Β-glucuronidase Expression Research Articles

GUS activity for miR165a/166b, REV, and WUS/CLV3 in in vitro direct Arabidopsis thaliana shoot regeneration

In Arabidopsis thaliana, the process of shoot regeneration in vitro requires the presence of specific miRNAs. We describe here the β-glucuronidase (GUS) expression domains for miR165a/166b, REV, and WUS/CLV3 during direct shoot regeneration. Increased GUS activity of miR166b and REV were first detected within the shoot apical meristem of explants, while no pmiR165a::GUS activity appeared there. The zone of pWUS::GUS activity covered the inner sides of developing protuberances, while that of pCLV3::GUS was more restricted. Once the primary shoot had emerged from the protuberance, pREV::GUS activity was turned on throughout the protuberance. pmiR165a::GUS activity was detected in a small number of protuberance surface cells, while pWUS::GUS activity was restricted to within a few cells beneath the protuberance surface. After the differentiation of leaf-like structures, GUS activity for miR165a and miR166b appeared largely on their abaxial surface, while pWUS::GUS activity was concentrated at the apex of the primary shoot, and no pCLV3::GUS activity was detectable. Following the formation of secondary shoots, pmiR165a::GUS activity was detected on their abaxial surface. GUS activity for miR166b, REV, and WUS/CLV3 were concentrated in the stem apical meristem. The observations suggested that each member of this set of genes might play a distinct role in both primary and secondary shoot regeneration.

Promoter regions of potato vacuolar invertase gene in response to sugars and hormones

Potato vacuolar acid invertase (StvacINV1) (β-fructofuranosidase; EC 3.2.1.26) has been confirmed to play an important role in cold-induced sweetening of potato tubers. However, the transcriptional regulation mechanisms of StvacINV1 are largely unknown. In this study, the 5'-flanking sequence of StvacINV1 was cloned and the cis-acting elements were predicted. Histochemical assay showed that the StvacINV1 promoter governed β-glucuronidase (GUS) expression in potato leaves, stems, roots and tubers. Quantitative analysis of GUS expression suggested that the activity of StvacINV1 promoter was suppressed by sucrose, glucose, fructose, and cold, while enhanced by indole-3-acetic acid (IAA), and gibberellic acid (GA3). Further deletion analysis clarified that the promoter regions from-118 to-551,-551 to-1021, and-1021 to-1521 were required for responding to sucrose/glucose, GA3, and IAA, respectively. These findings provide essential information regarding transcriptional regulation mechanisms of StvacINV1.

Expression of β-glucuronidase on the surface of bacteria enhances activation of glucuronide prodrugs

Extracellular activation of hydrophilic glucuronide prodrugs by β-glucuronidase (βG) was examined to increase the therapeutic efficacy of bacteria-directed enzyme prodrug therapy (BDEPT). βG was expressed on the surface of Escherichia coli by fusion to either the bacterial autotransporter protein Adhesin (membrane βG (mβG)/AIDA) or the lipoprotein (lpp) outermembrane protein A (mβG/lpp). Both mβG/AIDA and mβG/lpp were expressed on the bacterial surface, but only mβG/AIDA displayed enzymatic activity. The rate of substrate hydrolysis by mβG/AIDA-BL21cells was 2.6-fold greater than by pβG-BL21 cells, which express periplasmic βG. Human colon cancer HCT116 cells that were incubated with mβG/AIDA-BL21 bacteria were sensitive to a glucuronide prodrug (p-hydroxy aniline mustard β-D-glucuronide, HAMG) with an half maximal inhibitory concentration (IC50) value of 226.53±45.4 μM, similar to the IC50 value of the active drug (p-hydroxy aniline mustard, pHAM; 70.6±6.75 μM), indicating that mβG/AIDA on BL21 bacteria could rapidly and efficiently convert HAMG to an active anticancer agent. These results suggest that surface display of functional βG on bacteria can enhance the hydrolysis of glucuronide prodrugs and may increase the effectiveness of BDEPT.

Globulin Endosperm-Specific Promoter For High-Level Expression of Gus Gene IN Rice

The world population is expected to rise by an addition of 2 billion by 2030 and rice consumers are projected to increase by 1.8% annually. Hence, rice production must be increased between 25-45% to match-up the growing population since it is a staple food to more than half of the world’s population. The endosperm is a reliable tissue for the production of recombinant proteins over other tissues because it is more cost-effective, it is easier to scale-up agricultural yield, provides a larger storage ability and safe long-term storage. However only a few endosperm-specific promoters have been identified. The present research focused on the use of the Avena sativa strong endosperm-specific globulin (AsGpro) promoter (Accession number AY795082.1) for the expression of β-glucuronidase (GUS) in the endosperm of transgenic rice. Histochemical GUSPlus quantitative assays revealed that expression strength driven by the full length promoter region was 8 fold and 6 fold stronger that both rice actin and wheat high molecular weight glutenin promoters respectively. The AsGpro was up to 100 fold stronger than the CaMV35S constitutive promoter. The endosperm-specific globulin promoter can be used for the production of nutritional and pharmaceutical recombinant proteins using rice as a bioreactor which will help in alleviating the food crisis facing the world. Th

Identification of an Internal RNA Element Essential for Replication and Translational Enhancement of Tobacco Necrosis Virus AC

Different regulatory elements function are involved in plant virus gene expression and replication by long-distance RNA-RNA interactions. A cap-independent functional element of the Barley yellow dwarf virus (BYDV) – like translational enhancer (BTE) is present in Tobacco necrosis virus A (TNV-A), a Necrovirus member in the Tombusviridae family. In this paper, an RNA stretch flanking the 5′ proximal end of the TNV-AC coat protein (CP) gene was shown to be essential for viral replication in Chenopodium amaranticolor plants and tobacco cells. This internal sequence functioned in transient expression of β-glucuronidase (GUS) when present at either the 5′ or 3′ sides of the GUS open reading frame. Serial deletion analyses revealed that nine nucleotides from nt 2609 to 2617 (−3 to +6 of the CP initiation site) within TNV-AC RNA are indispensable for viral replication in whole plants and tobacco cells. Fusion of this RNA element in mRNAs translated in tobacco cells resulted in a remarkable enhancement of luciferase expression from in vitro synthesised chimaeric RNAs or DNA expression vectors. Interestingly, the element also exhibited increased translational activity when fused downstream of the reporter genes, although the efficiency was lower than with upstream fusions. These results provide evidence that an internal RNA element in the genomic (g) RNA of TNV-AC, ranging approximately from nt 2543 to 2617, plays a bifunctional role in viral replication and translation enhancement during infection, and that this element may use novel strategies differing from those previously reported for other viruses.

In vivo magnetic resonance imaging of mice liver tumors using a new gadolinium-based contrast agent

We compared the enhancement effect between a newly synthesized tissue-specific contrast agent, [Gd-DOTA-FPβG], and a commercially available agent, [Gd(DOTA)]−, in a murine model of liver tumor using a clinical magnetic resonance imaging scanner. The colon cancer cell lines with and without β-glucuronidase (βG) expression were implanted into the liver of mice. Self-synthesized gadolinium-based magnetic resonance contrast agent, [Gd(DOTA-FPβG)], was administered to measure enhancement on magnetic resonance images using a commercially available agent, [Gd(DOTA)]−, as control in a clinical 3.0 tesla (T) magnetic resonance scanner. In vivo fluorescence imaging and histopathology of the liver were also performed to compare and correlate with the magnetic resonance studies. The in vivo fluorescence imaging failed to depict a sufficiently intense signal for liver or liver tumor of mice without exposure of the liver following an incision on the abdominal wall. The tissue-specific magnetic resonance agent, [Gd(DOTA-FPβG)], caused significantly stronger enhancement in tumors expressing βG (CT26/mβG-eB7) than in tumors not expressing βG (CT26) (p < 0.05). In the magnetic resonance imaging studies using control agent [Gd(DOTA)]−, the tumors with and without βG expression depicted no significant difference in enhancement on the T1-weighted images. The [Gd(DOTA-FPβG)] also provided significantly more contrast uptake in the CT26/mβG-eB7 tumor than in the normal liver parenchyma, whereas the [Gd(DOTA)]− did not. This study confirms that better contrast enhancement can be readily detected in vivo by the use of a tissue-specific magnetic resonance contrast agent to target tumor cells with specific biomarkers in a clinical magnetic resonance imaging scanner.

Screening promoters for Anthurium transformation using transient expression

KEY MESSAGE : There are multiple publications on Anthurium transformation, yet a commercial product has not been achieved. This may be due to use of non-optimum promoters here we address this problem. Different promoters and tissue types were evaluated for transient β-glucuronidase (GUS) expression in Anthurium andraeanum Hort. 'Marian Seefurth' following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35S promoter from Cauliflower Mosaic Virus fused to a GUS reporter gene were bombarded into in vitro grown anthurium lamina, somatic embryos and roots. The number of GUS foci and the intensity of GUS expression were evaluated for each construct. Ubiquitin promoters from rice and maize resulted in the highest number of expressing cells in all tissues examined. Due to the slow growth of anthurium plants, development of transgenic anthurium plants takes years. This research has rapidly identified multiple promoters that express in various anthurium tissues facilitating the development of transformation vectors for the expression of desirable traits in anthurium plants.

Repression vs. activation ofMOX,FMD,MPP1andMAL1promoters by sugars inHansenula polymorpha: the outcome depends on cell's ability to phosphorylate sugar

A high-throughput approach was used to assess the effect of mono- and disaccharides on MOX, FMD, MPP1 and MAL1 promoters in Hansenula polymorpha. Site-specifically designed strains deficient for (1) hexokinase, (2) hexokinase and glucokinase, (3) maltose permease or (4) maltase were used as hosts for reporter plasmids in which β-glucuronidase (Gus) expression was controlled by these promoters. The reporter strains were grown on agar plates containing varied carbon sources and Gus activity was measured in permeabilized cells on microtitre plates. We report that monosaccharides (glucose, fructose) repress studied promoters only if phosphorylated in the cell. Glucose-6-phosphate was proposed as a sugar repression signalling metabolite for H.polymorpha. Intriguingly, glucose and fructose strongly activated expression from these promoters in strains lacking both hexokinase and glucokinase, indicating that unphosphorylated monosaccharides have promoter-derepressing effect. We also show that maltose and sucrose must be internalized and split into monosaccharides to exert repression on MOX promoter. We demonstrate that at yeast growth on glucose-containing agar medium, glucose-limitation is rapidly created that promotes derepression of methanol-specific promoters and that derepression is specifically enhanced in hexokinase-negative strain. We recommend double kinase-negative and hexokinase-negative mutants as hosts for heterologous protein production from MOX and FMD promoters.

Overexpression of a C2H2-type zinc finger protein gene, ZAT11, leads to enhanced primary root growth and increased nickel ion sensitivity in Arabidopsis

The C2H2-type zinc finger proteins play crucial roles in various developmental processes and environmental stress tolerance in plants. Zinc Finger of Arabidopsis Thaliana11 (ZAT11), has been previously reported as an active repressor. We show here that GFP-tagged ZAT11 is targeted to the nucleus. Transgenic plants overexpressing ZAT11 show enhanced primary root growth. Analysis of β-glucuronidase (GUS) expression in transgenic plants expressing a ZAT11 promoter–GUS reporter chimeric gene confirmed that ZAT11 was expressed in roots and its expression was particularly high in root tips. ZAT11-overexpressing transgenic plants were specifically sensitive to nickel ions (Ni2+). Transcript levels of a tonoplast-localized Ni2+ transporter gene, IREG2, were approximately 2-fold decreased in transgenic plants overexpressing ZAT11 and this correlated with a reduced capacity for Ni2+ accumulation in their roots. Altogether, our results reveal that ZAT11 functions as a positive regulator of primary root growth but a negative regulator of Ni2+ tolerance, suggesting a novel biological function of Arabidopsis C2H2-type zinc finger protein in metal detoxification.

Optimization of an Agrobacterium-mediated Transient Assay for Gene Expression Studies in Anthurium andraeanum

Gene function studies in anthurium (Anthurium andraeanum) have been hindered by the low efficiency of stable transformation, the long regeneration time required as well as the genotype-dependent nature of Agrobacterium (Agrobacterium tumemaciens)-mediated transformation protocols. Agrobacterium-mediated transient expression assays can serve as an attractive alternative for investigating gene function once such assays are optimized. The effects of host factors (genotype, explant type, and developmental maturity of explants), Agrobacterium factors (strain, growth phase, and its concentration), media conditions (infiltration medium used, acetosyringone concentration, type of surfactant, and its concentration), and other experimental factors (infiltration time, cocultivation time, and vacuum infiltration) were investigated on the efficiency of Agrobacterium transient transformation, with replications, using transient expression of β-glucuronidase as an indicator. Although the efficiency of transient transformation was initially found to be highly host genotype-dependent, the genotypic differences in transient transformation efficiency diminished as the protocol was optimized. Agrobacterium strain GV3101 grown to an optical density at 600 nM (OD600) of 1.5 and resuspended to a final OD600 of 0.8 in infiltration medium [0.5% glucose, 10 mm 2-(N-morpholino) ethanesulfonic acid] supplemented with 100 μM acetosyringone and 0.05% of a non-ionic surfactant (S240), for an infiltration period of 16 hours and a cocultivation timeframe of 2 days yielded transient transformation efficiencies as high as 100%.

The 5′UTR-intron of the Gladiolus polyubiquitin promoter GUBQ1 enhances translation efficiency in Gladiolus and Arabidopsis

BackgroundThere are many non-cereal monocots of agronomic, horticultural, and biofuel importance. Successful transformation of these species requires an understanding of factors controlling expression of their genes. Introns have been known to affect both the level and tissue-specific expression of genes in dicots and cereal monocots, but there have been no studies on an intron isolated from a non-cereal monocot. This study characterizes the levels of GUS expression and levels of uidA mRNA that code for β-glucuronidase (GUS) expression in leaves of Gladiolus and Arabidopsis using GUBQ1, a polyubiquitin promoter with a 1.234 kb intron, isolated from the non-cereal monocot Gladiolus, and an intronless version of this promoter.ResultsGladiolus and Arabidopsis were verified by Southern hybridization to be transformed with the uidA gene that was under control of either the GUBQ1 promoter (1.9 kb), a 5′ GUBQ1 promoter missing its 1.234 kb intron (0.68 kb), or the CaMV 35 S promoter. Histochemical staining showed that GUS was expressed throughout leaves and roots of Gladiolus and Arabidopsis with the 1.9 kb GUBQ1 promoter. GUS expression was significantly decreased in Gladiolus and abolished in Arabidopsis when the 5′UTR-intron was absent. In Arabidopsis and Gladiolus, the presence of uidA mRNA was independent of the presence of the 5′UTR-intron. The 5′-UTR intron enhanced translation efficiency for both Gladiolus and Arabidopsis.ConclusionsThe GUBQ1 promoter directs high levels of GUS expression in young leaves of both Gladiolus and Arabidopsis. The 5′UTR-intron from GUBQ1 resulted in a similar pattern of β-glucuronidase translation efficiency for both species even though the intron resulted in different patterns of uidA mRNA accumulation for each species.

Sequence analysis and potentials of the native RbcS promoter in the development of an alternative eukaryotic expression system using green Microalga Ankistrodesmus convolutus.

The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of β-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5′-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.

Low Co-Cultivation Temperature at 20&amp;#176;C Resulted in the Reproducible Maximum Increase in Both the Fresh Weight Yield and Stable Expression of GUS Activity after &amp;lt;i&amp;gt;Agrobacterium tumefaciens&amp;lt;/i&amp;gt;-Mediated Transformation of Tobacco Leaf Disks

The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.

Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris

The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD(600)) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L(-1)) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.

A Simple and Efficient Protocol for Plant Regeneration and Genetic Transformation of Tomato cv. Micro-Tom from Leaf Explants

A simplified protocol to obtain transgenic tomato plants was established. The effects of culture media composition and Agrobacterium concentration were evaluated. The highest shoot-forming capacity index (5.6) was observed when leaf explants were cultured for 6 weeks with 2 mg·L−1 zeatin, 0.1 mg·L−1 indoleacetic acid, and 300 mg·L−1 timentin. Shoot elongation and root formation were performed in one step on growth regulator-free media. The highest percentage (82%) of fully developed plantlets was obtained when shoots were cultured for 4 weeks with 0.5× Murashige and Skoog (MS) media and 15 g·L−1 sucrose. A 100% of plant survival rate was observed after 4 weeks of being transplanted to ex vitro conditions followed by fruit production (15 fruits/plant) after 2 more weeks. Transient expression of β-glucuronidase was visualized in 100% of the leaf explants infected with Agrobacterium at an OD600 = 0.5 and cocultured for 48 h with 2 mg·L−1 benzylaminopurine, 0.1 mg·L−1 naphthaleneacetic acid, and 100 μM acetosyringone. Stable transformation was confirmed by histochemical glucuronidase assay and polymerase chain reaction (PCR) analysis with a total efficiency of 19.1%. The complete protocol, from shoot induction to fruit production of soil-adapted transgenic plants can be accomplished in only 4 months, and it seems to be very useful for both micropropagation and genetic transformation purposes.

Culturing of immature inflorescences and Agrobacterium-mediated transformation of foxtail millet (Setaria italica)

In previous reports, we developed an Agrobacterium -mediated transformation system for foxtail millet. Here, we report optimization of the system through improvement of the regeneration system efficiency and optimization of conditions for gene delivery. Immature inflorescences explants of foxtail millet cv. Jigu 11 varying in length (0.5 to 1.0, 1.1 to 1.5, 1.6 to 2.0 and >2.0 cm) were cultured on modified MS medium for callus induction and regeneration. The highest embryogenic callus-formation efficiency (90.72%) was achieved with 0.5 to 1.0 cm long inflorescences and 25 days old calli induced from 0.5 to 1.0 cm long immature inflorescences gave rise to the highest differentiation frequency (90.93%). In addition, factors affecting T-DNA delivery were examined by transient β-glucuronidase (GUS) expression. Calli induced from younger explants (0.5 to 1.0 cm immature inflorescences) were optimal. Agrobacterium tumefaciens strain LBA4404 performed significantly better than EHA105. Co-cultivation at 22°C with 0.15 g/l dithiothreitol (DTT) in the infection solution and co-cultivation medium led to higher GUS transient expression efficiency than with other treatments. Using this optimized procedure, the lysine-rich protein encoding gene SBgLR from potato was transformed into foxtail millet cv. Jigu 11 with 5.5% transformation efficiency. The procedure described here will be useful for genetic improvement of foxtail millet. Key words : Foxtail millet, regeneration, Agrobacterium-mediated transformation, temperature, dithiothreitol (DTT).

Transformation of Solanum tuberosum plastids allows high expression levels of β-glucuronidase both in leaves and microtubers developed in vitro

Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of β-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of β-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on β-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8days increased β-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.

Live Diatom Silica Immobilization of Multimeric and Redox-Active Enzymes

Living organisms are adept in forming inorganic materials (biominerals) with unique structures and properties that exceed the capabilities of engineered materials. Biomimetic materials syntheses are being developed that aim at replicating the advantageous properties of biominerals in vitro and endow them with additional functionalities. Recently, proof-of-concept was provided for an alternative approach that allows for the production of biomineral-based functional materials in vivo. In this approach, the cellular machinery for the biosynthesis of nano-/micropatterned SiO₂ (silica) structures in diatoms was genetically engineered to incorporate a monomeric, cofactor-independent ("simple") enzyme, HabB, into diatom silica. In the present work, it is demonstrated that this approach is also applicable for enzymes with "complex" activity requirements, including oligomerization, metal ions, organic redox cofactors, and posttranslational modifications. Functional expression of the enzymes β-glucuronidase, glucose oxidase, galactose oxidase, and horseradish peroxidase in the diatom Thalassiosira pseudonana was accomplished, and 66 to 78% of the expressed enzymes were stably incorporated into the biosilica. The in vivo incorporated enzymes represent approximately 0.1% (wt/wt) of the diatom biosilica and are stabilized against denaturation and proteolytic degradation. Furthermore, it is demonstrated that the gene construct for in vivo immobilization of glucose oxidase can be utilized as the first negative selection marker for diatom genetic engineering.

Analysis of banana fruit-specific promoters using transient expression in embryogenic cells of banana cultivar Robusta (AAA Group)

The study reports the transient expression of gusA gene in embryogenic cells using three banana derived fruit-specific promoters. Banana embryogenic cells were transformed with a pCAMBIA-1301 derived plasmid construct harboring gusA gene driven by either chitinase, glucanase or expansin promoters derived from banana. The transient expression of β-glucuronidase was studied 5 days after co-cultivation with Agrobacterium harboring the expression plasmids. The transformed embryogenic cells were treated with different inducers of ethylene such as ethephon, methyl jasmonate, methyl salicylate, abscisic acid and indole acetic acid. The maximum expression of 64099.78 pmoles 4-MU/h/mg total protein was noted with expansin promoter when the cells were treated with the combination of ethephon (0.25 mM) and MJ (10 mM). The results suggest that these promoters can be used to achieve fruit-specific expression of useful transgenes in banana. The results should prove to be an important guide for short term expression studies for promoter validation and gene screening.

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: How to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed ( Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.