利用抑制消减杂交分离受褐飞虱取食下调的水稻基因

Isolation of Rice Genes Down-regulated by Brown Planthopper Infestation by Suppression Subtractive Hybridization

为了分离受褐飞虱取食抑制的水稻基因,采用抑制消减杂交的方法,以正常生长的水稻幼苗为目标群体,以褐飞虱胁迫32 h的水稻幼苗作为对照群体,构建了含200个重组质粒的SSH cDNA文库。随机挑选50个重组质粒进行反向Northern差异筛选后,再经Northern杂交验证,得到2个受褐飞虱取食抑制的基因:一个是Lhca,编码水稻光系统Ⅰ天线蛋白;另一个基因(BpHd002)与肌苷-5’-单磷酸脱氢酶基因有同源性。以BpHd002为探针筛选水稻幼苗cDNA文库分离出该基因的全长cDNA(BpHd002A)。其长度为1 285bp,含有一由519 bp组成的完整的阅读框,编码的蛋白质具有两个CBS结构域。

Suppression subtractive hybridization (SSH) was used to isolate rice genes down-regulated by BPH infestation. The SSH cDNA library constructed with the tester cDNA from rice seedlings, and driver cDNA from rice seedlings fed by BPH consists of 200 clones. Fifty clones were randomly picked and screened by reverse Northern dot blot and Northern blot (Fig. 1). As a result, two cDNA fragments have been isolated: one is Lhca, which encodes rice pho-tosystem I antenna protein; the other is a novel rice gene, and designated as BpHd002 (Brown planthopper depressed). The cDNA library for rice seedlings was screened using BpHd002 as probe, and a full-length cDNA (BpHd002A) has been isolated (Fig.2). The cDNA is 1 285 bp in length; the putative open reading frame is 519 bp long, which encodes a protein with two CBS domains, homologous with inosine-5'-mono-phosphate dehydrogenase.