RESUMEN
The transcriptional activity of the sorghum sbeIIb gene, encoding starch branching enzyme IIb, is seed specific, with expression in both the endosperm and the embryo. In comparison, expression of barley sbeIIb is confined to the endosperm, whereas that of barley sbeIIa occurs in endosperm, embryonic and vegetative tissues. It has been suggested that the second intron of barley sbeIIb may be instrumental in conferring endosperm-specific expression. Therefore, to further investigate the regulatory mechanisms of barley and sorghum sbe, we examined the tissue-specific activity of the sorghum sbe promoter in transient assays of green fluorescent protein (gfp) reporter constructs. We found that, when linked to the barley sbeIIb second intron, the sorghum sbeIIb promoter could not drive gfp transcription in sorghum or barley embryonic cells. Similar results were obtained for the barley sbeIIa promoter. Database searches showed that sequences homologous to the barley sbeIIb intron also exist in introns and flanking regions of some other grass genes. Deletion mutagenesis of the sorghum sbeIIb promoter identified the minimal promoters required for high- and low-level expression, respectively, but did not reveal any putative promoter elements crucial for expression. A sequence with similarity to the SURE element, implicated in sugar signaling, was located in the distal promoter region of sorghum sbeIIb, upstream of the minimal promoters. SURE elements are present in the proximal promoter regions of the sugar-regulated barley iso1 gene, and barley sbeIIb. In keeping in line with these observations, RNA-gel blot analyses demonstrated that expression of barley sbeIIb was sugar inducible, whereas that of sorghum sbeIIb was not.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/genética , Regulación de la Expresión Génica de las Plantas , Hordeum/genética , Intrones/genética , Sorghum/genética , Transcripción Genética , Secuencia de Bases , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Hordeum/enzimología , Datos de Secuencia Molecular , Semillas/metabolismo , Sorghum/enzimologíaRESUMEN
A genomic clone for starch branching enzyme (SBE) IIb was isolated from a sorghum bacterial artificial chromosome (BAC) library. The promoter and 5' flanking sequence, the first four exons and introns as well as the last exon and the 3' untranslated region were sequenced. The tentative transcription start site of sorghum sbeIIb was mapped based on alignment with the maize sbeIIb gene. The exon-intron structure of the 5' portion of sorghum sbeIIb was similar to that of maize sbeIIb but differed from that of barley sbeIIb. Specifically, the intronic BbI element involved in the endosperm specific expression of barley sbeIIb was lacking in the sorghum gene. A cDNA clone for sorghum sbeIIb was reverse PCR amplified and found to encode an 803 amino acids long protein. The amino acid sequence of sorghum SBEIIb was compared to that of sorghum SBEI and corresponding enzymes in barley. The overall identity in amino acid sequence was 54% in the central portion of the enzymes. A major difference between the SBEII and SBEI isoforms was a 67 amino acids-long C-terminal extension in the SBEIs. The spatial and temporal expression patterns of sorghum sbeIIb was determined and compared with those of the sorghum gene for SBEI and the barley genes for SBEIIB and SBEI. All four genes exhibited a seed specific expression. However, while barley sbeIIb and sbeI transcripts were detected exclusively in the endosperm, the sorghum genes were expressed also in the embryo. The activity of sorghum sbeIIb and sbeI exhibited a late onset, with a peak of transcription at around 22 days after pollination. This is similar to the pattern of barley sbeI but different from that of barley sbeIIb, which showed a peak of transcription at 12 days after pollination.