Characterization of a strong and constitutive promoter from the Arabidopsis serine carboxypeptidase-like gene AtSCPL30 as a potential tool for crop transgenic breeding

Abstract

Background: Transgenic technology has become an important technique for crop genetic improvement. The application of well-characterized promoters is essential for developing a vector system for efficient genetic transformation. Therefore, isolation and functional validation of more alternative constitutive promoters to the CaMV35S promoter is highly desirable.

Results: In this study, a 2093-bp sequence upstream of the translation initiation codon ATG of AtSCPL30 was isolated as the full-length promoter (PD1). To characterize the AtSCPL30 promoter (PD1) and eight 5' deleted fragments (PD2-PD9) of different lengths were fused with GUS to produce the promoter::GUS plasmids and were translocated into Nicotiana benthamiana. PD1-PD9 could confer strong and constitutive expression of transgenes in almost all tissues and development stages in Nicotiana benthamiana transgenic plants. Additionally, PD2-PD7 drove transgene expression consistently over twofold higher than the well-used CaMV35S promoter under normal and stress conditions. Among them, PD7 was only 456 bp in length, and its transcriptional activity was comparable to that of PD2-PD6. Moreover, GUS transient assay in the leaves of Nicotiana benthamiana revealed that the 162-bp (- 456~ - 295 bp) and 111-bp (- 294~ - 184 bp) fragments from the AtSCPL30 promoter could increase the transcriptional activity of mini35S up to 16- and 18-fold, respectively.

Conclusions: As a small constitutive strong promoter of plant origin, PD7 has the advantage of biosafety and reduces the probability of transgene silencing compared to the virus-derived CaMV35S promoter. PD7 would also be an alternative constitutive promoter to the CaMV35S promoter when multigene transformation was performed in the same vector, thereby avoiding the overuse of the CaMV35S promoter and allowing for the successful application of transgenic technology. And, the 162- and 111-bp fragments will also be very useful for synthetic promoter design based on their high enhancer activities.

Keywords: AtSCPL30; CaMV35S; Constitutive promoter; GUS analysis; Nicotiana benthamiana; Stress treatment.

Fig. 1

The constructs of the AtSCPL30 promoter reporter plasmids and the picture of restriction digestion analysis. a The AtSCPL30 promoter (PD1) and eight 5′ deleted fragments (PD2-PD9) were ligated upstream of the GUS reporter gene of the pCAMBIA1391Z vector. The numbers indicate the nucleotide position from the translational initiate code ATG (A as + 1). b The fused plasmids were confirmed by restriction digestion analysis with BamHI/NcoI. Marker, DL2000

Fig. 2

Histochemical GUS staining in tissues of PD1-PD9 and CaMV35S transgenic Nicotiana benthamiana. The 2-, 7-, 14-, and 21-day-old seedlings, leaves of 90-day-old plants, and roots, stems and leaves of 60-day-old plants were incubated in staining solution at 37 °C for 3 h. The roots, stems, flowers, fruits and seeds of 90-day-old plants were stained at 37 °C for 24 h. 35S, transgenic tobacco of the CaMV35S promoter. Scale bar of 2-day-old seedlings: 0.1 cm; the other scale bar: 0.5 cm

Fig. 3

GUS staining and fluorescent quantitative analysis of PD1-PD9 Nicotiana benthamiana transgenic plants under normal and stress conditions. For NaCl and PEG treatments of whole plants, 60-day-old transgenic plants were immersed in a liquid 1/2 MS medium supplemented with either 200 mM NaCl or 18% (w/v) PEG 6000 at 25 °C for 24 h. For 4 °C treatment of whole plants, 60-day-old plants were immersed in a liquid 1/2 MS medium at 4 °C for 24 h. The plants grown in liquid 1/2 MS medium at 25 °C for 24 h were treated as a control. a GUS histochemical staining. The leaves of PD1–PD9 and CaMV35S promoter transgenic plants were stained for 3 h. Samples were then observed and photographed after decolorization. b GUS activity assays of leaves. Values represent the means ± SD from 15 independent transgenic plants (5 individual plants/line, 3 lines for each construct). Different lowercase letters above the bars indicate significant differences at P < 0.05

Fig. 4

GUS transient assays in the leaves of Nicotiana benthamiana plants. a The plasmids used in the transient assay. Mini35S represents the truncated CaMV35S promoter (− 46 ~ + 10 bp). The test constructs consisted of PD7~ 8-mini35S, PD8~ 9-mini35S and PD7~ 9-mini35S, in which the 162-bp (− 456 ~ − 295 bp), 111-bp (− 294 ~ − 184 bp) and 273-bp (− 456 ~ − 184 bp) fragments identified in the AtSCPL30 promoter were fused to the mini35S promoter to drive GUS expression. b The fused plasmids were confirmed by restriction digestion analysis with BamHI/PstI. M, Molecular marker DL2000; 1, PD7~ 9-mini35S; 2, PD7~ 8-mini35S; 3, PD8~ 9-mini35S. c Histochemical GUS staining resulting from non-transformed leaves (WT) and transiently transformed leaves with constructs mini35S, PD7~ 8-mini35S, PD8~ 9-mini35S and PD7~ 9-mini35S under normal conditions. d GUS activity in the transiently transformed tobacco leaves with constructs mini35S, PD7~ 8-mini35S, PD8~ 9-mini35S and PD7~ 9-mini35S under normal conditions. The results are the mean ± SD from three experiments (n = 23). Different lowercase letters above the bars indicate significant differences at P < 0.05

Fig. 5

GUS histochemical staining of transformed maize calli. Resistant calli were incubated in staining solution at 37 °C for 24 h. Finally, the samples were observed and photographed after decolorization

Fig. 6

Diagrams of PD7 promoter fragment and the 163- and 106-bp fragments for enhanced gene expression. Potential cis-acting elements associated with increased gene expression in the PD7 sequence predicted by PlantCARE and PLACE are shown on the border. CAAT box, common cis-acting element in promoter and enhancer regions; DOFCOREZM, core site required for binding of Dof proteins in maize. Dof1 and Dof2 transcription factors are associated with the expression of multiple genes involved in carbon metabolism in maize