核桃NBS类抗病基因类似物的序列特征及其与炭疽病的抗性

【目的】利用同源序列法分离核桃的NBS（Nucleotide binding site）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；根据已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MEGA 5.2及DNAman 7.0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%—100%同源性，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-loop、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)和同义替换率(dS)的比值dN/dS在0.00—0.95，为纯化选择。氨基酸序列相似性表明核桃优系不同NBS亚类间的相似度为28.3%—63.5%，与已知抗病基因相应区域的氨基酸相似性为22.0%—48.5%。【结论】核桃NBS类抗病基因类似物与炭疽病抗性相关联，NBS序列与其他物种抗病基因有较高同源性，包含抗病基因保守的功能域，在进化上为纯化选择。

Sequence Analysis of NBS-Type RGAs and Their Relationship with Anthracnose Resistance in Walnut

【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 conservative structures. Phylogenetic analysis revealed that the NBSs were clustered into seven subgroups at nucleotide level. They were grouped into two clades (TIR and non-TIR) and were subdivided into 7 groups based on their amino acid sequence similarity. Ratio of non-synonymous to synonymous nucleotide substitution (dN/dS) among NBS-RGAs varied from 0.00 to 0.95 (lower than one) for different classes, suggesting a purifying selection. Similarity percentages of deduced amino acid among these 7 NBS subgroups ranged from 28.3% to 63.5% with identifies to R genes ranging from 22.0% to 48.5%. 【Conclusion】 NBS-type RGAs isolated in the study were associated with anthracnose resistance, they were highly similar to cloned R genes and contained some conserved motifs. Walnut NBS-type RGAs undergo a purifying selection.