菠菜NBS-LRR类抗病基因同源序列的克隆及分析  

Cloning and Analysis of NBS-LRR Type Disease-resistant Gene Analogs in Spinach oleracea L.

作　　者：折红兵 范桂彦 张合龙 王晓武[1] 武剑[1] 钱伟 徐兆生 SHE Hong-bing;FAN Gui-yan;ZHANG He-long;WANG Xiao-wu;WU Jian;QIAN Wei;XU Zhao-sheng(Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China;China Vegetable Seed Technology Co.,Ltd.,Beijing 100081,China)

机构地区：[1]中国农业科学院蔬菜花卉研究所,北京100081 [2]中蔬种业科技(北京)有限公司,北京100081

出　　处：《中国蔬菜》2017年第5期26-33,共8页China Vegetables

基　　金：国家"十二五"科技支撑计划项目(2012BAD02B04;2014BAD01B08);国家自然科学基金项目(31401872);中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS);农业部园艺作物生物学与种质创制重点实验室项目

摘　　要：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(resistancegeneanalogs,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。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-assistedselection f resistance breeding,2pares of degenerate primers were designed from the conserved motifs of NBSLRR ype plant resistance gene.There are3types of spinach material-commercial hybrid‘RZ51-147’,inbred ine‘12S3’and‘12S4’.Two pares of primers were used for amplifying the disease-resistant gene analog(s RGAs)y polymerase chain reaction(PCR)from the genomic DNA of these3material.After cloning and sequencing he PCR products,23resistance 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 these23RGAs shared high homology with some putative isease-resistant genes or the other relative protein genes in Beta vulqaris.For example,the nucleotides of SP1,SP2,SP5,SP6,SP10,SP11,SP12,SP13were over86%identical to disease resistance protein RF45from ugar beet,and the nucleotides of SP3,SP4,SP8,SP9,SP24were over85%identical to sugar beet disease esistance RPP13protein.In addition,these5RGAs shared32.20%-33.52%amino acid sequence homology ith sunflower downy mildew resistance gene PI8,and clustered a group with PI8gene in phylogenetic tree,suggesting that these5RGAs might be related to downy mildew gene.Homologous evolution analysis demonstrated hat 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.

关 键 词：菠菜 抗病基因同源序列 NBS-LRR 菠菜霜霉病 

分 类 号：S436.36[农业科学—农业昆虫与害虫防治]