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

Roman A Komakhin,Denis A Vysotskii,Svetlana R Strelnikova,Ekaterina M Vetchinkina,Vera V Komakhina,Vera D Voblikova,Rahim R Shukurov,Alexey V Babakov

doi:10.1186/s12896-016-0273-x

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.

Highlights

In 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
When studying antifungal peptides of chickweed (Stellaria media), we found that the expression of their genes proSmAMP1 and pro-SmAMP2 is high and that it increases when S. media is treated by the elicitor methyl jasmonate or infected by pathogenic fungi [26]
Its sequencing showed that the fragment includes part of the coding sequence of the pro-SmAMP2 gene and 2160 bp 5′-flanking region from the translation start site (ATG codon) including 40 bp of pro-SmAMP2 gene 5′-untranslated region (UTR)

Summary

Introduction

In 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. In the present study we focused on the nucleotide sequence identification and the functional characteristics of the pro-SmAMP2 promoter in transgenic plants. The CaMV 35S promoter usually provides a high expression level of heterologous genes in plants, but it has some significant drawbacks. The widespread use of the CaMV 35S promoter increases the probability to inactivate expression of heterologous genes under its control as a result of the homologous recombination [10, 11]

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Discussion

Conclusion