Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation

Abstract

BackgroundThe ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues.ResultsPvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues.ConclusionsThe PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.

Cis-regulatory elements used to control gene expression in plants

“…The described promoter is ten-fold more active in monocots than the CaMV 35S promoter (Christensen et al 1992). Polyubiquitin promoters PvUbi1 and PvUbi2, isolated from switchgrass, analyzed by Mann et al (2011), demonstrated particularly high constitutive expression. They are more efficient than ZmUbi1, CaMV 35S and OsAct1 (actin promoter derived from rice).…”

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

“…Each gene was targeted for deletion using two gRNAs. To provide expression in cereals, we created vectors with ZmUbi, switchgrass (Panicum virgatum) ubiquitin 1 (PvUbi1), and switchgrass ubiquitin 2 (PvUbi2) promoters (Mann et al, 2011). The sequences of PvUbi1 and PvUbi2 were modified to remove AarI, SapI, and BsaI sites, and the modified promoters were confirmed to drive high levels of GFP expression in wheat scutella (Supplemental Figure 9).…”

In vitro transformation of pearl millet (Pennisetum glaucum (L). R. BR.): Selection of chlorsulfuron-resistant plants and long term expression of the gus gene under the control of the emu promoter

“…By using immature and mature embryos, shoot tips and embryogenic calli for in vitro transformation of S. bicolor, Tadesse et al (2003) observed that the strength of the CaMV 35S promoter ranked after those of the ubi1, act1D, adh1 promoters. Two switchgrass (Panicum virgatum L.) ubiquitin genes promoters PvUbi1 and PvUbi2, showed strong expression of the GUS gene in switchgrass and rice calli, equaling or surpassing the expression levels of the CaMV 35S promoter (Mann et al, 2011). Furthermore, in these studies, GUS expression levels driven by the different promoters vary according to the plant species and the type of explants used for in vitro transformations.…”