Abstract
BackgroundTransgenic 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.ResultsIn 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.ConclusionsAs 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.
Highlights
Transgenic technology has become an important technique for crop genetic improvement
Some cisacting elements that enable the tissue-specific, inducible, and enhanced or suppressed expression of AtSCPL30 were identified, including five types of light-responsive elements (TCT-motif, GAG-motif, GATABOX, G-box and GT1CONSENSUS), six kinds of phytohormone-responsive elements (ABRE, NTBBF1ARROLB, WRKY71OS, TCAelement, SARE and CATATGGMSAUR), two kinds of elicitor-responsive elements (Box-W1 and EIRE), two kinds of elements required for tissue- or organ-specific expression (OSE1ROOTNODULE and TATCCAOSAMY), several elements involved in defense or stress (HSE, PREATPRODH, LTR, GT1GMSCAM4 and TC-rich repeats), five new signal element (POLASIG1), A negative cis-element conserved in plastid-related genes (S1FBOXSORPS1L21), five elements required for etiolation-induced expression of erd1 (ACGT ATERD1) and 14 potential core sites required for the binding of Dof transcription factors that regulate the intensity of gene expression (DOFCOREZM)
Expression patterns and activities of the AtSCPL30 promoter and its 5′ deletion segments in Nicotiana benthamiana transgenic plants under normal conditions To profile the expression of Nicotiana benthamiana transgenic plants driven by PD1-PD9 and the cauliflower mosaic virus 35S (CaMV35S) promoter under normal conditions, the radicle of seeds germinated for 2 days; 7, 14- and 21-day-old seedlings; the roots, stems, and leaves of 60- and 90-day-old plants; and the flowers, fruits and seeds from 90-day-old plants were subjected to GUS histochemical staining (Fig. 2)
Summary
Transgenic technology has become an important technique for crop genetic improvement. Strong constitutive promoters facilitate the high expression of Bacillus thuringiensis gene, leading to an enhanced insect resistance [12], and the improtance of which has been verified by several studies in monocot or dicot plants, such as the cauliflower mosaic virus 35S (CaMV35S) promoter, the rice Actin and cytochrome c gene promoter, and the maize ubiquitin promoter [13,14,15,16,17,18]. Among these promoters, the CaMV35S promoter and maize ubiquitin promoter are used most frequently to drive transgene expression in plants [19, 20]. A shortage of available plant-derived promoters for high-level stable expression of foreign genes limits the development of genetically modified crops through transgenic technology
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