【摘要】 磷酸甘露糖变位酶(PMM)是褐藻胶和岩藻聚糖合成过程中的关键酶之一。本研究利用c DNA末端快速克隆(RACE)技术,获得2条海带PMM基因(Sjpmm1,Sjpmm2)序列。其中,Sjpmm1的开放阅读框(ORF)长759 bp,其编码的Sj PMM1为卤酸脱卤酶(HAD)超家族成员,含252个氨基酸,分子量约为28.51 k Da;而Sjpmm2的ORF长1866 bp,其编码的Sj PMM2属于磷酸己糖变位酶超家族的成员,含621个氨基酸,分子量约为66.49 k Da。海带PMM的二级结构均以?-螺旋为主。进化分析表明,Sjpmm1来自于原始真核生物,而Sjpmm2来源于质体的第一次内共生作用。实时定量PCR分析发现,海带受到高温或低温胁迫时,Sjpmm1和Sjpmm2转录水平上升,以合成岩藻聚糖抵抗环境影响。此外,利用p MAL-c5X载体对Sj PMM1进行体外表达,得到高浓度的可溶性融合蛋白,为后续的Sj PMM功能分析提供基础。更多还原

【Abstract】 Phosphomannomutase(PMM) is an important enzyme involved in the synthesis of fucoidan and alginate. Two PMM genes of Saccharina japonica(Sjpmm1 and Sjpmm2) were cloned by rapid-amplification of c DNA ends(RACE). The open reading frame(ORF) length of Sjpmm1 is 759 bp, encoding 252 amino acids(Sj PMM1) with a molecular weight(MW) of 28.51 k Da which belongs to the haloacid dehalogenase(HAD) superfamily. While the ORF length of Sjpmm2 is 1866 bp, encoding 621 amino acids(Sj PMM2) with a MW of 66.49 k Da which belongs to the phosphohexomutase superfamily. The α-helix is the major secondary structure for both Sj PMM1 and Sj PMM2. The phylogenetic analysis showed that Sjpmm1 evolved from ancient eukaryotes, while Sjpmm2 originated from primary endosymbiosis. In addition, real-time PCR analysis indicated that temperature could increase the transcriptional level of Sjpmm, which may lead to the upregulation of fucoidan. Furthermore, a high concentration of the Sj PMM1 fusion protein was expressed with the p MAL-c5 X vector, contributing to the further function studies.更多还原