High GUS Activity Research Articles

Promoter analysis of the WRKY transcription factors CaWRKY1a and CaWRKY1b homoeologous genes in coffee (Coffea arabica)

The regulation of the CaWRKY1 homoeologous genes were analyzed through the characterization of their promoters. The pW1a promoter is proposed as a new tool for coffee plant biotechnologies. WRKY transcription factors are important elements of the plant immune response. The CaWRKY1 gene from Coffea arabica is induced by several biotic and abiotic stresses, including challenge by the rust fungus Hemileia vastatrix. Two homoeologous CaWRKY1 genes, named CaWRKY1a and CaWRKY1b, were previously identified in the C. arabica allotetraploid genome. To gain insight into the transcriptional regulation of these genes, their promoter sequences, named pW1a and pW1b, respectively, were cloned and characterized in this study. In silico analysis revealed some important defense-associated regulatory elements, including W-boxes and as-1 elements. Promoter activities were analyzed in transient assays conducted by agroinfiltration of tobacco leaves. Exogenous salicylic acid (SA) treatments increased promoter activities corroborating the presence of as-1 regulatory elements. Transactivation assays with the CaWRKY1 protein showed the reduction of both pW1a and pW1b promoter activities, indicating that the CaWRKY1 protein may negatively regulate its own promoters. Stable transgenic C. arabica lines expressing a pW1a::GUS construct were obtained by Agrobacterium-mediated transformation and high GUS activity was observed in leaves subjected to mechanical wounding. Hence, the ability of pW1a to drive transgene expression in coffee plants as well as to enhance expression in response to stresses opens possibilities for using this promoter as a new tool for biotechnological approaches in coffee plants.

Reproductive Organ and Vascular Specific Promoter of the Rice Plasma Membrane Ca2+ATPase Mediates Environmental Stress Responses in Plants

BackgroundPlasma membrane Ca2+ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca2+) from the cell, hence regulating Ca2+ level within cells. Though plant Ca2+ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.ResultsThe 1478 bp promoter sequence of rice plasma membrane Ca2+ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca2+ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The −1478 to −886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for −1210 and −886 bp flanking region. The −1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The −1210 and −886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the −886 bp and −519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.ConclusionsThe rice plasma membrane Ca2+ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.

Expression and Promoter Analysis of Six Heat Stress‐Inducible Genes in Rice

During the long evolutionary process, plant gradually formed a series of strategies and mechanisms to cope with stress environment such as drought, heat, cold, and high salinity. Six highly heat responsive genes were identified in rice by microarray data analysis. The qRT-PCR analysis confirmed that the expression of these six genes were highly heat inducible and moderately responded to salt stress, polyethylene glycol, and abscisic acid treatment, but little affected by cold treatment. Promoters of the three highly heat-inducible genes (OsHsfB2cp, PM19p, and Hsp90p) were used to drive GUS gene expression in rice. The results of the GUS gene expression, histochemical staining, and GUS activities in panicles and flag leaves of the transgenic rice plants confirmed high heat-induced GUS activities and moderate drought-induced activities. The three promoters exhibited similar high activity lever in rice leaf under heat, but OsHsfB2cp and PM19p showed much higher activities in panicles under heat stress. Our work confirmed that the OsHsfB2c and PM19 promoters were highly heat inducible and further characterization and reconstruction of cis-elements in their promoters could lead to the development of highly effective heat-inducible promoters for plant genetic engineering.

MiRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea maysL.)

BackgroundMicroRNAs are a class of small, non-coding RNAs that regulate gene expression by binding target mRNA, which leads to cleavage or translational inhibition. The NAC proteins, which include NAM, ATAF, and CUC, are a plant-specific transcription factor family with diverse roles in development and stress regulation. It has been reported that miR164 negatively regulates NAC1 expression, which in turn affects lateral root development in Arabidopsis; however, little is known about the involvement of the maize NAC family and miR164 in lateral root development.ResultsWe collected 175 maize transcripts with NAC domains. Of these, 7 ZmNACs were putative targets for regulation by miR164. We isolated one gene, called TC258020 (designated ZmNAC1) from 2 maize inbred lines, 87-1 and Zong3. ZmNAC1 had a high expression level in roots and showed higher abundance (1.8 fold) in Zong3 relative to 87-1, which had less lateral roots than Zong3. There was a significant correlation between the expression level of ZmNAC1 and the lateral root density in the recombinant inbred line (RIL) population. Transgenic Arabidopsis that overexpressed ZmNAC1 had increased lateral roots in comparison to the wild type. These findings suggest that ZmNAC1 played a significant role in lateral root development. An allelic expression assay showed that trans-regulatory elements were the dominant mediators of ZmNAC1 differential expression in 87-1 and Zong3, and further analysis revealed that miR164 was a trans-element that guided the cleavage of endogenous ZmNAC1 mRNA. Both mature miR164 and miR164 precursors had higher expression in 87-1 than Zong3, which was the opposite of the expression pattern of ZmNAC1. Additionally, the allelic assay showed that the cis-regulatory element most likely affected Zm-miR164b's expression pattern. A β-glucuronidase (GUS) assay showed that the Zm-miR164b promoter had higher GUS activity in 87-1 than in Zong3. In addition, we detected miR164b expression in the RIL population, and the results indicated that miR164b had a higher expression level in the RILs containing 87-1 promoter than those containing Zong3 promoter.ConclusionOur results indicate one possible pathway in maize by which differences in miR164b promoter activity resulted in a different expression pattern for mature miR164 which negatively regulates ZmNAC1 expression in 87-1 and Zong3, thereby contributing to a significantly different lateral root phenotype.

Vascular-specific expression of GUS and GFP reporter genes in transgenic grapevine (Vitis vinifera L. cv. Albariño) conferred by the EgCCR promoter of Eucalyptus gunnii

In the view of the economic importance of grapevine and the increasing threaten represented by vascular diseases, transgenic grapevine with enhanced tolerance could represent an attractive opportunity. Hitherto, constitutive promoters have been used generally to study the effects of transgene expression in grapevine. Given the fact that constitutive gene expression may be harmful to the host plant, affecting plant growth and development, the use of tissue -specific promoters restricting gene expression to tissues of interest and at given developmental stages could be more appropriate. For this purpose, we decided to study in grapevine the activity of the Eucalyptus gunnii CCR promoter that was previously reported to be vascular-preferential. We transformed grapevine with the "Sonication assisted Agrobacterium-mediated transformation" (SAAT) method and a construct where both GUS and GFP (green fluorescent protein) marker genes were under control of the EgCCR promoter. High GUS and GFP activities were found to be associated with the newly formed vascular tissues in stems, leaves and petioles of transformed grapevine, suggesting a preferential activity of the EgCCR promoter in the vascular tissues of grapevine. These results suggest the tissue-specificity of this promoter from eucalyptus is conserved in grapevine and that it could be used to drive expression of defense genes in order to enhance resistance against vascular pathogens.

The promoter from tomato sucrose synthase gene TOMSSF drives stamen-specific gene expression in Chrysanthemum

Anther-specific promoters have been extensively studied in terms of the genetic engineering of male-sterile plants. Here, we fused the 5′ upstream promoter regions of a sucrose synthase gene from tomato (TOMSSF) to the β-glucuronidase (GUS) gene and used the construct to transform Chrysanthemum plants. Histochemical analysis of two transformants showed high GUS activity in ray florets and tubular florets. Analysis of the staining pattern in these florets reveled that staining was stamen-specific. GUS gene expression was highest in the stamen and remained at a steady-state level throughout the experiment. These results suggest that the TOMSSF promoter has high activity in the stamen of tubular florets and would be useful as a high stamen-specific promoter in Chrysanthemum.

Characterization of a Novel Rice Metallothionein Gene Promoter: Its Tissue Specificity and Heavy Metal Responsiveness

The rice (Oryza sativa L.) metallothionein gene OsMT-I-4b has previously been identified as a type I MT gene. To elucidate the regulatory mechanism involved in its tissue specificity and abiotic induction, we isolated a 1 730 bp fragment of the OsMT-I-4b promoter region. Histochemical β-glucuronidase (GUS) staining indicated a precise spacial and temporal expression pattern in transgenic Arabidopsis. Higher GUS activity was detected in the roots and the buds of flower stigmas, and relatively lower GUS staining in the shoots was restricted to the trichomes and hydathodes of leaves. No activity was observed in the stems and seeds. Additionally, in the root of transgenic plants, the promoter activity was highly upregulated by various environmental signals, such as abscisic acid, drought, dark, and heavy metals including Cu²(+) , Zn²(+) , Pb²(+) and Al³(+) . Slight induction was observed in transgenic seedlings under salinity stress, or when treated with Co²(+) and Cd²(+) . Promoter analysis of 5'-deletions revealed that the region -583/-1 was sufficient to drive strong GUS expression in the roots but not in the shoots. Furthermore, deletion analysis indicated important promoter regions containing different metal-responsive cis-elements that were responsible for responding to different heavy metals. Collectively, these findings provided important insight into the transcriptional regulation mechanisms of the OsMT-I-4b promoter, and the results also gave us some implications for the potential application of this promoter in plant genetic engineering.

Characterization of an expansin gene and its ripening-specific promoter fragments from sour cherry (Prunus cerasus L.) cultivars

The presence of expansins was investigated in various developmental and ripening stages of cherry fruits by SDS-PAGE and immunoblotting. An expansin gene and three fragments (242, 607 and 929 bp) of its promoter region were cloned. The genomic clone of the expansin gene contained three introns, two exons spanning a 1.6 and a 1.0 kb upstream region. Semi-quantitative PCR analysis showed that this gene was ripening specific. Chimeric promoter—GUS constructs were made and truncated forms of the expansin promoter were introduced into tomatoes by agroinjection and fruits were analyzed for GUS expression by histochemical GUS staining and enzyme activity assays. The 0.60 kb expansin promoter efficiently induced GUS expression in transgenic tomatoes, whereas constructs with the 0.25 kb promoter did not display significant GUS staining. The highest GUS activity was detected in tomatoes containing the 1.0 kb promoter construct. Both large base pair promoter constructs drove the expression of the GUS gene at an equal or higher rate than the tomato E8 promoter.

Characterization of Fruit-Type Sucrose Synthase Gene Promoters Isolated from Tomato and Japanese Pear

We isolated the 5' upstream promoter regions of the fruit-type sucrose synthase (SS) gene from tomato and Japanese pear by inverse PCR. The 5' region of the tomato SS gene (TOMSSF) contained an intron approximately 1.6 kbp long in the 5'untranslated region, whereas the equivalent 5' region in the Japanese pear SS gene (PypSUS1) had no intron. Each region was fused to the β-glucuronidase (GUS) gene and then each construct (TOMSSF 5'::GUS or PypSUS1 5'::GUS) was used to transform tomato. Histochemical analysis of GUS activity of all transformants showed high GUS activity in the fruit. Analysis of the staining pattern in the fruits of all transformants showed staining specific to vascular tissues and testae. The highest level of GUS activity and GUS mRNA was found in fruits at earlier stages of development in all transformants. However, the increase in the level was not observed in the ripening fruits of PypSU1 5'::GUS lines, indicating that the expression patterns of PypSUS1 5'::GUS tomato were different from those of PypSUS1 in Japanese pear. It may ascribe to the differences in genetic background between tomato and Japanese pear that relate to the mechanisms contributing to the sucrose synthesis during ripening.

Cloning and molecular characterisation of the maize retinoblastoma gene ( ZmRBR2)

Retinoblastoma-related (RBR) proteins have been described in plant cells, and have been shown to be important in controlling plant growth, gametophyte and leaf development, and maintenance of stem cell identity. Whereas all dicotyledonous plants studied so far contain a single RBR gene, monocotyledonous species contain at least two genes and maize is the only known plant species with three RBR genes in its genome suggesting a recent gene duplication event. We report the genomic structure of the ZmRBR2 gene and its expression pattern. The ZmRBR2 transcript contained a 283-bp 5′-UTR and a putative canonical TATA box. The gene had a low GC content and showed conserved and unique features with other plant RBR genes. ZmRBR2 gene promoter-GUS fusions, introduced in Arabidopsis thaliana plants, drove high GUS activity in cotyledons, the shoot apical meristem and in flowering tissue, especially in anthers and pollen. ZmRBR2 transcript levels were consistently 50% lower in several tissues than those of ZmRBR1, suggesting a differential regulation of these two genes. Our results are consistent with the ubiquitous tissue distribution and function of plant RBR proteins and emphasize differences between the monocot and dicot RBR pathways.

Introns control expression of sucrose transporter LeSUT1 in trichomes, companion cells and in guard cells

In solanaceous plants such as tomato and tobacco, the sucrose transporter SUT1 is crucial for phloem loading. Using GUS as a reporter, the promoter and other regulatory cis elements required for the tomato LeSUT1 expression were analyzed by heterologous expression of translational chimeric constructs in tobacco. Although LeSUT1 is highly expressed at the RNA level, GUS expression under the control of a 1.8 kb LeSUT1 promoter resulted in few plants expressing GUS. In GUS-positive transformants, expression levels were low and limited to leaf phloem. Increasing or decreasing the length of LeSUT1 promoter did not lead to a significant increase in positive transformants or higher expression levels. Translational fusion of GUS to the LeSUT1 C-terminus in a construct containing all exons and introns and the 3'-UTR led to a higher number of positive transformants and many plants with high GUS activity. LeSUT1 expression was detected in ab- and adaxial phloem companion cells, trichomes and guard cells. The role of individual introns in LeSUT1 expression was further analyzed by placing each LeSUT1 intron into the 5'-UTR within the 2.3 kb LeSUT1 promoter construct. Results showed remarkable functions for the three introns for SUT1 expression in trichomes, guard cells and phloem cells. Intron 3 is responsible for expression in trichomes, whereas intron 2 is necessary for expression in companion cells and guard cells. The combination of all introns is required for the full expression pattern in phloem, guard cells and trichomes.

Increased expression of OsPT1, a high-affinity phosphate transporter, enhances phosphate acquisition in rice

Most high-affinity phosphate transporter genes (OsPTs) in rice were highly induced in roots when phosphate was depleted. OsPT1, however, was highly expressed in primary roots and leaves regardless of external phosphate concentrations. This finding was confirmed histochemically using transgenic rice plants that express the GUS reporter gene under the control of the OsPT1 promoter, which exhibited high GUS activity even in the phosphate sufficient condition. Furthermore, transgenic rice plants overexpressing the OsPT1 gene accumulated almost twice as much phosphate in the shoots as did wild-type plants. As a result, transgenic plants had more tillers than did wild-type plants, which is a typical physiological indicator for phosphate status in rice.

Improved translation efficiency in chrysanthemum and torenia with a translational enhancer derived from the tobacco alcohol dehydrogenase gene

We investigated the efficiency of the 5� -untranslated region (UTR) of the tobacco alcohol dehydrogenase gene (NtADH-5� UTR) as a translational enhancer in chrysanthemum (Chrysanthemum morifolium) and torenia (Torenia fournieri). Three constructs were introduced: control, ADH� , cauliflower mosaic virus 35S RNA (CaMV 35S) promoter::b-glucuronidase (GUS); NtADH-5� UTR with a spacer, ADHS, CaMV 35S promoter::NtADH-5� UTR::25-bp spacer::GUS; and NtADH-5� UTR with no spacer, ADH� , CaMV 35S promoter::NtADH-5� UTR::GUS. The highest GUS activity in ADHS and ADHfor chrysanthemum was about 45 and 190 times, respectively, and for torenia was 12 and 22 times, respectively, than that for the ADHplants. NtADH-5� UTR enhanced translational efficiency in both species. With the lowest translational efficiency set to 1, the relative translational efficiencies were 1 to 15 (ADH� ), 47 to 568 (ADHS), and 267 to 1360 (ADH� ) for chrysanthemum, and 1 to 3 (ADH� ), 47 to 114 (ADHS), and 85 to 226 (ADH� ) for torenia. NtADH-5� UTR would facilitate practical breeding and fundamental genetic research in chrysanthemum and torenia.

Structural and functional characteristics of two novel members of pathogensis-related multigene family of class 10 from yellow lupine+.

PR-10 proteins (pathogensis-related), ubiquitous within the plant kingdom, are usually encoded by multigene families. To date we have identified 10 homologous pr-10 genes in a yellow lupine cDNA library. Here, the structure and expression of two newly identified yellow lupine pr-10 genes (LlYpr10-2b and LlYpr10-2f) are presented. Many potential regulatory sites were found in both gene promoters including common ones as well as those unique for each gene. However, promoter deletion analysis in transgenic tobacco plants revealed similar patterns of reporter gene (gus) expression. Shortened fragments of both gene promoters studied caused high GUS activity in leaves (along vascular bundles), stamen stigma, anthers and pollen grains. When conjugated with longer LlYpr-10.2 promoter fragments, GUS was additionally present in petal edges. Only a long fragment of the LlYpr10-2b gene promoter caused GUS expression in the stem. In yellow lupine the pr-10.2 genes are present in all studied organs, but their level of expression depends on the stage of development and is affected by wounding, oxidative stress and salicylic acid treatment. Silencing of the Llpr-10.2b gene in 4-week-old yellow lupine plants did not lead to any visible symptoms, which suggests that the function of the silenced gene is supplemented by its close homologues, still present in the studied plants.

A reliable transformation method and heterologous expression of β-glucuronidase in Lentinula edodes

A simple and reliable mushroom transformation procedure based on electroporation of basidiospores or mycelial fragments was developed. This method eliminated the problem of protoplast preparation, the transformation efficiency were 30–150 transformants per μg DNA and the hygromycin resistant marker gene and gus were expressed in Lentinula edodes successfully. No false positive antibiotic-resistant cultures were detected by PCR amplification and the β-glucuronidase (GUS) expression was maintained stable during mitotic cell division without selection pressure for more than 6 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. Using the glyceraldehyde-3-phosphate dehydrogenase ( gpd) promoter with the first intron of gpd gene, the average GUS activity in L. edodes reached 144.6 ± 3.9 U mg − 1 soluble protein, while only 30.1 ± 0.7 U mg − 1 soluble protein was detected for those without the intron. The percentage of GUS in total soluble protein was 5.67 × 10 − 4 (0.06%) for the transformant with the highest GUS activity. This rapid and convenient electroporation procedure offers a new approach for the genetic manipulation and tool to tag genes of important edible mushroom species.

Functional analysis of BADH gene promoter from Suaeda liaotungensis K.

A 1,993 bp region upstream of the gene encoding the betaine aldehyde dehydrogenase (BADH) was isolated from Suaeda liaotungensis K., and the analysis of the promoter sequence has revealed the existence of several putative cis-elements by the PLACE database. In this study, according to the characteristic of the BADH promoter, five chimeric constructs varied in the length of promoter fragments from -1,993, -1,466, -1,084, -573 and -300 to +62 bp relative to the transcriptional start site were placed to the upstream of the beta-glucuronidase (GUS) coding region and transferred to Nicotiana tabacum L.cv.89 by Agrobacterium tumefaciens-mediated leaf-disc transformation. The functional properties of each promoter fragment were examined by GUS histochemical staining and fluorescence quantitative analyses in the transgenic tobacco leaves treated with different NaCl concentrations for 48 h. The results show that healthy transgenic plants had decreased GUS activity in leaves, whereas a higher GUS activity was observed when the transgenic plants were challenged with sodium chloride (NaCl). Induction levels were proportional to the concentration of NaCl treatment, allowing fine-tuning of protein expression. GUS enzyme activity was enhanced 6.3-fold in transgenic tobacco leaves containing -300 bp promoter fragment in the presence of 400 mmol/l NaCl compared to the noninductive leaves. This suggests that the smallest promoter fragment (-300 to +62 bp) possesses all the essential cis-acting elements and is sufficient for NaCl induction.

Comparative expression analysis of three genes from the Arabidopsis vacuolar Na+/H+ antiporter (AtNHX) family in relation to abiotic stresses

Na+/H+ antiporters (NHX) are ubiquitous transmembrane proteins that play a key role in salt tolerance of plants. In this study, the sequence of 3 Arabidopsis NHX genes (AtNHX2–4) were compared with other AtNHX members. Putative cis-elements analysis identified elements that have been associated with stress responses. The activities of the promoters AtNHX2–4 were studied in transgenic plants carrying corresponding promoter-β-glucuronidase (GUS) fusions. The AtNHX2 promoter-GUS analysis indicated that AtNHX2 was expressed in constitutive pattern with high GUS activity in roots and leaves. AtNHX2 promoter activity was not up-regulated by NaCl or abscisic acid (ABA), in contrast the AtNHX1 promoter which was previously studied. The AtNHX3 and AtNHX4 promoters showed tissue-specific activities. Strong GUS activity was detected in roots and vascular bundles of the stele in plants carrying an AtNHX4 promoter-GUS fusion, and GUS activity increased under salt stress suggesting a function related to salt tolerance. Transgenic plants carrying the AtNHX3 promoter-GUS fusion showed strong GUS activity in petals, stamens and tops of siliques, suggesting a possible role of AtNHX3 in flower and seed development. Results of histochemical analysis suggested that AtNHX2–4 are involved in divergent functions and are differentially regulated under abiotic stress. The structure of AtNHX4 was predicted to include 12 transmembrane regions and a NHX domain. Overexpression of AtNHX4 in Arabidopsis transgenic lines confers greater salt tolerance than in wild type plants. These results suggest that AtNHX4 may encode a putative vacuolar NHX that plays an important role in salt tolerance.

Ubiquitous presence of β-glucuronidase (GUS) in plants and its regulation in some model plants

The enzyme beta-glucuronidase (GUS) is well characterized in animals and microbes. However, this enzyme is not well studied in plants and is widely assumed to be absent in them. In this study we document the ubiquitous presence of GUS in the model plants Arabidopsis thaliana, Oryza sativa, Nicotiana tabacum and Zea mays and record its expression pattern. The pH of the assay buffer was found to be critical with pH 4.0 being optimum for detection in all the species. GUS in plants appears to be associated with growth. In general, younger regions of the organs showed more GUS activity than the older and more mature tissues. In Brassica juncea roots stained for GUS, intense blue color could be seen in the trichoblast cells and the growing root hair cells as compared to the non-root hair forming epidermal cells or the fully elongated root hairs. Cotton fibers showed high GUS activity during the initial phase of elongation while the seed coat, from which the fibers formed, did not stain for GUS activity. The activity in the fibers disappeared after they were fully elongated. The level of GUS activity increased 2.58 folds in leaf tissues of N. tabacum when cultured in MS medium supplemented with 6-benzylaminopurine, while gibberellic acid enhanced GUS activity 2.9 folds in the inter-nodal regions of rice in 12-h treatment. In addition, elongation of stem, root and root hairs in tobacco seedlings was strongly inhibited by the specific inhibitor of GUS, saccharo-1-4-lactone in a reversible manner. Taken together, these evidences suggest a probable association of plant GUS in cell growth.

Essential role of the Box II cis element and cognate host factors in regulating the promoter of Rice tungro bacilliform virus

Rice tungro bacilliform virus (RTBV) is a double-stranded DNA virus with a single, tissue-specific promoter that is expressed primarily in phloem tissues. Rice transcription factors RF2a and RF2b bind to Box II, a cis element adjacent to the TATA box, and control gene expression from the promoter. Mutations were made in the promoter to delete or mutate Box II and the mutated promoters were fused to a reporter gene; the chimeric genes were expressed in transient BY-2 protoplast assays and in transgenic Arabidopsis plants. The results of these studies showed that Box II is essential to the activity of the RTBV promoter. A chimeric beta-glucuronidase (GUS) reporter gene containing the Box II sequence and a minimal promoter derived from the Cauliflower mosaic virus 35S promoter were co-transfected into protoplasts with gene constructs that encoded RF2a or RF2b. The reporter gene produced threefold higher GUS activity when co-transfected with RF2a, and 11-fold higher activity when co-transfected with RF2b, than in the absence of added transcription factors. Moreover, chimeric reporter genes were activated by approximately threefold following induction of expression of the RF2a gene in transgenic Arabidopsis plants. The work presented here and earlier findings show that Box II and its interactions with cognate rice transcription factors, including RF2a and RF2b, are essential to the activity of the RTBV promoter and are probably involved in expression of the RTBV genome during virus replication.

Isolation of a cotton reversibly glycosylated polypeptide ( GhRGP1) promoter and its expression activity in transgenic tobacco

Reversibly glycosylated polypeptides (RGPs) are thought to be involved in polysaccharide metabolism. A cDNA of the cotton (Gossypium hirsutum) RGP gene, designated GhRGP1, has previously been characterized, and is preferentially expressed in fiber cells. In order to investigate its temporal and spatial control, we isolated a 624bp fragment upstream of the GhRGP1 coding sequence using a polymerase chain reaction (PCR)-based genomic walking method, transcriptionally fused the 624bp promoter sequence to the beta-glucuronidase (GUS) gene, and analyzed the stable gene expression in tobacco (Nicotiana tabacum). In 4-week-old transgenic tobacco plants, the highest expression level was observed in roots, and the GUS activity was 1.13- and 6.65-fold higher than that in stems and leaves, respectively. In the reproductive growth stage, the GUS expression level was highest in the pistils and the GUS activity in the stigmas and styles were 17.6-fold higher than that in the ovaries. High GUS activity was also detected in the anthers. In addition, histochemical staining for GUS activity on transgenic tobacco plants further indicated a higher expression in the trichomes, seeds and vascular tissues of stems. Abiotic stress treatments on transgenic tobacco plants showed that wounding and dehydration induced GUS expression. These results demonstrated the spatial and temporal regulation of a cotton RGP promoter in a model plant, and provided an important insight into the factors that control the fiber development and stress responses of the gene.