Analysis of rice Act1 5' region activity in transgenic rice plants.

The 5' region of the rice actin 1 gene (Act1) has been developed as an efficient regulator of foreign gene expression in transgenic rice plants. To determine the pattern and level of rice Act1 5' region activity, transgenic rice plants containing the Act1 5' region fused to a bacterial beta-glucuronidase (Gus) coding sequence were generated. Two independent clonal lines of transgenic rice plants were analyzed in detail. Quantitative analysis showed that tissue from these transgenic rice plants have a level of GUS protein that represents as much as 3% of total soluble protein. We were able to demonstrate that Act1-Gus gene expression is constitutive throughout the sporophytic and gametophytic tissues of these transgenic rice plants. Plants from one transgenic line were analyzed for the segregation of GUS activity in pollen by in situ histochemical staining, and the inheritance and stability of Act1-Gus expression were assayed in subsequently derived progeny plants.     

Electroporation and PEG delivery of DNA into maize microspores

Summary The ability to deliver and detect reporter gene activity in maize microspores was tested. Tested expression vectors contained the chloramphenicol acetyl transferase (CAT) gene and one of the following promoter-intron combinations: 1) cauliflower mosaic virus (CaMV 35S), 2) CaMV 35S + maize alcohol dehydrogenase 1 intron 6 (Adh1-I6), 3) maize alcohol dehydrogenase 1 + intron 1 (Adh1-I1), or 4) maize ubiquitin 1 + intron 1 (Ubiq 1-I1) promoter + intron. The expression vectors were delivered into maize microspores using electroporation or polyethylene glycol (PEG). Both methods were effective for delivering free DNA into microspores. Although all four promoters were active in maize protoplasts, only two promoters were active in maize microspores. The CaMV 35S and the Adh1 promoters did not promote gene expression in maize microspore. The CaMV 35S + Adh1-I6 and Ubiq1-I1 promoters produced high levels of CAT activity in maize microspores.     

The effect of different promoter-sequences on transient expression of gus reporter gene in cultured barley (Hordeum vulgare L.) cells

The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli -glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.NRCC No. 36482     

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).     

Intragenic control of expression of a rice MADS box gene OsMADS1

OsMADS1 is a rice MADS box gene necessary for floral development. To identify the key cis-regulatory regions for its expression, we utilized transgenic rice plants expressing GUS fusion constructs. Histochemical analysis revealed that the 5.7-kb OsMADS1 intragenic sequences, encompassing exon 1, intron 1, and a part of exon 2, together with the 1.9-kb 5' upstream promoter region, are required for the GUS expression pattern that coincides with flower-preferential expression of OsMADS1. In contrast, the 5' upstream promoter sequence lacking this intragenic region caused ectopic expression of the reporter gene in both vegetative and reproductive tissues. Notably, incorporation of the intragenic region into the CaMV35S promoter directed the GUS expression pattern similar to that of the endogenous spatial expression of OsMADS1 in flowers. In addition, our transient gene expression assay revealed that the large first intron following the CaMV35S minimal promoter enhances flower-preferential expression of GUS. These results suggest that the OsMADS1 intragenic sequence, largely intron 1, contains a key regulatory region(s) essential for expression.     

Expression Enhancement of a Rice Polyubiquitin Gene Promoter

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes.     

The Commelina yellow mottle virus promoter is a strong promoter in vascular and reproductive tissues.

Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects the monocot Commelina diffusa. Although CoYMV and cauliflower mosaic virus (CaMV; another double-stranded DNA virus) probably replicate by a similar mechanism, the particle morphology and host range of CoYMV place it in a distinct group. We present evidence that a prompter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different from that conferred by the CaMV 35S promoter. When the CoYMV promoter is used to drive expression of the beta-glucuronidase reporter gene in stably transformed tobacco plants, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers. Activity is also detected throughout the anther, with highest activity in the tapetum. In contrast, the CaMV 35S promoter is active in most cell types. The CoYMV promoter is a strong promoter, and when the activity of the CoYMV promoter is compared with that of a duplicated CaMV 35S promoter, it is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells. These properties of the CoYMV promoter make it potentially useful for high-level expression of engineered genes in vascular cells.     

Use of the lacZ reporter gene as an internal control for GUS activity in microprojectile bombarded plant tissue

A protocol is described for simultaneous histochemical detection of LacZ and Gus activity in plant tissues after microprojectile bombardment. The suitability of two different Gus substrates (Salmon-glcA, Magenta-β- -glcA) is compared. This detection system is used to assay the number of cells expressing either or both reporter gene. This technique is used as a qualitative assay to demonstrate the tissue specificity of a Hrgp promoter in maize embryos, and to measure ABA responsiveness of a Lea promoter in Arabidopsis. The promoter to be studied is linked to the gus reporter gene and the lacZ reporter gene linked to the CaMV 35S promoter is used as a constitutive internal control. The use of an internal control drastically reduces the data variation inherent to microprojectile bombardment.     

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the -glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.     

Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derivedEmu andCaMV 35S in cells of barley,maize and tobacco

The particle gun approach was used for the quantification of promoter efficiency in a test system for transient gene expression. β-Glucuronidase was used as reporter gene for determining promotote strength. The variability inherent in this gene transfer system was considerably reduced by calculating a transformation efficiency factor given by the expression of a cotransferred second reporter gene (firefly luciferase). The calibration of β-glucuronidase activity by the transformation efficiency factor caused a lower statistical variance of the values and allowed reliable results to be obtained with a smaller set of repetitions. The CaMV 35S promoter (as a control) and the monocot-specific promoters for maize polyubiquitin1, rice actin 1 and the maize-derivedEmu were characterized and compared with respect to expression strength, as tested under identical conditions in suspension cell cultures of maize, barley and tobacco. Compared to the 35S promoter, the monocot-specific promoters show up to 15-fold higher expression in maize and barley but give only weak expression in tobacco. No expression was found for the rice actin 1 promoter in tobacco. The level of reporter gene expression is influenced by the osmotic potential in the agar medium. For theEmu promoter, the calibrated β-glucuronidase activities remained mearly constant at low sucrose concentrations. Above 8% sucrose, the calibrated activities increased steadily with increasing osmotic conditions, reaching a three-to four-fold higher level at the highest sucrose concentration (32%) as compared to the standard concentration (4% sucrose) in the medium.     

Systemic induction of a potato pin2 promoter by wounding,methyl iasmonate,and abscisic acid in transgenic rice plants

To address the question whether common signal(s) and transduction pathways are used to mediate a systemic wound response in monocot and dicot plants, a fusion of the potato proteinase inhibitor II gene (pin2) promoter and the bacterial -glucuronidase gene (Gus)-coding region was introduced into rice. In transgenic rice plants, the expression of the pin2-Gus fusion gene displays a systemic wound response, although the expression level is relatively low. Incorporation of the first intron from the rice actin 1 gene (Act1) into the 5-untranslated region of the pin2-Gus construct results in high-level, systemically wound-inducible expression of the modified construct in transgenic rice plants. Histochemical analysis shows that this high-level, wound-inducible expression is associated with the vascular tissue in both leaves and roots. Furthermore, the expression of the pin2-Act1 intron-Gus fusion gene in transgenic rice plants can be systemically induced by both methyl jasmonate (MJ) and the phytohormone abscisic acid (ABA). These results suggest that the signal(s) mediating the observed systemic wound response and certain steps of the transduction pathways are conserved between dicot and monocot plants. Transient expression assays show that the pin2-Act1 intron-Gus construct is also actively expressed in transformed cells and tissues of several other monocot plants. Thus, the wound-inducible pin2 promoter in combination with the rice Act1 intron 1 might be used as an efficient regulator for foreign gene expression in transgenic monocot plants.