To establish the size and organization of the rice alpha-amylase multigene family, we have isolated 30 alpha-amylase clones from three independent genomic libraries. Partial characterization of these clones indicates that they fall into 5 hybridization groups containing a total of 10 genes. Two clones belonging to the Group 3 hybridization class have more than one gene per cloned fragment. The nucleotide sequence of one clone from Group 1, lambda OSg2, was determined and compared to other known cereal alpha-amylase sequences revealing that lambda OSg2 is the genomic analog of the rice cDNA clone, pOS103. The rice alpha-amylase genes in Group 1 are analogous to the alpha-Amy1 genes in barley and wheat. lambda OSg2 contains sequence motifs common to most actively transcribed genes in plants. Two consensus sequences, TAACAAGA and TATCCAT, were found in the 5' flanking regions of alpha-amylase genes of rice, barley and wheat. The former sequence may be specific to alpha-amylase gene while the latter sequence may be related to a 'CATC' box found in many plant genes. Another sequence called the pyrimidine box (TCCTTTTTC) was found in the alpha-amylase genes as well as other genes regulated by gibberellic acid (GA). Comparisons based on amino acid sequence alignment revealed that the multigene families in rice, barley and wheat shared a common ancestor which contained three introns. Some of the descendants of the progenitor alpha-amylase gene appear to have lost the middle intron while others maintain all three introns.
A rice beta-glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation. This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots. Expression in the roots appears to be constitutive. Shoots express Gns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogen Sclerotium oryzae or from the non-pathogen Saccharomyces cereviseae. Shoots also express Gns1 at higher levels in response to wounding. Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid. The beta-glucanase shows 82% amino acid similarity to the barley 1,3;1,4-beta-D-glucanases, and from hybridization studies it is the beta-glucanase gene in the rice genome closest to the barley 1,3;1,4-beta-glucanase EI gene. The mature peptide has a calculated molecular mass of 32 kDa. The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide. The gene exhibits a very strong codon bias of 99% G + C in the third position of the codon in the mature peptide coding region, but only 61% G + C in the signal peptide region.
The nucleotide sequence of a cDNA clone isolated from developing wheat embryos and encoding the E protein is reported. The entire coding region for E and the 3' non-translated flank are contained within this clone. The amino m acid sequence deduced for Em is very rich in glycine (18 mol%) as well as both basic and acidic residues. The molecular weight of the protein is ca. 9,900 daltons. The deduced sequence is supported by direct amino acid sequencing of cyanogen bromide cleavage fragments obtained from purified E protein. E is shown by Southern blots to be a product of a gene family of approximately ten members.
Steady-state levels of mRNA from individual alpha-amylase genes were measured in the embryo and aleurone tissues of rice (Oryza sativa) and two varieties of barley (Hordeum vulgare L. cv. Himalaya and cv. Klages) during germination. Each member of the alpha-amylase multigene families of rice and barley was differentially expressed in each tissue. In rice, alpha-amylase genes displayed tissue-specific expression in which genes RAmy3B, RAmy3C, and RAmy3E were preferentially expressed in the aleurone layer, genes RAmy1A, RAmy1B and RAmy3D were expressed in both the embryo and aleurone, and genes RAmy3A and RAmy2A were not expressed in either tissue. Whenever two or more genes were expressed in any tissue, the rate of mRNA accumulation from each gene was unique. In contrast to rice, barley alpha-amylase gene expression was not tissue-specific. Messenger RNAs encoding low- and high-pI alpha-amylase isozymes were detectable in both the embryo and aleurone and accumulated at different rates in each tissue. In particular, peak levels of mRNA encoding high-pI alpha-amylases always preceded those encoding low-pI alpha-amylases. Two distinct differences in alpha-amylase gene expression were observed between the two barley varieties. Levels of high-pI alpha-amylase mRNA peaked two days earlier in Klages embryos than in Himalaya embryos. Throughout six days of germination, Klages produced three times as much high-pI alpha-amylase mRNA and nearly four times as much low-pI alpha-amylase mRNA than the slower-germinating Himalaya variety.
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