The promoter fragments were extracted from the pGEM-T vector (including the Pptha1 promoter), with a BamHI- SalI sequential digestion, and transcriptionally fused to the uidA reporter gene in the promoterless binary vector pBI101.1 [ 23].
The binary vector constructs including pBI121 as negative control were introduced into Agrobacterium tumefaciens strain C58C1 by electroporation.
As noted in previous research, an ideal binary vector system should include the following features: an easy route for cloning the gene of interest under control of a wide variety of promoter and terminator sequences, the ability to simultaneously express several target genes, and a wide choice of selection markers and reporter genes [16].
This vector includes a binary variable for the Original Member Countries (OMCs)l, binary variables for Denmark and the United Kingdom that accessed the EEC in 1973, binary variables for Portugal and Spain that become members of the EEC in 1986, and binary variables for Austria, Finland, and Sweden that become members of the EU in 1995.
For SiLEA14 overexpression in foxtail millet, the coding region of SiLEA14 fused with a flag tag at the 3′end was amplified and ligated into the SacI/ KpnI sites of the binary vector pCOU [ 61], which includes the ubiquitin promoter to drive transgene expression.
The construct was cloned into the binary vector pART27 using the NotI sites [ 33].
In addition to the 20-length binary vector of amino acid encodings, every residue position including the central Ser/Thr was encoded as a 5-length binary vector or alternatively encoded as a vector of raw Jpred prediction scores [0.0 1.0], resulting in a vector of length 20 k + 5 k + 1).
Digested PCR products were then ligated into the binary vector to get the final construct.
The binary vector pTM050 contained the hACHE-S gene with a single-base mutation.
The P35S::LIF2:GFP and P35S::GFP:LIF2 constructs were then introduced into the pCambia1300 binary vector, generating the binary pCaLIF2 GFP and pCaGFP LIF2 plasmids.
