Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system

Abstract

A novel automated image collection and analysis system was used to compare two new soybean (Glycine max (L.) Merr.) promoters with the cauliflower mosaic virus 35S (CaMV35S) promoter, which was used as an expression standard. For expression comparisons, various permutations of a soybean polyubiquitin (Gmubi) promoter, a soybean heat shock protein 90-like (GmHSP90L) promoter and the CaMV35S promoter were placed upstream of a green fluorescent protein (gfp) gene. DNA constructs were introduced via particle bombardment into excised cotyledons of germinating lima bean (Phaseolus lunatus L.) seeds, which were arranged in Petri dishes for automated image capture and image analysis. The automated system allowed monitoring and quantification of gfp gene expression in the same piece of tissue over time. The Gmubi promoter, with its intronic region intact, showed the highest expression that was over five times stronger than the CaMV35S promoter. When an intronic region was removed from the Gmubi promoter, GFP expression was reduced, but was still over two times greater than with the CaMV35S promoter. The full-length soybean GmHSP90L promoter was four times stronger than the CaMV35S promoter. Truncation of the GmHSP90L promoter resulted in stepwise decreases in promoter strength, which appear to correspond to removal of regulatory elements. Automated image capture and analysis allowed the rapid and efficient evaluation of these new promoters.

Promoter diversity in multigene transformation

“…This has been used to increase the expression of the bacterial genes crtB and crtI resulting in greater accumulation of b-carotene (Aluru et al 2008). Many plant promoters can also be enhanced by including an intron (e.g., Vain et al 1996;Mitsuhara et al 1996;Fiume et al 2004;Chiera et al 2007).…”

The Arabidopsis Tandem Zinc Finger Protein AtTZF1 Traffics between the Nucleus and Cytoplasmic Foci and Binds Both DNA and RNA

“…The recipes of three solutions were modified as follows: WI solution (0.5 M mannitol, 20 mM MES, and 20 mM KCl, pH 5.7), W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KCl, and 5 mM MES, pH 5.7), MMg solution (0.5 M mannitol, 20 mM MgCl 2 , and 5 mM MES, pH 5.7). Transient expression in lima bean (Phaseolus lunatus) cotyledons using particle bombardment was conducted as described (Chiera et al, 2007). For stable transformation, floral dip or vacuum infiltration was used as described (Bechtold and Pelletier, 1998;Clough and Bent, 1998).…”

Genetically Modified Soybean for Insect-Pests and Disease Control

“…Some nematode-induced promoters could represent excellent vectors for the administration of lethal doses to nematode feeding sites in infected roots. Hence, a soybean heat shock protein 90-like promoter (GmHSP90L) and a soybean polyubiquitin promoter (Gmubi) have shown higher expression levels of the gfp gene than the cauliflower mosaic virus 35S promoter (CaMV35S), when transformed in cotyledons of germinating lima bean (Phaseolus lunatus) seeds (CHIERA, 2007). An intronic region (Gumpri) from the Gmubi promoter seems to be essential for increasing gene expression levels.…”

“…Also, The expression of the same promoter in tobacco was not only reduced when the intron was not present, but the tissue-specific expression was altered (PLESSE et al, 2001). Moreover, the GmHSP90L promoter showed stronger expression levels than CaMV35S, but the expression level decreased after promoter truncation, revealing that this region might be related to GmHSP90L promoter regulatory elements (CHIERA, 2007 ITOH et al, 2003), many developmental processes and cell cycle control (MOON et al, 2004). Thus, the E2 enzyme, a member of the Leubc4 family, has a major role in cellular metabolism and giant cell formation.…”