Expression Of sGFP Research Articles

Improvement of Agrobacterium-mediated transformation for tannin-producing sorghum.

Sorghum (Sorghum bicolor L.) ranks as the fifth most widely planted cereal in the world and is used for food as well as a biomass plant for ethanol production. Use of the TX430 non-tannin sorghum variety has enhanced Agrobacterium-mediated sorghum transformation. These protocols could not be applied, however, to other tannin producing sorghum varieties such as the BTx623 model cultivar for sorghum with full genome information of sorghum. Here we report an improved protocol for Agrobacterium-mediated genetic transformation of tannin-producing sorghum variety BTx623. We successfully developed modification of root regeneration condition for generation of transgenic plant of BTx623. We inoculated immature embryos with Agrobacterium tumefaciens strain EHA105 harboring pMDC32-35S-GFP to generate transgenic plants. In the root regeneration step, we found that regeneration from transformed calli was affected by tannin. For root regeneration, shoots that appeared were not transferred to agar plate, but instead transferred to vermiculite in a plastic pod. Direct planting of regenerated shoots into vermiculite prevented the toxic effect of tannin. Root regeneration efficiency from calli emerged shoots in vermiculite was 78.57%. Presence of sGFP transgene in the genome of transgenic plants was confirmed by PCR and sGFP expression was confirmed in transgenic plants. This improved protocol of Agrobacterium-mediated transformation for tannin-producing sorghum BTx623 could be a useful tool for functional genomics using this plant.

Truncated, strong inducible promoter Pmcl1 from Metarhizium anisopliae.

In this study, Metarhizium collagen -like protein (MCL1) promoter from Metarhizium anisopliae was analysed and truncated into different sizes through series of targeted and random deletions based on the presence of various transcription factor-binding sites. Synthetic Green Fluorescent Protein (sGFP) was being utilized as a reporter gene to study the relative expression driving capability of unmodified and truncated promoters. Conserved promoter sequence analysis revealed similarity between the paralogous promoters from M. brunneum and M. acridum. sGFP expression in the haemolymph was directed with the help of mcl1 signal peptide sequence. Deleting the promoter region from - 2764 to - 1583bp increases the promoter mcl1 (Pmcl1) activity by twofolds, while deletions of the regions upstream of - 1150bp and - 840bp caused a decrease of sGFP expression level (80% and 70%, respectively). Transcriptional binding sites predicted for the deleted region revealed the loss of upstream repressing sequences such as Matalpha2 along with ROX1 and Rap1repressor-binding sites located - 2234bp, - 1754bp and - 1724bp from the TSS. Compared with Pmcl1-wild type (2.7kbp), Pmcl1-1583bp had a shorter sequence and showed statistically significant expression in M. anisopliae. This study introduces a highly efficient strong inducible promoter for over-expression of target genes in M. anisopliae.

Green Fluorescent Protein Expression in Pseudogymnoascus destructans to Study Its Abiotic and Biotic Lifestyles.

Pseudogymnoascus destructans (Pd) is the etiologic agent of bat White-nose syndrome, a disease that has caused the unprecedented reduction in the hibernating bat populations across eastern North America. The Pd pathogenesis appears to be a complex adaptation of fungus in its abiotic (caves and mines) and biotic (bats) environments. There is a general lack of experimental tools for the study of Pd biology. We described the successful expression of codon-optimized synthetic green fluorescent protein sGFP in Pd. The sGFP(S65T) gene was first fused in frame with the Aspergillus nidulans promoter in the tumor-inducing plasmid pRF-HUE, and the resulting plasmid pHUE-sGFP(S65T) was transformed into Pd by Agrobacterium tumefaciens-mediated transformation system. The integration of sGFP(S65T) in Pd genome was analyzed by PCR, and single integration frequency of approximately 66% was confirmed by Southern hybridization. Fluorescent microscopy and flow cytometric analyses of two randomly selected transformants with single integration revealed high expression of sGFP in both spores and hyphal structures. The biology of mutants as judged by sporulation, growth rate, and urease production was not altered indicating sGFP is not toxic to Pd. Thus, we have generated a valuable tool that will facilitate the elucidation of Pd biology, ecology, and pathogenicity in real time.

Gene Transfer to Rice Mediated by Agrobacterium tumefaciens: Transient Expression of sgfp in Rice Calluses of indica cv. Fatmawati and japonica cv. Nipponbare

Transient expression of synthetic green fluorescent protein (sgfp) mediated by Agrobacterium is rapid and useful approach for visual monitoring the genetic transformation event in transformed cells/tissues of examined genotype. The significant differences in transient expression of two genotypes (indica cv. Fatmawati and japonica cv. Nipponbare) were found with regard to the osmotic treatment (0.4 M mannitol), solid subcultured callus, and 10 min. air drying. While, no significant differences in sgfp expression were observed in two genotypes on without air-drying and 5 min. air-drying of calluses prior immersed in Agrobacterium suspension. Surprisingly, the sgfp expression could not be detected in suspension-subcultured callus of both cultivars. The highest sgfp expression was achieved in Nipponbare callus treated with 10 min. air drying. The level of green fluorescent spots was higher in Nipponbare than that in Fatmawati, however, plants regenerated from Fatmawati were considerably comparable with those of Nipponbare. Seventeen and 16 putative transgenic rice plants expressing sgfp transgene were obtained from Nipponbare and Fatmawati, respectively.

Introduction of the Serine Green Fluorescent Protein (sGFP) Gene into Pyricularia grisea Race dc4 Isolated from Digitaria ciliaris using Agrobacterium tumefaciens-mediated Genetic Transformation

Gene serin Green Fluorescent Protein (sGFP) has been used to monitor gene expression specific tagged proteins that has implication for fungal cell study. This research aimed to introduce sGFP gene into genome of P. grisea dc4 from D. ciliaris using A. tumefaciens. Plasmid sGFP was introduced into A. tumefaciens by triparental mating method (TPM). Genetic transformation was performed by co-cultivating spore P. grisea dc4 with A. tumefaciens LBA4404–pCAMB-sGFP. Pyricularia grisea dc4 transformant was selected by using selection medium that contains 300 µg/ml hygromycin. The integration of sGFP gene into genome was confirmed by PCR using sGFP’s spesific primer pair, sGFP-Nos terminator primer pair and β-Tubulin primer pair as internal control. Expression of sGFP from P. grisea dc4 transformant were detected with blue light fluorescent microscope.

Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes

Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS-ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi-in vitro, whereas normal PT growth was shown in its sense control in vitro/semi-in vitro. PT growth was impaired in a manner dependent on the dose of AS-ODNs against both ANX1 and ANX2 above 10 μm. The treatment with AS-ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS-ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS-ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS-ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.

Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus

It is important to develop native and highly efficient promoters for effective genetic engineering of filamentous fungi. Although Aspergillus terreus is an important industrial fungus for the production of itaconic acid and lovastatin, the available genetic toolbox for this microorganism is still rather limited. We have cloned the 5' upstream region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd; 2,150bp from the start codon) from A. terreus CICC 40205 and subsequently confirmed its promoter function using sgfp (synthetic green fluorescent protein) as the reporter. The sequence of the promoter PgpdAt was further analysed by systematic deletion to obtain an effective and compact functional promoter. Two truncated versions of PgpdAt (1,081 and 630bp) were also able to drive sgfp expression in A. terreus. The activities of these three PgpdAt promoters of varying different lengths were further confirmed by fluorescence, western blot and transcription. The shortest one (630bp) was successfully applied as a driver of vgb expression in the genetic engineering of A. terreus. The function of expressed haemoglobin was demonstrated by the CO (carbon monoxide)-difference spectrum and enhanced oxygen uptake rate, glucose consumption and itaconic acid titer. Our study was successful in developing and validating an efficient and compact native promoter for genetic engineering of A. terreus.

Development of an efficient Agrobacterium-mediated transformation system and production of herbicide-resistant transgenic plants in garlic (Allium sativum L.).

The genetic improvement of garlic plants (Allium sativum L.) with agronomical beneficial traits is rarely achieved due to the lack of an applicable transformation system. Here, we developed an efficient Agrobacterium-mediated transformation procedure with Danyang, an elite Korean garlic cultivar. Examination of sGFP (synthetic green fluorescence protein) expression revealed that treatment with 2-(N-morpholino) ethanesulfonic acid (MES), L-cysteine and/or dithiothreitol (DTT) gives the highest efficiency in transient gene transfer during Agrobacterium co-cultivation with calli derived from the roots of in vitro plantlets. To increase stable transformation efficiency, a two-step selection was employed on the basis of hygromycin resistance and sGFP expression. Of the hygromycin-resistant calli initially produced, only sGFP-expressing calli were subcultured for selection of transgenic calli. Transgenic plantlets produced from these calli were grown to maturity. The transformation efficiency increased up to 10.6% via our optimized procedure. DNA and RNA gel-blot analysis indicated that transgenic garlic plants stably integrated and expressed the phosphinothricin acetyltransferase (PAT) gene. A herbicide spraying assay demonstrated that transgenic plants of garlic conferred herbicide resistance, whilst nontransgenic plants and weeds died. These results indicate that our transformation system can be efficiently utilized to produce transgenic garlic plants with agronomic benefits.

Overexpression ofOsRab7B3, a Small GTP-Binding Protein Gene, Enhances Leaf Senescence in Transgenic Rice

Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress-inducible gene that plays an important role in the leaf senescence process.

Selection of co-transformed Dendrobium Sonia 17 using hygromycin and green fluorescent protein

Dendrobium Sonia 17 calluses were used for co-transformation study using particle bombardment. The bombarded transformed callus tissues were selected using half-strength MS medium containing 25 mg dm-3 hygromycin. Expression of green fluorescent protein (GFP) was observed in the callus and protocorm-like body (PLBs) tissues survived on the selection medium. The presence of green fluorescence protein (sgfp), hygromycin-B-phosphotransferase (hptII) and β-glucuronidase (uidA) genes in the transformed tissues were verified using PCR, Southern blot and dot blot analyses. Based on the results from PCR and expression of sgfp and uidA genes in the calluses and PLBs survived from hygromycin selection, we reported the co-transformation of sgfp, hptII and uidA genes into Dendrobium Sonia 17. GFP-expressing tissues were also observed in the regenerated transformed plantlets.

Selection of co-transformed Dendrobium Sonia 17 using hygromycin and green fluorescent protein

Dendrobium Sonia 17 calluses were used for co-transformation study using particle bombardment. The bombarded transformed callus tissues were selected using half-strength MS medium containing 25 mg dm-3 hygromycin. Expression of green fluorescent protein (GFP) was observed in the callus and protocorm-like body (PLBs) tissues survived on the selection medium. The presence of green fluorescence protein (sgfp), hygromycin-B-phosphotransferase (hptII) and β-glucuronidase (uidA) genes in the transformed tissues were verified using PCR, Southern blot and dot blot analyses. Based on the results from PCR and expression of sgfp and uidA genes in the calluses and PLBs survived from hygromycin selection, we reported the co-transformation of sgfp, hptII and uidA genes into Dendrobium Sonia 17. GFP-expressing tissues were also observed in the regenerated transformed plantlets.

An efficient protocol for particle bombardment-mediated transformation of Centella asiatica callus

In this study, we report an optimization of particle bombardment transformation system for Centella asiatica callus. A total of eight parameters affecting the genetic transformation system were optimized using the synthetic green fluorescent protein (sGFP) as a reporter driven by the CaMV 35S promoter. The results indicated that DNA delivery conditions of 9-cm target distance, 1,100 psi helium pressure, 1.0 μm gold particles size, 27 mmHg chamber vacuum pressure, 2 times number of bombardment, spermidine as precipitation agent, 60 h post-bombardment incubation time and 2 μg plasmid DNA concentration were optimal for C. asiatica callus transformation. The expression of sGFP was monitored using fluorescence microscope and further confirmed using RT-PCR. This optimized genetic transformation system is applicable for rapid transient gene analysis and transgenic C. asiatica production.

Green Fluorescent Protein (GFP) in Vector Systems Played Sense Role of Epigenetic in Plants

AbstractThe green fluorescent protein (GFP) of jellyfish (Aequorea victoria) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. However, sGFP(S65T) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(S65T) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(S65T) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system.

Transient Expression of Green Fluorescent Protein in Rice Calluses : Optimization of Parameters for Helios Gene Gun Device

An optimized condition for particle bombardment is necessary for efficient genetic transformation. Parameters for Helios gene gun, the new system for nucleic acid delivery which is mainly consists of hand-held device sold by Bio-Rad Laboratories (California USA), were examined based on transient expression of synthetic green fluorescent protein (sgfp) in rice calluses of indica cv. Fatmawati and japonica cv. Nipponbare. In the experimental conditions that we examined, parameters found to be the most favorable conditions for transient expression of sgfp in rice callus cells were as follows: 200−250 psi helium pressure, 0.6 μm gold particle size, 0.25 mg gold particles per shot, and 1.5 μg plasmid-DNA per shot. Desiccation of callus cells for eight min was also found appropriate. The level of transient sgfp expression was not significantly influenced by the pre-culture for 4 to 12 d before bombardment or by callus age between 10 and 33 wk old in Fatmawati. These parameters for this particular device should improve the transient expression, thus enabling stable expression of introduced genes via Helios gene gun using callus as a target tissue.

Improvement of protein quality in transgenic soybean plants

Glycinin is one of the abundant storage proteins in soybean seeds. A modified Gy1 (A1aB1b) proglycinin gene with a synthetic DNA encoding four continuous methionines (V3-1) was connected between the hpt gene and the modified green fluorescent protein sGFP(S65T) gene, and a resultant plasmid was introduced into soybean by particle bombardment in order to improve nutritional value of its seeds. After the selection with hygromycin, the efficiency of gene introduction was evaluated. More than 60 % of the regenerated plants tolerant to hygromycin yielded the hpt and V3-1 fragment by polymerase chain reaction (PCR) analysis, and the expression of sGFP was detected in about 50 % of putative transgenic soybeans. Southern hybridization confirmed the presence of transgenes in T0 plants and the transgenic soybeans hybridized with the hpt and V3-1 genes were analyzed showed different banding patterns. Most of the transgenic plants were growing, flowering normally and produced seeds. Analysis of seed obtained from transgenic soybean plants expressing hpt and V3-1 genes showed higher accumulation of glycinin compared with non-transgenic plants. In addition, protein expression in transgenic soybean plants was observed by using 2D-electrophoresis.

Molecular Control of Gene Co-suppression in Transgenic Soybean via Particle Bombardment

Molecular co-suppression phenomena are important to consider in transgene experiments. Embryogenic cells were obtained from immature cotyledons and engineered with two different gene constructs (pHV and pHVS) through particle bombardment. Both constructs contain a gene conferring resistance to hygromycin (hpt) as a selective marker and a modified glycinin (11S globulin) gene (V3-1) as a target. sGFP(S65T) as a reporter gene was, however, inserted into the flanking region of the V3-1 gene (pHVS). Fluorescence microscopic screening after the selection of hygromycin, identified clearly the expression of sGFP(S65T) in the transformed soybean embryos bombarded with the pHVS construct. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Seeds of transgenic plants obtained from the pHV construct frequently lacked an accumulation of endogenous glycinin, which is encoded by homologous genes to the target gene V3-1. Most of the transgenic plants expressing sGFP(S65T) showed highly accumulation of glycinin. The expression of sGFP(S65T) and V3-1 inherits into the next generations. sGFP(S65T) as a reporter gene may be useful to increase the transformation efficiency of transgenic soybean with avoiding gene co-suppression.

Efficient production of transgenic soybean (Glycine max [L] Merrill) plants mediated via whisker-supersonic (WSS) method

The present study was designed to evaluate the transformation efficiency and proof the capability of whisker supersonic (WSS) method as an alternative option for soybean (Glycine max [L] Merrill) transformation. We compared soybean transformation efficiency obtained by WSS-mediated with that of particle bombardment transformation by carrying out molecular analysis of the T0 plants in two independent experiments. For this, we used for both transformation techniques the same genotype, the same plasmid and the same selection method. To assess the efficiency of soybean genetic transformation, we evaluated the efficiency of multi gene transformation by the selection with hygromycin and the expression of green fluorescent protein [sGFP (S65T)] resulted from both techniques. Regenerable embryogenic cells were induced from immature cotyledons of soybean c.v Jack on MSD40 media within 3 weeks then proliferated on FN lite liquid media and engineered with pUHG gene construct through both WSS and particle bombardment-mediated transformation. The pUHG was constructed with pUC 19 and contain the hpt gene conferring resistance to hygromycin as a selective marker and sGFP(S65T) as a reporter gene. Fluorescence microscopy screening after the selection of hygromycin, identified the clearly expression of sGFP(S65T) in the transformed soybean embryos. Stable integration of the transgenes was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The average transformation efficiency achieved with WSS was higher than that obtained by particle bombardment and hence it may represent an alternative method for soybean transformation.

Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes

The green fluorescent protein (GFP) has been established as the premier in vivo reporter for investigations of gene expression, protein localization, and cell and organism dynamics. The fungal transformation vector pCT74, with sGFP under the control of the ToxA promoter from Pyrenophora tritici-repentis, effectively expresses GFP in a diverse group of filamentous ascomycetes. Due to the versatility of ToxA promoter-driven expression of GFP, we constructed an additional set of fluorescent protein expression vectors to expand the color palette of fluorescent markers for use in filamentous fungi. EYFP, ECFP and mRFP1 were successfully expressed from the ToxA promoter in its fungus of origin, P. tritici-repentis, and a distant relative, Verticillium dahliae. Additionally the ToxB promoter from P. tritici-repentis drove expression of sGFP in V. dahliae, suggesting a similar potential to the ToxA promoter for heterologous expression in ascomycetes. The suite of fungal transformation vectors presented here promise to be useful for a variety of fungal research applications.

Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes

The green fluorescent protein (GFP) has been established as the premier in vivo reporter for investigations of gene expression, protein localization, and cell and organism dynamics. The fungal transformation vector pCT74, with sGFP under the control of the ToxA promoter from Pyrenophora tritici-repentis, effectively expresses GFP in a diverse group of filamentous ascomycetes. Due to the versatility of ToxA promoter-driven expression of GFP, we constructed an additional set of fluorescent protein expression vectors to expand the color palette of fluorescent markers for use in filamentous fungi. EYFP, ECFP and mRFP1 were successfully expressed from the ToxA promoter in its fungus of origin, P. tritici-repentis, and a distant relative, Verticillium dahliae. Additionally the ToxB promoter from P. tritici-repentis drove expression of sGFP in V. dahliae, suggesting a similar potential to the ToxA promoter for heterologous expression in ascomycetes. The suite of fungal transformation vectors presented here promise to be useful for a variety of fungal research applications.

Recovery of herbicide-resistant Azuki bean [ Vigna angularis (Wild.), Ohwi & Ohashi] plants via Agrobacterium -mediated transformation

Transgenic azuki bean [ Vigna angularis (Willd.) Ohwi & Ohashi] plants expressing the hygromycin phosphotransferase ( hpt ), green fluorescent protein ( sgfp ) and phosphinothricin acetyltransferase ( bar ) genes were obtained by Agrobacterium- tumefacients - mediated transformation. A total of 210 epicotyl explants were inoculated with A. tumefaciens strain EHA105, harboring the binary plasmid pZHBG on MS co-cultivation medium supplemented with 100 mM acetosyringone and 10 mg/l of BA. Following selection on MS medium with 15 mg/l of hygromycin, the regenerated adventitious shoots that formed on the induced calli were further screened for sgfp expression before transferred to rooting medium. 31 transgenic plants were obtained with transformation frequency of 14%. The presence of transgenes in transformed azuki bean plants was confirmed by polymerase chain reaction (PCR) and southern blot analysis. Transcription of the bar and hpt genes was assessed by reverse transcription polymerase chain reaction (RT-PCR) analysis. sgfp- positive transgenic plants exhibited functional expression of the bar gene as determined by assaying for resistance to bialaphos applied directly to leaves. This result demonstrates the feasibility of introducing potentially useful agronomic traits into azuki bean through genetic engineering. Key Words: Agrobacterium tumefaciens, bar gene, bialaphos, transgenic, Vigna angulazris . African Journal of Biotechnology Vol.4(1) 2005: 61-67