坛紫菜核糖体蛋白S7基因的克隆与表达分析

为了研究坛紫菜在高温胁迫条件下的分子应答机制,应用引物退火控制(ACP)技术对耐高温型纯系(Z-61)叶状体在高温胁迫条件下的差异表达基因进行筛选时,获得了一条核糖体蛋白S7基因的全长序列,命名为Phrps7(GenBank登录号:JF719273).该基因序列全长702 bp,包含一个585 bp的开放阅读框,其编码195个氨基酸的核糖体S7蛋白( PhRPS7),蛋白分子式为C984H1604N294O283S2,由5条α螺旋,6个片层及6个环状连接组成,与多个物种的RPS7蛋白具有较高的序列一致性(＞55％).系统进化树分析表明,PhRPS7蛋白与真菌的亲缘关系较近,而与其它物种的亲缘关系较远.实时荧光定量PCR分析结果表明,Phrps7基因的表达水平与高温胁迫密切相关,在高温胁迫刚开始时(0 ～6d),Phrps7基因的表达水平极显著上调,而随着胁迫的继续(＞6 d),表达量又有所下调,但仍显著高于胁迫开始前.

Molecular cloning and expression analysis of ribosomal protein S7 gene from Porphyra haitanensis

Porphyra haitanensis is an economically important marine crop in southern China.To study the molecular mechanism of high temperature tolerance in P. haitanensis,the technology of annealling control primer(ACP) was used to screen the differentially expressed genes in F₄ generation gametophytic blades of a high temperature tolerance line Z-61.The primer combination of dT-RSL and RSL7 amplified one differentially expressed gene fragment.After sequence analysis,the gene fragment was proved to be the full length sequence of ribosomal protein S7 gene,and then it was named Phrps7 (acceession number JF719273).The Phrps7 gene of 702 bp contains an open reading frame of 585 bp encoding a PhRPS7 protein of 195 amino acid.The PhRPS7 protein assembled by 5 helix,6 sheet and 6 cycle shared high sequence identity with RPS7 proteins from other organisms(>55%),and its molecular formula was C₉₈₄H₁₆₀₄N₂₉₄O₂₈₃S₂.Phylogenetic analysis showed that the PhRPS7 protein has closer relation with RPS7 proteins from epiphyte than those from other organisms.The results of real-time quantitative PCR indicated that the expressed level of Phrps7 has high relationship with high temperature stress.At the beginning of high temperature stress(0-6 d),the expression of Phrps7 gene was up-regulated significantly,but at the later stage of high temperature stress(>6 d),the expression of Phrps7 gene was down-regulated,however,it was still higher than that at the beginning.