Particle Gun Bombardment Research Articles

Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor.

Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.

Geranylgeranyl diphosphate synthase from Scoparia dulcis and Croton sublyratus. Plastid localization and conversion to a farnesyl diphosphate synthase by mutagenesis.

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene-producing plants, Scoparia dulcis and Croton sublyratus, have been isolated using the homology-based polymerase chain reaction (PCR) method. Both clones contained highly conserved aspartate-rich motifs (DDXX(XX)D) and their N-terminal residues exhibited the characteristics of chloroplast targeting sequence. When expressed in Escherichia coli, both the full-length and truncated proteins in which the putative targeting sequence was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to produce geranylgeranyl diphosphate (GGPP). The structural factors determining the product length in plant GGPPSs were investigated by constructing S. dulcis GGPPS mutants on the basis of sequence comparison with the first aspartate-rich motif (FARM) of plant farnesyl diphosphate synthase. The result indicated that in plant GGPPSs small amino acids, Met and Ser, at the fourth and fifth positions before FARM and Pro and Cys insertion in FARM play essential roles in determination of product length. Further, when a chimeric gene comprised of the putative transit peptide of the S. dulcis GGPPS gene and a green fluorescent protein was introduced into Arabidopsis leaves by particle gun bombardment, the chimeric protein was localized in chloroplasts, indicating that the cloned S. dulcis GGPPS is a chloroplast protein.

Rapid Development of Homozygous Transgenic Rice using Anther Culture Harboring Rice chitinase Gene for Enhanced Sheath Blight Resistance.

Transgenic rice was developed from both calli and immature embryos of popular cultivar ‘Swarna’ with a rice chitinase gene (chi11) by particle gun bombardment. Homozygous dihaploid transgenics were produced in less than a year through the anther culture of primary transgenics. Stable integration and expression of the chi11 transgene were confirmed by Southern and western analyses, respectively of primary as well as anther culture-derived dihaploid transgenics. The homozygous transgenics with functional transgene and varied levels of chitinase activity showed enhanced resistance to sheath blight fungus. The bioassay data were correlated with the molecular and biochemical results.

Stable chloroplast transformation in Chlamydomonas reinhardtii using microprojectile bombardment.

The chloroplasts of Chlamydomonas reinhardtii were transformed using a vector (paadAGUS4.1) that contained a spectinomycin-resistance gene (aadA) as a selectable gene, and bacterial uidA (GUS) as a reporter gene, and pea 4.1 kb D-loop containing sequence. The vector was introduced into the alga through particle gun bombardment. The transformed colonies were screened for the presence of foreign genes by Southern hybridization using GUS, aadA and 4.1 pea Ori probes. Expression of aadA and GUS genes was detected in all colonies that were grown on spectinomycin. A detailed restriction analysis followed by southern hybridization of total genomic DNA using pea 4.1 kb D-loop as probe indicated that the D-loop sequence can serve in site-specific integration of foreign DNA due to high homology. Restriction analysis of different colonies showed that the foreign DNA was probably present in a mixture population of autonomous segment and integrated in the native chloroplast genome.

Plastidic Pathway of Serine Biosynthesis: MOLECULAR CLONING AND EXPRESSION OF 3-PHOSPHOSERINE PHOSPHATASE FROM ARABIDOPSIS THALIANA

In plants, Ser is biosynthesized by two different pathways: a photorespiratory pathway via Gly and a plastidic pathway via the phosphorylated metabolites from 3-phosphoglycerate. In contrast to the better characterization of the photorespiratory pathway at a molecular level, the molecular regulation and significance of the plastidic pathway are not yet well understood. An Arabidopsis thaliana cDNA encoding 3-phosphoserine phosphatase, the enzyme that is responsible for the conversion of 3-phosphoserine to Ser in the final step of the plastidic pathway of Ser biosynthesis, was cloned by functional complementation of an Escherichia coli serB- mutant. The 1.1-kilobase pair full-length cDNA, encoding 295 amino acids in its open reading frame, contains a putative organelle targeting presequence. Chloroplastic targeting has been demonstrated by particle gun bombardment using an N-terminal 60-amino acid green fluorescence protein fusion protein. Southern hybridization suggested the existence of a single-copy gene that mapped to chromosome 1. 3-Phosphoserine phosphatase enzyme activity was detected in vitro in the overexpressed protein in E. coli. Northern analysis revealed preferential gene expression in leaf and root tissues of light-grown plants with an approximately 1.5-fold abundance in the root compared with the leaf tissues. This indicates the possible role of the plastidic pathway in supplying Ser to non-photosynthetic tissues, in contrast to the function of the photorespiratory pathway in photosynthetic tissues. This work completes the molecular cloning and characterization of the three genes involved in the plastidic pathway of Ser biosynthesis in higher plants.

Effects of overproduction of tobacco MnSOD in maize chloroplasts on foliar tolerance to cold and oxidative stress

Transgenic maize plants have been generated by particle gun bombardment that overproduce a Nicotiana plumbaginifolia L. manganese superoxide dismutase (MnSOD). To target this mitochondrial enzyme into chloroplasts, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene, whereas expression of the chimeric gene was driven by the CaMV 35S promoter. Transgenic MnSOD activity contributed to 20% of the total SOD activity. The presence of transgenic MnSOD had clear effects on foliar tolerance to chilling and oxidative stress. The results suggest that overproduction of MnSOD in the chloroplasts increases the antioxidant capacity of the leaves.

Effects of overproduction of tobacco MnSOD in maize chloroplasts on foliar tolerance to cold and oxidative stress

Transgenic maize plants have been generated by particle gun bombardment that overproduce a Nicotiana plumbaginifolia L. manganese superoxide dismutase (MnSOD). To target this mitochondrial enzyme into chloroplasts, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene, whereas expression of the chimeric gene was driven by the CaMV 35S promoter. Transgenic MnSOD activity contributed to 20% of the total SOD activity. The presence of transgenic MnSOD had clear effects on foliar tolerance to chilling and oxidative stress. The results suggest that overproduction of MnSOD in the chloroplasts increases the antioxidant capacity of the leaves.

Overproduction of Arabidopsis thaliana FeSOD confers oxidative stress tolerance to transgenic maize.

Transgenic maize (Zea mays L.) plants have been generated by particle gun bombardment that overproduce an Arabidopsis thaliana iron superoxide dismutase (FeSOD). To target this enzyme into chloroplasts, the mature Fesod coding sequence was fused to a chloroplast transit peptide from a pea ribulose-1,5-bisphosphate carboxylase gene. Expression of the chimeric gene was driven by the CaMV 35S promoter. Growth characteristics and in vitro oxidative stress tolerance of transgenic lines grown in control and chilling temperatures were evaluated. The transgenic line with the highest transgenic FeSOD activities had enhanced tolerance toward methyl viologen and had increased growth rates.

Expression of an engineered cysteine proteinase inhibitor (Oryzacystatin-IΔD86) for nematode resistance in transgenic rice plants

We have used a genotype-independent transformation system involving particle gun bombardment of immature embryos to genetically engineer rice as part of a programme to develop resistance to nematodes. Efficient tissue culture, regeneration, DNA delivery and selection methodologies have been established for elite African varieties (‘ITA212’, ‘IDSA6’, ‘LAC23’, ‘WAB56-104’). Twenty-five transformed clones containing genes coding for an engineered cysteine proteinase inhibitor (oryzacystatin-IΔD86, OC-IΔD86), hygromycin resistance (aphIV) and β-glucuronidase (gusA) were recovered from the four varieties. Transformed plants were regenerated from all clones and analysed by PCR, Southern and western blot. Detectable levels of OC-IΔD86 (up to 0.2% total soluble protein) in plant roots were measured in 12 out of 25 transformed rice lines. This level of expression resulted in a significant 55% reduction in egg production by Meloidogyne incognita.

Transformation of Digitaria sanguinalis: a model system for testing maize streak virus resistance in Poaceae.

Digitaria sanguinalis, an African grass, was micropropagated from immature inflorescences and callus was transformed by particle gun bombardment. Integration and expression of the bar and uidA genes was shown in the T0 generation. Transformants were shown to be fertile by the production of viable seeds. The time taken from bombardment to the setting of seed was approximately four months. D. sanguinalis and its transformants were shown to be sensitive to maize streak virus (MSV). Therefore this is an ideal model system for testing genetically engineered resistance to MSV.

Recovery of transgenic asparagus plants by particle gun bombardment of somatic cells

Transgenic asparagus (Asparagus officinalis L.) plants were obtained through particle gun bombardment of suspension cells using the pKGUS plasmid containing both neomycin phosphotransferase II (nptII) and β-glucuronidase (uidA) genes. In experiments to optimize bombardment parameters, the combination of 1.1 μm tungsten particles and 1000 psi bombardment pressure was best; 65 cells or cell clumps per plate of 105–106 suspension culture derived cells expressed uidA in transient assays. No significant genotypic effects were detected for the number of cells or cell clumps expressing uidA among four genotypes. A two-step selection procedure was established for the recovery of transformed plants. The bombarded cells were first placed on embryo induction medium with 25 mg l−1 kanamycin for 4 weeks, and the resistant calli were then transferred to the same medium with 50 mg l−1 kanamycin for embryo development. A total of 10 plants from six kanamycin resistant calli were recovered. The expression of uidA was detected in the stems and cladodes of these 10 plants. Southern analysis confirmed the integration of nptII and uidA into the asparagus genome, and revealed that the 10 transgenic plants from six resistant calli were from one transformation event. In addition to the detection of junction fragments where the pKGUS plasmid integrated into the genome, Southern analysis also provided evidence that most of the pKGUS plasmid DNA was integrated intactly and organized as head-to-tail concatemers in these plants. uidA assays of the hybrid seeds from the crosses between one transgenic male asparagus T0 plant and nontransformed female asparagus suggested that uidA activity was inherited as a single locus with Mendelian segregation.

The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase.

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

Genetic transformation ofEucalyptus globulusthrough biolistics: complementary development of procedures for organogenesis from zygotic embryos and stable transformation of corresponding proliferating tissue

Stable transformation of Eucalyptus globulus after biolistic DNA delivery was investigated using zygotic embryos as target material. Conditions for plant regeneration were first investigated. Six-day-old cultured embryos, which had shown to be a good target for DNA transient expression, appeared to be suitable for regeneration. Whole plants were recovered through organogenesis after particle gun bombardment. Transformation experiments were performed with a linear T-DNA fragment harbouring GUS and NPTII genes, using the biological and physical conditions defined for optimum transient expression. After 2 months on a culture medium, neoformed GUS-positive calli were obtained from the 'T-DNA' bombarded embryos. GUS-expressing calli were also recovered after selection with kanamycin following bombardment. The integration of both GUS and NPTII genes into the Eucalyptus genome was confirmed by Southern blot analysis, demonstrating stable transformation of Eucalyptus globulus cells for the first time.

Stable transformation of Gladiolus by particle gun bombardment of cormels

Transgenic plants of Gladiolus were produced following particle bombardment of cormel slices. Plant cells were cotransformed with the gene for phosphinothricin acetyltransferase under control of the cauliflower mosaic virus 35S promoter and the uidA gene coding for β-glucuronidase (GUS) under control of the actin promoter isolated from rice. The optimum concentration for the first selection of transformed plants was 8 mg/l phosphinothricin which resulted in 14% of the bombarded cormel slices regenerating plants that were transformed as confirmed by polymerase chain reaction amplification. Polymerase chain reaction amplification, Southern hybridization and histochemical staining for GUS gene expression on plants after two selective screenings with phosphinothricin indicated that regenerated plants were transformed. Histochemical staining for GUS gene expression showed that the actin promoter resulted in GUS gene expression primarily in callus cells and root meristems. Leaves were typically chimeric for GUS gene expression.

Development of a pollen-mediated transformation method forNicotiana glutinosa

The development is described of a new procedure to genetically transform plant species using the male gametophyte as a natural transformation vector. Our system avoids the need for complicated regeneration procedures thus making it broadly applicable. Naked plasmid DNA encoding kanamycin resistance and GUS activity was introduced by particle gun bombardment into mature pollen grains ofNicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, designated VIP A and VIP B, were obtained in two independent experiments. In VIP A, TR2′-driven GUS activity was observed in vascular bundles, trichomes and in a small number of pollen grains. DNA gel blot analysis indicated that the introduced DNA was integrated independently into the genome of VIP A and VIP B. It was shown that male and female gametophyte development and seed set were highly aberrant in both VIP A and VIP B and that the offspring of self- and cross-pollinations did not contain the transgenes. This might be caused by a recombination event during the integration of the naked DNA resulting in a deletion of part of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VIP B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.

Regulated transcription of the maize Bronze-2 promoter in electroporated protoplasts requires the C1 and R gene products.

The putative maize transcription factor genes R and C1 are required for expression of reporter genes with promoters from the Bz1 and A1 genes, which encode enzymes required for anthocyanin biosynthesis in maize. Bz2 is another anthocyanin biosynthetic gene; we show that expression of a reporter gene from the Bz2 promoter also requires R and C1 when the fusion construct is introduced into maize kernels by particle gun bombardment. When electroporated into maize protoplasts from a suspension cell line not synthesizing anthocyanins, reporter genes with Bz2, Bz1, and A1 promoters are expressed only when both R and C1 expression plasmids are co-electroporated. Electroporation of R and C1 expression plasmids also induces the endogenous genes required for anthocyanin synthesis, resulting in pink protoplasts within 24 h. RNase protection analysis demonstrates that accumulation of mRNA from the endogenous Bz1 and Bz2 genes absolutely requires introduced R and C1. In time-course experiments there is a delay of 3-6 h before the Bz2 promoter is activated, supporting the proposed role for R- and C1-encoded proteins in transcriptional control. An excess of R relative to C1 suppresses expression of A1, Bz1, and Bz2 promoters, suggesting an interaction between the R and C1 proteins.