柱花草的NBS类抗病基因类似物的分离及分析

范文编号:SP029 范文字数:8071,页数:19

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
摘　要 1
目录 2
1 前言 2
2 材料和方法 3
2.1 材料 3
2.2 方法 4
2.2.1 DNA提取 4
2.2.2 PCR扩增 5
2.2.3 TA克隆 6
2.2.4数据处理 7
3 结果分析 7
3.1 DNA的提取效果分析 7
3.2 柱花草中NBS抗病基因的分离与鉴定。 7
3.3 柱花草中NBS抗病基因序列和氨基酸序列的分析 9
4 讨论 15
4.1 柱花草RGAs的分离与鉴定 15
4.2 柱花草RGAs的多样性及进化关系分析 15
参　考　文　献 16
致    谢 18

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