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1.
水稻抗白叶枯病基因Xa4位点跨叠BAC克隆群的构建   总被引:2,自引:0,他引:2  
水稻白叶枯病抗性基因Xa4已被定位于第11染色体长臂末端的分子标记VG181和L1044之间,并与抗性基因同源序列片段RS13共分离。利用这3个标记筛选IRBB56的BAC文库,共得到128个阳性BAC克隆,其中RS13获得18个阳性克隆,这18个克隆中有4个和6个我隆分别同时为G181和L1044的阳性克隆,选其中的12克隆进行分析,构建了一个从G181到L1044区间的BAC跨叠克隆,全长420kb,并且56M22、106P13和104B153个BAC克隆可覆盖整个跨叠克隆群。这一研究结果为进一步分离Xa4基因打下基础。  相似文献   

2.
水稻NBS-LRR类R基因同源序列   总被引:28,自引:0,他引:28  
根据多数抗病基因(R)编码蛋白质的核苷酸结合区(nucleotide binding site, NBS)和富含亮氨酸重复(leucine-rich repeat,LRR)保守区域特点,设计PCR特异扩增引物,从水稻中克隆了大小约为520 bpDNA片段23个.通过序列同源比较分析发现, 它们编码的蛋白质氨基酸序列包括有NBS-LRR类基因所具有的kinase-1a,kinase-2a, kinase-3a和保守的domain 2区域,它们属于R基因同源序列(R gene homologous sequence, 简称RS).聚类结果发现它们分为4类.遗传定位结果表明它们分布在1,3,4,7~11染色体上,其中10个RS位于已知R基因所在的染色体区间.用水稻抗白叶枯病基因Xa4的近等基因系和基因累加系对克隆的NBS-LRR同源序列进行RFLP分析,发现序列RS13可能来自Xa4基因家族.  相似文献   

3.
为发掘甘薯近缘野生种三裂叶薯(Ipomoea triloba)的NBS-LRR类抗病基因,从基因数据库中对三裂叶薯基因组序列进行了筛选、鉴定和分析。结果表明,从三裂叶薯的98 025个基因中,筛选到282个编码NBS-LRR类蛋白的基因,其中N型80个,NL型83个,CN型28个,CNL型57个,TN型10个,TNL型23个,RN型1个。三裂叶薯的16条染色体上均含有NBS-LRR家族基因,数量最多的染色体含有65个,最少的只有1个。三裂叶薯基因组共有55个基因簇,包含了63.5%的NBS-LRR家族基因。在NBS-LRR抗病基因家族中,CNL和TNL亚家族分别对应到7和11个保守结构域。这为三裂叶薯抗性资源的利用提供了科学参考。  相似文献   

4.
小麦Mlo及NBS—LRR类抗病基因同源序列的分离与鉴定   总被引:4,自引:0,他引:4  
根据GenBank中公布的大麦白粉病抗性控制基因MlocDNA序列及一个来源于栽培一粒小麦(Triticum monococcumL.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选。结果获得两个表达基因的cDNA克隆。其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%。另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类。白粉菌诱导前后,该片段RT-PCR扩增产物存在差异。表明该片段可能与小麦抗病性相关。利用“中国春”缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上。  相似文献   

5.
为研究云南野生蔷薇属中的NBS类抗病基因,根据已知抗病基因NBS LRR序列中的保守区域设计简并引物,利用RT PCR技术从云南悬钩子蔷薇中进行体外扩增,获得了对应区域的cDNA片段,回收、克隆这些特异片段,测序分析,共得到4个含有NBS LRR保守结构域的抗病基因同源序列(RGAs),分别命名为AC9、AC39、AC50和AC68。它们与已报道的11个NBS类抗病基因相应区段的氨基酸序列相似性为5.4%~79.2%,其中这4个RGAs片段与Mi、RPS2、Pib和RPM1基因聚为一类。表明这4条RGAs序列可进一步用作悬钩子蔷薇抗病候选基因的分子筛选及遗传图谱的构建。  相似文献   

6.
为研究云南野生蔷薇属中的NBS类抗病基因,根据已知抗病基因NBSLRR序列中的保守区域设计简并引物,利用RTPCR技术从云南悬钩子蔷薇中进行体外扩增,获得了对应区域的cDNA片段,回收、克隆这些特异片段,测序分析,共得到4个含有NBSLRR保守结构域的抗病基因同源序列(RGAs),分别命名为AC9、AC39、AC50和AC68。它们与已报道的11个NBS类抗病基因相应区段的氨基酸序列相似性为5.4%~79.2%,其中这4个RGAs片段与Mi、RPS2、Pib和RPM1基因聚为一类。表明这4条RGAs序列可进一步用作悬钩子蔷薇抗病候选基因的分子筛选及遗传图谱的构建。  相似文献   

7.
根据GenBank中公布的大麦白粉病抗性控制基因Mlo cDNA序列及一个来源于栽培一粒小麦(Triticum monococcum L.)的假定抗病基因序列分别设计引物,以携带小麦抗白粉病基因的近等基因系为材料进行RT-PCR筛选.结果获得两个表达基因的cDNA克隆.其中一个与大麦白粉病抗性控制基因Mlo的同源性达83%.另一个为非通读序列,含有两个可能的开放阅读框,分别包含抗病基因NBS保守结构域2和3以及与水稻抗稻瘟病基因Pib蛋白末端相似的13个LRR区域,推测该序列属于NBS-LRR类.白粉菌诱导前后,该片段RT-PCR扩增产物存在差异,表明该片段可能与小麦抗病性相关.利用"中国春"缺体-四体系,将该NBS-LRR类序列定位在小麦1D染色体上.  相似文献   

8.
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   

9.
Yang XL  Bai DZ  Qiu W  Dong HQ  Li DQ  Chen F  Ma RL  Hugh TB  Gao JF 《遗传》2012,34(7):887-894
在已知中国美利奴羊MHC(Major histocompatibility complex)区段BAC(Bacterial artificial chromosome)克隆序列信息和预测的基因注释前提下,用位于中国美利奴羊基因组BAC文库MHC区段的6个BAC克隆酶切片段为探针,以噬菌斑原位杂交筛选法筛选中国美利奴羊混合组织cDNA文库(库库杂交),对分离到的cDNA阳性克隆进行全序列测定,并与相应的已知序列信息和基因注释的BAC克隆比对以及在NCBI Blastn数据库中序列相似性检索,旨在验证基因注释结果的准确性和对基因(序列)功能的初步分析。实验中,经过两轮杂交共筛选出27个cDNA阳性克隆(序列),并发现这些序列均可定位到相应的BAC克隆上,且25条序列处在注释基因的外显子部分;在NCBI数据库中经Blastn序列相似性检索发现,23条序列与牛基因的序列相似性最高,且与免疫功能密切相关。  相似文献   

10.
番茄Pto基因是一类可以编码丝氨酸/苏氨酸激酶(STK)序列的广谱抗性候选基因,其序列克隆与鉴定为深入了解番茄的抗病机制奠定了基础。在该研究中,一对依据Pto基因的保守序列设计的简并引物被用来扩增巴西橡胶中Pto基因抗病同源序列,扩增得到了一个约550 bp的基因片段,其随后被克隆并测序。序列分析发现,其中的7个抗病同源序列与Pto基因高度同源(BLASTX E value3e-53),所以其被认为是Pto基因抗病同源序列(Pto-RGCs)。通过巴西橡胶的Pto-RGCs多序列比对表明,这些序列包含了多个STKs保守的次级结构域。此外,系统发育分析也表明,巴西橡胶的Pto-RGCs属于Pto基因同源的R基因。该研究结果中Pto-RGCs可为巴西橡胶抗病的发展提供一个有效的基因资源。  相似文献   

11.
Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava ( Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.Communicated by M.-A. Grandbastien  相似文献   

12.
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   

13.
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   

14.
The rice gene Xa21 represents a unique class of plant disease resistance (R) genes with distinct protein structure and broad-spectrum specificity; few sequences or genes of this class have been cloned and characterized in other plant species. Degenerate primers were designed from the conserved motifs in the kinase domains of Xa21 and tomato Pto, and used in PCR amplification to identify this class of resistance gene candidate (RGC) sequences from citrus for future evaluation of possible association with citrus canker resistance. Twenty-nine RGC sequences highly similar to the kinase domain of Xa21 (55%–60% amino-acid identity) were cloned and characterized. To facilitate recovery of full-length gene structures and to overcome RGC mapping limitations, large-insert genomic clones (BACs) were identified, fingerprinted and assembled into contigs. Southern hybridization revealed the presence of 1–3 copies of receptor-like kinase sequences (i.e., clustering) in each BAC. Some of these sequences were sampled by PCR amplification and direct sequencing. Twenty-three sequences were thus obtained and classified into five groups and eight subgroups, which indicates the possibility of enhancing RGC sequence diversity from BACs. A primer-walking strategy was employed to derive full-length gene structures from two BAC clones; both sequences 17o6RLK and 26m19RLK contained all the features of the rice Xa21 protein, including a signal peptide, the same number of leucine-rich-repeats, and transmembrane and kinase domains. These results demonstrate that PCR amplification with appropriately designed degenerate primers is an efficient approach for cloning receptor-like kinase class RGCs. Utilization of BAC clones can facilitate this approach in multiple ways by improving sequence diversity, providing full-length genes, and assisting in understanding gene structures and distribution.Communicated by P. Langridge  相似文献   

15.
Western white pine (Pinus monticola Dougl. ex. D. Don., WWP) shows genetic variation in disease resistance to white pine blister rust (Cronartium ribicola). Most plant disease resistance (R) genes encode proteins that belong to a superfamily with nucleotide-binding site domains (NBS) and C-terminal leucine-rich repeats (LRR). In this work a PCR strategy was used to clone R gene analogs (RGAs) from WWP using oligonucleotide primers based on the conserved sequence motifs in the NBS domain of angiosperm NBS-LRR genes. Sixty-seven NBS sequences were cloned from disease-resistant trees. BLAST searches in GenBank revealed that they shared significant identity to well-characterized R genes from angiosperms, including L and M genes from flax, the tobacco N gene and the soybean gene LM6. Sequence alignments revealed that the RGAs from WWP contained the conserved motifs identified in angiosperm NBS domains, especially those motifs specific for TIR-NBS-LRR proteins. Phylogenic analysis of plant R genes and RGAs indicated that all cloned WWP RGAs can be grouped into one major branch together with well-known R proteins carrying a TIR domain, suggesting they belong to the subfamily of TIR-NBS-LRR genes. In one phylogenic tree, WWP RGAs were further subdivided into fourteen clusters with an amino acid sequence identity threshold of 75%. cDNA cloning and RT-PCR analysis with gene-specific primers demonstrated that members of 10 of the 14 RGA classes were expressed in foliage tissues, suggesting that a large and diverse NBS-LRR gene family may be functional in conifers. These results provide evidence for the hypothesis that conifer RGAs share a common origin with R genes from angiosperms, and some of them may play important roles in defense mechanisms that confer disease resistance in western white pine. Ratios of non-synonymous to synonymous nucleotide substitutions (Ka/Ks) in the WWP NBS domains were greater than 1 or close to 1, indicating that diversifying selection and/or neutral selection operate on the NBS domains of the WWP RGA family.Communicated by R. Hagemann  相似文献   

16.
高覆盖率水稻BAC库的构建及抗病基因相关克隆的筛选   总被引:20,自引:2,他引:18  
利用含Xa4、xa5和xa13 3个水稻白叶枯病抗性基因的累加系IRBB56构建了一个水稻细菌人工染色体文库,该文库包含55296个克隆,平均插入升段为132kb。按水稻基因组为450Mb计,该文库覆盖14倍基因组,筛选出任一水稻基因或序列的概率为99.99%。用均匀分布的3个叶绿体基因和4个线粒体基因克隆作探针筛选文库,结果显示该文库中含细菌器基因组DNA同源序列的克隆数小于1%、用分布于水稻3条不同染色体、分别与Xa4、xa5和xa13连锁的DNA标记筛选文库,分别检测出11-106个阳性克隆,为克隆这些基因打下了基础。该文库对水稻基因组的高度覆盖率和较大的插入片段,非常适合于物理作图和基因的分离和克隆。  相似文献   

17.
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   

18.
A complete set of candidate disease resistance ( R) genes encoding nucleotide-binding sites (NBSs) was identified in the genome sequence of japonica rice ( Oryza sativa L. var. Nipponbare). These putative R genes were characterized with respect to structural diversity, phylogenetic relationships and chromosomal distribution, and compared with those in Arabidopsis thaliana. We found 535 NBS-coding sequences, including 480 non-TIR (Toll/IL-1 receptor) NBS-LRR (Leucine Rich Repeat) genes. TIR NBS-LRR genes, which are common in A. thaliana, have not been identified in the rice genome. The number of non-TIR NBS-LRR genes in rice is 8.7 times higher than that in A. thaliana, and they account for about 1% of all of predicted ORFs in the rice genome. Some 76% of the NBS genes were located in 44 gene clusters or in 57 tandem arrays, and 16 apparent gene duplications were detected in these regions. Phylogenetic analyses based both NBS and N-terminal regions classified the genes into about 200 groups, but no deep clades were detected, in contrast to the two distinct clusters found in A. thaliana. The structural and genetic diversity that exists among NBS-LRR proteins in rice is remarkable, and suggests that diversifying selection has played an important role in the evolution of R genes in this agronomically important species. (Supplemental material is available online at .)Communicated by R. HagemannThe first three authors contributed equally to this work  相似文献   

19.
Disease resistance has not yet been characterized at the molecular level in cucurbits, a group of high-value, nutritious, horticultural plants. Previously, we genetically mapped the Fom-2 gene that confers resistance to Fusarium wilt races 0 and I of melon. In this paper, two cosegregating codominant markers (AM, AFLP marker; FM, Fusarium marker) were used to screen a melon bacterial artificial chromosome (BAC) library. Identified clones were fingerprinted and end sequenced. Fingerprinting analysis showed that clones identified by each marker assembled into two separate contigs at high stringency. GenBank searches produced matches to leucine-rich repeats (LRRs) of resistance genes (R genes); to retroelements and to cellulose synthase in clones identified by FM; and to nucleotide-binding sites (NBSs) of R genes, retroelements, and cytochrome P-450 in clones identified by AM. A 6.5-kb fragment containing both NBS and LRR sequences was found to share high homology to TIR (Toll-interleukin-1 receptor)-NBS-LRR R genes, such as N, with 42% identity and 58% similarity in the TIR-NBS and LRR regions. The sequence information may be useful for identifying NBS-LRR class of R genes in other cucurbits.  相似文献   

20.
Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a range of pathogens and insect pests. Consequently, NBS-encoding genes have been the focus of a number of recent studies in molecular disease resistance breeding programs. However, little is known about NBS-encoding genes in Lotus japonicus. In this study, a full set of disease resistance (R) candidate genes encoding NBS from the complete genome of L. japonicus was identified and characterized using structural diversity, chromosomal locations, conserved protein motifs, gene duplications, and phylogenetic relationships. Distinguished by N-terminal motifs and leucine-rich repeat motifs (LRRs), 92 regular NBS genes of 158 NBS-coding sequences were classified into seven types: CC-NBS-LRR, TIR-NBS-LRR, NBS-LRR, CC-NBS, TIR-NBS, NBS, and NBS-TIR. Phylogenetic reconstruction of NBS-coding sequences revealed many NBS gene lineages, dissimilar from results for Arabidopsis but similar to results from research on rice. Conserved motif structures were also analyzed to clarify their distribution in NBS-encoding gene sequences. Moreover, analysis of the physical locations and duplications of NBS genes showed that gene duplication events of disease resistance genes were lower in L. japonicus than in rice and Arabidopsis, which may contribute to the relatively fewer NBS genes in L. japonicus. Sixty-three NBS-encoding genes with clear conserved domain character were selected to check their gene expression levels by semi-quantitative RT-PCR. The results indicated that 53 of the genes were most likely to be acting as the active genes, and exogenous application of salicylic acid improved expression of most of the R genes.  相似文献   

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