云南悬钩子蔷薇NBS-LRR类抗病基因同源克隆与分析

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。     

甘薯NBS类抗病基因类似物的分离与序列分析

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.     

陆地棉抗病基因同源序列的克隆与分析

根据已知抗病基因NBS保守区的P-loop和GLPL区设计一对简并引物F1/R1,以7个抗黄萎病陆地棉品种和2个感黄萎病品种的基因组DNA为模板进行PCR扩增.在9个品种中均扩增出500 bp左右的条带.对目的条带进行回收,连接、转化克隆得到350个阳性克隆,进行测序.在8个棉花品种中克隆到74条具有完整开放读码框的棉花RGAs序列.这74条序列共有64种不同的基因型,有10条与其他品种中的RGAs序列相同.用MEGA软件对8个棉花品种的74条RGA序列以及12个已知的抗病基因的NBS区域进行聚类分析,可分为4类;4类RGAs之间的相似性较低,各类之内的RGAs虽然来自不同品种,氨基酸序列的相似度却非常高.推测各大类中相似性较高的序列分别属于同一个基因家族,从位点上说可能处于同一个基因簇.     

海岛棉抗病基因类似物与防卫基因类似物的分离及特征分析

利用已克隆的抗病基因(resistance-gene, R基因)与病程相关蛋白基因β-1,3-葡聚糖酶基因(PR2)的保守序列设计简并引物, 从海岛棉品种海7124基因组中成功分离了79条核苷酸结合位点(NBS, nucleotide binding site)类R基因类似物(RGAs, resistance gene analogs), 21条丝氨酸/苏氨酸蛋白激酶(STK, Serine/Threonine kinase)类RGAs和11条防卫基因类似物(DGAs, defense gene analogs). 将NBS类RGAs与棉花中已经克隆的该类RGAs进行序列分析, 鉴定了1个新的亚族. 具有连续ORF的48条NBS类RGAs和20条STK类RGAs分别可以划分为TIR-NBS与non-TIR-NBS两个亚类和A与B两个亚类. DGA中有4条具有连续的ORF. 对这些具有连续ORF的序列进行表达分析发现, 6条NBS类与1条STK类RGA受黄萎病菌诱导表达, 1条DGA经黄萎病菌诱导后表达量明显提高. 随机选取的4条TIR类与4条non-TIR类NBS类RGAs中有3条呈现2~3条杂交带, 其余的都有5~10条或以上的杂交带. 可见, NBS类RGAs大多数以多基因家族的形式存在. 另外, 有3条non-TIR类NBS类RGAs出现了一样的杂交带型, 可能这3条RGAs在基因组中成簇分布.     

野生稻NBS类抗病基因同源序列的分离与序列分析

为了挖掘野生稻中的抗病资源,根据已克隆的植物抗病基因核苷酸结合位点序列中的保守结构域设计3对简并引物,从疣粒、药用、高秆、宽叶和斑点野生稻基因组DNA中分离出13条NBS类抗病基因类似物,其中11条具有连续的ORF,具有NBS类R基因的保守基元P-loop、kinas-2、kinas-3a和GLPL。在NCBI上进行同源性搜索发现,其中12条RGAs的核苷酸序列与水稻已知的NBS类R基因具有66%～94%的同源性,与其他植物已知R基因具有67%～84%的同源性;其对应的氨基酸序列与水稻已知的NBS类R基因具有43%～93%的同源性,与其他植物已知R基因具有37%～79%的同源性。另外1条的核苷酸序列与水稻假定的NBS类R基因具有76%的同源性,其氨基酸序列与水稻假定的NBS类R基因具有74%的同源性。根据序列分析结果设计6对不同基因特异性引物,并利用RT-PCR技术进行表达分析,结果表明,RN1BD5、RN1BD10、RN1GG2和RN1YY6均能表达,说明这些片段可能是功能性抗病基因的部分序列;而RN1KY9和RN1GG5没有表达,可能是假基因。     

小麦NBS类抗病基因同源cDNA序列的克隆与特征分析

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。     

水稻白叶枯病候选抗性基因SHNLR的RGAs克隆及分析

旨在从含有疣粒野生稻抗白叶枯病基因的新种质SH5、SH76基因组中克隆抗病基因。利用RGAs法得到1个NBS-LRR类同源基因,暂命名为SHNLR(登录号为JF934724)。结果表明,SHNLR的开放阅读框长度为3 105 bp,编码1 034个氨基酸,含有CC、NB-ARC与LRR结构域,具备CC-NBS-LRR类植物抗病基因的结构特征。BLASTn和BLASTp比对显示SHNLR是单拷贝基因,未发现同源性较高且功能已知的基因,仅NBS保守域序列与番茄Prf基因的相似度最高。对SHNLR基因电子定位,发现其位于水稻第11号染色体的长臂末端,但与11号染色体上已定位或克隆的8个白叶枯病抗性基因具有不同序列或处于不同的位置。半定量RT-PCR分析表明,SHNLR在抗病新种质叶片中的表达明显受到白叶枯病菌Zhe173的诱导。因此推测SHNLR可能是1个与抗白叶枯病相关的R基因。     

海岛棉NBS类型抗病基因类似物的起源、多样性及进化

利用已克隆植物的R基因NBS序列中保守模体合成简并引物,以海岛棉品系Pima 90(Gossypium barba—dense)基因组DNA为模板进行PCR扩增,通过T／A克隆、测序和序列比较分析共得到31条RGAs,其中19条具有连续的ORF。利用海岛棉的31条RGAs与GenBank中陆地棉种质系M—249(Gossypium hirsutum)的RGAs进行了比较分析,RGAs可分为两大类：其中第Ⅰ类全部为陆地棉的RGAs;第Ⅱ类分别包括了陆地棉和海岛棉的RGAs。同时对海岛棉RGAs的核苷酸和氨基酸序列进行系统发育树分析,表明海岛棉RGAs可分为TIR(Drosophila Toll or human inter—leukin receptor—1ike)和non—FIR两类,与前人所报道的R基因进化一致。对19条具有连续ORF的RGAs进行了结构分析,结果表明它们包括P—loop、Kin—2、“PLAL”及Meyers等所定义的RNBS—A、B、C3个模体。结果表明,可能海岛棉NBS类型抗病基因类似物和其他物种具有同样的起源和进化机制。     

小麦Mlo及NBS—LRR类抗病基因同源序列的分离与鉴定

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。     

小麦Mlo及NBS-LRR类抗病基因同源序列的分离与鉴定

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.     

Isolation, genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine ( Pinus monticola Dougl. ex. D. Don.)

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.     

Isolation of TIR and nonTIR NBS–LRR resistance gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt)

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.     

Genetic diversity of NBS–LRR class disease-resistance gene analogs in cultivated and wild eggplants

The genes encoding the nucleotide-binding site (NBS) and leucine-rich repeat (LRR) motifs constitute a large gene family in plants and have attracted much interest, because most of the plant disease-resistance genes that have been cloned are from this gene family. In this study, degenerate oligonucleotide primers, designed on the basis of conserved regions of the NBS domains from known plant resistance genes, were used to isolate resistance gene analogs (RGAs) from cultivated and wild eggplants, i.e., S. melongena, S. aethiopicum gr. Gilo, S. linnaeanum, S. integrifolium, S. sisymbriifolium, and S. khasianum. Sequence analysis indicated that the cloned eggplant RGAs belong to the non-TIR–NBS–LRR type, which are very similar to the R genes or the RGAs identified in other plant species, especially Solanaceae plants, suggesting the existence of common ancestors. Wide genetic diversity of eggplant RGAs was observed both in interspecific and intraspecific sequences, and eight distinct families of eggplant RGAs were identified. Further studies revealed a high average ratio of synonymous to non-synonymous substitution and a low level of recombination. These results suggest that NBS-encoding sequences of RGAs in cultivated and wild eggplants are subject to gradual accumulation of mutations leading to purifying selection. This is the first report of NBS–LRR class RGAs in eggplants.     

Isolation,genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine (<Emphasis Type="Italic">Pinus monticola </Emphasis>Dougl. ex. D. Don.)

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     

Barley disease resistance gene analogs of the NBS-LRR class: identification and mapping

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.     

Genomic distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane

A large sugarcane EST (expressed sequence tag) project recently gave us access to 261,609 EST sequences from sugarcane, assembled into 81,223 clusters. Among these, we identified 88 resistance gene analogs (RGAs) based on their homology to typical pathogen resistance genes, using a stringent BLAST search with a threshold e-value of e(-50). They included representatives of the three major groups of resistance genes with NBS/LRR, LRR or S/T KINASE domains. Fifty RGAs showed a total of 148 single-dose polymorphic RFLP markers, which could be located on the sugarcane reference genetic map (constructed in cultivar R570, 2n=approximately 115). Fifty-five SSR loci corresponding to 134 markers in R570 were also mapped to enable the classification of the various haplotypes into homology groups. Several RGA clusters were found. One cluster of two LRR-like loci mapped close to the only disease resistance gene known so far in sugarcane, which confers resistance to common rust. Detailed sequence comparison between two NBS/LRR RGA clusters in relation to their orthologs in rice and maize suggests their polyphyletic origins, and indicates that the degree of divergence between paralogous RGAs in sugarcane can be larger than that from an ortholog in a distant species.     

Molecular characterization of NBS-LRR-RGAs in the rose genome

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.