海带磷酸甘露糖变位酶(PMM)基因的克隆与表达分析  

Cloning and expression of phosphomannomutase from Saccharina japonica(Laminariales, Phaeophyceae)

作　　者：张朋艳[1,2,3] 于雪[1,2,3] 姚建亭[1,2] 段德麟[1,2] 

机构地区：[1]中国科学院海洋研究所实验海洋生物学重点实验室,山东青岛266071 [2]青岛海洋科学与技术国家实验室实验海洋生物学与生物技术实验室,山东青岛266071 [3]中国科学院大学,北京100049

出　　处：《海洋科学》2016年第3期32-39,共8页Marine Sciences

基　　金：国家科技支撑计划项目(2013BAB01B01);国家海洋公益性行业科研专项(201405040)~~

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。Phosphomannomutase （PMM） is an important enzyme involved in the synthesis of fucoidan and alginate. Two PMM genes of Saccharina japonica （Sjpmml and Sjpmm2） were cloned by rapid-amplification of cDNA ends （RACE）. The open reading frame （ORF） length of Sjpmml is 759 bp, encoding 252 amino acids （SjPMM1） with a molecular weight （MW） of 28.51 kDa which belongs to the haloacid dehalogenase （HAD） superfamily. While the ORF length of Sjpmm2 is 1866 bp, encoding 621 amino acids （SjPMM2） with a MW of 66.49 kDa which belongs to the phosphohexomutase superfamily. The a-helix is the major secondary structure for both SjPMM1 and SjPMM2. The phylogenetic analysis showed that Sjpmml 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 SjPMM1 fusion protein was expressed with the pMAL-c5X vector, contributing to the further function studies.

关 键 词：海带 磷酸甘露糖变位酶 岩藻聚糖 褐藻胶 实时定量PCR分析 

分 类 号：X55[环境科学与工程—环境工程]