RESUMO
Differential display was used to isolate cDNA clones showing differential expression in response to ABA, drought and cold in barley seedling shoots. One drought-regulated cDNA clone (DD12) was further analyzed and found to encode a branched-chain amino acid aminotransferase (HvBCAT-1). A genomic clone was isolated by probing the Morex BAC library with the cDNA clone DD12 and the structure of Hvbcat-1 was elucidated. The coding region is interrupted by six introns and contains a predicted mitochondrial transit peptide. Hvbcat1 was mapped to chromosome 4H. A comparison was made to rice and Arabidopsis genes to identify conserved structural patterns. Complementation of a yeast (Saccharomyces cerevisiae) double knockout strain revealed that HvBCAT-1 can function as the mitochondrial (catabolic) BCATs in vivo. Transcript levels of Hvbcat-1, increased in response to drought stress. As the first enzyme in the branched-chain amino acid (BCAA) catabolic pathway, HvBCAT-1 might have a role in the degradation of BCAA. Degradation of BCAA could serve as a detoxification mechanism that maintains the pool of free branched-chain amino acids at low and non toxic levels, under drought stress conditions.
Assuntos
Regulação da Expressão Gênica de Plantas , Hordeum/genética , Proteínas de Plantas/genética , Transaminases/genética , Água/metabolismo , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Aminoácidos de Cadeia Ramificada/metabolismo , Mapeamento Cromossômico , Cromossomos de Plantas , Clonagem Molecular , Sequência Conservada , Desidratação , Teste de Complementação Genética , Hordeum/enzimologia , Dados de Sequência Molecular , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , Transaminases/química , Transaminases/metabolismoRESUMO
Dehydrins (DHNs) compose a family of intrinsically unstructured proteins that have high water solubility and accumulate during late seed development, low temperature or water deficit conditions, and are thought to play a protective role in freezing and drought tolerance in plants. Twelve Dhn genes were previously described in the barley genome. Here, we report an additional member of this multigene family, Dhn13. The Dhn13 gene is located in chromosome 4 near marker MWG634 and encodes a 107-amino acid KS-type DHN. Semi-quantitative reverse transcriptase PCR data indicated that Dhn13 is constitutively expressed in seedling tissues and embryos of developing seeds. Microarray data were consistent with these results and showed a considerable increase of Dhn13 transcripts when plants were subjected to chilling and freezing temperatures. The highest transcript levels where observed in anthers. The presence of ABRE, MYC, DRE, and POLLEN1LELAT52 regulatory elements in the putative Dhn13 promoter region is in agreement with expression data.
Assuntos
Regulação da Expressão Gênica de Plantas , Hordeum/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Genes Reguladores/genética , Dados de Sequência Molecular , Família Multigênica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/genética , Alinhamento de Sequência , Análise de Sequência de DNA , TemperaturaRESUMO
A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.