【作者】 王海燕；

【导师】 刘大群； 杨文香；

【作者基本信息】 河北农业大学 ， 植物病理学， 2003， 硕士

【摘要】 以抗病基因中的几个保守序列为依据合成寡核苷酸引物，并形成不同引物组合，通过对小麦抗叶锈近等基因系材料ThLr35及其感病亲本Thatcher基因组DNA进行扩增，同时结合ISSR标记，寻找预期的RGA片段，为克隆小麦抗叶锈基因Lr35目的基因奠定了基础。主要实验结果如下： 1．根据抗病基因NBS-LRR保守序列不同区域设计4个简并引物，形成不同组合。其中，引物组合RAF1：R2、RAF8：R2、RAF8：R5和组合RAF1：R5均能获得有效扩增，而且有一条约630bp条带几乎在各种引物组合中都存在。将根据保守序列GG(V／I)GKTT(P-loop)和GLPLAL(domain5)设计的引物组合RAF1：R2扩增获得的该DNA条带回收，并克隆，测序，BLASTn同源性比较表明，与GeneBank中一211Kb片段(AF326781)同源性达79％，而该211Kb片段为分离获得的450Kb小麦抗叶锈基因Lr10大片段的一部分。 2．根据蛋白激酶类保守序列DVKPEN(domainⅥ)和GTPWYIAPE(subdomainⅧ)设计一对简并引物RAF3：R4扩增，获得不同大小的4条片段，多次重复，表现良好的稳定性；而根据LRR类保守序列设计引物扩增，未获得有效扩增产物，说明小麦基因组中可能含有蛋白激酶类结构，而不含胞外LRR结构。 3．用小麦抗叶锈基因Lr35 ISSR标记回收片段作模板，以不同类保守序列引物进行扩增，NBS-LRR类引物组合RAF1：R2可获得一条597bp特异性条带，将其回收，克隆，测序。根据BLASTn同源性比较，与GeneBank中小麦属BAC克隆(AF459639)和一211Kb双倍体小麦(AF326781)同源性高达90％，可将该片段初步确定为Lr35相关片段。 4．根据RAF1：R2引物扩增产物的测序结果，设计两对特异性引物，摸索不同的PCR反应条件，在ThLr35基因组DNA中最终分别获得约1kb和2kb片段，而在感病亲本Thatcher中未获得相应扩增片段，可能为Lr35相关片段。更多还原

【Abstract】 A study based on the homology-based candidate gene method for cloning resistance gene was conducted by scanning genomic DNA in wheat near-isogenic lines (ThLr35) with degenerated oligonucleotide primers designed according to different conserved domains in cloned resistance genes. The main results are as follows:Four oligonucleotide primers were designed based on the conserved NBS-LRR domain of R genes. No specific amplified products ranging from approximately 200-2000bp were obtained from different primer combinations such as RAF1:R2, RAF8.-R2, RAF8.-R5 and RAF1 :R5. In the meanwhile, a fragment of 633bp which was common in four combinations was cloned.lt showed 79% homology to a part of 211kb fragment (AF326781) in GeneBank which is in turn a part of 450kb fragment containing Lr10.Another primer combination RAF3:R4 was designed corresponding to conserved sequence DVKPEN and GTPWYIAPE of protein kinase. Four main fragments were amplified, the optimized reaction system was established. But no product was obtained using primers designed according to the conserved LRR domains of R genes. The result indicates that conserved motif NBS-LRR and STK might exist in wheat genome, LRR might not exist. In other words, wheat resistance genes to leaf rust might not belong to the type of LRR resistance gene, but NBS-LRR and STK.A 597bp PCR product appeared in ThLr35, but not in the susceptible background parent Thatcher using Lr35 ISSR marker as a template. It has a high homology (90%) with a wheat BAC clone (AF459639) and a 211Kb fragment (AF326781), which contains Lr10, implying this gene may be involved in wheat leaf rust resistance gene Lr35.With primers designed according to the sequence of the DNA fragment, twofragments about 1.0kb and 2.0kb were amplified from the genomic DNA of ThLr35. Whether these fragments are related to Lr35 or not should be further testified.更多还原