中文摘要：

【目的】利用同源序列法分离核桃的NBS（NucleotidebindingSite）类抗病基因类似物，为核桃抗炭疽病分子辅助育种及抗病基因的克隆提供基础。【方法】以35个核桃优系为试材，利用接种法鉴定供试材料对炭疽病的抗性；根据己知植物抗病基因的保守结构域P-loop和GLPL设计简并引物，以核桃优系的基因组DNA为模板，通过PCR扩增NBS类抗病基因同源序列片段，并分析所得NBS类抗病基因类似物与核桃优系炭疽病抗性的关系；利用BLASTN／X程序对所得NBS序列在GenBank数据库中进行同源性搜索；利用MEGA5．2及DNAman7．0等软件对其进行序列相似性分析和系统进化研究。【结果】35个供试核桃优系中，有20个优系为抗炭疽病类型（R），其相对抗性指数在0．63—0．82；有5个优系为中抗类型（M），相对抗性指数在0．27—0．56；有10个为感病类型（s），相对抗性指数在0．00—0．21。PCR结果显示，从20个抗炭疽病优系的基因组扩增得到20条NBS类抗病基因类似序列；而在其它15个优系（5个中抗优系和10个感病优系）中未扩增到条带，表明核桃NBS序列与炭疽病抗性相关联。BLASTN显示，所得NBS序列在核苷酸水平与GenBank中已知的核桃NBS序列（jrRGAPGs）存在89％-ioo％~源性，与其它物种NBS序列的同源性在69％以上；BLASTX显示，所得NBS序列与已知核桃NBS抗病蛋白同源性为77％-99％，与其它物种的NBS抗病蛋白同源性在49％-66％。氨基酸序列多重比对分析表明，所得核桃NBS序列均包含有抗病基因所具有的典型功能域，如P-Ioop、kinase-2、kinase-3和GLPL等，在典型功能域的核苷酸多态性（Pi）明显低于非保守区，有较高保守性。系统发育分析表明，在核苷酸水平上可将所得核桃优系的NBS序列区分为7类，在氨基酸水平上可分TIR和non—TIR两大类7个亚类；所得核桃NBS序列非同义替换率（dN）和同

英文摘要：

[ Objective ] Isolating NBS-type （Nucleotide binding site） resistance gene analogs （RGAs） from walnut （Juglans regia L.） using homology-based method would provide a foundation for molecular-assisted selection and for cloning R gene during walnut breeding. [Method] Thirty-five walnut superiors were used as plant materials in the study and their resistance to anthracnose was identified with inoculation. NBS-type RGAs were isolated by a PCR strategy using degenerate primers specific to P-loop and GLPL, conserved motifs of NBS domain in plant R gene. The relationship between NBSs and walnut anthracnose resistance was analyzed. Sequence identification of obtained sequences was performed against known RGAs deposited in GenBank using BLASTN/X algorithms. Similar and phylogenetic analysis were elaborated using MEGA 5.2 and DNAman 7.0 software. [Result] Out of 35 tested walnut superiors, 20 superiors were identified as resistant （R） with a relative resistance index （RRI） ranging from 0.63 to 0.82; 5 of them were medium resistant （M）, whose RRI ranged 0.27-0.56; the rest 10 superiors as susceptible （S） with the RRI ranging 0.00-0.21. NBSs were amplified only in 20 resistant superiors and no bands were found in the remaining 15 superiors （5 medium resistant and 10 susceptible）, indicating that the NBS-RGAs were associated with the resistance to walnut anthracnose （Colletotrichum gloeosporioides）. BLASTN showed the obtained NBSs shared high similarities to cloned jrRGAPGs with a identity ranging 89%-100%; and they shared than 69% homology to other species from GenBank; BLASTX revealed 77%-99% and 49%-66% similarity to the NBS proteins from jrRGAPGs and other species, respectively. Multiple alignment analysis revealed that these NBS-type RGAs contained some well-characteristics motifs of NBS genes, including P-loop, kinase-2, kinase-3 and GLPL. The nucleotide polymorphism and diversity （Pi） were highly conserved at each motif than non-conservative fragments, indicating their conservativ