Synthetic Green Fluorescent Protein Research Articles

Engineering a Yellow Thermostable Fluorescent Protein by Rational Design.

Thermal green protein (TGP) is an extremely stable, highly soluble synthetic green fluorescent protein. The quantum yield of TGP is lower than the closest related natural fluorescent protein, monomeric Azami-Green. We improved the thermal recovery of TGP through the introduction of a chromophore mutation, Q66E. Furthermore, we developed a yellow thermal protein (YTP) via mutation of histidine 193 to tyrosine. Incorporation of Q66E into YTP (YTP-E) improved chemostability and pH stability. Both YTP and YTP-E have superior thermostability compared to TGP or TGP-E. These proteins offer a new option for green or yellow fluorescence under harsh chemical or thermal conditions.

Complexation of Green and Red Kaede Fluorescent Protein Chromophores by a Zwitterion to Probe Electrostatic and Induction Field Effects.

The photophysics of green fluorescent protein (GFP) and red Kaede fluorescent protein (rKFP) are defined by the intrinsic properties of the light-absorbing chromophore and its interaction with the protein binding pocket. This work deploys photodissociation action spectroscopy to probe the absorption profiles for a series of synthetic GFP and rKFP chromophores as the bare anions and as complexes with the betaine zwitterion, which is assumed as a model for dipole microsolvation. Electronic structure calculations and energy decomposition analysis using Symmetry-Adapted Perturbation Theory are used to characterize gas-phase structures and complex cohesion forces. The calculations reveal a preponderance for coordination of betaine to the phenoxide deprotonation site predominantly through electrostatic forces. Calculations using the STEOM-DLPNO-CCSD method are able to reproduce absolute and relative vertical excitation energies for the bare anions and anion-betaine complexes. On the other hand, treatment of the betaine molecule with a point-charge model, in which the charges are computed from some common electron density population analysis schemes, show that just electrostatic and point-charge induction interactions are unable to account for the betaine-induced spectral shift. The present methodology could be applied to investigate cluster forces and optical properties in other gas-phase ion-zwitterion complexes.

Snake Toxins Labeled by Green Fluorescent Protein or Its Synthetic Chromophore are New Probes for Nicotinic acetylcholine Receptors.

Fluorescence can be exploited to monitor intermolecular interactions in real time and at a resolution up to a single molecule. It is a method of choice to study ligand-receptor interactions. However, at least one of the interacting molecules should possess good fluorescence characteristics, which can be achieved by the introduction of a fluorescent label. Gene constructs with green fluorescent protein (GFP) are widely used to follow the expression of the respective fusion proteins and monitor their function. Recently, a small synthetic analogue of GFP chromophore (p-HOBDI-BF2) was successfully used for tagging DNA molecules, so we decided to test its applicability as a potential fluorescent label for proteins and peptides. This was done on α-cobratoxin (α-CbTx), a three-finger protein used as a molecular marker of muscle-type, neuronal α7 and α9/α10 nicotinic acetylcholine receptors (nAChRs), as well as on azemiopsin, a linear peptide neurotoxin selectively inhibiting muscle-type nAChRs. An activated N-hydroxysuccinimide ester of p-HOBDI-BF2 was prepared and utilized for toxin labeling. For comparison we used a recombinant α-CbTx fused with a full-length GFP prepared by expression of a chimeric gene. The structure of modified toxins was confirmed by mass spectrometry and their activity was characterized by competition with iodinated α-bungarotoxin in radioligand assay with respective receptor preparations, as well as by thermophoresis. With the tested protein and peptide neurotoxins, introduction of the synthetic GFP chromophore induced considerably lower decrease in their affinity for the receptors as compared with full-length GFP attachment. The obtained fluorescent derivatives were used for nAChR visualization in tissue slices and cell cultures.

Sand-Wounding of Shoot and Petiole Explants Enhances Transformation Efficiency in Sugar Beet (Beta vulgaris L.) by Agrobacterium-mediated Transformation

Genetic transformation of recalcitrant species, like sugar beet, has been always a challenging task. In this study, a selectable marker gene (phosphomannose isomerase) and a reporter gene (synthetic green fluorescent protein) were transferred to sugar beet via Agrobacterium-mediated transformation by using sand-wounded shoot and petiole explants of two different diploid sugar beet genotypes. Considering the overall means of genotypes and explant types, a clear genotypic variation was evident as ELK345 produced a mean of 5.7% transient expression and 1.97% stable transformation efficiency while it was 4.3% and 1.28% for the genotype M1195. When explant types were compared independently from other parameters, it was shown that petiole explants were more productive than shoot explants, with an overall mean of 5.3% versus 4.7% and 1.77% versus 1.48% transient expressions and stable transformation efficiencies, respectively. On the other hand, when a comparison was made without taking into account the genotypes, the effect of wounding treatment on transient expression resulted in about 25% (4.7% vs. 5.9%) and 20% (4.3% vs. 5.1%) increases in petiole and shoot explants, respectively. Such a promoting effect of wounding was even more prominent when the stable transformation efficiencies were compared; petiole explants achieving almost a 60% increase (1.38% vs. 2.16%) while shoot explants doubled (0.98% vs. 1.97%) their efficiencies. The optimized protocol reported here can be employed for further increasing the transformation efficiency in recalcitrant plant species including sugar beet. The advantages of using GFP as a reporter and mannose as a positive selection are also discussed.

Comparative analysis of overexpressed Fragaria vesca S-adenosyl-l-methionine synthase (FvSAMS) and decarboxylase (FvSAMDC) during salt stress in transgenic Nicotiana benthamiana

We investigated the effect of overexpressing Fragaria vesca L. cv. Rügen S-adenosyl-l-methionine synthase (FvSAMS) and decarboxylase (FvSAMDC) genes on control and salt stressed Nicotiana benthamiana Domin plants. According to previous studies the overproduction of both proteins enhances the abiotic stress tolerance of plants, but the two enzymes have not yet been studied in one experimental system. We found that the transgenic plants subjected to long-term salt stress displayed higher levels of tolerance than the wild type (WT). In contrast to several earlier studies no antagonistic effect between ethylene and polyamine biosynthesis was observed in our experimental system. Overexpression of FvSAMDC had higher impact on the plant physiological parameters both in control and salt stress conditions, than that of FvSAMS. Based on the data measured in the FvSAMDC lines there appears to be a positive correlation between the free polyamine levels and the proline content as well as the amount of ethylene, while there is a negative correlation between the free polyamine levels and the lignin content in the plants exposed to salt stress. The transformation vectors contained the CaMV35S promoter, the coding sequence of FvSAMS and FvSAMDC fused with synthetic green fluorescent protein (sGFP). We detected the subcellular localization of both enzymes and examined the possible stress induced changes in their distribution. In the case of FvSAMS::sGFP nuclear, nucleolar, cytoplasmic (near to the plasmalemma), plastid membrane, whereas in FvSAMDC::sGFP nuclear and homogenous cytoplasmic localization was detected. Therefore, SAM is assumed to be produced in situ for numerous biochemical reactions.

Dual-Self-Restricted GFP Chromophore Analogues with Significantly Enhanced Emission.

The tremendous gap of fluorescence emission of synthetic green fluorescent protein (GFP) chromophore to the protein itself makes it impossible to use for applications in molecular and cellular imaging. Here, we developed an efficient methodology to enhance the photoluminescence response of synthetic GFP chromophore analogues by constructing dual-self-restricted chromophores. Single self-restricted chromophores were first generated with 2,5-dimethoxy substitution on the aromatic ring, which were further modified with phenyl or 2,5-dimethoxy phenyl to form dual-self-restricted chromophores. These two chromophores showed an obvious solvatofluorochromic color palette across blue to yellow with a maximum emission Stokes shift of 95 nm and dramatically enhanced fluorescence emission in various aprotic solvents, especially in hexane, where the QY reached around 0.6. Importantly, in acetonitrile and dimethyl sulfoxide, the fluorescence QYs of both chromophores were over 0.22, which were the highest reported so far in high polar organic solvents. Meanwhile, the fluorescence lifetimes also improved obviously with the maximum of around 4.5 ns. Theoretical calculations revealed a more favorable Mülliken atomic charge translocation over the double-bond bridge and illustrated much higher energy barriers for the Z/E photoisomerization together with larger bond orders compared with the GFP core chromophore, p-HBDI. Our work significantly improved the fluorescence emission of synthetic GFP chromophore analogues in polar solvents while reserved the multicolor emitting function, providing a solid molecular motif for engineering high-performance fluorescent probes.

The subcellular localization of two isopentenyl diphosphate isomerases in rice suggests a role for the endoplasmic reticulum in isoprenoid biosynthesis.

Both OsIPPI1 and OsIPPI2 enzymes are found in the endoplasmic reticulum, providing novel important insights into the role of this compartment in the synthesis of MVA pathway isoprenoids. Isoprenoids are synthesized from the precursor's isopentenyl diphosphate (IPP) and dimethylallyl diphosphosphate (DMAPP), which are interconverted by the enzyme isopentenyl diphosphate isomerase (IPPI). Many plants express multiple isoforms of IPPI, the only enzyme shared by the mevalonate (MVA) and non-mevalonate (MEP) pathways, but little is known about their specific roles. Rice (Oryza sativa) has two IPPI isoforms (OsIPPI1 and OsIPPI2). We, therefore, carried out a comprehensive comparison of IPPI gene expression, protein localization, and isoprenoid biosynthesis in this species. We found that OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues, and its expression in de-etiolated leaves mirrored the accumulation of phytosterols, suggesting a key role in the synthesis of MVA pathway isoprenoids. We investigated the subcellular localization of both isoforms by constitutively expressing them as fusions with synthetic green fluorescent protein. Both proteins localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids, due to an N-terminal transit peptide which is not present in OsIPPI1. Despite the plastidial location of OsIPPI2, the expression of OsIPPI2 mRNA did not mirror the accumulation of chlorophylls or carotenoids, indicating that OsIPPI2 may be a redundant component of the MEP pathway. The detection of both OsIPPI isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. Our work shows that the ER plays an as yet unknown role in the synthesis of MVA-derived isoprenoids, with important implications for the metabolic engineering of isoprenoid biosynthesis in higher plants.

Gateway binary vectors with organelle-targeted fluorescent proteins for highly sensitive reporter assay in gene expression analysis of plants

Fluorescent proteins are valuable tools in the bioscience field especially in subcellular localization analysis of proteins and expression analysis of genes. Fusion with organelle-targeting signal accumulates fluorescent proteins in specific organelles, increases local brightness, and highlights the signal of fluorescent proteins even in tissues emitting a high background of autofluorescence. For these advantages, organelle-targeted fluorescent proteins are preferably used for promoter:reporter assay to define organ-, tissue-, or cell-specific expression pattern of genes in detail. In this study, we have developed a new series of Gateway cloning technology-compatible binary vectors, pGWBs (attR1-attR2 acceptor sites) and R4L1pGWB (attR4-attL1 acceptor sites), carrying organelle-targeted synthetic green fluorescent protein with S65T mutation (sGFP) (ER-, nucleus-, peroxisome-, and mitochondria-targeted sGFP) and organelle-targeted tag red fluorescent protein (TagRFP) (nucleus-, peroxisome-, and mitochondria-targeted TagRFP). These are available for preparation of promoter:reporter constructs by an LR reaction with a promoter entry clone attL1-promoter-attL2 (for pGWBs) or attL4-promoter-attR1 (for R4L1pGWBs), respectively. A transient expression experiment with particle bombardment using cauliflower mosaic virus 35S promoter-driven constructs has confirmed the correct localization of newly developed organelle-targeted TagRFPs by a co-localization analysis with the previously established organelle-targeted sGFPs. More intense and apparent fluorescence signals were detected by the nucleus- and peroxisome-targeted sGFPs than by the normal sGFPs in the promoter assay using transgenic Arabidopsis thaliana. The new pGWBs and R4L1pGWBs developed here are highly efficient and may serve as useful platforms for more accurate observation of GFP and RFP signals in gene expression analyses of plants.

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.

In-vitro Transcribed mRNA Delivery Using PLGA/PEI Nanoparticles into Human Monocyte-derived Dendritic Cells.

Induction of protein synthesis by the external delivery of in-vitro transcription-messenger RNA (IVT-mRNA) has been a useful approach in the realm of cell biology, disease treatment, ‎reprogramming of cells, and vaccine design. Therefore, the development of new formulations for ‎protection of mRNA against nucleases is required to maintain its activity in-vivo. It was the aim of the present study to ‎investigate the uptake, toxicity, transfection efficiency as well as phenotypic consequences of ‎a nanoparticle (NP) in cell culture. NP consists of poly D, L-lactide-co-glycolide (PLGA) and polyethyleneimine (PEI) ‎for delivery of in-vitro transcription-messenger RNA (IVT- mRNA) encoded green fluorescent protein (GFP) in human monocyte-derived ‎dendritic cells (moDCs). Nanoparticles that were synthesized and encapsulated with synthetic GFP mRNA, exhibited size distribution in this formulation, with mean particle sizes ranging between 415 and 615 nm. Zeta potential was positive (above 12-13 mV) and the encapsulation efficiency exceeded 73.5%. Our results demonstrated that PLGA/PEI NPs encapsulation of GFP mRNA had ‎no toxic effect on immature monocyte-derived ‎dendritic cells and was capable of delivering of IVT-mRNA into moDCs and was highly effective. The expression of GFP protein 48 h after transfection was confirmed by flow cytometry, microscopic examination and western blotting assay. This NP can make a way to target moDCs to express a variety of antigens by IVT- mRNA. The ‎present study introduced the PLGA/PEI NP, which provided effective delivery of ‎IVT-mRNA that encodes the GFP protein.

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.

Synthetic green fluorescent protein (GFP) chromophore analog for rapid, selective and sensitive detection of cyanide in water and in living cells

Here, we report Green Fluorescent Protein (GFP) chromophore analog as a turn-on fluorescent chemodosimeter (THBI) for selective detection of cyanide in water, on solid state and in living cells. The detection limit was found to be 0.17 μM (4.5 ppb). The time dependent study revealed that there is a rapid enhancement in fluorescence intensity (in less than 5s) and was constant over the period of 1 h. Cell imaging data exhibited that THBI was successfully crossed cell membrane and visualized fluorescence response in live HCT cells.

Synthetic green fluorescent protein chromophore analogues with a positive charge at the phenyl-like group.

Synthetic green fluorescent protein (GFP) chromophore analogues with a positive charge at the phenyl-like group have the highly electrophilic amidine carbon, smaller LUMO-HOMO energy gap, red-shifted electronic absorptions and fluorescent emissions, and accelerated E-Z thermoisomerization rates. They are water-labile and their hydrolysis results in ring-opening of the imidazolinone moiety with a half life around 25-37h in D2O at 25°C.

P gas , a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

The dynamic control of gene expression is important for adjusting fluxes in order to obtain desired products and achieve appropriate cell growth, particularly when the synthesis of a desired product drains metabolites required for cell growth. For dynamic gene expression, a promoter responsive to a particular environmental stressor is vital. Here, we report a low-pH-inducible promoter, Pgas, which promotes minimal gene expression at pH values above 5.0 but functions efficiently at low pHs, such as pH 2.0. First, we performed a transcriptional analysis of Aspergillus niger, an excellent platform for the production of organic acids, and we found that the promoter Pgas may act efficiently at low pH. Then, a gene for synthetic green fluorescent protein (sGFP) was successfully expressed by Pgas at pH 2.0, verifying the results of the transcriptional analysis. Next, Pgas was used to express the cis-aconitate decarboxylase (cad) gene of Aspergillus terreus in A. niger, allowing the production of itaconic acid at a titer of 4.92 g/liter. Finally, we found that Pgas strength was independent of acid type and acid ion concentration, showing dependence on pH only.IMPORTANCE The promoter Pgas can be used for the dynamic control of gene expression in A. niger for metabolic engineering to produce organic acids. This promoter may also be a candidate tool for genetic engineering.

Self-restricted oxazolone GFP chromophore for construction of reaction-based fluorescent probe toward dopamine

The β-barrel provides a confined environment for chromophores of the green fluorescent protein (GFP) family, defining their emission profiles by the chromophore/β-barrel interactions. Here, we describe the generation of self-restricted oxazolone GFP chromophore (GFPc) for construction of reaction-based fluorescent probe toward dopamine by mimicking the confinement effect of the β-barrel. Through standard synthetic method, the first self-restricted GFPc oxazolone analogue (MBDO) and the conventional pyrenyl-based chromophore (PDO) were prepared respectively. Under the same condition, MBDO shows much better emission response with fluorescent quantum yield (QY) over one order of magnitude higher than that of PDO due to the generation of the self-restricted effect. And, the fluorescent QY of MBDO reaches above 30% in dimethyl sulfoxide, which is the largest ever recorded for unlocked GFPc analogues in highly polar solvents. Moreover, theoretical calculations further reveal that the enhanced emission of MBDO is due to the inhibition of conformational motions around the exocyclic CC bonds. Combination the enhanced emission and the reactivity of the lactone, MBDO is applied to construct reaction-based fluorescent probe toward dopamine via a ring-opening reaction of the lactone. Prospectively, the destruction of the oxazolone would break the effective conjugated structure of the chromophore, which can decrease the corresponding fluorescence. This work puts forward a novel approach to generate highly emissive GFPc oxazolone analogue, which can be used to fabricate reaction-based fluorescent probe toward dopamine, potentially promoting the biochemical applications using synthetic GFP chromophore analogues.

Emission enhancement and application of synthetic green fluorescent protein chromophore analogs

Emission enhancement and application of GFPc analogs are achieved via chemical modification or physical encapsulation due to conformational motion inhibition.

Plastoglobule-Targeting Competence of a Putative Transit Peptide Sequence from Rice Phytoene Synthase 2 in Plastids.

Plastoglobules (PGs) are thylakoid membrane microdomains within plastids that are known as specialized locations of carotenogenesis. Three rice phytoene synthase proteins (OsPSYs) involved in carotenoid biosynthesis have been identified. Here, the N-terminal 80-amino-acid portion of OsPSY2 (PTp) was demonstrated to be a chloroplast-targeting peptide by displaying cytosolic localization of OsPSY2(ΔPTp):mCherry in rice protoplast, in contrast to chloroplast localization of OsPSY2:mCherry in a punctate pattern. The peptide sequence of a PTp was predicted to harbor two transmembrane domains eligible for a putative PG-targeting signal. To assess and enhance the PG-targeting ability of PTp, the original PTp DNA sequence (PTp) was modified to a synthetic DNA sequence (stPTp), which had 84.4% similarity to the original sequence. The motivation of this modification was to reduce the GC ratio from 75% to 65% and to disentangle the hairpin loop structures of PTp. These two DNA sequences were fused to the sequence of the synthetic green fluorescent protein (sGFP) and drove GFP expression with different efficiencies. In particular, the RNA and protein levels of stPTp-sGFP were slightly improved to 1.4-fold and 1.3-fold more than those of sGFP, respectively. The green fluorescent signals of their mature proteins were all observed as speckle-like patterns with slightly blurred stromal signals in chloroplasts. These discrete green speckles of PTp-sGFP and stPTp-sGFP corresponded exactly to the red fluorescent signal displayed by OsPSY2:mCherry in both etiolated and greening protoplasts and it is presumed to correspond to distinct PGs. In conclusion, we identified PTp as a transit peptide sequence facilitating preferential translocation of foreign proteins to PGs, and developed an improved PTp sequence, a stPTp, which is expected to be very useful for applications in plant biotechnologies requiring precise micro-compartmental localization in plastids.

Green fluorescent protein chromophore derivative suppresses ultraviolet A-induced JNK-signalling and apoptosis in keratinocytes and adverse effects in zebrafish embryos.

Solar ultraviolet (UV) light has been recognized as the important environmental hazard and contributes to diverse skin damage such as cell death, photoageing and even carcinogenesis. Revelation of harmful responses attributed to UVA radiation has promoted the development of photoprotective agents against UVA-induced skin damage. In the present study, we tried to evaluate the potential protective effects of a synthetic green fluorescent protein (GFP) chromophore derivative, 4-chlorobenzyldene-1, 2-dimethylimidazolinone (Cl-BDI, called TC-22) on UVA- and UVB- induced stress responses in skin. The HaCaT keratinocytes were used to evaluate the cellular effects. Zebrafish (Danio rerio), which is regarded as a useful and cost-effective alternative to some mammalian models, was applied as the in vivo animal model. In HaCaT keratinocytes, TC-22 was able to obviously decrease UVA-induced cell death. Dissection of the UVA-induced signalling pathways revealed that TC-22 could suppress the activation of JNK and caspase 3, but not of ERK and p38. Reduction of UVA-induced cleavage of caspase 3 and sub-G1 phase accumulation by pretreatment of TC-22 was also observed. In zebrafish, we showed that UVA irradiation could decrease the survival and hatching rate, suppress heart beats of embryos and enhance the pigmentation of larvae. Pretreatment of TC-22 could significantly reverse UVA-induced the suppression in hatching of eggs and heart beating of embryos and also lowered the UVA-induced pigmentation in zebrafish. Collectively, we demonstrate that TC-22, a GFP chromophore derivative, can ameliorate the UVA-induced stress responses in both epidermal keratinocytes and zebrafish, suggesting the potential use of TC-22 in photoprotection in the future.

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.

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.