GUS Fusion Gene Research Articles

Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco

<p>Reporter gene assays are commonly used to study the expression pattern of a gene and the promoter activity. The purpose of this study was to assemble the chimeric gene constructs consisting of CaMV 35S promoter orOsAER1 gene promoter connected to the β-glucuronidase (GUS) reporter gene encoding the β-glucuronidase enzyme and to obtain an efficient method for Agrobacterium tumefaciens-mediated transient transformation of tobacco sprouts. The CaMV 35S promoter fragment reamplified from pCAMBIA1301 binary vector and the OsAER1 gene promoter fragment amplified from rice cv. Awan Kuning were ligated into pCAMBIA1300int::gus::tNOS to produce binary vectors pCAMBIA1300int::p35S::gus::tNOS and pCAMBIA1300int::prOsAER1::gus::tNOS. The vectors were used for transient transformation of 5–day old tobacco seedlings. The transformation was carried out using two bacterial cultures with densities of OD600 = 0.5 or OD600 = 1.0 combined with a vacuum for 15 or 30 minutes. Tobacco seedlings transformed with pCAMBIA1300int::p35S::gus::tNOS showed higher transformation efficiency as compared tothe ones transformed with pCAMBIA1300int::prOsAER1::gus::tNOS. A higher efficiency was obtained from transformation using bacterial culture with density of OD600 = 0.5 in combination with a vacuum for 30 minutes. Expression of GUS gene in the tobacco sprouts transformed with CaMV 35S promoter construct was observed through out the sprouts area (root, hypocotyl, cotyledon, and leaf), where as expression of GUS gene was observed in root, hypocotyl, and cotyledon, but not in leaf on tobacco sprouts transformed with OsAER1 promoter construct. These results indicate that the transient transformation is a quick and simple method for testing a chimeric gene construct.</p>

High throughput screening of β-glucuronidase (GUS) reporter in transgenic microalgae transformed by Agrobacterium tumefaciens

GUS (β-glucuronidase) is a chemiluminescent reporter gene that has been used in E. coli, fungi, yeast, mammalian cells, plant cells and microalgae. Currently, the GUS gene fusion system used for detection of GUS enzyme activity is carried out by cell lysis and requires 500 μl cell lysate. Hence, GUS activity cannot be assessed by high throughput screening (HTS) method. This study addresses this by using a HTS technique to quickly isolate transgenic strains (shown using Chlorella vulgaris) expressing high GUS activity in a 96 well microplate format. In this technique, quantitative results were obtained without carrying out cell lysis and all the experiments were carried out in a 96 well microplate. The method developed is cost effective, less labor intensive and can be carried out in a timely manner.For this, a new GUS reporter vector pBIN + TetR + TetO was developed, followed by transformation (Agrobacterium tumefaciens), screening and characterization of the transgenic C. vulgaris. In the screening study, strain number 18 showed the highest fluorescence intensity (16,988 ± 1168). The GUS enzyme was found to be stable for >8 h for intact cell and lysed cell studies. The optimum concentration of Triton X-100 to release the product (4-Methylumbelliferone) into buffer was 0.1% and the fluorescence intensity was 28,397 ± 787. The values of Km and Vmax of the recombinant GUS for lysed cells were 0.1304 ± 0.0101 mM and 0.35 ± 0.004 pmol 4-MU/min/ml of crude cell lysate respectively.

Isolation and characterization of a novel pollen-specific promoter in maize (Zea mays L.).

ZmSTK2_USP, located on the long arm of chromosome 4, belongs to the serine/threonine kinase gene in maize. The sequence analysis of 2100 bp upstream from the start codon ATG has shown that it contains cis-element motifs and two types of anther/pollen-specific promoter elements (GTGA and AGAAA), suggesting that it is the pollen-specific promoter. To investigate the function of ZmSTK2_USP promoter, the GUS gene fusion system was employed. In proZmSTK2_USP-GUS genetically modified plants, GUS activity was detected in mature pollen grains and pollen tubes but not found in other floral and vegetative tissues. These results show that proZmSTK2_USP is the pollen-specific promoter and drives pollen-specific activity during the middle stage of pollen development until pollen maturation.

Isolation and characterization of Calcineurin B-like gene (PbCBL1) and its promoter in birch-leaf pear (Pyrus betulifolia Bunge).

Calcium plays a critical role in regulating abiotic stress responses in plants. Calcineurin B-like (CBL) proteins are calcium sensors in calcium signaling pathways. However, the molecular mechanisms underlying calcium signaling remain to be elucidated. In this study, the CBL1 gene, which codes for the CBL protein, was isolated from the birch-leaf pear. One 2,969-bp sequence was cloned using PCR, and using the cloned 2,027-bp sequence was isolated from pear genomic DNA via genome walking. Sequencing analysis revealed that the 4,996-bp sequence was a PbCBL1 gene consisting of eight exons and seven introns, and the 2,027-bp sequence was identified as the promoter of the PbCBL1 gene, which contains the basic promoter elements TATA and CAAT boxes. In addition, some other cis-acting elements including heat, cold, drought, and hormone responsive elements were also present. To further investigate the activity of this promoter, the sequence was used to drive a GUS fusion gene into leaf discs of tobacco (Nicotiana benthamiana) with Agrobacterium-mediated transformation method. GUS gene expression could be regulated by the PbCBL1 promoter following induction by GA, ABA, SA, and MeJA. Furthermore, the results of real-time RT-qPCR indicate that the PbCBL1 gene can respond to changes in the intracellular calcium concentration, and that it can be induced by cold, heat, drought, and stress by several hormones including GA, ABA, SA, and MeJA. PbCBL1 gene may be involved in several signal transduction pathways, and play an important role in the condition of adversity stress in pear.

Class I patatin genes from potato (Solanum tuberosum L.) cultivars: molecular cloning, sequence comparison, prediction of diverse cis-regulatory motifs, and assessment of the promoter activities under field and in vitro conditions

Patatin, the most abundant glycoprotein (∼40 kDa) in potato (Solanum tuberosum L.) tuber is encoded by a large multigene family comprising of two classes. Class I patatin gene promoters are attractive molecular tools for studying multiple facets of transcriptional regulation. Only a few members of this class are relatively well understood at the molecular level. Here, we report the isolation of two new partial class I patatin genes with corresponding 5′-flanking regions, designated as StPN02 and StPK01, from two commercially important Indian potato cultivars, ‘Kufri Jyoti’ and ‘Kufri Chipsona-1’, respectively. Their 5′-flanking regions were found to vary significantly because of insertions/deletions in addition to point mutations. A major 487-bp insertion in the 5′-flanking region of StPN02 was a distinctive from other family members. Many of the diverse cis-regulatory sequence motifs predicted in this study have not been previously documented. The 1,215 and 2,225 bp 5′-flanking regions of StPN03 (derived from StPN02) and StPK01, respectively, were used in generating patatin promoter–GUS gene fusions for assessment of promoter activities in potato. Under field conditions, both StPK01 and StPN03 promoters showed tuber-specificity. The former was significantly stronger than the latter as examined by histochemical and fluorometric β-glucuronidase assays. Under in vitro conditions, these promoters were highly inducible by sucrose in stems and leaves. Apart from the applied aspects of this research, this study may help in unraveling the molecular mechanisms involved in the regulation of patatin gene expression.

The non-catalytic N-terminal domain of ACS7 is involved in the post-translational regulation of this gene in Arabidopsis.

Post-transcriptional control of the expression of the 1-aminocyclopropane-1-carboxylate synthase (ACS) gene family is important for maintaining appropriate levels of ethylene production. However, the molecular mechanism underlying the post-transcriptional regulation of type 3 ACS proteins remains unclear. Multiple sequence alignment revealed that the N-terminus of type 3 ACSs was longer than that of other ACSs. Fusing the N-terminal 54 residues of ACS7, the sole type 3 ACS in Arabidopsis, to the β-glucuronidase (GUS) reporter significantly decreased the stability of N(7(1-54))-GUS fusion protein. Among these 54 residues, residues 1-14 conferred this negative effect on the GUS fusion gene. Consistently, a truncated form of ACS7 lacking residues 1-14 was more stable than full-length ACS7 when transgenically expressed in Arabidopsis and led to a more severe ethylene response phenotype in the light-grown transgenic seedlings. Interestingly, the ACS7 N-terminus had no effect on the stability of N(7)-GUS and ACS7 proteins at the etiolated seedling stage. Both exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) treatment and salt stress could rescue the levels of accumulation of N(7)-GUS fusion protein in light-grown seedlings. These results suggest that the non-catalytic N-terminus of ACS7 is involved in its own post-translational regulation. The proteasome inhibitor MG132 suppressed degradation of full-length ACS7 in vivo, but had little effect on the N-terminal truncated form of ACS7, indicating that the N-terminus mediates the regulation of ACS7 stability through the ubiquitin-26S proteasome pathway.

Isolation and characterization of Histone1 gene and its promoter from tea plant (Camellia sinensis)

One 1.2 kbp long sequence was cloned by using PCR with primers that were designed from cDNA sequence of CsH1 gene (Genbank: EU716314) from tea plant (Camellia sinensis). According to the 1.2 kbp sequence, a 0.6 kbp sequence was isolated from tea plant genomic DNA using DNA Walking Method. Sequence analysis revealed that the 1.2 kbp sequence is a CsH1 gene consisting of 1 exon and 2 introns, the border of exton and intron sequences conforming to the GT-AG rule, and the 0.6 kbp sequence was found to be the promoter of CsH1 gene which contains basic promoter elements, TATA-box and CAAT-box. Abscisic acid responsiveness cis-acting element, elictor-responsive element, GA response element, light response cis-acting element and TC-rich repeats were also represented. To further study the activity of this promoter, the sequence was used to drive a GUS fusion gene in Agrobacterium-mediated transformation of tea plant somatic embryos, leaf discs and calli of tobacco (Nicotiana tabacum L.) where a high level of GUS expression was both observed in the tobacco calli and tea plant somatic embryos. These results suggest that the CsH1 gene promoter isolated is capable of conferring nuclear gene expression.

A Basic Helix-Loop-Helix Gene from Poplar is Regulated by a Basic Leucine-Zipper Protein and is Involved in the ABA-Dependent Signaling Pathway

Basic helix-loop-helix (bHLH) transcription factors (TF) comprise a large group of proteins that are involved in many developmental and physiological processes in plants. In this study, a bHLH gene (PkbHLH2), along with its promoter, was cloned from Populus koreana Rehd. A PkbHLH2 promoter::GUS gene fusion construct was generated to investigate the expression of PkbHLH2. The results demonstrated that PkbHLH2 was expressed mainly in leaf stalks, leaf veins and roots. Yeast one-hybrid assays showed that a bZIP gene product (PkbZIP2) can bind specifically to the ABA-responsive elements (ABRE) that exist in the promoter region of PkbHLH2, regulating the expression of PkbHLH2. In addition, the “GC” of the ABRE core motif “ACGTG” was very important for PkbZIP2 recognition, because its mutation to “TT” completely prevented the interaction between PkbZIP2 and ABRE. Furthermore, both PkbHLH2 and PkbZIP2 can be up-regulated by abscisic acid (ABA) and osmotic stress, and share similar expression patterns when exposed to ABA and osmotic stress. These results suggest that PkbZIP2 is an upstream regulator of PkbHLH2, which can control the expression of PkbHLH2 through an ABA-dependent signaling pathway.

Characterization of the Hormone and Stress-Induced Expression of FaRE1 Retrotransposon Promoter in Strawberry

Retrotransposons are the most abundant mobile elements in the plant genome and seem to play an important role in genome reorganization induced by environmental challenges. Their success in this function depends on the ability of their promoters to regulate plant adaptation to biotic and abiotic stresses. In this study, the promoter region of FaRE1 was amplified in the strawberry genome, and promoter::GUS fusion was constructed. We produced transgenic strawberry plants carrying FaRE1 promoter::GUS-fusion genes, and monitored GUS reporter activity. Histochemical and fluorimetric GUS analysis these plants showed the characteristics of the FaRE1 promoter were activated by either hormones treatments with ABA, NAA, and 2,4-D or cold stress. In addition, we found the GUS reporter was activated in the leaves of transgenic strawberry plants using 5-azaC. These results suggest that the promoter of FaRE1 may act as different signal transduction pathways, allowing FaRE1 retrotransposon to be activated in response to multiples challenges.

MIZU-KUSSEI1 plays an essential role in the hydrotropism of lateral roots in Arabidopsis thaliana

Root hydrotropism is the directional growth of roots toward a source of moisture in response to a water potential gradient. Root hydrotropism is therefore one of the most important factors for efficient water acquisition under water-limited conditions. However, the mechanisms underlying hydrotropism are not well understood. A previous study of the hydrotropism of primary roots in Arabidopsis thaliana identified the MIZU-KUSSEI (MIZ) 1 and MIZ2 genes as essential for this process. However, despite the important role of lateral roots in water acquisition under water deficient conditions, the mechanism of hydrotropism in lateral roots has not been investigated to date. The present study established an experimental system for the induction of lateral root hydrotropism in Arabidopsis and analyzed its characteristics in comparison with those of primary roots. Lateral roots were found to exhibit hydrotropism irrespective of their length. Expression of a pMIZ1:: GUS fusion gene at the tip of lateral roots and assessment of lateral root hydrotropism in the ahydrotropic mutant miz1, which did not display hydrotropism, demonstrated that the lateral roots of Arabidopsis have the capacity to respond to water potential gradients, and that this activity is regulated by MIZ1.

Genomic organization, induced expression and promoter activity of a resistance gene analog (PmTNL1) in western white pine (Pinus monticola)

Cronartium ribicola causes white pine blister rust (WPBR) in subgenus Strobus. Various genetic and molecular approaches were used to detect white pine genes contributing to host resistance. The molecular role of the NBS-LRR family is highly related to plant immuno-activity against various pathogens and pests. In the present study, genomic organization of a resistance gene analog (RGA), designated as PmTNL1, and its allelic variants were characterized in Pinus monticola. PmTNL1 showed high identity with TIR-NBS-LRR proteins from other plants. qRT-PCR revealed that the PmTNL1 transcript was expressed at low basal levels in different tissues and exhibited similar patterns during compatible and incompatible interactions of P. monticola with C. ribicola at early stages post inoculation. In comparison, PmTNL1 was up-regulated significantly in diseased P. monticola tissues with WPBR symptoms. Expression of the PmTNL1 promoter::GUS fusion gene in transgenic Arabidopsis demonstrated that GUS signal appeared only inside phloem tissues of young seedlings and at hydathodes and branching and organ-connecting points in mature Arabidopsis plants. Similar to the endogenous expression pattern for this gene in pine, GUS activity was up-regulated significantly around vascular tissues locally at pathogen infection sites, but little or no induction was observed in response to abiotic stresses. A DNA marker was developed based on variation of the LRR-coding region, and PmTNL1 was mapped to one genetic linkage group using a pedigree with major dominant gene (Cr2) conferring HR resistance to C. ribicola. These results suggest that PmTNL1 may play an important role in white pine partial resistance against C. ribicola.

Spatial association of photosynthesis and chemical defense inArabidopsis thalianafollowing herbivory byTrichoplusia ni

Because they share common precursors and require significant amounts of energy, photosynthesis and defense against herbivores and pathogens may be inversely related. This relationship was examined in Arabidopsis thaliana exposed to herbivory by Trichoplusia ni neonates. The spatial pattern of photosynthesis was compared statistically with that of induction of the defense-related cinnamate-4-hydroxylase (C4H) gene across individual leaves exposed to herbivory in transgenic plants harboring a C4H:GUS gene fusion. In portions of the leaf where C4H:GUS expression was upregulated, photosynthesis was depressed, while non-photochemical quenching was increased, suggesting a trade-off between these two processes. However, photosynthetic damage spread further into surrounding areas than the induction of C4H:GUS expression. Photosynthetic depression was observed up to 1 mm from the edges of holes, whereas C4H:GUS induction typically was limited to about 0.5 mm or less from edges. Other mechanisms may be responsible for the spread of photosynthetic damage beyond where C4H-related defense was induced. Alternatively, C4H induction may reflect a subset of defensive responses more limited in their spatial distribution than the downregulation of photosynthesis. The suppression of photosynthesis in remaining leaf tissue represents a 'hidden cost' of herbivore damage.

Zn stress-induced inhibition of bean PvSR2– GUS fusion gene splicing is gene-specific in transgenic tobacco

The stress-related gene no. 2 of Phaseolus vulgaris (PvSR2) is metal inducible and contains a single intron. Here, we report that Zn stress inhibited the splicing of the PvSR2-beta-glucuronidase (GUS) fusion gene in a concentration- and time-dependent manner in tobacco seedlings. The inhibition appears to be specific for the PvSR2-GUS transgene: splicing of four endogenous tobacco genes was unaffected by Zn stress. Our results provide in vivo evidence that Zn stress-dependent intron retention is transgene specific in plants.

Life with and without AtTIP1;1, an Arabidopsis aquaporin preferentially localized in the apposing tonoplasts of adjacent vacuoles

The Arabidopsis thaliana Tonoplast Intrinsic Protein 1;1 (AtTIP1;1) is a member of the tonoplast aquaporin family. The tissue-specific expression pattern and intracellular localization of AtTIP1;1 were characterized using GUS and GFP fusion genes. Results indicate that AtTIP1;1 is expressed in almost all cell types with the notable exception of meristematic cells. The highest level of AtTIP1;1 expression was detected in vessel-flanking cells in vascular bundles. AtTIP1;1-GFP fusion protein labelled the tonoplast of the central vacuole and other smaller peripheral vacuoles. The fusion protein was not found evenly distributed along the tonoplast continuum but concentrated in contact zones of tonoplasts from adjacent vacuoles and in invaginations of the central vacuole. Such invaginations may result from partially engulfed small vacuoles. A knockout mutant was isolated and characterized to gain insight into AtTIP1;1 function. No phenotypic alteration was found under optimal growth conditions indicating that AtTIP1;1 function is not essential to the plant and that some members of the TIP family may act redundantly to facilitate water flow across the tonoplast. However, a conditional root phenotype was observed when mutant plants were grown on a glycerol-containing medium.

Characterization of a Highly Active Promoter, PBbgpd, in Beauveria bassiana

The promoter of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene from Aspergillus nidulans (PgpdA) is widely used to direct expression of target genes constitutively in fungi. However, in some species, a heterogeneous promoter is found to be of low efficiency. To obtain a high-efficiency promoter for transformation of Beauveria bassiana, an entomopathogenic fungus widely used as an mycoinsecticide, a glyceraldehyde-3-phosphate dehydrogenase gene (Bbgpd) promoter, was cloned and characterized. Four deletion constructs (-2118, -1153, -726, and -354) of the 5'-upstream sequence of Bbgpd linked to a bar::gus fusion gene (phosphinothricin-resistance::beta-glucuronidase fused gene), which were used as selected marker gene and report gene, respectively, were generated. GUS activities of transgenic strains harboring -726, -1153, and -2118 deletion constructs were much stronger than that of the promoter of Aspergillus nidulans gpdA (PgpdA), with a twofold to threefold increase over that in the PgpdA construct. The -726 fragment was necessary to direct GUS expression in B. bassiana. No -354 transgenic progenies were obtained, possibly because it failed to initiate the transcription of bar::gus fusion gene. A remarkable increase of GUS activity was found between the -1153 and -726 constructs, indicating that some active transcriptional elements were located in this region. With a high expression level and relatively short sequence, PBbgpd can be used to drive target genes in B. bassiana transgenic research.

A gene essential for hydrotropism in roots

Roots display hydrotropism in response to moisture gradients, which is thought to be important for controlling their growth orientation, obtaining water, and establishing their stand in the terrestrial environment. However, the molecular mechanism underlying hydrotropism remains unknown. Here, we report that roots of the Arabidopsis mutant mizu-kussei1 (miz1), which are impaired in hydrotropism, show normal gravitropism and elongation growth. The roots of miz1 plants showed reduced phototropism and a modified wavy growth response. There were no distinct differences in morphological features and root structure between miz1 and wild-type plants. These results suggest that the pathway inducing hydrotropism is independent of the pathways used in other tropic responses. The phenotype results from a single recessive mutation in MIZ1, which encodes a protein containing a domain (the MIZ domain) that is highly conserved among terrestrial plants such as rice and moss. The MIZ domain was not found in known genomes of organisms such as green algae, red algae, cyanobacteria, or animals. We hypothesize that MIZ1 has evolved to play an important role in adaptation to terrestrial life because hydrotropism could contribute to drought avoidance in higher plants. In addition, a pMIZ1::GUS fusion gene was expressed strongly in columella cells of the root cap but not in the elongation zone, suggesting that MIZ1 functions in the early phase of the hydrotropic response.

A novel wound-responsive cis-element, VWRE, of the vascular system-specific expression of a tobacco peroxidase gene, tpoxN1

The wound-induced expression of tpoxN1, encoding a tobacco peroxidase, is unique because of its vascular system-specific expression and insensitivity to known wound-signal compounds such as jasmonic acid, ethylene, and plant hormones [Sasaki et al. (2002) Plant Cell Physiol 43:108-117]. To study the mechanism of expression, the 2-kbp tpoxN1 promoter region and successive 5'-deletion of the promoter were introduced as GUS fusion genes into tobacco plants. Analysis of GUS activity in transgenic plants indicated that a vascular system-specific and wound-responsive cis-element (VWRE) is present at the -239/-200 region of the promoter. Gel mobility shift assays suggested that a nuclear factor(s) prepared from wounded tobacco stems binds a 14-bp sequence (-229/-215) in the -239/-200 region in a sequence-specific manner. A mutation in this 14-bp region of the -239 promoter fragment resulted in a considerable decrease in wound-responsive GUS activity in transgenic plants. An 11-bp sequence, which completely overlaps with the 14-bp sequence, was found in the 5' distal region (-420/-410) and is thought to contribute to the wound-induced expression together with the 14-bp. The -114-bp core promoter of the tpoxN1 gene was indispensable for wound-induced expression, indicating that the 14-bp region is a novel wound-responsive cis-element VWRE, which may work cooperatively with other factors in the promoter.

RNA Interference of the Arabidopsis Putative Transcription Factor TCP16 Gene Results in Abortion of Early Pollen Development

Pollen development is a fundamental and essential biological process in seed plants. Pollen mother cells generated in anthers undergo meiosis, which gives rise to haploid microspores. The haploid cells then develop into mature pollen grains through two mitotic cell divisions. Although several sporophytic and gametophytic mutations affecting male gametogenesis have been identified and analyzed, little is known about the underlying molecular mechanism. In this study, we investigated the function of the TCP16 gene, which encodes a putative transcription factor. Expression analysis of the promoter::GUS fusion gene revealed that TCP16 transcription occurred predominantly in developing microspores. GUS expression began at the tetrad stage and markedly increased in an early unicellular stage. Transgenic plants harboring a TCP16 RNA interference (RNAi) construct generated equal amounts of normal and abnormal pollen grains. The abnormal pollen grains exhibited morphological abnormality and degeneration of genomic DNA. The defective phenotype of the RNAi plants was first detectable at the middle of the unicellular stage. Our results therefore suggest that TCP16, a putative transcription factor, plays a crucial role in early processes in pollen development.

Transgenic sterility in Populus: expression properties of the poplar PTLF, Agrobacterium NOS and two minimal 35S promoters in vegetative tissues

Transgenic sterility is a desirable trait for containment of many kinds of transgenes and exotic species. Genetically engineered floral sterility can be imparted by expression of a cytotoxin under the control of a predominantly floral-tissue-specific promoter. However, many otherwise desirable floral promoters impart substantial non-floral expression, which can impair plant health or make it impossible to regenerate transgenic plants. We are therefore developing a floral sterility system that is capable of attenuating undesired background vegetative expression. As a first step towards this goal, we compared the vegetative expression properties of the promoter of the poplar (Populus trichocarpa Torr. & Gray) homolog of the floral homeotic gene LEAFY (PTLF), which could be used to impart male and female flower sterility, to that of three candidate attenuator-gene promoters: the cauliflower mosaic virus (CaMV) 35S basal promoter, the CaMV 35S basal promoter fused to the TMV omega element and the nopaline synthase (NOS) promoter. The promoters were evaluated via promoter::GUS gene fusions in a transgenic poplar hybrid (Populus tremula L. x P. alba L.) by both histochemical and fluorometric GUS assays. In leaves, the NOS promoter conveyed the highest activity and had a mean expression level 5-fold higher than PTLF, whereas the CaMV 35S basal promoter fused to the omega element and the CaMV 35S basal promoter alone directed mean expression levels that were 0.5x and 0.35x that of PTLF, respectively. Differential expression in shoots, leaves, stems and roots was observed only for the NOS and PTLF promoters. Strongest expression was observed in roots for the NOS promoter, whereas the PTLF promoter directed highest expression in shoots. The NOS promoter appears best suited to counteract vegetative expression of a cytotoxin driven by the PTLF promoter where 1:1 toxin:attenuator expression is required.

Enhanced Induction of Vir Genes Results in the Improvement of Agrobacterium - Mediated Transformation of Eggplant

Induction of Agrobacterium vir genes is one of the basic requirements for T-DNA transfer and integration into plant genome. Here we study the vir gene induction by various explant types of eggplant in order to develop a transformation protocol with improved efficiency using binary vector constructs - harbouring a hygromycin phosphotransferase gene (hpt) as a selection marker and a gfp:gus fusion gene as a reporter. A protocol for efficient Agrobacterium-mediated transformation of eggplant (Solanum melongena L cv Pusa Purple Long) has been developed by optimizing factors. Leaf, cotyledon and hypocotyl explants were tested for their ability to induce Agrobacterium vir-genes using a VirE:lacZ fusion construct and were shown to be poor inducers of the same. Addition of 100 µM acetosyringone during infection and co-cultivation steps of transformation could enhance the vir gene induction as well as a 2–3 fold increase in transformation frequency. Transformed explants showed the expression of reporter genes gus and gfp. The transgenics were analysed by peR and Southern blot hybridization, and were shown to have T-DNA integrated into their genome. The data suggest that eggplant is a relatively poor inducer of Agrobacterium vir genes, probably due to minimal phenolic production, and by modulating vir gene induction using phenolics like acetosyringone eggplant transformation can be improved.