (5′-GGGGATGAAAGTAGCATCCTG-3′) and oBuS015 Mm BMP4 ECR2 rv (5′-TTCCACTTTGCTTCCCAAACTGG-3′); for pAB019 pE5 Hs HOPX_4q12N3.1, oAB019 Hs HOPX fw (5′-GTGTTGGGTTAGTTTGAGC-3′) and oAB019 Hs HOPX rv (5′-GTTCTGTTGGGGATATGTCC-3′); for pCK033 p5E desma MCS, oCK024 Dr desma MCS fw (5′-gatatcACTGATgctagcTCCTTGAGGCACTTTCGG-3′) and oCK025 Dr desma MCS rv (5′-gtcgacTACGCTGTGTGAATGCTGG-3′).

Functional complementation assay was performed by using the Multisite Gateway Technology (Invitrogen). Promoters were PCR-amplified from genomic DNA and inserted into the pENTR5’-TOPO vector using the pENTR5′-TOPO TA Cloning Kit (Invitrogen). CDSs of candidate genes were amplified by PCR on cDNA extracted from mature pollen, using forward and reverse primers containing attB1 and attB2 sites, respectively (Supplementary Data 11). Gateway BP reaction was used to insert amplified CDS into the pDONR207 vector. We used Gateway LR reaction to clone the ANX2 (AT5G28680) promoter region in combination with the CDS of PRK2, PRK4, and RALFL15, as well as the regulatory sequence of Raba4D (AT3G12160) in front of RKF2, TRX5, and PLOU (AT2G16030) into the pDestination vector pB7m24GW3. Transformation of Agrobacterium tumefaciens (strain GV3101) were then performed by incubation with 1 µg of the produced vector (5 min on ice), followed by 5 min freezing in liquid nitrogen. After 1 min recovery at 37 °C, 1 mL of Luria-Bertani (LB) medium were added for a 2 h incubation at 28 °C (with shaking), plated on LB containing gentamycin, rifampicin + spectinomycin, and incubated 48 h at 28 °C. Plants were transformed by dipping in A. tumefaciens solution. Primers used to generate constructs are listed in Supplementary Data 11.