木榄甘油醛-3-磷酸脱氢酶全长cDNA的克隆及其序列分析

Molecular Cloning and Sequence Analysis of Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) cDNA from Bruguiera gymnorrhiza

甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)是糖酵解过程和卡尔文循环中的关键酶,在糖代谢和能量代谢过程中起着重要的作用。为揭示GAPDH在植物耐盐过程中的作用,研究了从前期构建的盐胁迫下木榄幼叶cDNA文库中克隆到编码GAPDH的cDNA全序列,并对其编码蛋白的理化性质、结构特点和空间分布特点进行了分析。序列分析结果表明:木榄GAPDH的cDNA全长为1482bp,编码区为1218bp,编码一个由405个氨基酸残基组成蛋白质,理论Mr为43.4k,理论等电点(pI)为8.65。基于该蛋白的氨基酸序列分析基础上的系统发育分析表明木榄的进化地位与毛果杨较为接近。结构分析和预测结果显示:木榄的GAPDH为无信号肽和跨膜区的亲水性蛋白,含有GAPDH的保守结构域NADB-Rossmann(71-222aa)和Gp_dh_C(227-383aa)。同源模建分析结果表明:该蛋白与菠菜相比具有1个高度保守的活性区,该活性区由第70～405氨基酸残基组成。

Glyceraldehyde- 3 - phosphate dehydrogenase （GAPDH） plays a very important role in sugar metabolism and energy metabolism. To make better understanding of the molecular mechanisms of plant salt-tolerance, the full-length GAPDH cDNA was cloned and sequenced from the leave cDNA library of Bruguiera gymnorrhiza seedling under salt stress. Meanwhile, the physicochemical characteristics, structure and function characteristics of GAPDH were analyzed and predicted by informatics software. The full cDNA is 1 482 bp long and contains an ORF with 1 218 bp, which is encoding a protein composed of 405 amino acid residues. The theoretical molecular weight and pI of this protein is about 43. 4 kD and pH 8. 65, respectively. Phylogenetic analysis based on the sequence of GAPDH showed that B. gymnorrhiza had a closer relationship with Populus trichocarpa. Bioinformatics analysis also indicated that the protein of GAPDH is a soluble protein without any signal peptide and transmembrane domain. There are two conserved domains, named as NADB_Rossmann （71- 222aa） and Gp_dh_C （227 - 383aa） in the structure of this protein. Homology modeling of the GAPDH in B. gymnorrhiza based on the SD structure of the one in Spinacia oleracea confirmed that the polypeptides between 70th to 405th amino acids was highly conserved.