摘要：
In order to understand better the resistant mechanism of spinach (Spinach oleracea L.) downy mildew and lay foundation for further clone of spinach downy mildew resistance genes and marker-assisted-selection of resistance breeding,2 pares of degenerate primers were designed from the conserved motifs of NBS-LRR type plant resistance gene.There are 3 types of spinach material-commercial hybrid ‘RZ51-147’,inbred line‘ 12S3’ and‘ 12S4’ Two pares of primers were used for amplifying the disease-resistant gene analogs(RGAs) by polymerase chain reaction (PCR) from the genomic DNA of these 3 material.After cloning and sequencing the PCR products,23 resistance gene analogs with uninterrupted open reading frames (ORFs) and conserved domains of NBS were obtained from spinach,and deposited in GenBank with the accession number of KX914865-KX914887.Homology research showed that most of these 23 RGAs shared high homology with some putative disease-resistant genes or the other relative protein genes in Beta vulqaris.For example,the nucleotides of SP1,SP2,SP5,SP6,SP10,SP11,SP12,SP13 were over 86％ identical to disease resistance protein RF45 from sugar beet,and the nucleotides of SP3,SP4,SP8,SP9,SP24 were over 85％ identical to sugar beet disease resistance RPP13 protein.In addition,these 5 RGAs shared 32.20％-33.52％ amino acid sequence homology with sunflower downy mildew resistance gene PI8,and clustered a group with PI8 gene in phylogenetic tree,suggesting that these 5 RGAs might be related to downy mildew gene.Homologous evolution analysis demonstrated that all of RGAs were ranked into non-TIR-NBS-LRR type R gene except SP7,which was consistent with the result based on multiple alignment of deduced amino acid sequences.%依据已知NBS-LRR类抗病基因的P-loop和GLPL两个保守结构域设计简并引物,对抗菠菜霜霉病商业杂交种RZ51-147、自交系12S3和12S4的基因组DNA进行PCR扩增、克隆和序列测定,共获得23条具有完整开放阅读框且含有NBS结构的抗病基因同源序列(resistance gene analogs,RGAs),编号为SP1～ SP17、SP19 ～ SP24,其在NCBI中的登录号为KX914865 ～ KX914887.核苷酸比较分析结果表明,这23条RGAs大多与甜菜中推测的一些抗病蛋白基因或其他相关蛋白基因具有较高的同源性,其中SP1、SP2、SP5、SP6、SP10、SP11、SP12、SP13等8条序列均与甜菜RF45类抗病蛋白有着86％以上的同源性;SP3、SP4、SP8、SP9、SP24等5条序列与甜菜RPP13类抗病蛋白的同源性在85％以上,与向日葵抗霜霉病基因PI8相应区域的氨基酸序列相似性为32.20％～ 33.52％,且在进化树上聚为一类,推测其可能与抗霜霉病相关.同源进化分析结果表明,除SP7外,其余序列均为non-TIR-NBS-LRR类抗病基因,并与推导的氨基酸序列多重比较结果一致.
