植物抗病基因同源序列及其在抗病基因克隆与定位中的应用

近10年来已有20多个植物抗病基因被克隆,测序,这些抗病基因所编码的蛋白中大多含有核苷酸结合位点,富含亮氨酸重复序列,蛋白激酶,亮氨酸拉链结构,跨膜结构域,Toll白介素－1区域等保守结构域。利用这些保守结构域合成PCR引物,已扩增出大量的植物抗病基因同源序列（RGA）。对RGA与抗病基因的关系进行了分析,讨论了RGA在研究抗病基因进化中的作用,指出RGA在抗病基因定位和转基因中具有重要意义。     

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。     

植物病原物无毒基因及其功能

植物抗病基因与病原物无毒基因产物间直接或间接相互作用导致产生的基因对基因抗性是植物抗病性的重要形式。无毒基因已在多种植物病原物 ,包括真菌、细菌、病毒和卵菌等中得到克隆。绝大多数已克隆无毒基因之间 ,及其与已知蛋白之间 ,均无显著序列同源性。然而 ,多数已克隆植物抗病基因有较高序列一致性 ,产物往往具有相似的结构域。由序列一致性很高的抗病基因产物与没有明显序列同源性的无毒基因产物相互作用 ,介导产生的过敏性细胞坏死和抗病性 ,在产生速度、强度和组织特异性等方面均可能有显著差异。无毒基因具有双重功能 :在含互补抗病基因植物中表现无毒效应 ,而在不含互补抗病基因植物中显示小种、菌株、致病型、或种特异性毒性效应     

巴西橡胶Pto类抗病同源序列的克隆与系统发育重建

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 value <3e-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.     

巴西橡胶Pto类抗病同源序列的克隆与系统发育重建（英文）

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。     

用同源序列的染色体定位寻找水稻抗病基因DNA片段

根据已知植物抗病基因的序列以及蛋白激酶序列中的高保守区域设计合成了特异性和简并引物,用聚合酶链反应从水稻（ＯｒｙｚａｓａｔｉｖａＬ．）ＤＮＡ中扩增同源片段,获约１００个大小不同的克隆。以这些克隆作探针进行限制性片段长度多态性（ＲＦＬＰ）分析,已将２６个克隆定位在两个水稻分子标记连锁图１２条染色体的３４个位点上。其中１０个克隆与８个已定位的水稻抗病基因在分子标记连锁图上的位置对应或毗邻。用其中部分与抗稻瘟病基因在染色体位置相对应的克隆作探针,分析抗稻瘟病近等基因系,ＲＦＬＰ带型在抗性基因系和感病亲本间表现出多态性,表明这些克隆与抗病基因在染色体位置上有较好的对应关系。     

小粒野生稻STK类抗病基因同源序列的克隆与分析

根据丝氨酸/苏氨酸蛋白激酶类(serine-threonine kinase,STK)抗病基因结构中保守结构域,设计引物,以小粒野生稻基因组DNA为模板,扩增获得10条STK类抗病基因同源序列.同源性分析表明其都具有STK保守结构域,与已克隆的STK类抗病基因有不同程度的相似性,为进一步克隆小粒野生稻中的STK类抗病基因提供依据.     

三浅裂野牵牛NBS类抗病基因同源序列的克隆与分析

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.     

植物NBS类抗病基因的进化

NBS(Nucleotide-binding site)类抗病基因是植物中最重要的一类抗病基因, 其进化模式、结构特点和功能调控一直是抗病基因研究领域的热点。这类基因具有保守的结构域, 广泛存在于植物基因组中, 在不同植物基因组中数目差异较大且具有较低的表达量。此外, 同源NBS类抗病基因之间通过频繁的序列交换产生广泛的序列多样性, 且抗病基因位点具有较差的线性。依据基因之间序列交换的频率, 抗病基因可分为TypeⅠ和TypeⅡ两类。文章从抗病基因的结构、数量、分布、序列多样性、进化模式以及表达调控等方面进行了综述, 旨在为后续NBS类抗病基因的相关研究提供参考。     

小麦NBS-LRR类抗病基因同源序列的分离与鉴定

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。     

植物抗病基因的研究进展

近年来,已有１０多个植物抗病基因被克隆并定序。植物抗病基因编码的蛋白,大多含有富氨酸重复单位（ＬＲＲ）和核苷酸结合位点（ＮＢＳ）等结构。在植物与病原物的互作中,这些蛋白可作为受体识别由病原物无毒基因编码的激发子,从而激发一系列防卫反应,使植物表现出抗病性。克隆的植物抗病基因可用于培育基因工程植株而大大加快育种速度。本文对目前植物抗病基因研究中存在的问题及发展前景也进行了探讨。     

The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes

Late blight caused by the oomycete Phytophthora infestans is the most destructive disease in potato cultivation worldwide. New, more virulent P. infestans strains have evolved which overcome the genetic resistance that has been introgressed by conventional breeding from wild potato species into commercial varieties. R genes (for single-gene resistance) and genes for quantitative resistance to late blight are present in the germplasm of wild and cultivated potato. The molecular basis of single-gene and quantitative resistance to late blight is unknown. We have cloned R1, the first gene for resistance to late blight, by combining positional cloning with a candidate gene approach. The R1 gene is member of a gene family. It encodes a protein of 1293 amino acids with a molecular mass of 149.4 kDa. The R1 gene belongs to the class of plant genes for pathogen resistance that have a leucine zipper motif, a putative nucleotide binding domain and a leucine-rich repeat domain. The most closely related plant resistance gene (36% identity) is the Prf gene for resistance to Pseudomonas syringae of tomato. R1 is located within a hot spot for pathogen resistance on potato chromosome V. In comparison to the susceptibility allele, the resistance allele at the R1 locus represents a large insertion of a functional R gene.     

植物类病变坏死突变体及其基因的研究进展

在植物中类病变坏死突变现象广泛存在,突变体植株在无病害侵染也未受逆境或损伤的条件下自发形成与病原物侵染后坏死类似的症状,它们与植物抗病及细胞程序化死亡相关,并可能增强系统抗病性。综述了几种植物的典型类病变坏死突变体及其基因的研究情况,探讨了其发生的机制并对今后的应用前景进行了展望。     

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。     

Protein-protein interactions in pathogen recognition by plants

Protein-protein interactions have emerged as key determinants of whether plant encounters with pathogens result in disease or successful plant defense. Genetic interactions between plant resistance genes and pathogen avirulence genes enable pathogen recognition by plants and activate plant defense. These gene-for-gene interactions in some cases have been shown to involve direct interactions of the products of the genes, and have indicated plant intracellular localization for certain avirulence proteins. Incomplete specificity of some of the interactions in laboratory assays suggests that additional proteins might be required to confer specificity in the plant. In many cases, resistance and avirulence protein interactions have not been demonstrable, and in some cases, other plant components that interact with avirulence proteins have been found. Investigation to date has relied heavily on biochemical and cytological methods including in vitrobinding assays and immunoprecipitation, as well as genetic tools such as the yeast two-hybrid system. Observations so far, however, point to the likely requirement for multiple, interdependent protein associations in pathogen recognition, for which these techniques can be insufficient. This article reviews the protein-protein interactions that have been described in pathogen recognition by plants, and provides examples of how rapid future progress will hinge on the adoption of new and developing technologies.     

Chemical induction of disease resistance in rice is correlated with the expression of a gene encoding a nucleotide binding site and leucine-rich repeats

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is an agricultural chemical primarily used to prevent rice blast disease. Probenazole-treated rice acquires resistance to blast fungus irrespective of the rice variety. The chemical is applied prophylactically, and is thought to induce or bolster endogenous plant defenses. However, the mechanisms underlying this effect have not been established. To understand the mode of the chemical's action, we screened for novel probenazole-responsive genes in rice by means of differential display and identified a candidate gene, RPR1. RPR1 contains a nucleotide binding site and leucine-rich repeats, thus sharing structural similarity with known disease resistance genes. The expression of RPR1 in rice can be up-regulated by treatment with chemical inducers of systemic acquired resistance (SAR) and by inoculation with pathogens. RPR1-related sequences in rice varieties seem to be varied in sequence and/or expression, indicating that RPR1 itself is not a crucial factor for induced resistance in rice. However, Southern blot analysis revealed the existence of homologous sequences in all varieties examined. While the role of RPR1 has yet to be clarified, this is the first report of the identification of a member of this gene class and its induction during the systemic expression of induced disease resistance.     

Isolation,Cloning and Characterization of Resistance Gene Analogues in Pearl Millet Based on Conserved Nucleotide‐binding Sites

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.     

植物抗病信号转导途径

植物抗病信号转导途径的研究一直是植物病理学关注的热点,近年来国内外不少实验室正在大量分离与植物抗病有关的突变体,克隆与抗病调节有关的基因并研究其功能。实验表明,一些植物抗病信号途径中的正、负调节因子及不同信号转导途径之间形成复杂的调控网络。在着重对R基因介导的抗病信号途径、细胞程序化死亡、水杨酸信号途径、茉莉酸和乙烯信号途径和诱导系统抗性途径研究进展加以综述的同时,对各抗病信号途径之间信号交换的复杂性进行了讨论。     

转几丁质酶基因研究进展

几丁质酶基因是近年来作物抗病基因工程研究的热点之一。已对10多种作物进行了转几丁质酶基因研究,大多数转基因作物能增强作物对真菌或昆虫的抗性,转几丁质酶基因于生防因子的研究也越来越多,为生物防治提供了一条新途径。