RESUMO
In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.
Assuntos
Clonagem Molecular/métodos , DNA Bacteriano/genética , DNA de Plantas/genética , Genoma de Planta , Solanum lycopersicum/genética , Marcadores Genéticos , Vetores Genéticos , Plasmídeos , Polimorfismo Genético , Recombinação Genética , Mapeamento por RestriçãoRESUMO
The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.
Assuntos
Cladosporium/patogenicidade , Genes de Plantas , Glicoproteínas de Membrana/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Sequência de Aminoácidos , Sequência de Bases , Cladosporium/genética , DNA de Plantas/genética , Proteínas Fúngicas/genética , Dosagem de Genes , Genes Fúngicos , Solanum lycopersicum/fisiologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/fisiologia , Dados de Sequência Molecular , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Sequências Repetitivas de Aminoácidos , Homologia de Sequência de Aminoácidos , Virulência/genéticaRESUMO
A PCR-based method is described for the production of cDNA libraries and total cDNA probes from a few milligrams of tissue. Using a model system, we show how a PCR library and PCR probes can be used to identify genes expressed at different levels in two tissues. Small amounts of tissue derived from two plants, one infected with arabis mosaic virus and the other uninfected, were used to make a library and probes. This library and the probes were used to identify viral genes expressed only in the infected plant.
