Novel strong promoter of antimicrobial peptides gene pro-SmAMP2 from chickweed (Stellaria media)

Abstract

BackgroundIn a previous study we found that in chickweed the expression level of the pro-SmAMP2 gene was comparable or even higher to that of the β-actin gene. This high level of the gene expression has attracted our attention as an opportunity for the identification of novel strong promoters of plant origin, which could find its application in plant biotechnology. Therefore, in the present study we focused on the nucleotide sequence identification and the functional characteristics of the pro-SmAMP2 promoter in transgenic plants.ResultsIn chickweed (Stellaria media), a 2120 bp promoter region of the pro-SmAMP2 gene encoding antifungal peptides was sequenced. Six 5′-deletion variants −2120, −1504, −1149, −822, −455, and −290 bp of pro-SmAMP2 gene promoter were fused with the coding region of the reporter gene gusA in the plant expression vector pCambia1381Z. Independent transgenic plants of tobacco Nicotiana tabacum were obtained with each genetic structure. GUS protein activity assay in extracts from transgenic plants showed that all deletion variants of the promoter, except −290 bp, expressed the gusA gene. In most transgenic plants, the GUS activity level was comparable or higher than in plants with the viral promoter CaMV 35S. GUS activity remains high in progenies and its level correlates positively with the amount of gusA gene mRNA in T3 homozygous plants. The activity of the рro-SmAMP2 promoter was detected in all organs of the transgenic plants studied, during meiosis and in pollen as well.ConclusionOur results show that the рro-SmAMP2 promoter can be used for target genes expression control in transgenic plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0273-x) contains supplementary material, which is available to authorized users.

Stellaria media (L.) Vill.- A plant with immense therapeutic potentials: phytochemistry and pharmacology

“…The extracts significantly inhibited the microbial growth of the gram positive and negative bacteria [45]. In a recent study, peptides genepro-SmAMP2 and β-actin gene isolated from S. media significantly displayed strong inhibitory activity against the tested bacterial isolates whereas Sm-AMP-X exhibited significant inhibitory activity against phytopathogenic fungi due to the N-and C-terminal regions of the specified peptide [46,47]. 2.9.…”

New efficient gene promoters from Stellaria media for plant genetic engineering

“…The strains were injected into leaves with a needle-free syringe. Agrobacterial transformation of plants was performed by the "lawn" method, which had been used earlier (Komakhin et al, 2016). Genetic constructs for transient expression containing the reporter gene gus controlled by the plant promoters were incorporated into plasmid pCambia1381Z (Cambia, Australia).…”

“…At the present time in plant biotechnology there is a deficit of regulatory elements for effective expression of recombinant genes due, first, to the lack of native promoters of plant genes with necessary parameters (Naqvi et al, 2010) and, secondly, to complexity in using available promoters for coordinated transcriptional control (Shrestha et al, 2018). We had previously found that hevein-like peptide genes from chickweed (Stellaria media) are a source of strong constitutive promoters for biotechnology of cultivated plants (Shukurov et al, 2012;Komakhin et al, 2016;Vysotskii et al, 2016;Vetchinkina et al, 2016). E. g., in transient expression experiments in Nicotiana benthamiana pro-SmAMP1 and pro-SmAMP2 promoters were 2-4 times more effective than the CaMV35S promoter; in rape (Brassica napus) and sugar beet (Beta vulgaris) plants pro-SmAMP1 and pro-SmAMP2 were comparable to CaMV35S (Madzharova et al, 2018).…”

Primary Structure Analysis of Antifungal Peptides from Cultivated and Wild Cereals

“…Curiously, only for cultivated cereals were these peptides obtained first. It allowed a reconsideration of the modern classification of hevein AMPs to diverge them into two subfamilies: six-cysteine-containing peptides, such as peptides from amaranth ( Amaranthus caudatus, A. retroflexus ) [ 73 , 74 , 75 ], common chickweed ( Stellaria media ) [ 76 , 77 ], and 10-cysteine-containing peptides [ 78 , 79 ]. The most famous examples are wheat antimicrobial peptides (WAMPs) from T. kiharae and lyme grass antimicrobial peptides (LAMP) from L. arenarius families [ 80 , 81 ].…”