盐地碱蓬亲环素CYP1基因的克隆、表达及耐盐性分析

The Cloning and Ectopic Prokaryotic Expression of Suaeda salsa CYP-1 Gene Involved in Salt Tolerance

为探索盐地碱蓬亲环素与其耐盐性的关系,使用RT-PCR和RACE完成盐地碱蓬亲环素基因的全长克隆与测序,并命名为SsCYP-1。SsCYP-1基因全长为875 bp,编码区为531 bp,编码176个氨基酸。多重序列BLAST结果表明,SsCYP-1基因可归于亲环素家族,并具有特定的肽酰脯氨酸顺反异构酶活性结构域特征。为验证盐地碱蓬亲环素的耐盐特性,构建了SsCYP-1表达载体并在大肠杆菌(E.coli)BL21(Rosetta)中通过IPTG诱导表达,通过PAGE检测目的蛋白为19 KD,与Editseq软件预测的目的蛋白大小基本一致。对重组菌PET-SsCYP1进行耐盐分析表明其在高盐浓度培养下生长速度远高于对照菌pETDuet-1。据此,推测该基因在盐地碱蓬生长中可能具有相关的耐盐性作用。

In order to verify the relationship between cyclophilins from Suaeda salsa and salt tolerance, the cyclophilin gene of Suaeda salsa was cloned and sequenced, with the designation as SsCYP-1 with the method of RT-PCR and RACE together. The full length of cyclophilin eDNA was 875 bp with the putative ORF of 531 bp, coding for 176 amino acids. Multiple alignment based on BLAST showed that the target protein harbored the typical domain for the peptidyl-prolyl cis- transisomerase of the cyclophilin family. To investigate the salt tolerance of Suaeda salsa, the SsCYP-1 expression vector was constructed and expressed in Escherichia eoli （E. coli） BI21 （Rosetta） by IPTG induction, followed by the PAGE detection of the target protein with the molecular weight of 19 KDa which was in accordance with the same size predicted by the EditSeq software. The salt-tolerance determination was established directly according to the growth speed of the re- combinant strains transformed with SsCYP-1 expression plasmids. The result indicated that PET-SsCYP1 strains propaga- ted much faster than those wild-type strains （ pETDuet-1 ） in the high-concentrated sah solutions, which has revealed the possible important roles of cyclophilins in the salt tolerance of bacteria.