（毕业论文 字数：5619 页数：10）摘 要：利用已知的植物R基因NBS序列中保守区域设计简并引物, 以热研2号柱花草（Stylosanthes guianensis cv.Reyan No 2）基因组DNA为模板进行 PCR扩增，获得500 bp的PCR产物，通过克隆、测序和序列比得到了3条 RGAs，分别命名为 SG1, SG2和 SG3，它们全部具有连续的 ORF。利用柱花草的3条 RGAs在GenBank上进行相似性搜索，同源性比较分析表明，它们与已克隆的抗病基因或抗病基因片段有不同程度的同源性。对它们的推断的氨基酸序列进行结构分析表明，它们包括了“P-loop”、“Kinase-2”、“kinase-3”、“GLPL”4个抗病基因所共有的保守模体。进行系统发育树分析表明, 这3个RGAs分为 TIR (Drosophila Toll or human interleukin receptor-like) 和 non-TIR两类, 与前人所报道的R 基因进化一致。说明柱花草NBS类RGAs可能和其他物种具有同样的起源和进化机制。
关键词：柱花草 NBS RGAs 序列分析
Isolation and Analysis of the NBS Class Resistance Gene Analogs from Stylosanthes spp
Abstract: Degenerate oligonucleotide primers based on conserved motifs in the nucleotide binding site (NBS) region of known resistance proteins were used to amplify sequences from genomic DNA of Stylosanthes guianensis cv.Reyan No.2. Amplified products of about 500 bp were cloned and sequenced. Three resistant gene analogs with opening reading frame were obtained, named as SG1, SG2 and SG3 respectively. Searching in the GenBank database using BLAST revealed that three classes of sequences showed high level of amino acid similarity to several cloned NBS class resistance genes and many RGAs known from other plant species. Analysis of the amino acid sequences deduced from the three sequenced RGAs, four motifs of NBS conserved regions exist in the three sequenced RGAs, which including P-loop GGVGKTT, kinase-2VLDD, kinase-3 GSRII and membrane spanning region GLPL. Three resistant gene analogs were divided to TIR and non-TIR by analysing phylogenetic tree, and which consistent with the evolutin mechanism of R gene reported by predecessor. NBS class resistance gene analogs from Stylosanthes were probably similar to origin and evolution of other species resistance gene.
Keyword: Stylosanthes guianensis; NBS; RGAs; sequence analysis
目录
1 前言
2 材料和方法
3 结果分析
4 讨论
1 前言
植物在生长过程中常会受到病原微生物的侵袭，为了抵御外来微生物的危害，植物在长期的进化过程中，形成了一系列复杂的抗病防御机制，如：由于植物先天性结构屏障和固有的毒性化合物的存在而表现的非寄主抗性，以及植物由于抗病基因(Resistance gene，R)的存在而表现出的局部或系统抗性[1]。因此， 克隆与分析植物抗病基因，对阐明植物抗病性的分子机制及改良作物抗病性具有重要的意义。
目前已从植物中分离到近50个抗病基因，通过对其产物分析发现这些抗病基因编码的产物存在极其相似的结构域，如核苷酸结合位点(nucleotide binding site, NBS)、富含亮氨酸重复序列(leucine-rich repeats, LRR)、丝氨酸/苏氨酸激酶(serine-threonine kinase, STK)、亮氨酸拉链结构(leucine zippers,LZ)、跨膜结构域(transmembrane domain, TM)、Toll白介素-1区域(Toll-interleukin-1region, TIR)等[1]。由于所有NBS类抗病基因都含有许多保守模体（conserved motif），如NBS区域中的“Ploop”、“GLPL”等，这为基于同源序列的NBS类抗病候选基因的克隆提供另论上的可能性。Kanazin[2]和Yu[3]报道用抗病基因产物的保守序列为引物扩增R基因类似序列（resistance gene analogues, RGA）后，目前已在大豆（Glycine max L.）、水稻（Oryza sativa L.）、花生（Arachis hypogaea L.）等近20种植物中分离到了许多抗病基因类似物(resistance gene analogues, RGAs)序列，并对其起源、多样性和进化机制进行了研究。
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