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

Abstract

Background: 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. Therefore, in the present study we focused on the nucleotide sequence identification and the functional characteristics of the pro-SmAMP2 promoter in transgenic plants.

Results: In 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.

Conclusion: Our results show that the рro-SmAMP2 promoter can be used for target genes expression control in transgenic plants.

Keywords: Expression control; Nicotiana tabacum; Promoter; Stellaria media; Transgenic plants; pro-SmAMP2.

Fig. 1

The nucleotide sequence of 5′-flanking promoter region of pro-SmAMP2 gene and location of cis-acting elements (color-coded and labeled). Vertical arrows mark the starting points of the 5′-deletion variant nucleotide sequences. TSS is the transcription start site at −40 bp from ATG codon behind the region highlighted in red. Translation initiation site ATG +41 is labeled in italics

Fig. 2

The scheme of constructs with deletion variants of pro-SmAMP2 gene promoter region designed on basis of plant expression vector pCambia 1381Z for analysis of gus reporter gene expression. Plant expression vector pMOG-35SintGUS containing CaMV 35S viral promoter was used as a control. The translated region of gus gene is colored black; Int - modified castor bean catalase intron within the translated region of gus gene; PIV2 - modified potato ST-LS1 gene intron within the translated region of gus gene. Promoters are depicted as arrows with the appropriate signatures

Fig. 3

The GUS activity in tobacco transformants T₀. a Plants were growing under aseptic conditions 1.5 months after rooting on the medium with a selective agent. The average values of activity from three mature leaves of each transformant ± SE (the standard error) are presented. b Plants were grown for six weeks in soil after being transplanted from aseptic conditions. The average activity values of three samples from one leaf of each transformant ± SE are presented

Fig. 4

The GUS activity in leaves of transgenic tobacco plants from three consecutive generations (T1, T2, T3) measured in six weeks of growth in soil after transplanting from aseptic conditions. The average activity values of three samples from one leaf of each transformant ± SE are presented. The average activity among the plants of one population ± confidence interval (p = 5 %) are shown as gray bars. a T₁. b T₂. c T₃

Fig. 5

Southern blotting analysis of total DNA from leaves of two plants Т₃p2120 No. 8-2-2 (lanes 1 and 2) and Т₃p455 No. 14-3-3 (lanes 3 and 4) restricted with EcoRI. “C-“- total DNA from leaves of non-transgenic tobacco plants restricted with EcoRI. M - marker (Fermentas, #Sm333). “C+” - plasmid pMOG-35SintGus restricted with HindIII. The 740 bp fragment of gus gene was used as probe

Fig. 6

The GUS activity in the leaves of T₃ generation homozygous transgenic tobacco plants grown in soil from seeds. Eight plants were studied under long day (16/8 h) and six plants under short day (12/12 h) conditions for both variants T₃p2120 and T₃p455. The average levels of GUS activity in adjacent leaves from medium tiers ± SE are presented

Fig. 7

Gus gene expression in leaves of T₃p2120 and T₃p455 tobacco plants measured by real-time PCR. a Gus gene expression normalized to the expression of actin gene. Average values for individual transgenic plants ± SE are presented as gray bars. For transgenic variants, the average values ± confidence interval (p = 5 %) are presented as black bars. b The dependence of GUS activity level on gus gene expression. Correlation coefficients (r) and regression coefficients (b) ± SE are shown

Fig. 8

The GUS activity in different organs of Т₃р2120 (line No. 8-2-2) and Т₃p455 (line No. 10-8-9) transgenic tobacco plants at various stages of their development. a Roots, stems, and leaves at the age of 2 weeks after transplanting from aseptic conditions, (b) bud, (c) anthers, (d) microsporocytes, additionally stained by acetocarmine, (e) pollen