草鱼钠磷协同转运载体Slc34a2基因的克隆及组织表达分析

MOLECULAR CHARACTERIZATION AND TISSUE DISTRIBUTION OF SODIUM-DEPENDENT PHOSPHATE COTRANSPORTER GENE (SLC34A2) IN GRASS CARP CTENOPHARYNGODON IDELLA

  • 摘要: 为了揭示草鱼对磷的吸收机制,运用RT-PCR和快速扩增cDNA末端方法,从草鱼(Ctenopharyngodon idella)肠中克隆获得钠磷协同转运载体基因Slc34a2,该基因全长为2446 bp,包含了1938 bp的开放阅读框,47 bp的5非编码区(Untranslated region,UTR)和461 bp的3UTR,编码645个氨基酸。草鱼SLC34A2蛋白的分子式为C3215H5125N801O902S30,分子量大小为70.39 kD,等电点为5.68,总平均疏水指数为0.458。对草鱼SLC34A2蛋白结构和功能预测分析,发现SLC34A2蛋白有11个跨膜域,1个半胱氨酸富集区,且N-端在胞外而C-端在胞内,也在第二个细胞外环中发现4个N-糖基化位点。用邻接法构建系统进化树,发现草鱼Slc34a2基因与硬骨鱼类聚类为一支,且草鱼SLC34A2蛋白与鲤(Cyprinus carpio)和斑马鱼(Danio rerio)SLC34A2的相似性最高,分别为90.3%和87.0%。实验采用了实时荧光定量PCR对草鱼Slc34a2 mRNA进行组织表达分析,结果表明Slc34a2 mRNA在组织中广谱表达,且在肠中表达最高,其次是肝脏、鳃、肾脏、脾脏、皮肤、肌肉、脑和头肾。实验为以后研究提高鱼对磷的利用和减少磷的排放奠定分子基础。

     

    Abstract: To reveal the mechanism of grass carp (Ctenopharyngodon idella) on absorbing phosphate, a sodium-dependent phosphate cotransporter gene (Slc34a2) was cloned from grass carp intestine. The full-length cDNA of Slc34a2 was consisted of 2446 bp with a 1938 bp open reading frame, a 47 bp 5'UTR (untranslated region) and a 461 bp 3' UTR, which encoded 645 amino acids with an estimated formula of C3215H5125N801O902S30 that has 70.39 kD molecular weight, a 5.68 isoelectric point, and 0.458 grand average of hydropathicity. The putative grass carp SLC34A2 protein had eleven membrane-spanning domains with the extracellular N-termini and intracellular C-termini and acysteinerich region in C-termini domain as well as four N-glycosylation sites in second extracellular loop. The phylogenetic tree based onneighbor-joining method revealed that the SLC34A2 of grass carp was clustered with other teleost fish. The deduced amino acid sequence showed 90.3% and 87.0% sequence identity to Cyprinus carpio and Danio rerio, respectively. The highest level of Slc34a2 mRNA was in the intestine, followed by the liver, gill, kidney, spleen, skin, muscle, brain and head-kidney. Through cloning the full-length of Slc34a2 grass carp, this paper studied the characteristics of SLC34A2 structure, function and tissues distribution for laying the molecular foundation for further efforts to improve phosphate utilization and minimize the excretion of phosphorus.

     

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