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1.
Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species. 总被引:3,自引:0,他引:3
Most known plant disease-resistance genes (R genes) include in their encoded products domains such as a nucleotide-binding site (NBS) or leucine-rich repeats (LRRs). Sequences with unknown function, but encoding these conserved domains, have been defined as resistance gene analogues (RGAs). The conserved motifs within plant NBS domains make it possible to use degenerate primers and PCR to isolate RGAs. We used degenerate primers deduced from conserved motifs in the NBS domain of NBS-LRR resistance proteins to amplify genomic sequences from Lens species. Fragments from approximately 500-850 bp were obtained. The nucleotide sequence analysis of these fragments revealed 32 different RGA sequences in Lens species with a high similarity (up to 91%) to RGAs from other plants. The predicted amino acid sequences showed that lentil sequences contain all the conserved motifs (P-loop, kinase-2, kinase-3a, GLPL, and MHD) present in the majority of other known plant NBS-LRR resistance genes. Phylogenetic analyses grouped the Lens NBS sequences with the Toll and interleukin-1 receptor (TIR) subclass of NBS-LRR genes, as well as with RGA sequences isolated from other legume species. Using inverse PCR on one putative RGA of lentil, we were able to amplify the flanking regions of this sequence, which contained features found in R proteins. 相似文献
2.
Recently, a number of disease-resistance genes related to a diverse range of pathogens were isolated from a wide variety of plant species. The majority of plant disease-resistance genes encoded a nucleotide-binding site (NBS) domain. According to the comparisons of the NBS domain of cloned R -genes, it has shown highly conserved amino acid motifs in this structure, which made it possible to isolate resistance gene analogs (RGAs) by PCR using degenerate primers. We have designed three pairs of degenerate primers based on two conserved motifs in the NBS domain of resistance proteins encoded by R -genes to amplify genomic sequences from ryegrass ( Lolium sp.). Sixteen NBS-like RGAs were isolated from turf and forage type grasses. The sequence analysis of these RGAs revealed that there existed a high similarity (up to 85%) between RGA sequences among ryegrass species and other plants. The alignment of the predicted amino acid sequences of RGAs showed that ryegrass RGAs contained four conserved motifs (P-Loop, kinase-2, kinase-3a, GLPL) present in other known plant NBS-leucine rich repeat resistance genes. These ryegrass RGAs all belonged to non-toll and interleukin-1 receptor subclass. Phylogenetic analysis of ryegrass RGAs and other cloned R -genes indicated that gene mutation was the predominant source of gene variations, and the sequence polymorphism was due to purifying selection rather than diversifying selection. We further analyzed the source of gene variation in other monocots, rice, barley, wheat, and maize based on the data published before. Our analysis indicated that the source of RGA diversity in these monocots was the same as in ryegrass. Thus, monocots were probably the same as dicots in the source of RGA diversity. Ryegrass RGAs in the present paper represented a large group of resistance gene homologs in monocots. We discussed the origin and the evolution of R -genes in grass species. 相似文献
3.
小麦NBS类抗病基因同源cDNA序列的克隆与特征分析 总被引:2,自引:0,他引:2
根据已克隆植物抗病(R)基因NBS保守结构域设计简并引物,采用RT-PCR和cDNA末端快速扩增技术(RACE),在小麦抗叶锈病近等基因系材料TcLr19中进行抗病同源基因cDNA全长的扩增。获得了1个通读的NBS类抗病同源基因S11A11cDNA序列,该序列全长2923bp,编码878个氨基酸序列。生物信息学分析结果表明,该片段含有NB-ARC保守结构域和多个LRR结构域。聚类分析表明,S11A11编码的蛋白与小麦抗叶锈病基因Lr1编码的蛋白亲缘关系较近,而与Lr10亲缘关系较远。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr19小麦中成功获得了抗病基因同源序列,为最终克隆小麦抗叶锈病目的基因奠定了基础。 相似文献
4.
小麦NBS-LRR类抗病基因同源序列的分离与鉴定 总被引:7,自引:0,他引:7
根据已知植物抗病基因的保守区域设计引物,从抗锈病小麦品种西农88基因组DNA扩增出3条与植物抗病基因同源的序列,分别为WRGA1、WRGA2和WRGA14。这三条同源片段均含有典型的NBS-LRR类抗病基因所拥有的保守性结构域Kinase-2a、Kinase-3a和疏水结构域(HD).它们与部分已知NBS-LRR类抗病基因的氨基酸序列同源性为46.0%-9.9%,三个片段间在氨基酸水平上的同源性为80.7%-56.8%。Northern杂交表明WRGA1在小麦中受水杨酸正调控,属诱导型表达。 相似文献
5.
Cloning and characterisation of a family of disease resistance gene analogs from wheat and barley 总被引:7,自引:0,他引:7
S. Seah K. Sivasithamparam A. Karakousis E. S. Lagudah 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(5-6):937-945
The most common class of plant disease resistance (R) genes cloned so far belong to the NBS-LRR group which contain nucleotide-binding
sites (NBS) and a leucine-rich repeat (LRR). Specific primer sequences derived from a previously isolated NBS-LRR sequence
at the Cre3 locus, which confers resistance to cereal cyst nematode (CCN) in wheat (Triticum aestivum L.) were used in isolating a family of resistance gene analogs (RGA) through a polymerase chain reaction (PCR) cloning approach.
The cloning, analysis and genetic mapping of a family of RGAs from wheat (cv ‘Chinese Spring’) and barley (Hordeum vulgare L. cvs ‘Chebec’ and ‘Harrington’) are presented. The wheat and barley RGAs contain other conserved motifs present in known
R genes from other plants and share between 55–99% amino acid sequence identity to the NBS-LRR sequence at the Cre3 locus. Phylogenetic analysis of the RGAs with other cloned R genes and RGAs from various plant species indicate that they
belong to a superfamily of NBS-containing genes. Two of the barley derived RGAs were mapped onto loci on chromosomes 2H (2),
5H (7) and 7H (1) using barley doubled haploid (DH) mapping populations. Some of these loci identified are associated with
regions carrying resistance to CCN and corn leaf aphid.
Received: 6 January 1998 / Accepted: 1 April 1998 相似文献
6.
Most plant disease-resistance genes (R-genes) isolated so far encode proteins with a nucleotide binding site (NBS) domain and belong to a superfamily. NBS domains related to R-genes show a highly conserved backbone of an amino acid motif, which makes it possible to isolate resistance gene analogues (RGAs) by degenerate primers. Degenerate primers based on the conserved motif (P-loop and GLPL) of the NBS domain from R -genes were used to isolate RGAs from the genomic DNA of sweet potato cultivar Qingnong no.2. Five distinct clusters of RGAs (22 sequences) with the characteristic NBS representing a highly diverse sample were identified in sweet potato genomic DNA. Sequence identity among the 22 RGA nucleotide sequences ranged from 41.2% to 99.4%, while the deduced amino acid sequence identity from the 22 RGAs ranged from 20.6%to 100%. The analysis of sweet potato RGA sequences suggested mutation as the primary source of diversity. The phylogenetic analyses for RGA nucleotide sequences and deduced amino acids showed that RGAs from sweet potato were classified into two distinct groups--toll and interleukin receptor-1 (TIR)-NBS-LRR and non-TIR-NBS-LRR. The high degree of similarity between sweet potato RGAs and NBS sequences derived from R-genes cloned from tomato, tobacco, flax and potato suggest an ancestral relationship. Further studies showed that the ratio of non-synonymous to synonymous substitution within families was low. These data obtained from sweet potato suggest that the evolution of NBS-encoding sequences in sweet potato occur by the gradual accumulation of mutations leading to purifying selection and slow rates of divergence within distinct R-gene families. 相似文献
7.
Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping 总被引:11,自引:0,他引:11
Madsen LH Collins NC Rakwalska M Backes G Sandal N Krusell L Jensen J Waterman EH Jahoor A Ayliffe M Pryor AJ Langridge P Schulze-Lefert P Stougaard J 《Molecular genetics and genomics : MGG》2003,269(1):150-161
The majority of verified plant disease resistance genes isolated to date are of the NBS-LRR class, encoding proteins with a predicted nucleotide binding site (NBS) and a leucine-rich repeat (LRR) region. We took advantage of the sequence conservation in the NBS motif to clone, by PCR, gene fragments from barley representing putative disease resistance genes of this class. Over 30 different resistance gene analogs (RGAs) were isolated from the barley cultivar Regatta. These were grouped into 13 classes based on DNA sequence similarity. Actively transcribed genes were identified from all classes but one, and cDNA clones were isolated to derive the complete NBS-LRR protein sequences. Some of the NBS-LRR genes exhibited variation with respect to whether and where particular introns were spliced, as well as frequent premature polyadenylation. DNA sequences related to the majority of the barley RGAs were identified in the recently expanded public rice genomic sequence database, indicating that the rice sequence can be used to extract a large proportion of the RGAs from barley and other cereals. Using a combination of RFLP and PCR marker techniques, representatives of all barley RGA gene classes were mapped in the barley genome, to all chromosomes except 4H. A number of the RGA loci map in the vicinity of known disease resistance loci, and the association between RGA S-120 and the nematode resistance locus Ha2 on chromosome 2H was further tested by co-segregation analysis. Most of the RGA sequences reported here have not been described previously, and represent a useful resource as candidates or molecular markers for disease resistance genes in barley and other cereals. 相似文献
8.
Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes. 相似文献
9.
Targeted isolation,sequence analysis,and physical mapping of nonTIR NBS-LRR genes in soybean 总被引:2,自引:0,他引:2
Peñuela S Danesh D Young ND 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,104(2-3):261-272
Most cloned plant disease resistance genes (R-genes) code for proteins belonging to the nucleotide binding site (NBS) leucine-rich
repeat (LRR) superfamily. NBS-LRRs can be divided into two classes based on the presence of a TIR domain (Toll and interleukin receptor-like sequence) or a coiled coil motif (nonTIR) in their N-terminus. We used conserved motifs specific
to nonTIR-NBS-LRR sequences in a targeted PCR approach to generate nearly 50 genomic soybean sequences with strong homology
to known resistance gene analogs (RGAs) of the nonTIR class. Phylogenetic analysis classified these sequences into four main
subclasses. A representative clone from each subclass was used for genetic mapping, bacterial artificial chromosome (BAC)
library screening, and construction of RGA-containing BAC contigs. Of the 14 RGAs that could be mapped genetically, 12 localized
to a 25-cM region of soybean linkage group F already known to contain several classical disease resistance loci. A majority
of the genomic region encompassing the RGAs was physically isolated in eight BAC contigs, together spanning more than 1 Mb
of genomic sequence with at least 12 RGA copies. Phylogenetic and sequence analysis, together with genetic and physical mapping,
provided insights into the genome organization and evolution of this large cluster of soybean RGAs.
Received: 8 May 2001 / Accepted: 30 June 2001 相似文献
10.
Irigoyen ML Loarce Y Fominaya A Ferrer E 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2004,109(4):713-724
Degenerate primers based on conserved regions of the nucleotide binding site (NBS) domain (encoded by the largest group of cloned plant disease resistance genes) were used to isolate a set of 15 resistance gene analogs (RGA) from the diploid species Avena strigosa Schreb. These were grouped into seven classes on the basis of 60% or greater nucleic acid sequence identity. Representative clones were used for genetic mapping in diploid and hexaploid oats. Two RGAs were mapped at two loci of the linkage group AswBF belonging to the A. strigosa × A. wiestii Steud map, and ten RGAs were mapped at 15 loci in eight linkage groups belonging to the A. byzantina C. Koch cv. Kanota × A. sativa L. cv. Ogle map. A similar approach was used for targeting genes encoding receptor-like kinases. Three different sequences were obtained and mapped to two linkage groups of the hexaploid oat map. Associations were explored between already known disease resistance loci mapped in different populations and the RGAs. Molecular markers previously linked to crown rust and barley yellow dwarf resistance genes or quantitative trait loci were found in the Kanota × Ogle map linked to RGAs at a distance ranging from 0 cM to 20 cM. Homoeologous RGAs were found to be linked to loci either conferring resistance to different isolates of the same pathogen or to different pathogens. This suggests that these RGAs identify genome regions containing resistance gene clusters. 相似文献
11.
Isolation and linkage analysis of expressed disease-resistance gene analogues of sugar beet (Beta vulgaris L.). 总被引:9,自引:0,他引:9
Sandra Hunger Gabriele Di Gaspero Slike M?hring Diana Bellin Ralf Sch?fer-Pregl Dietrich C Borchardt Charles-Eric Durel Martin Werber Bernd Weisshaar Francesco Salamini Katharina Schneider 《Génome》2003,46(1):70-82
12.
13.
甘薯NBS类抗病基因类似物的分离与序列分析 总被引:12,自引:0,他引:12
利用已克隆植物抗病基因NBS(Nucleotide binding site)序列中的保守模体(motif)“P-loop”和“GLPL”合成简并引物,以甘薯(Ipomoea batatas)栽培品种青农2号基因组DNA为模板进行PCR扩增,通过T/A克隆、测序和序列分析,共得到15条具有连续ORF的抗病基因类似物(Resistance gene analogues,RGAs)序列,它们之间核苷酸序列间的相似性系数在41.2%-99.4%之间,而相应推测的氨基酸序列间的相似性系数在20.6%-100%之间,同时对分离的RGAs的核苷酸和氨基酸序列进行系统发育树分析,表明甘薯RGAs可分为TIR(Drosophila Toll or human interleukin receptor-like)和nonTIR两类.对甘薯RGAs和5个已克隆植物NBS的氨基酸序列进行结构分析表明,它们包括“P-loop”、“Kinase-2”、“Kinase-3a”、“GLPL”4个抗病基因所共有的保守模体.这些表明甘薯与其它物种的NBS类RGAs可能具有同样的起源和进化机制. 相似文献
14.
Xu Q Wen X Deng X 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2005,111(5):819-830
Toll and interleukin-1 receptor (TIR) and nonTIR nucleotide binding site–leucine rich repeat (NBS–LRR) resistance gene analogues
(RGAs) were obtained from chestnut rose (Rosa roxburghii Tratt) by two PCR-based amplification strategies (direct amplification and overlap extension amplification) with degenerate
primers designed to the conserved P-loop, kinase-2, and Gly-Leu-Pro-Leu (GLPL) motifs within the NBS domain of plant resistance
gene (R gene) products. Thirty-four of 65 cloned PCR fragments contained a continuous open reading frame (ORF) and their predicted
protein products showed homology to the NBS–LRR class R proteins in the GenBank database. These 34 predicted protein sequences exhibited a wide range (19.5–99.4%) of sequence identity
among them and were classified into two distinct groups by phylogenetic analysis. The first group consisted of 23 sequences
and seemed to belong to the nonTIR NBS–LRR RGAs, since they contained group specific motifs (RNBS-A-nonTIR motif) that are
often present in the coiled-coil domain of the nonTIR NBS–LRR class R genes. The second group comprised 11 sequences that contained motifs found in the TIR domain of TIR NBS–LRR class R genes. Restriction fragment length polymorphic (RFLP) markers were developed from some of the RGAs and used for mapping powdery
mildew resistance genes in chestnut rose. Three markers, RGA22C, RGA4A, and RGA7B, were identified to be linked to a resistance
gene locus, designated CRPM1 for chestnut rose powdery mildew resistance 1, which accounted for 72% of the variation in powdery mildew resistance phenotype in an F1 segregating population. To our
knowledge, this is the first report on isolation, phylogenetic analysis and potential utilization as genetic markers of RGAs
in chestnut rose. 相似文献
15.
Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.) 总被引:1,自引:0,他引:1
Lanaud Claire Risterucci Ange Marie Pieretti Isabelle N'Goran Jeanne A.K. Fargeas Dominique 《Molecular breeding : new strategies in plant improvement》2004,13(3):211-227
Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management. 相似文献
16.
Sarosh Bejai Ramachandra Niranjan Raj Sathyanarayana Sivaramakrishnan Subramonium Shekar Hunthrike Shetty 《Journal of Phytopathology》2011,159(5):382-389
Sequence analysis of plant disease resistance genes shows similarity among themselves, with the presence of conserved motifs common to the nucleotide‐binding site (NBS). Oligonucleotide degenerate primers designed from the conserved NBS motifs encoded by several plant disease resistance genes were used to amplify resistance gene analogues (RGAs) corresponding to the NBS sequences from the genomic DNA of various plant species. Using specific primers designed from the conserved NBS regions, 22 RGAs were cloned and sequenced from pearl millet (Pennisetum glaucum L. Br.). Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into nine distinct classes. GenBank database searches with the consensus protein sequences of each of the nine classes revealed their conserved NBS domains and similarity to other known R genes of various crop species. One RGA 213 was mapped onto LG1 and LG7 in the pearl millet linkage map. This is the first report of the isolation and characterization of RGAs from pearl millet, which will facilitate the improvement of marker‐assisted breeding strategies. 相似文献
17.
Huettel B Santra D Muehlbauer J Kahl G 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,105(2-3):479-490
Resistance gene analogues (RGAs) of Cicer were isolated by different PCR approaches and mapped in an inter-specific cross segregating for fusarium wilt by RFLP and CAPS analysis. Initially, two pairs of degenerate primers targeting sequences encoded at nucleotide-binding sites (NBS), which are conserved in plant disease resistance genes such as RPS2, L6 and N, were selected for amplification. Cloning and sequence analysis of amplified products from C. arietinum DNA revealed eight different RGAs. Additionally, five RGAs were identified after characterisation of the presumptive RGA alleles from C. reticulatum. Therefore, a total of 13 different RGAs were isolated from Cicer and classified through pair-wise comparison into nine distinct classes with sequence similarities below a 68% amino acid identity threshold. Sequence comparison of seven RGA alleles of C. arietinum and C. reticulatum revealed polymorphisms in four RGAs with identical numbers of synonymous and non-synonymous substitutions. An NlaIII site, unique in the RGA-A allele of C. arietinum, was exploited for CAPS analysis. Genomic organisation and map position of the NBS-LRR candidate resistance genes was probed by RFLP analysis. Both single-copy as well as multi-copy sequence families were present for the selected RGAs, which represented eight different classes. Five RGAs were mapped in an inter-specific population segregating for three race-specific Fusarium resistances. All RGAs mapped to four of the previously established eight linkage groups for chickpea. Two NBS-LRR clusters were identified that could not be resolved in our mapping population. One of these clusters, which is characterised by RFLP probe CaRGA-D, mapped to the linkage group harbouring two of three Fusarium resistance genes characterised in the inter-specific population. Our study provides a starting point for the characterisation and genetic mapping of candidate resistance genes in Cicer that is useful for marker-assisted selection and as a pool for resistance genes of Cicer. 相似文献
18.
We amplified resistance gene analogues (RGAs) from the genomic DNA of 10 rice lines having varying degree of resistance to Magnaporthe grisea by using degenerate primers and various RGAs were mapped in silico on different rice chromosomes. The amplified products were grouped into 3–8 restriction fragment length polymorphic classes by using Mbo1 and Alu1 restriction enzymes. Of 98 RGAs obtained in this study, 65 RGA clones showed more than 95% homology with various RGAs sequences present in the GenBank. Phylogenetic analysis of these RGAs formed 11 groups. Using sequence homology approach, RGAs isolated in this study were physically mapped on 23 loci on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12. Twenty RGAs were mapped near to the chromosomal regions containing known genes/QTLs for rice blast, bacterial leaf blight and sheath blight resistance. Thirty‐nine RGA sequences also contained open reading frame representing signature of potential disease resistance genes. 相似文献
19.
Molecular characterization of NBS-LRR-RGAs in the rose genome 总被引:2,自引:0,他引:2
To isolate resistance gene analogues (RGAs) from roses we used various degenerate oligonucleotide primers targeting conserved motifs within the NBS region of nucleotide binding site (NBS)-leucine-rich repeat (LRR) resistance genes. A large RGA sublibrary consisting of 7000 clones was constructed. This sublibrary contains at least 40 unique RGA families of the TIR (toll-/interleukin-1 receptor) and the LZ (leucine zipper) type, which were further analysed. Phylogenetic studies revealed close relationships of some rose RGAs to R genes and RGAs from other plants and gave rise to the assumption that rose R genes evolved from different starting points, prior to and subsequent to speciation. Southern blot analyses showed that the RGAs were organized as single, low and multicopy loci in the rose genome. None of the analysed sequences detected any hybridization signal in Prunus cérasus indicating that the analysed RGAs are not conserved across genera. The efficiency and selectivity of the different degenerate primers used for the RGA isolation is discussed in detail. 相似文献
20.
NBS类植物抗病基因保守结构域的克隆为利用简并引物扩增抗病基因同源序列提供了可能.根据抗病基因Gro1-4、Gpa2、N等的P-loop和GLPL保守结构域设计简并引物,分离甘薯近缘野生种三浅裂野牵牛NBS类型抗病基因同源序列,共获得6条相关序列,核苷酸序列的相似性为48%~97%,推测氨基酸序列的相似性在25.2%~95.1%之间.系统进化分析表明,6条三浅裂野牵牛RGA序列可分为2个不同的类群:TIR-NBS和non-TIR-NBS.三浅裂野牵牛RGA序列与源自甘薯的RGA序列有很高的相似性,这在一定程度上反映了三浅裂野牵牛与甘薯之间的亲缘关系.分离的6条RGA序列分别命名为ItRGA1~ItRGA6,GenBank登录号分别为DQ849027~DQ849032. 相似文献