RESUMO
The cloning of promoter sequences of two invertase genes from potato (Solanum tuberosum L.) is described. Histochemical analysis of series of reporter transgenic lines reveals phloem-specific expression from both promoters, with one expressed preferentially in internal phloem and the other in external phloem of stem vascular bundles.
Assuntos
Regulação da Expressão Gênica de Plantas , Glicosídeo Hidrolases/genética , Solanum tuberosum/genética , Fusão Gênica Artificial , Clonagem Molecular , Genes Reporter , Glicosídeo Hidrolases/metabolismo , Histocitoquímica , Estruturas Vegetais/anatomia & histologia , Estruturas Vegetais/enzimologia , Estruturas Vegetais/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Solanum tuberosum/anatomia & histologia , Solanum tuberosum/enzimologia , beta-FrutofuranosidaseRESUMO
The organisation of two invertase genes (invGE and invGF) linked in direct tandem repeat within the potato genome is detailed. The genes exhibit a similar intron/exon structure which differs from previously described plant invertase genes; while intron locations are conserved between the genes, minor differences in exon length are seen. Both genes encode enzymes with putative extracellular location. Biochemical analysis of gene expression showed expression in floral tissues for both genes, with expression of the upstream gene (invGE) also detected in leaf tissue. Promoter sequences from both genes have been fused to the beta-glucuronidase (GUS) reporter gene (uidA) and transformed into potato. One promoter-GUS reporter construct was also transformed into tobacco. Histochemical analysis of transgenic lines defined specific expression from the downstream (invGF) promoter in potato and tobacco pollen, with expression first detected in the late uninucleate stage of tobacco microspore development. The invGE promoter determined expression in pollen and other floral tissues, but also at lateral nodes in stem, root and tuber. An association of invertase expression with generative tissue, both in vegetative and sexual modes of growth, is indicated.
Assuntos
Glicosídeo Hidrolases/genética , Solanum tuberosum/genética , Sequência de Bases , Clonagem Molecular , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucuronidase/genética , Glucuronidase/metabolismo , Íntrons , Isoenzimas/genética , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Plantas Tóxicas , RNA de Plantas/genética , RNA de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodução/genética , Solanum tuberosum/enzimologia , Solanum tuberosum/crescimento & desenvolvimento , Distribuição Tecidual , Nicotiana/genética , beta-FrutofuranosidaseRESUMO
We show that two invertase genes in potato, like most other plant invertase genes, include a very short second exon of 9 bp which encodes the central three amino acids of a motif highly conserved in invertases of diverse origin. This mini-exon is one of the smallest known in plants and pre-mRNA from these genes may be susceptible to alternative splicing, because of a potential requirement for specialized interaction with the splicing machinery to ensure correct processing for the production of a mature mRNA. No evidence of aberrant post-transcriptional processing was observed during normal invertase gene expression in potato. The fidelity of post-transcriptional processing of the pre-mRNA from one of the genes was perturbed by cold stress, resulting in the deletion of the mini-exon from some transcripts. This alternative splicing event occurred under cold stress in both leaf and stem, but was not induced by wounding. This adds an example of exon skipping and the induction of alternative processing by cold stress to the small number of transcripts which have been shown to exhibit alternative splicing in plants. The differential sensitivity of post-transcriptional processing to cold stress observed for the two transcripts examined will permit further dissection of the nucleotide sequence requirements for their accurate splicing.