Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.     

Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants

GUS (uidA) reporter gene expression for two sugarcane polyubiquitin promoters, ubi4 and ubi9, was compared to expression from the maize Ubi-1 promoter in stable transgenic rice (only ubi9) and sugarcane (ubi4 and ubi9). Ubi9 drove high-level GUS expression, comparable to the maize Ubi-1 promoter, in both callus and regenerated plants of rice transformed by Agrobacterium. This high level expression was inherited in R1 plants. Expression from ubi4 and ubi9 was quite high in sugarcane callus transformed via particle bombardment. Expression dropped to very low or undetectable levels in the resulting plants; this drop in expression resulted from PTGS. PTGS in regenerated sugarcane plants also occurred with the maize Ubi-1 promoter. In sugarcane callus, ubi4 was HS inducible, but ubi9 was not. This physiological difference corresponds to a MITE insertion that is present in the putative HSEs of ubi9 but not present in ubi4.     

Factors affecting transient gene expression in cultured radiata pine cotyledons following particle bombardment

Transfer and expression of the β–glucuronidase gene ( gusA ) in cultured cotyledons of radiata pine ( Pinus radiata D. Don ) were obtained by particle bombardment. Conditions for optimum transient expression were established by using plasmid pB[/12], delivered by gold particles, 1.6 μm in diameter, into 8-day-old cultured cotyledons. Helium pressure of 7.6 MPa, bombardment distance between the stopping screen and the target tissues of 6 cm, and 0.8 μg of plasmid DNA per bombardment proved to be the best parameters for transient expression; using these parameters 79% of bombarded cotyledons showed GUS activity, with 4.3 blue spots per cotyledon. This system was used for studying the expression of several gus-driven promoters the expression of the sunflower ubiquitin gene promoter was higher (99% of positive cotyledons, with 14.2 blue spots per cotyledon) than that of the CaMV 35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower gusA expression, as determined histochemically. These results were confirmed by using the gus fluorometric assay. Use of the sunflower ubiquitin gene promoter resulted in gusA expression up to 20 days after bombardment, with a significant level of gus expressing loci per bombarded cotyledon, whereas with the CaMV 35S promoter gusA expression was lost 12 days after bombardment.     

Optimised DNA delivery into Coffea arabica suspension culture cells by particle bombardment

An optimised bombardment protocol to introduce DNA into Coffea arabica suspension culture cells was developed. Osmotic preconditioning of cells and physical bombardment parameters including Helium pressure, gap and target distances affecting DNA delivery were evaluated by monitoring transient expression of the uidA gene driven by the CaMV35S promoter. The highest transient GUS expression was obtained when cells were subjected to a 0.5 M mannitol–sorbitol pre-treatment 4 h prior to bombardment and a Helium pressure of 1550 psi, a 9-mm gap distance and 12 cm target distance as physical bombardment parameters. The optimised protocol was tested with two coffee promoters: -tubulin and arabicin, which presented similar activity to the CaMV35S promoter in suspension culture cells by fluorometric GUS assays. GUS expression was reduced in bombarded tissue culture leaves, and only the CaMV35S and arabicin promoters showed histochemical activity in coffee endosperms. This is the first report of optimization of particle bombardment on coffee suspension culture cells, equivalent CaMV35S activity for a coffee promoter and transient -glucoronidase expression in coffee endo-sperms.     

Expression of a polyubiquitin promoter isolated from Gladiolus

A polyubiquitin promoter (GUBQ1) including its 5′UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5′ and 3′ intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5′UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5′UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.     

Evaluation of five promoters for use in transformation of oil palm (Elaeis guineensis Jacq.)

The efficiency of GUS (-Glucuronidase) gene expression in embryogenic callus and young leaflets of mature and seedling palm after microprojectile bombardment with five constructs (pEmuGN, pAHC25, pAct1-F4, pGH24 and pBARGUS) was evaluated to identify the most suitable promoter(s) to use in transformation attempts in oil palm. Expression of the GUS gene driven by theEmu, Ubi1, Act1 35S orAdh1 was assayed, both histochemically and fluorometrically, from a total of 200 plates of tissues in eight independent experiments two days after bombardment. A completely randomized experimental design was used for each experiment, and the data analysed by ANOVA and Duncan's Multiple Range Test. The expression level of GUS driven by theEmu orUbi1 promoters was significantly higher than that of the Act], 35S and Adhl promoters in many experiments, and that of theAdhl was significantly lower than those of the other four promoters. Both histochemical and fluorometric data indicate that in embryogenic callus, the expression of theEmu promoter was higher than that of theUbi1 whereas in young leaflets from mature palm the Ubi1 expression was stronger. The performances of the five promoters were also tested in tobacco callus using a fluorometric GUS assay. The activity of the 35S promoter was highest, and significantly different from that of all the other promoters except theEmu, and that of theAct1 promoter was lowest. These results indicate that either theUbil orEmu promoter should facilitate the expression of desired genes in oil palm and aid in development of an efficient stable transformation system.Abbreviations GUS -Glucuronidase - EC embryogenic callus - YLMP young leaflet from mature palm - YLSP young leaflet from seedling palm - MU methyl umbelliferone - MUG 4-methyl--D-glucuronide - X-glue 5-bromo-4-chloro-3-indoyl-glucuronide - Ubil maize ubiquitin 1 - Actl rice actin 1 - Adh1 maize alcohol dehydrogenase 1 - Emu a recombinant truncated maize alcohol dehydrogenase 1 - ANOVA Analysis of variance - DMRT Duncan's Multiple Range Test Communicated by W A. Parrott     

Transient expression of a reporter gene in bulbscales and immature embryos of three Lilium species is affected by 5'upstream sequences and culture conditions

In Lilium , a transformation system has not yet been developed. For efficient selection of cells expressing transferred genes following particle bombardment, the effects of 5'upstream regions on the transient expression of the β-glucuronidase gene ( gusA ) were estimated in bulbscales and immature embryos of lily. When four plasmids having the gusA gene under the control of the cauliflower mosaic virus (CaMV) 35S, maize alcohol dehydrogenase gene and rice actin gene ( Actl ) promoters, and the castor bean catalase introm were introduced by particle bombardment, the patterns of transient expression in the bulbscales showed differences among three Lilium species, L. x formolongi, L. dauricum and L. japonicum . In immature embryos of L x formolongi , transient expression was significantly influenced by age of embryos after self-pollination, duration of culture before bombardment, and culture conditions. Moreover, the transient gusA expression driven by six different 5'upstream regions, including the maize ubiquitin gene promoter and a modified CaMV 35S promoter were compared in both bulbscales and immature embryos. Use of the Actl and modified CaMV 35S promoters resulted in the greatest number of cells that transiently expressed gusA in both types of tissue of L. x formolongi . These two promoters are efficient for use in lily transformation.     

Regulation of the maize ubiquitin (Ubi-1) promoter in developing maize (Zea mays L.) seeds examined using transient gene expression in kernels grown in vitro

Kernel culture was assessed for evaluating novel gene expression in developing maize (Zea mays L.) seeds by comparing the transient expression of maize ubiquitin (Ubi-1) promoter-driven β-glucuronidase (GUS) delivered by particle bombardment in kernels grown in culture with those grown in planta. With kernels from either source, GUS expression, as determined by histochemical staining, was widespread in young, actively growing kernels, but it diminished with kernel age and by 25 days after pollination was found only in the embryo. Transient expression of Ubi-1 in kernels grown in vitro was not affected by wounding, ethylene treatment, pathogen invasion, or heat shock. In contrast, the plant hormones indole-3-acetic acid and kinetin both stimulated transient Ubi-1 expression in the endosperm, particularly at the periphery. Transient gene expression in developing maize seeds grown in vitro should allow for facile and rapid evaluation of the tissue-specificity and environmental responses of novel gene constructs in developing maize seeds. Received: 31 July 1997 / Revision received: 13 October 1997 / Accepted: 31 October 1997     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 μm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 μg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter (Δ1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intron-mediated enhancement of gene expression in maize (Zea mays L.) and bluegrass (Poa pratensis L.)

We report a strength comparison of a large variety of monocot and dicot intron-containing fragments inserted in the 5 untranslated leader, between the CaMV 35S promoter and the uidA gene (coding for the ß-glucuronidase: GUS). Relative strengths of the intron-containing fragments were evaluated by comparing transient GUS expression after particle bombardment in embryogenic maize and bluegrass suspension cultures. Our results confirm a dramatic dependence on the presence of an intron for chimeric gene expression in both species. On average, the maize first intron of ubi1 provided the highest enhancement of gene expression in maize and bluegrass (71- and 26-fold enhancement, respectively). Half of the introns tested affected gene expression differently in bluegrass and maize. This suggests that the intron-mediated enhancement of gene expression generally obtained with maize may not be fully applicable to all monocots. We also report enhancement of gene expression (92-fold) in a monocot species by a dicot intron (chsA intron).     

Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris)

Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species.     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

Comparisons of the efficiency of some promoters in silver birch (Betula pendula)

The efficiency of several promoters (pin2 from potato, ubiquitin from sunflower, rolC from Agrobacterium rhizogenes, act1 from rice and CaMV 35S from cauliflower mosaic virus) fused to the uidA reporter gene was measured after biolistic bombardment of birch leaves (Betula pendula L.). The highest level of β-glucuronidase (GUS) activity was achieved with the pin2 promoter and the lowest activity with the CaMV 35S promoter. The activity of the potato wound-inducible promoter (pin2) was also tested in stably transformed birch. The promoter showed induced activity after mechanical wounding and feeding by leaf weevils. The systemic effect was confirmed by enhanced GUS activity in non-wounded leaves. The results of this study indicated that the potato wound-inducible promoter maintains its function in birch and would be a suitable promoter in studies of insect-birch interaction at the molecular level. Received: 17 October 1996 / Revision received: 7 February 1997 / Accepted: 1 March 1997     

Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

 Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B₁- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B₁- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T₀ leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T₁ progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T₁ progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T₅ progeny in one line, T₄ progeny in one line, T₃ progeny in three lines and T₂ or T₁ progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B₁- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T₁ progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T₂ or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T₂ or later generations. We conclude that the B₁- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering. Received: 28 May 1998 / Accepted: 19 December 1998     

Transient gene expression and influence of promoters on foreign gene expression in Arabidopsis thaliana

Summary The influence of a variety of parameters was investigated on polyethylene glycol (PEG)-mediated transient nptII and gus gene expression in mesophyll protoplasts of Arabidopsis thaliana ecotype, Estland, in order to develop a suitable transient gene expression system. The investigation revealed that a combination of 20% PEG, incubation time of 15 min, 20–30 μg plasmid concentration per ml along with 50 μg carrier DNA m/l, and inclusion of calcium and magnesium ions during transfection followed by a culture period of 24 h registered maximum NPTII activity. Of the various promoters used for driving expression of the gus gene, the ubiquitin promoter from A. thaliana was the most efficient followed by 35S promoter of the CaMV and the actin promoter of rice. For comparison, similar studies in protoplasts of rice, wheat, and Brassica also revealed the differences in strength of these promoters. Arabidopsis ubiquitin promoter was the most effective in Brassica, and the rice actin1 promoter was the most effective in rice and wheat.     

A synthetic xylanase as a novel reporter in plants

Transient gene expression assays are often used to screen promoters before stable transformation. Current transient quantification methods have several problems, including a lack of reporter gene stability and expense. Here we report a synthetic, codon-optimised xylanase gene (sXynA) as a reporter gene for quantitative transient analyses in plants. Azurine-crosslinked xylan (AZCL-xylan) was used as a substrate for assaying xylanase activity. The enzymatic nature of the protein allows for sensitive assays at the low levels of transgene protein found in transiently transformed tissue extracts. The xylanase (XYN) protein is stable, activity slopes are linear over long time periods and assays are cost-effective. Coupled with the GUSPlus reporter gene, the XYN reporter allows sensitive and accurate quantification of gene control sequences in transient expression systems.Abbreviations Act1 Rice actin promoter - AZCL-xylan Azurine cross-linked xylan - AU absorbance units - Blt4.9 Barley lipid transfer protein promoter - GEB GUS extraction buffer - GFP Green fluorescent protein - GluB-1 Rice glutelin B-1 promoter - GUS -Glucuronidase - LUC Luciferase - sXynA Synthetic xylanase A gene - Ubi-1 Maize ubiquitin promoter - XAB Xylanase assay buffer - XYN Xylanase Communicated by P. Lakshmanan