The BamHI -SacI fragment was cloned by replacement of the uidA coding sequence into the binary vector pBI101 generating the pBI101- PsEND1::barnase-barstar construct.
A 6,334-bp 6,334-bpDNA fragenomicontaining the entire GhDNA coding region and upstream and downstream sequences ofragmentwas subcontainingm the BAC clonentire7658 and inserted into the binary vector pCAMBIA1300 to Ghd10ate the transformation vecodingGhd10 foregionin the complementandon test.
The PCR-amplified merE DNA fragment was subcloned into a binary vector, pMAT137, to generate the plasmid pMAE2 (Figure 2A).
The resulting vector was digested with NotI and inserted into the binary vector pWBvec8 [51] to generate pEC129 that contained the hygromycin selectable marker under the control of the CaMV35S promoter oriented so that the UBI and CaMV35S promoters were adjacent to one another and transcribed in opposite orientations with the Tml 3' end adjacent to the left border.
Each BamHI– SmaI fragment was subcloned into corresponding restriction sites on the binary vector pBI101.3 (Clontech) to generate the AtACBP6pro::GUS fusion plasmids (pAT452, pAT590, pAT591 and pAT592) that were used in Agrobacterium tumefaciens transformation of wild-type Arabidopsis Col-0 by the floral dip method.
To generate the ST111 binary vector, the ST77 binary vector was digested with NcoI and XbaI (to release the hTG coding region) and the resulting vector backbone was ligated with the synthesized hMBP-Sigma gene that was also previously digested with NcoI and XbaI.
The cDNA was cloned into the modified binary vector pBI121 to generate an overexpression construct driven by the fruit-specific TFM7 promoter (Accession no.X95261) [ 25].
The PCR product was digested with BamH I and Xho I and then ligated into the binary vector pMD1 to generate p21HAK, for expression under control of the 35S promoter.
The binary vector pBLTI121-sGFP was generated by inserting cDNA encoding sGFP without the ATG [ 85] as a SpeI and StuI fragment into the binary plant transformation vector pBLTI121 [ 9].
pMD18-T PtrABF was restricted with SalI and KpnI, and the resulting product was inserted into XhoI- KpnI sites of the binary vector pBI121 to generate pBI121- PtrABF, which was introduced into A. tumefaciens strain EHA105 by heat shock after fidelity verification by sequencing.
