吴茱萸SOD基因片段克隆和SNP分析

摘要

Two pairs of degenerate primers were designed according to the two conserved regions on the a-mino acid sequences of Cu/Zn-SOD gene and Mn/Fe-SOD gene from kinds of plants reported in GenBank in order to clone the core fragments of these two genes in ten Evodia rutaecarpa' s plants. The length of Cu/Zn-SOD gene and Mn/Fe-SOD gene were 375 bp and 429 bp respectively. Every nucleotide sequence of Cu/ZnSOD gene and Mn/Fe-SOD gene encoded 125 amino acids and 143 amino acids respectively. The biology softwares of DAMBE and DNA Star5. 0 were used to analyze these sequences. The nucleotide similarity with other plants was above 74% and the amino acid similarity was above 79% at ten Cu/Zn-SOD gene fragments than other plants. The nucleotide similarity was above 78% and the amino acid similarity was above 81% in ten Mn/Fe-SOD gene fragments. Twenty-one sites of single nucleotides were discovered in the ten Cu/Zn-SODgene fragments. Six amino acids sites transformed because of twenty-one single nucleotides variation. Seventeen variation sites of single nucleotides were discovered in the ten Mn/Fe-SOD gene fragments. Three amino acid sites transformed because of the variation of seventeen single nucleotides. Accoring to the calculation result , the polymorphism frequences in Cu/Zn-SOD gene and Mn/Fe-SOD gene were 1SNP/17. 9 bp and 1SNP/ 25. 2 bp, the nucleotide degrees of variation were 5. 6% and 4. 0% , the amino acid degrees of variation were 4.8% and 2. 1% , respectively. Genetic diversity of cloned ten Cu/Zn-SOD gene fragments was analyzed by using the clustering procedure of Neighbor-Joining.%根据GenBank已报道的多种植物Cu/Zn-SOD基因和Mn/Fe-SOD基因的保守区域,分别设计了两对用于克隆10种吴茱萸属植物Cu/Zn-SOD基因和Mn/Fe-SOD基因核心片段的简并引物.试验所得的Cu/Zn-SOD基因片段长度为375 bp,Mn/Fe-SOD基因片段长度为429 bp,分别编码125个和143个氨基酸残基.使用DAM-BE和DNAStar5.0生物统计软件对序列进行同源性比对,10种吴茱萸属植物与其他多种植物的Cu/Zn-SOD基因核苷酸序列同源性大于74％,氨基酸序列同源性大于79％；Mn/Fe-SOD基因的核苷酸序列同源性大于78％,氨基酸序列同源性大于81％.在所克隆的Cu/Zn-SOD基因片段中有21个核苷酸SNPs位点,导致6个氨基酸位点变异,多态性频率为1SNP/17.9 bp,核苷酸和氨基酸变异度分别为5.6％和4.8％；在所克隆的Mn/Fe-SOD基因片段中有17个核苷酸SNPs位点,导致3个氨基酸位点变异,多态性频率为1SNP/25.2 bp,核苷酸和氨基酸变异度分别为4.0％和2.1％.采用Neighbour-Joining聚类法,对10种吴茱萸属植物的Cu/Zn-SOD基因片段的核苷酸序列进行聚类分析,构建了精确遗传聚类图谱.