Efficiency of the alpha-hairpinin SmAMP-X gene promoter from Stellaria media plant depends on selection of transgenic approach

Abstract

The antimicrobial activity of the alpha-HAIRPININ ANTIMICROBIAL PEPTIDE X (SmAMP-X gene, GenBank acc. No. HG423454.1) from Stellaria media plant has been shown in vitro. Here, we isolated the SmAMP-X gene promoter and found two genomic sequences for the promoter (designated pro-SmAMP-X and pro-SmAMP-X-Ψ2) with 83% identity in their core and proximal regions. We found that the abilities of these promoters to express the uidA reporter and the nptII selectable marker differ according to the structural organization of T-DNA in the binary vector used for plant transformation. Analysis of Agrobacterium-infiltrated Nicotiana benthamiana leaves, transgenic Arabidopsis thaliana lines, and transgenic Solanum tuberosum plants revealed that both promoters in the pCambia1381Z and pCambia2301 binary vectors generate 42–100% of the ß-glucuronidase (GUS) activity generated by the CaMV35S promoter. According to 5’-RACE (rapid amplification of cDNA ends) analysis, both plant promoters are influenced by the CaMV35S enhancer used to express selectable markers in the T-DNA region of pCambia1381Z and pCambia2301. The exclusion of CaMV35S enhancer from the T-DNA region significantly reduces the efficiency of pro-SmAMP-X-Ψ2 promoter for GUS production. Both promoters in the pCambia2300 vector without CaMV35S enhancer in the T-DNA region weakly express the nptII selectable marker in different tissues of transgenic N. tabacum plants and enable selection of transgenic cells in media with a high concentration of kanamycin. Overall, promoter sequences must be functionally validated in binary vectors lacking CaMV35S enhancer.