Transgene expression driven by heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase small-subunit gene promoters in the vegetative tissues of apple (Malus pumila Mill.)

Abstract

It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small-subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max [h.] Mers.), to drive expression of the beta-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approximately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple.

Genomic approaches to the discovery of promoters for sustained expression in cotton (Gossypium hirsutum L.) under field conditions: expression analysis in transgenic cotton and Arabidopsis of a Rubisco small subunit promoter identified using EST sequence analysis and cDNA microarrays

“…Plants transformed with a constitutive 35SGUS construct stained strongly in all tissues examined except for the filament and anther wall that were only weakly stained (not shown), while non-transformed control plants showed no staining in any tissue examined (e.g., Figure 3B and 3C). These results are consistent with the data from Arabidopsis (Gittins, et al 2000;Kirby and Kavanagh 2002) and previous studies in cotton (Song et al 2000) that show that expression of the RbcS promoter is restricted primarily to photosynthetic tissues and especially in leaves. Northern blot analysis of GUS transcripts in different tissues ( Figure 4A) confirmed this result.…”

Isolation and characterization of strong gene regulatory sequences from apple, Malus × domestica

“…The RbcS family is a photosynthesis-associated nuclear gene which is known to be highly expressed in green photosynthetic tissues and was therefore a suitable candidate to isolate regulatory sequences from. Gittins et al (2000) tested RbcS promoters of tomato and soybean in apple and concluded that they would be suitable for expression in green photosynthetic tissues. They found that RbcS promoters of the different origins were able to drive gus expression to half that of plants containing the CaMV35S promoter.…”

“…The promoter of the gene coding for the small subunit of the ribulose biphosphate carboxylase (RBC) has been studied extensively (Khoudi et al 1997;Nomura et al 2000;Gittins et al 2000;Outchkourov et al 2003) in both homologous and heterologous crops and was found to be able to provide strong transcriptional activity. RBC is one of the most abundant proteins in the plant kingdom and can constitute 40-60% of total leaf protein.…”

“…Expression levels of heterologous genes driven by RbcS promoter sequences in different crops range between half that obtained by the CaMV35S promoter to seven to eightfold higher than CaMV35S (Outchkourov et al 2003;Khoudi et al 1997). In apple, Gittins et al (2000) tested heterologous RbcS promoters of tomato and soybean and concluded that they would be suitable for expression in green photosynthetic tissues. Also, the RbcS promoter of chrysanthemum was tested in our laboratory in apple, and strong transcriptional activity could be demonstrated (F.A.…”

Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

“…Gittins et al (2000) studied the ability of the Rubisco promoter in apple and got 12 transgenic lines with GUS activities ranging from 1,000-35,000 pmol MU min -1 mg -1 protein. Chitinase activity of 17 transgenic alfalfa plants containing the Rubisco promoter-ech42 construct varied from 0 to 2,000 pmol MU min -1 mg -1 protein (Samac et al, 2004).…”