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1.
Genes (Basel) ; 12(3)2021 03 19.
Article in English | MEDLINE | ID: mdl-33808690

ABSTRACT

Conifers are a group of woody plants with an enormous economic and ecological importance. Breeding programs are necessary to select superior varieties for planting, but they have many limitations due to the biological characteristics of conifers. Somatic embryogenesis (SE) and de novo organogenesis (DNO) from in vitro cultured tissues are two ways of plant mass propagation that help to overcome this problem. Although both processes are difficult to achieve in conifers, they offer advantages like a great efficiency, the possibilities to cryopreserve the embryogenic lines, and the ability of multiplying adult trees (the main bottleneck in conifer cloning) through DNO. Moreover, SE and DNO represent appropriate experimental systems to study the molecular bases of developmental processes in conifers such as embryogenesis and shoot apical meristem (SAM) establishment. Some of the key genes regulating these processes belong to the WOX and KNOX homeobox gene families, whose function has been widely described in Arabidopsis thaliana. The sequences and roles of these genes in conifers are similar to those found in angiosperms, but some particularities exist, like the presence of WOXX, a gene that putatively participates in the establishment of SAM in somatic embryos and plantlets of Pinus pinaster.


Subject(s)
Gene Expression Profiling/methods , Homeodomain Proteins/genetics , Sequence Analysis, DNA/methods , Tracheophyta/physiology , Cryopreservation , Gene Expression Regulation, Plant , In Vitro Techniques , Multigene Family , Organogenesis, Plant , Plant Breeding , Plant Proteins/genetics , Plant Somatic Embryogenesis Techniques , Regeneration , Sequence Analysis, RNA , Tracheophyta/genetics
2.
Plant Sci ; 301: 110691, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33218649

ABSTRACT

KNOTTED1-LIKE HOMEOBOX (KNOX) genes are a family of plant-specific homeobox transcription factors with important roles in plant development that have been classified into two subfamilies with differential expression domains and functions. Studies in angiosperms have shown that class I members are related to the maintenance of meristem homeostasis and leaf development, whereas class II members promote differentiation of tissues and organs. However, little is known about its diversification and function in gymnosperms. By combining PCR-based detection and transcriptome data analysis, we identified four class I and two class II KNOX genes in Pinus pinaster. Expression analyses showed that class I members were mainly expressed in meristematic regions and differentiating tissues, with practically no expression in lateral organs, whereas expression of class II members was restricted to lateral organs. Furthermore, overexpression of P. pinaster KNOX genes in Arabidopsis thaliana caused similar phenotypic effects to those described for their angiosperms counterparts. This is the first time to our knowledge that functional analyses of class II members are reported in a conifer species. These results suggest a high conservation of the KNOX gene family throughout seed plants, as the functional differentiation of both subfamilies observed in angiosperms might be partially conserved in gymnosperms.


Subject(s)
Pinus/genetics , Plant Proteins/genetics , Arabidopsis/genetics , Gene Expression , Homeodomain Proteins/genetics , Meristem/genetics , Organ Specificity , Phenotype , Transcription Factors/genetics
3.
Plant Cell Rep ; 39(4): 527-541, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31993729

ABSTRACT

KEY MESSAGE: Several members of WOX and KNOX gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer Pinus pinea. Similar to Arabidopsis thaliana, Pinus pinea shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in P. pinea cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in P. pinea. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2-6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes PpWUS, PpWOX5, PpKN2, PpKN3 and PipiRR1. WUS is functional in pines and has an important role in caulogenesis. Interestingly, WOX5 also seems to participate in the process, although its specific role has not been determined.


Subject(s)
Cotyledon/chemistry , Cotyledon/metabolism , Meristem/metabolism , Pinus/metabolism , Plant Growth Regulators/metabolism , Plant Proteins/metabolism , Aminobutyrates/pharmacology , Cells, Cultured , Chromatography, High Pressure Liquid , Cotyledon/drug effects , Cotyledon/genetics , Cytokinins/metabolism , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Indoleacetic Acids/metabolism , Meristem/chemistry , Meristem/genetics , Pinus/chemistry , Pinus/genetics , Plant Proteins/genetics , Plant Shoots/metabolism , Seeds/drug effects , Seeds/growth & development , Seeds/metabolism , Tandem Mass Spectrometry
4.
BMC Plant Biol ; 18(1): 198, 2018 Sep 17.
Article in English | MEDLINE | ID: mdl-30223777

ABSTRACT

BACKGROUND: Gene activity is largely controlled by transcriptional regulation through the action of transcription factors and other regulators. QsMYB1 is a member of the R2R3-MYB transcription factor family related to secondary growth, and in particular, with the cork development process. In order to identify the putative gene targets of QsMYB1 across the cork oak genome we developed a ChIP-Seq strategy. RESULTS: Results provide direct evidence that QsMY1B targets genes encoding for enzymes involved in the lignin and suberin pathways as well as gene encoding for ABCG transporters and LTPs implicated in the transport of monomeric suberin units across the cellular membrane. These results highlight the role of QsMYB1 as a regulator of lignin and suberin biosynthesis, transport and assembly. CONCLUSION: To our knowledge, this work constitutes the first ChIP-Seq experiment performed in cork oak, a non-model plant species with a long-life cycle, and these results will contribute to deepen the knowledge about the molecular mechanisms of cork formation and differentiation.


Subject(s)
Lignin/genetics , Lipids/genetics , Plant Proteins/genetics , Quercus/genetics , Transcription Factors/genetics , Binding Sites , Chromatin Immunoprecipitation , Enzymes/genetics , Enzymes/metabolism , Gene Expression Regulation, Plant , Lignin/biosynthesis , Plant Proteins/metabolism , Quercus/metabolism , Regulatory Sequences, Nucleic Acid , Seeds/genetics , Transcription Factors/metabolism
5.
Plant Physiol Biochem ; 123: 304-318, 2018 Feb.
Article in English | MEDLINE | ID: mdl-29278847

ABSTRACT

WUSCHEL-RELATED HOMEOBOX (WOX) genes are key players controlling stem cells in plants and can be divided into three clades according to the time of their appearance during plant evolution. Our knowledge of stem cell function in vascular plants other than angiosperms is limited, they separated from gymnosperms ca 300 million years ago and their patterning during embryogenesis differs significantly. For this reason, we have used the model gymnosperm Pinus pinaster to identify WOX genes and perform a thorough analysis of their gene expression patterns. Using transcriptomic data from a comprehensive range of tissues and stages of development we have shown three major outcomes: that the P. pinaster genome encodes at least fourteen members of the WOX family spanning all the major clades, that the genome of gymnosperms contains a WOX gene with no homologues in angiosperms representing a transitional stage between intermediate- and WUS-clade proteins, and that we can detect discrete WUS and WOX5 transcripts for the first time in a gymnosperm.


Subject(s)
Cycadopsida , Gene Expression Regulation, Plant/physiology , Genes, Plant/physiology , Homeodomain Proteins , Multigene Family/physiology , Pinus , Plant Proteins , Cycadopsida/genetics , Cycadopsida/metabolism , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Pinus/genetics , Pinus/metabolism , Plant Proteins/biosynthesis , Plant Proteins/genetics
6.
Article in English | MEDLINE | ID: mdl-27847150

ABSTRACT

Plant growth regulators (PGRs) are very different chemical compounds that play essential roles in plant development and the regulation of physiological processes. They exert their functions by a mechanism called cross-talk (involving either synergistic or antagonistic actions) thus; it is for great interest to study as many PGRs as possible to obtain accurate information about plant status. Much effort has been applied to develop methods capable of analyze large numbers of these compounds but frequently excluding some chemical families or important PGRs within each family. In addition, most of the methods are specially designed for matrices easy to work with. Therefore, we wanted to develop a method which achieved the requirements lacking in the literature and also being fast and reliable. Here we present a simple, fast and robust method for the extraction and quantification of 20 different PGRs using UHPLC-MS/MS optimized in complex matrices.


Subject(s)
Chromatography, High Pressure Liquid/methods , Pinus/chemistry , Plant Extracts/chemistry , Plant Growth Regulators/analysis , Tandem Mass Spectrometry/methods , Chromatography, High Pressure Liquid/economics , Liquid-Liquid Extraction/methods , Plant Growth Regulators/isolation & purification , Solvents/chemistry , Tandem Mass Spectrometry/economics
7.
ScientificWorldJournal ; 2013: 681792, 2013.
Article in English | MEDLINE | ID: mdl-24376383

ABSTRACT

An efficient transformation protocol based on kanamycin selection was developed for Agrobacterium-mediated transformation of maritime pine embryonal masses. The binary vector pBINUbiGUSint, which contained neomycin phosphotransferase II (nptII) as a selectable marker gene and ß -glucuronidase (uidA) as a reporter gene, was used for transformation studies. Different factors, such as embryogenic line, bacterial strain, bacterial concentration, and coculture duration, were examined and optimized. For selection of transformants, 15 mgL(-1) kanamycin was used. The highest transformation efficiency (11.4 events per gram of fresh mass) was achieved when a vigorously growing embryonal mass (embryogenic line L01) was cocultivated with Agrobacterium strain AGL1 at the optical density (OD(600 nm)) of 0.3 for 72 h. Evidence of the stable transgene integration was obtained by polymerase chain reaction for the nptII and uidA genes and expression of the uidA gene. Maturation capacity of the transgenic lines was negatively affected by the transformation process. Induction of axillary shoots by preculturing the embryos with benzyladenine allowed overcoming the low maturation rates of some transformed lines. The transgenic embryos were germinated and the axillar shoots were rooted. Transgenic plants were transferred to potting substrate showing normal growth.


Subject(s)
Agrobacterium/physiology , Kanamycin/metabolism , Pinus/genetics , Pinus/microbiology , Plants, Genetically Modified/microbiology , Plants, Genetically Modified/physiology , Transformation, Bacterial/genetics , Genetic Enhancement/methods
8.
Cryo Letters ; 33(6): 476-84, 2012.
Article in English | MEDLINE | ID: mdl-23250407

ABSTRACT

Pinus pinaster is one of the most economically important conifers in the world. Somatic embryogenesis is a powerful tool in breeding programmes because it allows the generation of a great number of different clonal lines from seeds of superior genotypes. Unfortunately, embryogenic competence decreases with the age of cultures. Therefore, it is necessary to have a cryopreservation protocol that ensures a continuous supply of juvenile mass while allowing good maturation and conversion rates into vigorously growing plants. In this work we studied the influence of several cryopreservation parameters, such as cryoprotectant solution and pre-cooling temperature, on embryogenic culture regrowth and embryo maturation. Recovery of rewarmed samples after cryopreservation in a -150 degree C freezer depended on the cooling temperature reached prior to plunging the tubes into liquid nitrogen. As a result, we present an optimised cryopreservation protocol that ensures high recovery and embryo maturation rates. The protocol presented is a simple and fast alternative and enabled successful cryopreservation and recovery of 100 percent of the lines tested. Cryopreserved lines presented the same maturation rates as non-cryopreserved controls.


Subject(s)
Cryopreservation/methods , Pinus/embryology , Seeds/growth & development , Cryoprotective Agents/metabolism , Dimethyl Sulfoxide/metabolism , Embryo Culture Techniques , Pinus/growth & development , Sucrose/metabolism
9.
J Plant Physiol ; 169(18): 1807-14, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-22959674

ABSTRACT

The molecular cloning and characterization of PipsRR1, a type-A response regulator in Pinus pinaster, is reported here. Type-A response regulators mediate downstream responses to cytokinin and act as negative feedback regulators of the signal transduction pathway. Some type-A response regulators in Arabidopsis have been related to de novo meristem formation. However, little information exists in Pinus spp. The PipsRR1 gene contains 5 exons, as do all type-A response regulators in Arabidopsis, and the deduced protein contains a receiver domain with the conserved DDK residues and a short C terminal extension. Expression analysis showed that the PipsRR1 gene is differentially expressed during the first phases of adventitious caulogenesis induced by benzyladenine in P. pinaster cotyledons, suggesting that PipsRR1 plays a role in caulogenesis in conifers. Additionally, a binary vector carrying the PipsRR1 promoter driving GFP:GUS expression was constructed to analyze the promoter activity in P. pinaster somatic embryos. The results of genetic transformation showed GUS activity during somatic embryo mass proliferation and embryo maturation.


Subject(s)
Benzene Derivatives/pharmacology , Cytokinins/metabolism , Gene Expression Regulation, Plant , Pinus/genetics , Plant Growth Regulators/metabolism , Plant Proteins/isolation & purification , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Cotyledon/drug effects , Cotyledon/genetics , Cotyledon/growth & development , Cotyledon/physiology , DNA, Plant/chemistry , DNA, Plant/genetics , Genes, Reporter , Molecular Sequence Data , Pinus/drug effects , Pinus/growth & development , Pinus/physiology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Shoots/drug effects , Plant Shoots/genetics , Plant Shoots/growth & development , Plant Shoots/physiology , Plant Somatic Embryogenesis Techniques , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis, DNA , Signal Transduction , Transformation, Genetic
10.
Methods Mol Biol ; 877: 385-99, 2012.
Article in English | MEDLINE | ID: mdl-22610642

ABSTRACT

An Agrobacterium-mediated transformation system for selected mature Quercus suber L. trees has been established. Leaf-derived somatic embryos in an early stage of development are inoculated with an AGL1 strain harboring a kanamycin-selectable plasmid carrying the gene of interest. The transformed embryos are induced to germinate and the plantlets transferred to soil. This protocol, from adult cork oak to transformed plantlet, can be completed in about one and a half years. Transformation efficiencies (i.e., percentage of inoculated explants that yield independent transgenic embryogenic lines) vary depending on the cork oak genotype, reaching up to 43%.


Subject(s)
Quercus/genetics , Transformation, Genetic/genetics , Genotype
11.
BMC Genomics ; 12: 366, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21762488

ABSTRACT

BACKGROUND: Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases. DESCRIPTION: EuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided. CONCLUSIONS: The EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.


Subject(s)
Databases, Genetic , Gene Expression Profiling , Pinus/genetics , Gene Library , Internet
12.
J Plant Physiol ; 168(7): 661-70, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21144618

ABSTRACT

The responses of juvenile plants of forest crops to drought stress are a key stage in the survival of forest populations. In this work, a suitable experimental system to study the early drought resistance mechanisms and signaling in a drought-tolerant clone (C14) of Eucalyptus globulus Labill is proposed. This system, using hydroponic culture and an osmotic agent, polyethylene glycol 8000, was demonstrated to induce severe stress in the root area, affecting the responses of the plantlets at the aerial level. These responses were very fast, beginning only 3h after the induction of stress, and the results highlight the roles of xylematic abscisic acid (ABA) and pH changes over other signals, such as cytokinins, as early chemical signals in rapid water stress. The relationship between these chemical factors, ABA and pH, and the physiological and water parameters observed were significant, supporting their proposed principal role. This work aids our understanding of underlying responses to hydrological limitations of forest crops, and provides valuable information for further physiological and molecular studies of water stress in this and other tree species.


Subject(s)
Droughts , Eucalyptus/metabolism , Plant Roots/metabolism , Abscisic Acid/isolation & purification , Abscisic Acid/metabolism , Cytokinins/isolation & purification , Cytokinins/metabolism , Gene Expression Regulation, Plant , Hydrogen-Ion Concentration , Photosynthesis , Signal Transduction
13.
Biotechnol Lett ; 31(9): 1477-83, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19543858

ABSTRACT

The bar gene was introduced into the cork oak genome. Cork oak embryogenic masses were transformed using the Agrobacterium strain AGL1 which carried the plasmid pBINUbiBar. This vector harbours the genes, nptII and bar, the latter under control of the maize ubiquitin promoter. The transgenic embryogenic lines were cryopreserved. Varying activities of phosphinothricin acetyl transferase were detected among the lines, which carried 1-4 copies of the insert. Molecular and biochemical assays confirmed the stability and expression of the transgenes 3 months after thawing the cultures. These results demonstrate genetic engineering of herbicide tolerance in Quercus spp.


Subject(s)
Herbicide Resistance , Herbicides/toxicity , Plants, Genetically Modified/genetics , Plants, Genetically Modified/physiology , Quercus/genetics , Quercus/physiology , Acetyltransferases/genetics , Acetyltransferases/metabolism , Gene Dosage , Genomic Instability , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/drug effects , Plasmids , Promoter Regions, Genetic , Quercus/drug effects , Rhizobium/genetics , Transformation, Genetic , Zea mays/genetics
14.
J Plant Physiol ; 166(10): 1069-76, 2009 Jul 01.
Article in English | MEDLINE | ID: mdl-19217688

ABSTRACT

Germination negatively affects adventitious shoot formation induced by cytokinins in pine cotyledons. To investigate the causes of this decrease in the organogenic response, uptake and metabolism of benzyladenine (BA) were studied in stone pine cotyledons (Pinus pinea) isolated from in vitro germinating embryos and cultured in bud induction medium. As embryos grew, cotyledons showed a progressive decrease in the amount of BA taken up from the medium. BA was barely metabolized; however, a BA metabolite previously undescribed in conifers was found. It was identified as a glucoside of the BA riboside, a type of metabolite recently described in other gymnosperms. Data revealed that differences in the organogenic capacity of P. pinea cotyledons associated with embryo germination are related primarily to their ability to absorb BA from the bud induction medium.


Subject(s)
Aminobutyrates/metabolism , Cotyledon/growth & development , Cotyledon/metabolism , Pinus/growth & development , Pinus/metabolism , Gene Expression Regulation, Plant/physiology , Plant Shoots/growth & development , Plant Shoots/metabolism
15.
J Plant Physiol ; 166(11): 1162-71, 2009 Jul 15.
Article in English | MEDLINE | ID: mdl-19232774

ABSTRACT

Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA) has been proposed as a model for the study of in vitro shoot organogenesis in conifers. This is because of its advantageous characteristics including the requirement of only one plant growth regulator (BA), the synchronous fashion of its induction, and the homogeneity and low degree of differentiation of cotyledons. Although optimal culture conditions have been developed and are currently in use, we still lack data for BA dynamics in cotyledons cultured under these conditions, and the morphological description of the early induction stages has not, until now, been approached from a histological perspective. Consequently, this is the focus of the present report. Additionally, we examined uptake and metabolism of BA in cotyledons from two selected families, previously characterized by, and selected for, the difference in the magnitude of their organogenic response. Media transfer experiments established that cotyledons should be in contact with 44.4 microM BA for at least 6h to obtain any caulogenic response (minimum shoot-induction period). Histological observations, carried out here for the first time in this species, determined that meristemoid structures had already begun to appear in explants within 12 h of culture. Moreover, results from the BA uptake and metabolism experiments indicated that the point at which explants reached the maximum concentration of active forms of BA (276.60 microM at 6 h) and the onset of the determination phase of shoot organogenesis were directly related. A direct relationship was also observed between the intensity of the caulogenic response in cotyledons from families 36 and 61 and the endogenous concentration of BA and its riboside at the start of the induction phase. Hence, family 36, characterized by its higher bud production, reached concentrations of 251.56 microM, while family 61, selected for its low bud-producing trait, only attained 175.80 microM. Finally, a correlation was observed between 6-benzylamino-9-[O-glucopyranosyl-(1-->3)ribofuranosyl]-purine values and the magnitude of the shoot organogenesis response.


Subject(s)
Aminobutyrates/pharmacology , Cotyledon/embryology , Pinus/drug effects , Pinus/embryology , Plant Growth Regulators/pharmacology , Cotyledon/drug effects , Cotyledon/metabolism , Gene Expression Regulation, Plant/drug effects , Plant Shoots/drug effects , Plant Shoots/embryology
16.
Tree Physiol ; 27(12): 1721-30, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17938103

ABSTRACT

As part of a study aimed at understanding the physiological and molecular mechanisms involved in adventitious shoot bud formation in pine cotyledons, we conducted a transcriptome analysis to identify early-induced genes during the first phases of adventitious caulogenesis in Pinus pinea L. cotyledons cultured in the presence of benzyladenine. A subtractive cDNA library with more than 700 clones was constructed. Of these clones, 393 were sequenced, analyzed and grouped according to their putative function. Quantitative real-time PCR analysis was performed to confirm the differential expression of 30 candidate genes. Results are contrasted with available data for other species.


Subject(s)
Cotyledon/genetics , Gene Expression Profiling/methods , Pinus/genetics , Plant Shoots/genetics , Benzyl Compounds , Cotyledon/growth & development , DNA, Complementary/chemistry , DNA, Complementary/genetics , Expressed Sequence Tags , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Plant/drug effects , Gene Library , Kinetin/pharmacology , Molecular Sequence Data , Nucleic Acid Hybridization/methods , Pinus/growth & development , Plant Growth Regulators/pharmacology , Plant Shoots/growth & development , Purines , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Analysis, DNA , Tissue Culture Techniques
17.
Transgenic Res ; 12(1): 127-30, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12650532

ABSTRACT

Rabbit hemorrhagic disease, which is caused by a calicivirus, is a lethal infection of adult animals that is characterized by acute liver damage and disseminated intravascular coagulation. In this study, we report the production of the major structural protein VP60 of rabbit hemorrhagic disease virus in transgenic tubers of potato plants and its use as an oral immunogen in rabbits.


Subject(s)
Antigens, Viral/immunology , Caliciviridae Infections/prevention & control , Hemorrhagic Disease Virus, Rabbit/immunology , Solanum tuberosum , Vaccines, Synthetic/immunology , Viral Structural Proteins/immunology , Viral Vaccines/immunology , Animals , Antigens, Viral/genetics , Caliciviridae Infections/immunology , Capsid/immunology , Enzyme-Linked Immunosorbent Assay , Hemorrhagic Disease Virus, Rabbit/genetics , Immunization , Plants, Genetically Modified , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Vaccines, Synthetic/genetics , Viral Structural Proteins/genetics , Viral Vaccines/genetics
18.
Physiol Plant ; 112(3): 388-396, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11473696

ABSTRACT

We have isolated and characterized a genomic clone encoding Scots pine (Pinus sylvestris) cytosolic glutamine synthetase (GS). The clone contains the 5' end half of the gene including part of the coding region and 980 bp upstream of the translation initiation codon. The major transcription start site (+1) was mapped around 180 nucleotides upstream of the translation initiation codon. Sequence analysis of the 5'-upstream region of the gene reveals the presence of putative regulatory elements including a poly-CT consensus sequence, a purine-rich tandem repeat and two AT-rich regions. Fusions of the upstream gene region to uidA were shown to be transiently expressed in the cotyledons of germinating pine seeds transformed by microprojectile bombardment. Stable transformation of Arabidopsis thaliana revealed the shoot apical meristem as the major region of heterologous permanent expression in Arabidopsis, in agreement with the expression of the GS gene in Pinus. Moreover, quantitative data derived from fluorometric beta-glucuronidase assays in control and continuous light-grown transgenic Arabidopsis plants indicate that the isolated upstream region of the gene contains regulatory sequences involved in the response to light.

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