柱花草的NBS類抗病基因類似物的分離及分析

食品科學(xué)論文編號:SP029 論文字?jǐn)?shù):8071,頁數(shù):19

目錄
摘　要 1
目錄 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擴(kuò)增 5
2.2.3 TA克隆 6
2.2.4數(shù)據(jù)處理 7
3 結(jié)果分析 7
3.1 DNA的提取效果分析 7
3.2 柱花草中NBS抗病基因的分離與鑒定。 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 討論 15
4.1 柱花草RGAs的分離與鑒定 15
4.2 柱花草RGAs的多樣性及進(jìn)化關(guān)系分析 15
參　考　文　獻(xiàn) 16
致    謝 18

摘　要：利用已知的植物R基因NBS序列中保守區(qū)域設(shè)計(jì)簡并引物, 以熱研2號柱花草（Stylosanthes guianensis cv.Reyan No 2）基因組DNA為模板進(jìn)行 PCR擴(kuò)增，獲得500 bp的PCR產(chǎn)物，通過克隆、測序和序列比得到了3條 RGAs，分別命名為 SG1,  SG2和 SG3，它們?nèi)�部具有連續(xù)的 ORF。利用柱花草的3條 RGAs在GenBank上進(jìn)行相似性搜索，同源性比較分析表明，它們與已克隆的抗病基因或抗病基因片段有不同程度的同源性。對它們的推斷的氨基酸序列進(jìn)行結(jié)構(gòu)分析表明，它們包括了“P-loop”、“Kinase-2”、“kinase-3”、“GLPL”4個(gè)抗病基因所共有的保守模體。進(jìn)行系統(tǒng)發(fā)育樹分析表明, 這3個(gè)RGAs分為 TIR (Drosophila Toll or human interleukin receptor-like) 和 non-TIR兩類, 與前人所報(bào)道的R 基因進(jìn)化一致。說明柱花草NBS類RGAs可能和其他物種具有同樣的起源和進(jìn)化機(jī)制。
關(guān)鍵詞：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