RESUMEN
The behavior of the autonomous maize transposable element En/Spm of maize was studied in Arabidopsis. Transgenic Arabidopsis plants carrying En-1 elements were propagated for 12 generations using a single seed descent procedure. The distribution and activity of the En-1 element was monitored using Southern DNA hybridisations in generations 1, 6 and 12. In the first generation the highest number of En-1 insertions per line was 7, which increased to 20 in generation 12. The average number of En-1 insertions increased only slightly in the population, due to a gradual accumulation of segregants that lost the transposable element. During the development of the En-1 mutagenised population the element remained active even in the high-copy lines. In situ hybridisation demonstrated that multiple En-1 insertions were distributed over all Arabidopsis chromosomes. From the initial En-1 mutagenised populations many unstable gene mutations were recovered, indicating that En-1 can be used as a efficient tool for gene tagging in Arabidopsis.
Asunto(s)
Arabidopsis/genética , Elementos Transponibles de ADN , Mutagénesis Insercional , Zea mays/genética , Southern Blotting , Cromosomas , Hibridación Fluorescente in Situ , Plantas Modificadas Genéticamente , Reacción en Cadena de la PolimerasaRESUMEN
The isolation and initial characterization of the Arabidopsis thaliana SPL3 gene are described. SPL3 belongs to a gene family encoding putative transcription factors characterized by a conserved DNA-binding domain, the SBP domain. SPL3 transcription is developmentally regulated and is localized mainly in vegetative and inflorescence apical meristems, floral meristems and in leaf and floral organ primordia. SPL3 recognizes a conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene AP1. Similarly to AP1, constitutive expression of SPL3 results in early flowering. However, constitutive expression of SPL3 in an ap1 mutant background showed that AP1 is not required for the early flowering phenotype of the SPL3 transgenic plants. The function of SPL3 during flowering as well as its possible functional redundancy are discussed.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/fisiología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas de Plantas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Secuencia de Bases , Regulación del Desarrollo de la Expresión Génica , Biblioteca Genómica , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas de Plantas/biosíntesis , Brotes de la Planta , Regiones Promotoras Genéticas , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/biosíntesis , Transcripción GenéticaRESUMEN
A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5'-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.
Asunto(s)
Elementos Transponibles de ADN , Nicotiana/genética , Plantas Modificadas Genéticamente/genética , Plantas Tóxicas , Tetrahidrofolato Deshidrogenasa/genética , Secuencia de Bases , Southern Blotting , Clonación Molecular , ADN , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/enzimología , Reacción en Cadena de la Polimerasa , Nicotiana/enzimología , Zea mays/genéticaRESUMEN
The autonomous element En-1 of the maize En/Spm transposable element system is capable of frequent somatic and germinal excision in the heterologous host Arabidopsis thaliana. The pattern of En-homologous transcripts generated in transgenic Arabidopsis resembles En transcription in maize. An excision reporter construct based on NPT-II gene (pKEn2) can be used reliably for the isolation of En-1 germinal revertants by seed germination on kanamycin-containing medium. Re-insertion after germinal excision is apparently frequent. A dSpm receptor element can be efficiently trans-activated in Arabidopsis either by En-1 or by expressing cDNAs of tnpA and tnpD. Excision and re-insertion of En/Spm take place with similar characteristics as in maize. This is the first description of En/Spm transposition in Arabidopsis and the parameters analysed here suggest that transposon tagging with En should be feasible in this species.
