RESUMEN
Suppression subtractive hybridization was carried out to enrich gene fragments over-expressed in rice leaves by subtraction to rice roots, from which two identical cDNA fragments were identified to encode putative phosphoenolpyruvate carboxylase. Then the corresponding full-length cDNA (Osppc) is isolated by RT-PCR and sequenced, which indicates an open reading frame of 2895bp is contained. Its deduced protein is encoded in 10 exons and shows high similarity to many other plant PEPCs. Comparing with maize and bacterial PEPCs, it is revealed that OSPPC shares many conserved domains and active sites that responsible for the structure, activity and regulation of this enzyme. Phylogenetic analysis demonstrates that OSPPC is grouped with C3 form PEPCs of wheat, maize and sorghum, which is consistent with the classification of rice. And a putative promoter element is predicted with DOF binding box, CAAT box and TATA box in the 5'-flanking sequence of Osppc gene. Moreover, Quantitative RT-PCR analyses are performed in hybrid rice and its parents, which show that Osppc is specifically expressed in leaf including leaf vein and sheath.
Asunto(s)
Oryza/genética , Fosfoenolpiruvato Carboxilasa/genética , Hojas de la Planta/genética , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Oryza/enzimología , Oryza/metabolismo , Fosfoenolpiruvato Carboxilasa/metabolismo , Filogenia , Hojas de la Planta/enzimología , Hojas de la Planta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
cDNA sequences of malate dehydrogenase (MDH) were cloned from various species, and MDH was identified to play an important role in cell energy metabolism. Here, we present the isolation and characterization of its homologue (OscMDH) in Oryza sativa. Comparison of the results to the genome details indicated that OscMDH consisted of seven exons. Sequence alignment showed that the deduced amino acid sequence of OscMDH shared a significant similarity with cMDH protein in Zea mays, as well as with other cMDH gene products. The different expression patterns of OscMDH in different tissues revealed that OscMDH mRNA was highly transcribed in either young panicle or immature seed, while its abundance was much low in green leaf and root. A nearly 56-kDa fusion protein generated by adding a Trx-His-tag at the N-terminal of OscMDH was induced by IPTG in Escherichia coli BL21 and an obvious MDH activity was detected in the protein by native PAGE analysis. All these results suggest that OscMDH encodes a cytosolic MDH in rice.
Asunto(s)
Malato Deshidrogenasa/genética , Oryza/genética , Actinas/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Malato Deshidrogenasa/metabolismo , Datos de Secuencia Molecular , Especificidad de Órganos , Oryza/enzimología , Oryza/metabolismo , Semillas/genética , Semillas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
A novel osClpD gene, encoding a highly conservative ClpD subfamily member, was first isolated and characterized from Oryza sativa. The full-length cDNA of osClpD gene was 3140 bp and contained a 2884 bp open reading frame encoding a 938 amino acid protein. The phylogenetic tree and blast search showed that OSClpD belonged to the ClpD subfamily of the Hsp100/Clp family, and contained all protein motifs characteristic for the ClpD subfamily of Hsp100/Clp proteins. The real-time quantitative PCR analysis proved that it was inducible by water deficit and temperature stress in vegetative tissues.
Asunto(s)
Adenosina Trifosfatasas/genética , Oryza/genética , Filogenia , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Análisis por Conglomerados , Cartilla de ADN , ADN Complementario/genética , Desecación , Endopeptidasa Clp , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , TemperaturaRESUMEN
A subtractive cDNA library was constructed using rice (Oryza sativa L.) callus cDNA as driver and differentiating callus cDNA as tester. A novel cDNA fragment encoding RNase L inhibitor (RLI) was isolated by screening the subtractive library, which had a higher expression level in differentiating callus than in callus. The full-length cDNA of rice-RLI was obtained by the method of rapid amplification of cDNA ends, which contained a 1812-bp open reading frame encoding a 604 amino acid polypeptide. Homologous analysis showed that rice-RLI contained the conserved motifs (two repeated P-loops, two ATP-binding boxes and an iron-sulfur binding motif). The fluorescence quantitative PCR analysis showed that it was a constitutive expressed gene but up-regulated in abiotic stress (low temperature and NaCl treatment) and down-regulated under the treatments of NAA and IAA.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Chaperoninas/genética , Regulación de la Expresión Génica , Oryza/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , ADN Complementario/genética , Fluorescencia , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Cloruro de Sodio , Temperatura , Factores de TiempoRESUMEN
Malate dehydrogenase (MDH) has been characterized as a key player in oxaloacetate (OAA) biosynthesis mechanism in citrate acid cycle that generates reducing powers for further assimilation in the whole cell. Here we present the cloning, characterization and prokaryotic expression of a putative Mdh (OsmMDH) in Oryza sativa. Sequence alignment shows that there is a high homology between the deduced amino acid sequence of OsmMDH and MDH portein in Eucalyptus gunnii (80%), as well as between the deduced amino acid sequence of OsmMDH and other MDHs. Moreover, pI and the mitochondrial location of OsmMDH are predicted. The tissue-specific expression pattern of OsmMDH reveals that it is abundant in young panicle and immature seed, while its expression level is mush lower in leaf and root. Its expression in E. coli BL21 as a fusion gene is studied further.
Asunto(s)
Malato Deshidrogenasa/genética , Mitocondrias/enzimología , Oryza/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Malato Deshidrogenasa/metabolismo , Datos de Secuencia Molecular , Oryza/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Some cDNA sequences of subunit V (PsaG) of Photosystem I (PS I) reaction center in several species have been cloned, and PsaG has been characterized as a connection of light-harvesting complex I (LHC I) protein to photosystem reaction center. Here we present the isolation and characterization of its salt-induced homologue (DsPsaG) in Dunaliella salina. Sequence alignment shows that there is a significant similarity between the deduced amino acid sequence of DsPsaG and PsaG protein in Chlamydomonas reinhardtii, as well as between the deduced amino acid sequence of DsPsaG and other PsaG gene products. The differential expression of DsPsaG at different time points after salt stress reveals that DsPsaG mRNA was absent without salt stress and was indeed salt-induced. Its expression reached its maximum level 5 days after stress. Our study suggests that DsPsaG should be a compensation of PsaG in D. salina when the alga was in a hyperosmotic condition. It can also be useful to effectively transfer light energy from LHC I to PS I reaction center.
Asunto(s)
Chlorophyta/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Secuencia de Aminoácidos , Secuencia de Bases , Chlorophyta/metabolismo , Datos de Secuencia Molecular , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Complejo de Proteína del Fotosistema I , Alineación de Secuencia , Cloruro de Sodio/metabolismoRESUMEN
In silico cloning was a new strategy of gene cloning developed with the development of genome, EST projects and bioinformatics. Using wheat glucose-6-phosphate dehydrogenase cDNA (clone: Tagpdl) sequence as a querying probe, one highly homologous BAC clone sequence was obtained from rice sequence database of GenBank and the putative cDNA sequence of rice glucose-6-phosphate dehydrogenase was assembled according to the wheat clone. Furthermore, the full-length cDNA of rice glucose-6-phosphate dehydrogenase was cloned by RT-PCR with two primers designed based on this assembled cDNA sequence. Since this fragment contained a complete ORF of 1515 bp with a stop codon in its upstream and poly(A) signal in its downstream, it could be concluded that a full-length gene (GenBank accession number AY078072), which was named as OsG6PDH. Homology analysis of OsG6PDH showed a 88% identity with wheat and the deduced amino acid showed 89%, 79% and 80% homology with G6PDH from wheat, tomato and tobacco respectively. OsG6PDH was expressed in inflorescence, embryo, root and leaf of rice, with a slightly higher in inflorescence and root. It was also discussed in this paper that the application of in silico cloning in the isolation of functional genes from rice.