用抑制差减杂交技术分离烯丙异噻唑诱导水稻特异表达的基因

烯丙异噻唑(PBZ)处理水稻根能使其产生对稻瘟病的系统获得性抗性，因此在东南亚稻区被广泛用于防治稻瘟病，然而关于其作用的分子机理还知之甚少．运用抑制差减杂交技术，试图通过分离鉴定受PBZ诱导调控的关键基因，探索其作用的分子机理．以PBZ处理后的水稻叶片cDNA为目标群体(tester)，以未处理水稻叶片cDNA为对照群体(driver)，用经过对照cDNA差减的、烯丙异噻唑处理的cDNA群体构建了一个含260个重组子的差减文库．通过差示筛选鉴定出了26个。PBZ诱导水稻特异表达和增强表达的候选克隆．对26个cDNA克隆进行了双向测序和同源性比较，发现其中3个克隆：rJAB1，rTAB2和蛋白磷酸酯酶2Aδ调节亚基同型物基因，位于抗病相关信号转导途径上，它们与哺乳动物和人类免疫途径上的信号因子有明显相似之处，因此推断可能与诱导抗性有关．另外8个克隆与已知基因同源性为70％～99％．经Northern杂交分析，其中rJAB1(编码c-jun激活区结合蛋白1)受烯丙异噻唑和稻瘟菌诱导表达；膜糖蛋白同源基因及肌动蛋白(actin)α1受烯丙异噻唑诱导表达，部分克隆为低丰度转录本．

Isolation of Genes with Enhanced Expressions in Rice Treated with Probenazole Using SSH Technique

Probenazole (PBZ; 3-allyloxy-1,2-benzisothiazole-1,1-dioxide), which is the active ingredient in Oryzemate, can induce rice to produce systemic acquired resistance (SAR) and has therefore been widely used in Southeast Asia for over 30 years to protect rice plants against the rice blast fungus Magnap orthe grisea . However, mechanisms underlying have not been convincingly elucidated. To understand the mechanisms, suppression subtractive hybridization (SSH) was adopted to isolate novel probenazole-responsive genes. Using cDNA from rice leaves treated with PBZ as tester and cDNA from control as driver to conduct subtractive hybridization,a subtractive cDNA library with two hundred sixty individual clones was constructed. Thirty six candidate clones, which were differentially expressed after the treatments,were identified by differential screening. Eleven cDNA clones were sequenced and blasted for homology in GenBank. Of these, three clones,rTAB2, rJAB1 and rPP2A, were considered to be related to signal-transduction pathways of the SAR,possibly induced by PBZ. To further isolate the upstream cis-elements of these genes, genomic clones were isolated from a rice genomic library with the fragment of rJAB1, rSarcosin and rDKFp586 as a probe, respectively.