Studies on the expression of linalool synthase using a promoter-β-glucuronidase fusion in transgenic Artemisia annua

Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to β-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652 bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.     

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene

We have successfully transferred and expressed a reporter gene driven by an -amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10–12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of -glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice -amylase gene (Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice -amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.     

Evaluation of seed storage-protein gene 5′ untranslated regions in enhancing gene expression in transgenic rice seed

5′ untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. Using the β-glucuronidase (GUS) reporter gene driven by the GluC promoter for the rice-seed storage-protein glutelin, we evaluated the potential of the 5′-UTRs of six seed storage-protein genes in enhancing the expression levels of the foreign gene in stable transgenic rice lines. All of the 5′-UTRs significantly enhanced the expression level of the GluC promoter without altering its expression pattern. The 5′-UTRs of Glb-1 and GluA-1 increased the expression of GUS by about 3.36- and 3.11-fold, respectively. The two 5′-UTRs downstream of the Glb-1, OsAct2 and CMV35S promoters also increased GUS expression level in stable transgenic rice lines or in transient expression protoplasts. Therefore, the enhancements were independent of the promoter sequence. Real-time quantitative RT-PCR analysis showed that the increase in protein production was not accompanied by alteration in mRNA levels, which suggests that the enhancements were due to increasing the translational efficiencies of the mRNA. The 5′-UTRs of Glb-1 and GluA-1, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds.     

Stability of β-glucuronidase gene expression in transgenic Tricyrtis hirta plants after two years of cultivation

Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated for two years. Four independent transgenic plants produced flowers 1–2 years after acclimatization, and all of them contained one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation.     

Tissue specific expression of β-glucuronidase gene driven by heterologous promoters in transgenic cauliflower plants

In this paper we compare five heterologous promoters fused to β-glucuronidase gene in their influence on localization of GUS activity in cauliflower (Brassica oleracea var. botrytis) tissues: roots, leaves, petioles and curds. A constitutive promoter CaMV 35S and four tissue specific promoters were used: extAP from rape, PsMTAP from pea, RBCS3CP from tomato and SRS1P from soybean, and introduced into cauliflower seedling explants using Agrobacterium rhizogenes mediated transformation. Quantitative and histochemical GUS assays confirmed tissue specific gus expression. It was found that extAP promoter was the most active in petioles but also caused a significant gus expression in curds. GUS activity was hardly observed in curd and restricted only to its epidermis when PsMTAP promoter drove the gene. RBCS3CP and SRS1P promoters controlled similar expression of the gus gene throughout the plant except for curd where RBCS3CP was almost inactive.     

β-glucuronidase gene expression and mRNA stability in oat protoplasts

Summary Protoplasts derived from oat (Avena sativa L.) suspension culture cells (7 days after subculturing) were electroporated with plasmid DNA containing the Escherichia coli uidA gene encoding the ß-glucuronidase reporter enzyme. Consistently high enzyme activity was observed with electroporation conditions of 500 F and 1125 volts/cm. Enzyme activity and mRNA accumulation time courses were determined. The maximum enzyme activity was detected at 24 hours after electroporation, while the maximum mRNA level was detected at 12 hours after electroporation. ß-glucuronidase mRNA was in vitro synthesized with and without a 5 methylated cap and then electroporated into protoplasts. Only capped mRNA produced significant enzyme activity. By electroporating radiolabeled, in vitro synthesized mRNA, the ß-glucuronidase mRNA half-life was estimated to be 35 minutes in oat protoplasts.Abbreviations GUS ß-glucuronidase - mRNA messenger RNA - ICP insecticidal crystal proteins - OCS octopine synthase - CAT chloramphenicol acetyltransferase - nt nucleotide - kb kilobase - MSOD3 Murashige and Skoog media with zero 2,4-dichlorophenoxy acetic acid and 3% sucrose - MU 4-methyl umbelliferone; ATA: aurintricarboxylic acid     

Tissue-specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants

The strawberry ( Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the ß-glucuronidase (GUS) reporter gene ( gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue . Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7- gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter.     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

Expression of a bacterial α-amylase gene in transgenic rice seeds

An alpha-amylase gene from Bacillus stearothermophilus under the control of the promoter of a major rice-seed storage protein was introduced into rice. The transgenic line with the highest alpha-amylase activity reached about 15,000 U/g of seeds (one unit is defined as the amount of enzyme that produces 1 mumol of reducing sugar in 1 min at 70 degrees C). The enzyme produced in the seeds had an optimum pH of 5.0-5.5 and optimum temperature of 60-70 degrees C. Without extraction or purification, the power of transgenic rice seeds was able to liquify 100 times its weight of corn powder in 2 h. Thus, the transgenic rice could be used for industrial starch liquefaction.     

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O₂ deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8 % of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8 % of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.     

Phytohormone-regulated β-amylase gene expression in rice

The expression of -amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination -amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the -amylase activity, while ABA inhibits the -amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of -amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.     

Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the -amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice -amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.     

Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants

Tomato (Lycopersicon esculentum Mill. cv. Better Boy) plants were transformed with a fused gene containing a 2.2-kb promoter fragment of the tomato prosystemin gene and the coding region of the β-glucuronidase (GUS) reporter gene. The transgenic plants exhibited a low constitutive level of prosystemin-β-glucuronidase gene expression, assayed by histochemical staining and GUS enzyme activity, that was associated in the vascular bundles of leaf main veins, petiolules, petioles and stems. The GUS activity in the vascular bundles in each tissue was increased by wounding and by treatment of the plants with methyl jasmonate, similar to the induction of prosystemin in wild-type plants. The increase in GUS activity in the vascular bundles of leaves in response to wounding correlated with the wound-inducible increase in prosystemin mRNA. Tissue printing, using rabbit anti-serum prepared against prosystemin, confirmed that inducible prosystemin protein was localized in vascular bundles of petiolules, petioles and stems of wild-type tomato plants. The evidence indicates that the 2.2-kb promoter region of the tomato prosystemin gene contains elements conferring its correct temporal and spatial expression in the vascular bundles of transgenic tomato plants. Received: 7 January 1997 / Accepted: 2 April 1997     

High-level expression of sucrose inducible sweet potato sporamin gene promoter: β-glucuronidase fusion gene in transgenic Nicotiana plumbaginifolia hairy roots

In Vitro Cellular & Developmental Biology - Plant - We developed transgenic Nicotiana plumbaginifolia hairy roots with sucrose-inducible minimal promoter (Spomin)-β-glucuronidase (GUS)...     

Regulation of the α-expansin gene OsEXPA8 expression affects root system architecture in transgenic rice plants

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix, and are encoded by a large gene family. However, data linked to loss of function of single genes which support the role of expansins in root growth remain limited. In this study, we used RNA interference to examine the biological functions of the rice α-expansin gene OsEXPA8. Repression of OsEXPA8 expression in rice impaired the root system architecture and plant growth significantly, leading to shorter primary roots and fewer lateral roots. Accordingly, the cell size of the root vascular bundle reduced drastically. Notably, OsEXPA8 silencing impaired root hair elongation; moreover, plant height was clearly reduced. Transient expression of OsEXPA8-GFP in onion epidermal cells verified that OsEXPA8 is located on the cell wall. OsEXPA8 was expressed predominantly in the root and shoot of one-week-old rice seedlings, and highly induced by NaCl but suppressed by nitrate and phosphate starvation. In addition, atomic force microscopy was used to explore alterations in cell wall nanomechanics caused by OsEXPA8 protein reduction, which showed that the wall stiffness (Young’s modulus) of OsEXPA8-silenced suspension cells was increased significantly. Taken together, our results suggest that OsEXPA8 is critical for root system architecture, which supports the hypothesis that expansins are involved in enhancing plant growth by mediating cell wall loosening.