RESUMO
The aspartic protease (FeAP9) gene from buckwheat resembles the exon-intron structure characteristic for typical aspartic proteinases, including the presence of the leader intron in the 5'-UTR. RT PCR experiments and gel protein blot analysis indicated that FeAP9 was present in all analyzed organs: developing seeds, seedlings, flowers, leaves, roots and stems. Using Real-time PCR, we found that FeAP9 expression is upregulated in buckwheat leaves under the influence of different abiotic stresses, including dark, drought and UV-B light, as well as wounding and salicylic acid.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Fagopyrum/enzimologia , Fagopyrum/fisiologia , Estresse Fisiológico , Ácido Aspártico Endopeptidases/genética , Sequência de Bases , Fagopyrum/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Dados de Sequência MolecularRESUMO
Two types of aspartic proteinase (AP) genes have been isolated from the cDNA library of developing buckwheat seeds. Analysis of their sequences showed that one of these, FeAP9, resembled the structure and shared high homology with the so-called typical plant APs characterized by the presence of a plant-specific insert (PSI), an element unique among APs. The other cDNA, FeAPL1, encoded an AP-like protein lacking that domain. Different expression profiles were observed for FeAP9 and FeAPL1. FeAPL1 mRNAs were restricted to the seeds only, whereas FeAP9 mRNAs were also present in the other plant tissues - leaves, roots, and flowers. Higher levels of FeAP9 were observed in senescent leaves compared with green leaves. The differential expression pattern of these two unique APs raises the interesting possibility that these proteinases have unique substrate specificity and may have different roles in plant development and other physiological processes.
Assuntos
Ácido Aspártico Endopeptidases/genética , Fagopyrum/enzimologia , Fagopyrum/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Sementes/enzimologia , Sementes/genética , Animais , Ácido Aspártico Endopeptidases/metabolismo , Autorradiografia , Clonagem Molecular , DNA Complementar/genética , Evolução Molecular , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Filogenia , RNA de Plantas/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sus scrofaRESUMO
Using the modified rapid amplification of cDNA ends (5'-RACE) approach, a fragment containing the 955 bp long 5'-regulatory region of the buckwheat storage globulin gene (FeLEG1) has been amplified from the genomic DNA of buckwheat. The entire fragment was sequenced, and the sequence was analyzed by computer prediction of cis-regulatory elements possibly involved in tissue-specific and developmentally controlled seed storage protein gene expression. The promoter obtained might be interesting not only for fundamental research but also as a useful tool for biotechnological application.
Assuntos
DNA de Plantas/isolamento & purificação , Fagopyrum/genética , Proteínas de Plantas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Sequência de Bases , Computadores , DNA Complementar/química , DNA de Plantas/química , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
An 8S storage globulin from buckwheat seed, which resembles the structure and features common to the vicilin-like family of seed storage proteins, was analyzed for this paper. It was found that expression of the 8S globulin gene precedes that of the 13S globulin (the main buckwheat storage protein) and starts from an early stage of buckwheat seed development (9-11 days after flowering), continuing to accumulate throughout seed development to contribute approximately 7% of total seed proteins. This protein fraction might be more interesting for biotechnological application than the 13S buckwheat legumin consisting of 23-25 kDa subunits reported to be the major buckwheat allergen. A partial cDNA was also isolated, showing high homology with cDNAs coding for vicilin-like storage proteins from various plant species, and its expression profile throughout seed development as well as in different buckwheat tissues was analyzed.
Assuntos
Fagopyrum/química , Proteínas de Plantas , Proteínas de Plantas/análise , Sementes/química , Sequência de Aminoácidos , Northern Blotting , DNA Complementar/isolamento & purificação , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Armazenamento de Sementes , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Alinhamento de SequênciaRESUMO
We have isolated and characterized a full-length cDNA for legumin-like storage polypeptide from buckwheat seed (Fagopyrum esculentum Moench) and compared its deduced amino acid sequence with those from different representatives of dicots, monocots and gymnosperms. The cDNA sequence was reconstructed from two overlapping clones isolated from a cDNA library made on mRNA of buckwheat seed at the mid-maturation stage of development. Analysis of the deduced amino acid sequence revealed that this specific buckwheat storage polypeptide should be classified in the methionine-rich legumin subfamily present in the lower angiosperm clades, a representative of which was first characterized in Magnolia salicifolia (clone B 14). The fact that a methionine-rich legumin coexists together with methionine-poor legumins in buckwheat should be an important element regarding the evolutionary position of buckwheat. This may also be supporting evidence that the B14 ortholog was not lost in evolution but was protected under pressure of an increased need for sulfur. Using primers designed from characterized cDNA, we also isolated its corresponding gene from buckwheat genomic DNA and analyzed the characteristic exon/intron structure. The firstly identified two-intron structure of buckwheat legumin gene is an important contribution to study of methionine-rich legumins in lower angiosperms.
