伴花生球蛋白cDNA克隆及其在花生发育种子中的表达

The cDNA Cloning of Conarachin Gene and Its Expression in Developing Peanut Seeds

利用伴花生球蛋白多克隆抗体,免疫筛选花生 品种汕油523成熟子叶中期cDNA文库得到6个阳性克 隆。经过DNA序列测定和同源性分析确定为2组(Ahy α和Ahyβ),2组序列之间的同源性为97％。Ahyβ与 花生过敏原Ara h 1 p17以及Ahy α与花生过敏原Ara h 1 p41b的核苷酸相同性达到99％以上。以Ahy-βcDNA 为探针的Northern blot分析结果表明,伴花生球蛋白 基因在发育的花生种子中大量表达,而在幼苗的叶片 中不表达。对成熟中期花生子叶表达序列标签(EST)分 析,获得了包括5种花生球蛋白、2种伴花生球蛋白、 6种conglutin蛋白的EST共70条,占总转录本的17％。

In order to clone the genes of peanut seed storage proteins, a cDNA library of mid-maturation stage cotyledons of peanut (Arachis hypogaea L. Shanyou 523) was constructed. After immunoscreening with polyclonal antibody against conarachin from peanut seeds, two cDNA sequences named Ahy α and Ahy β of conarachin genes were obtained. BLAST these sequences in GenBank showed that they share high homol-ogy with the genes of legume 7S globulins. One of the sequences (Ahy β) was found to be identical to part of the published complete cDNA sequence of the peanut allergen, Ara h1 p17. The other fragment (Ahy α) was almost identical to parts of the published complete cDNA sequence of the peanut allergen, Ara h1 p41b. Results of southern analysis show that they belong to a small gene family (the result is not shown). These sequences were expressed only in developing peanut seeds (Fig.2). To isolate the genes involved in seed storage protein and to understand the gene expression during peanut seed development, the sequences of 414 ESTs were determined and further analyzed by BLAST searches and categorized functionally. Five rESTs (representative ones) of arahin, two rESTs of conarachin and six rESTs of conglutin-like proteins were isolated. 17.0% of total transcripts isolated in this study are involved in peanut seed storage proteins (Table 1).