Yamauchi D, Nakamura K, Asahi T, Minamikawa T
Department of Biology, Tokyo Metropolitan University, Japan.
Eur J Biochem. 1988 Jan 4;170(3):515-20. doi: 10.1111/j.1432-1033.1988.tb13730.x.
By a method of Escherichia coli expression-vector-primed cDNA synthesis, a cDNA expression library was constructed from total poly(A)-rich RNA that was prepared from immature embryos of Canavalia gladiata. Essentially full-length cDNA clones for two seed proteins, canavalin and concanavalin A, were selected from the library by immunological screening of the colonies and in vitro RNA synthesis and translation. The complete amino acid sequence of canavalin was determined from the nucleotide sequence of the corresponding cDNA and was found to be very homologous to 7S seed proteins of other legumes. The nucleotide sequence of the cDNA predicts a 26-amino-acid extension in the precursor at the amino terminus of the mature canavalin. Canavalin mRNA and concanavalin A mRNA levels at successive stages of the seed development were estimated by RNA blot hybridization and results indicated that the two mRNA levels are differently regulated.
通过大肠杆菌表达载体引发的cDNA合成方法，从刀豆未成熟胚制备的富含多聚腺苷酸的总RNA构建了一个cDNA表达文库。通过对菌落进行免疫筛选以及体外RNA合成与翻译，从文库中筛选出了两种种子蛋白——伴刀豆球蛋白和刀豆球蛋白A的基本全长cDNA克隆。根据相应cDNA的核苷酸序列确定了伴刀豆球蛋白的完整氨基酸序列，发现它与其他豆科植物的7S种子蛋白高度同源。该cDNA的核苷酸序列预测成熟伴刀豆球蛋白前体在氨基末端有一个26个氨基酸的延伸。通过RNA印迹杂交估计了种子发育连续阶段的伴刀豆球蛋白mRNA和刀豆球蛋白A mRNA水平，结果表明这两种mRNA水平受到不同的调控。
