受白粉菌诱导表达的簇毛麦草酸氧化酶基因(Hv-OxOLP)的克隆和分析

为了筛选抗白粉病基因Pm21介导的抗病途径中的防卫基因，采用芯片杂交技术筛选携带或不携带Pm21基因的材料之间或抗病材料经白粉菌诱导与非诱导样品之间的差异表达基因，并采用RT-PCR方法分析了被筛选出的草酸盐氧化酶类似蛋白基因（Oxalate Oxidase Like Protein，OxOLP）的表达情况，进一步利用TAIL-PCR方法分离簇毛麦基因Hv-OxOLP中的全长序列。芯片杂交结果表明，探针Contig3156（一个推导的草酸盐氧化酶类似蛋白基因）在抗病簇毛麦中受白粉菌诱导的程度最大，在抗病易位系中该探针的杂交信号比在感病扬麦5号中的杂交信号强。半定量RT-PCR结果表明，在抗、感白粉病的材料中草酸盐氧化酶类似蛋白基因都受白粉菌诱导表达，但在抗病材料中达到表达峰值的时间早。用TAIL-PCR方法分离的Hv-OxOLP有一个内含子和两个外显子，ORF包含690个核苷酸，在启动子区包含两个W-box，在终止密码子后面还有一个polyA的加尾信号。作为一个受白粉菌诱导表达的基因，HpOxOLP在抗白粉病反应中可能起重要的作用。

Cloning and Analysis of an Hv-OxOLP Gene Induced by Ergsiphe graminis in H.villosa

To isolate defense genes in the resistance pathway mediated by Pm21(a gene shows high resistance to powdery mildew),microarray was used to screen the differentially expressed genes between plants with and without Pm21 and between the samples of resistant plants before and after inoculation with Ergsiphe graminis.RT-PCR was used to analyze the expression of the screened oxalate oxidase like protein gene(OxOLP) and the full length fragment of the Hv-OxOLP gene of Haynaldia.villosa was isolated by TAIL-PCR.The probe Contig3156,a putative OxOLP,was most upregulated by the Ergsiphe graminis inocalation in the resistant H.villosa and its expression level in a resistant translocation line(6VS/6AL) was also higher than that in the susceptible Yangmai 5.The result of RT-PCR indicated that the OxOLP was induced by Ergsiphe graminis in the resistant and susceptible plants,but the expression level of OxOLP reached the highest earlier in the resistant plants than in the susceptible plants.The complete sequence of the Hv-OxOLP in H.villosa was cloned using TAIL-PCR.The whole gene contained one intron and two extrons,and the ORF contained 690 nucleotides.In the promoter region of Hv-OxOLP,there was a TATA box and two W-boxes.Following the stop code,there was a polyA signal motif.As a gene induced by Ergsiphe graminis,Hv-OxOLP may play an important role in the resistance pathway to powdery mildew.