Adachi T, Izumi H, Yamada T, Tanaka K, Takeuchi S, Nakamura R, Matsuda T
Department of Food Science and Technology, School of Agriculture, Nagoya University, Japan.
Plant Mol Biol. 1993 Jan;21(2):239-48. doi: 10.1007/BF00019940.
Genomic and two novel cDNA clones for rice seed allergenic protein (RA) belonging to the alpha-amylase/trypsin inhibitor family were isolated and their nucleotide sequences determined. Ten cysteine residues deduced from nucleotide sequences were completely conserved among three cDNA clones including a clone, RA17, reported previously. One genomic clone, lambda 4, contained two RA genes, RAG1 and RAG2. Although RAG1 was cloned at the 5' portion only, two RA genes were arranged divergently. Nucleotide sequencing and DNA blotting analyses showed that RA are encoded by a multigene family consisting of at least four members. The transcriptional initiation site of RAG1 was localized at A, 26 bp upstream of the putative translational initiation codon, ATG, by the primer extension assay. The putative TATA box and CAAT box existed about 45 bp and 147 bp upstream of the transcription initiation site, respectively. A conserved sequence (ATGCAAAA) which was similar to the sequence (TGCAAAA) identified in rice glutelin promoters was observed in the 5' region of the two genes. In addition, RNA blotting analyses provided that RA genes specifically expressed in ripening seed and their transcripts accumulated maximally between 15 and 20 days after flowering.
分离出了水稻种子变应原蛋白（RA）的基因组克隆和两个新的cDNA克隆，该蛋白属于α-淀粉酶/胰蛋白酶抑制剂家族，并测定了它们的核苷酸序列。从核苷酸序列推导的10个半胱氨酸残基在包括先前报道的克隆RA17在内的三个cDNA克隆中完全保守。一个基因组克隆λ4包含两个RA基因，RAG1和RAG2。虽然RAG1仅在5'部分被克隆，但两个RA基因呈反向排列。核苷酸测序和DNA印迹分析表明，RA由一个至少由四个成员组成的多基因家族编码。通过引物延伸试验，RAG1的转录起始位点位于假定的翻译起始密码子ATG上游26bp处的A。假定的TATA盒和CAAT盒分别存在于转录起始位点上游约45bp和147bp处。在这两个基因的5'区域观察到一个与水稻谷蛋白启动子中鉴定的序列（TGCAAAA）相似的保守序列（ATGCAAAA）。此外，RNA印迹分析表明，RA基因在成熟种子中特异性表达，其转录本在开花后15至20天积累最多。
