High level transgenic expression of soybean (Glycine max) GmERF and Gmubi gene promoters isolated by a novel promoter analysis pipeline

Abstract

BackgroundAlthough numerous factors can influence gene expression, promoters are perhaps the most important component of the regulatory control process. Promoter regions are often defined as a region upstream of the transcriptional start. They contain regulatory elements that interact with regulatory proteins to modulate gene expression. Most genes possess their own unique promoter and large numbers of promoters are therefore available for study. Unfortunately, relatively few promoters have been isolated and characterized; particularly from soybean (Glycine max).ResultsIn this research, a bioinformatics approach was first performed to identify members of the Gmubi (G.max ubiquitin) and the GmERF (G. max Ethylene Response Factor) gene families of soybean. Ten Gmubi and ten GmERF promoters from selected genes were cloned upstream of the gfp gene and successfully characterized using rapid validation tools developed for both transient and stable expression. Quantification of promoter strength using transient expression in lima bean (Phaseolus lunatus) cotyledonary tissue and stable expression in soybean hairy roots showed that the intensity of gfp gene expression was mostly conserved across the two expression systems. Seven of the ten Gmubi promoters yielded from 2- to 7-fold higher expression than a standard CaMV35S promoter while four of the ten GmERF promoters showed from 1.5- to 2.2-times higher GFP levels compared to the CaMV35S promoter. Quantification of GFP expression in stably-transformed hairy roots of soybean was variable among roots derived from different transformation events but consistent among secondary roots, derived from the same primary transformation events. Molecular analysis of hairy root events revealed a direct relationship between copy number and expression intensity; higher copy number events displayed higher GFP expression.ConclusionIn this study, we present expression intensity data on 20 novel soybean promoters from two different gene families, ubiquitin and ERF. We also demonstrate the utility of lima bean cotyledons and soybean hairy roots for rapid promoter analyses and provide novel insights towards the utilization of these expression systems. The soybean promoters characterized here will be useful for production of transgenic soybean plants for both basic research and commercial plant improvement.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation

“…The transformations were conducted using a modified version of the binary vector backbone pNB96 (pSC218; Curtin et al, 2011) with an introduced inverted-repeat cassette to facilitate cloning of hairpin RNA fragments (pSC218GH) and a reading-frame cassette to incorporate both the Cas9 and target guide RNA entry vectors (pSC218GG). The inverted-repeat and reading-frame cassettes were derived from previously reported pTDT-DC-RNAi and pTDTO vectors (Valdés-López et al, 2008) and were driven by the constitutively expressed Glycine max Ubiquitin promoter (Hernandez-Garcia et al, 2010).…”

Truncated Ubiquitin 5′ Regulatory Region from Erianthus arundinaceus Drives Enhanced Transgene Expression in Heterologous Systems

“…Many polyubiquitin genes and some other highly expressed genes contain leader introns in the 5 0 untranslated region. These leader introns are frequently utilized to enhance transgene expression [38].…”

A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters

“…After 4 d, cotyledons from germinating seedlings were excised and used for particle bombardment as described previously [39–40]. In brief, cotyledons were placed, adaxial surface up, on a growth regulator free plant tissue culture medium to acclimate for 1–4 h. Cotyledons were then removed, placed on a stainless steel mesh supporting screen, and bombarded with tungsten particles coated with different DNA constructions using the Particle Inflow Gun [41].…”