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

为了揭示草鱼对磷的吸收机制,运用RT-PCR和快速扩增c DNA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3′UTR,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 k D,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 m RNA进行组织表达分析,结果表明Slc34a2 m RNA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。

To reveal the mechanism of grass carp （Ctenopharyngodon idella） on absorbing phosphate, a sodium -dependentphosphate cotransporter gene （Slc34a2） was cloned from grass carp intestine. T he full-length cDNA of Slc34a2w as consisted of 2446 bp with a 1938 bp open reading frame, a 47 bp 5＇UTR （untranslated region） and a 461 bp 3 fUIR , which encoded 645 am ino acids with an estim ated formula of C 32i5H 5125N 801O902S30 th at has 70.39 kD molecularw eight, a 5.68 isoelectric point, and 0.458 grand average of hydropathicity. The putative grass carp SLC 34A 2 proteinhad eleven m em brane-spanning dom ains with the extracellular N -termini and intracellular C-termini and acysteinerichregion in C-termini domain as well as four iV-glycosylation sites in second extracellular loop. The phylogenetic treebased onneighbor-joining method revealed that the SLC 34A 2 of grass carp was clustered with other teleost fish. T hededuced am ino acid sequence show ed 90.3% and 87.0% sequence identity to Cyprinus carpio and Danio rerio, respectively.T he 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 o fSLC 34A 2 structure, function and tissues distribution for laying the molecular foundation for further efforts to improvephosphate utilization and minimize the excretion of phosphorus.