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| 海带磷酸甘露糖变位酶(PMM)基因的克隆与表达分析 |
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张朋艳1,2,3, 于 雪1,2,3, 姚建亭1,2, 段德麟1,2
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1.中国科学院海洋研究所实验海洋生物学重点实验室;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;3.中国科学院大学
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| 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cDNA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SjPMM1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 kDa; 而Sjpmm2的ORF长1866 bp, 其编码的SjPMM2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 kDa。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pMAL-c5X 载体对SjPMM1 进行体外表达, 得到高浓度的可溶性融合蛋白, 为后续的SjPMM功能分析提供基础。 |
| 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 |
| DOI：10.11759/hykx20150113001 |
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| 基金项目: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) |
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| Cloning and expression of phosphomannomutase from Saccharina japonica (Laminariales, Phaeophyceae) |
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ZHANG Peng-yan,YU Xue,YAO Jian-ting,DUAN De-lin
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| 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 cDNA ends (RACE). The open reading frame (ORF) length of Sjpmm1 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 α-helix is the major secondary structure for both SjPMM1 and SjPMM2. 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 SjPMM1 fusion protein was expressed with the pMAL-c5X vector, contributing to the further function studies. |
| Key words: Saccharina japonica phosphomannomutase fucoidan alginate real-time PCR analysis |