裙带菜论文：裙带菜SAMS基因的克隆分析及原核表达.docVIP

裙带菜论文：裙带菜SAMS基因的克隆、分析及原核表达 【中文摘要】本实验采用改良的SDS提取方法从裙带菜配子体中提取总RNA。通过设计简并引物克隆裙带菜S-腺苷甲硫氨酸合成酶基因(UpSAMS)。获得的基因cDNA序列全长为1491bp,并编码397个氨基酸,预测蛋白分子量为43.2 KDa,等电点为5.244。起始密码子为ATG,终止密码子为TAA。5′非翻译区(UTR)长度为92 bp,3′UTR长度为205 bp,氨基酸序列中包含42个碱性氨基酸,57个酸性氨基酸,132个疏水性氨基酸和93个极性氨基酸。氨基酸序列还包含两个特定基序,第一个特定基序为GAGDQG,位置在氨基酸序列的127-132之间,第二个特定基序为GGGAFSgKD,位置在274-282之间。BLASTx比对显示裙带菜SAMS基因编码的蛋白(UpSams)序列与已发表的其他物种序列的相似度在68%-96%。结果表明裙带菜SAMS基因与藻类的相似度高于高等植物。进化树结果显示裙带菜SAMS基因首先和同属褐藻的海带和水云聚在一起,然后再与绿藻,红藻和硅藻聚合,最后再和十几种高等植物聚类。进化分析表明,裙带菜与其他海藻Sams亲缘关系要近于高等植物。疏水性分析结果显示UpSams亲水性最强是第339位的甘氨酸,疏水性最强是第314位赖氨酸。磷酸化分析显示丝氨酸有10处,苏氨酸有4处,酪氨酸有3处可能被磷酸化。不同的胁迫条件下,采用实时荧光定量聚合酶链式反应检测UpSAMS基因的相对表达量的变化水平,结果表明UpSAMS基因与抗逆相关。将UpSAMS基因转入大肠杆菌表达菌株BL21中进行原核表达,SDS电泳结果显示UpSAMS基因已经结合在pGEX4T-1表达载体上,并形成了融合蛋白。裙带菜SAMS基因的克隆分析和表达为今后进一步研究该基因的抗性机理奠定了重要的基础。 【英文摘要】Total RNA was extracted from the gametophytes of Undaria pinnatifida with improved SDS method. S-adenosylmethionine synthetase gene (UpSAMS) had been isolated from the economic seaweed U. pinnatifida by using degenerate primers. The cDNA sequence was 1,491 bp in length with an ORF of 1,194 nucleotides, encoding a deduced 397 amino acid residues, and had a predicted molecular weight of 43.2 kDa and was Iso-electronic point of 5.244. The sequence began with the presumed initial ATG codon and stop TAA codon,with the 5′UTR and 3′UTR 92 bp and 205 bp in length, respectively. UpSams comprised 42 strongly basic, 57 strongly acidic, 132 hydrophobic, and 93 polar amino acids. The amino acid sequence included two typical motifs, namely, signature motif-1 GAGDQG (at position 127?32) and signature motif-2 GGGAFSgKD (at position 274?82). BLASTx result revealed the Sams sequence of U. pinnatifida (UpSams) shared 68%?6% identity with the previously published Sams sequences of other species. U. pinnatifida was clustered with L. digitata and E. siliculosus of brown algae; with green and red algae; with diatoms and dinoflagellate; and with terrestrial plants. Phylogenetic analysis indicated that the phyloge