2.1 Construct development and wheat transformation
The binary vector pPZP211 vector was purchased commercially (Creative Biogene). A rice endosperm-specific promoter GluB-4 [17] and a Nos terminator (GenBank: AJ007623 ) were inserted upstream and downstream of the multiple cloning site (MCS) atEcoR I/Kpn I and Xba I/Hind III sites, respectively. The codon-optimized complete CDS of TM-1 protein ofMycoplasma gallisepticum (MG; GenBank: E09896 ) was followed by the endoplasmic reticulum-retention signal KDEL (Lys-Asp-Glu-Leu) linked to C-terminal of TM-1. The CTB (cholera toxin B subunit) coding sequence followed by a furin enzymatic cleavage site (RRKRSV) were inserted immediately upstream of TM-1. The entire PGluB-4-CTB-furin-TM-1-KDEL-Tnos cassette was synthesized (GenScript, NJ, USA) and subcloned in pPZP211 at Kpn I/Xba I sites (Figure 1A).
Wildtype wheat (Triticum aestivum L. cv Bobwhite) was used to generate transgenic plants in this study. PDS-1000/He particle gun or biolistics (Biorad, Germany) was utilized for direct bombardment. The 20ug plasmid DNA was coated with gold particles of size (0.6 µm) following procedure as described by Sanford et al., 1993, and with 950 psi rapture disks for bombardment. The immature embryos were isolated 14 days after anthesis (size 0.5-1.5mm) under aseptic conditions and were placed on induction media for 5-6 days in dark at 26C before bombardment for callus induction.
The total DNA was extracted from wheat seeds for confirmation of genetic integration of PGluB-4-TM-1-Tnos construct in wheat seed genome by Polymerase Chain Reaction (PCR) and sequencing with primers targeting transgene TM-1 (Seq Forward: 5’-attgcaaagctaccttttttctatta-3’; Seq Reverse: 5’-atcgcgcgcggtgtcatctatgtt-3’; Fig. 1A).