樟叶越桔甘油醛-3-磷酸脱氢酶基因VdGAPDH1的cDNA克隆与序列分析

摘要

应用转录组测序分析和RT-PCR技术首次从樟叶越桔Vaccinium dunalianum中克隆了全长1570 bp的甘油醛-3-磷酸脱氢酶基因VdGAPDH1，其完整的开放阅读框推测编码由337个氨基酸残基组成的VdGAPDH1。VdGAPDH1理论相对分子量为36.57 kD，等电点为7.06，负电荷残基（Asp+Glu）总数为42个，正电荷残基（Arg+Lys）总数为42个，不稳定系数为23.27，属稳定性蛋白质；其二级结构主要由随机卷曲、延伸链、α-螺旋和β-转角等构件组成。VdGAPDH1为无信号肽、无跨膜结构的亲水性蛋白质，推测其为NAD+-GAPDH的新成员，与已知细胞质型GAPDH之间存在高度保守性。VdGAPDH1包含2个保守超家族结构域Gp_dh_N和Gp_dh_C，Gp_dh_N为辅酶NAD+结合结构域，Gp_dh_C是行使糖运输和代谢的催化功能域。推测VdGAPDH1基因产物在樟叶越桔细胞质中参与糖酵解过程。该研究为后期VdGAPDH1基因的生理功能及其表达调控等研究奠定了基础。%In this study, the gene VdGAPDH1 with a full-length cDNA comprised 1570 nucleotides was primordial obtained by transcriptome sequencing and RT-PCR amplification from V. dunalianum. The open reading frame encoded a putative VdGAPDH1 comprising 337 amino acid residues with molecular weight of 36.57 kD and isoelectric point of 7.06. The total number of positively charged residues (Arg+Lys) was 42, and the total number of negatively charged residues (Asp+Glu) was also 42. VdGAPDH1 with unstable coefifcient 23.27 belongs to the stable protein. The main parts of predicted secondary structures of VdGAPDH1 wereα-helices, random coils and extended strand. It was predictive VdGAPDH1 belongs to hydrophilic protein without any signal peptide and trans-membrane structure. VdGAPDH1 was considered to be a new number of NAD+-GAPDH super family proteins. VdGAPDH1 shares with highly conservation in sequences with cytoplasmic GAPDH in many plants. There are two conserved super family structures, named as Gp_dh_N and Gp_dh_C in the structure of VdGAPDH1 protein. Homology modeling of the GAPDH in B. gymnorrhiza based on the 3D structure of the one in Spinaciaoleracea conifrmed that the polypeptides between 70th to 405th amino acids was highly conserved. Plays a very important role in sugar metabolism and energy metabolism. The VdGAPDH1 gene product consists of two super family structures Gp_dh_N with function combining and Gp_dh_C, This work also suggested that VdGAPDH1 involve in process of glycolysis in the cytoplasm in V. dunalianum. The study results established the foundation for the expression, regulation and functional analysis of VdGAPDH1.