山葡萄VaCBF3基因克隆及生物信息学分析

为预测山葡萄VaCBF3基因的功能,利用RT-PCR方法从低温处理的山葡萄中克隆出抗寒转录因子VaCBF3基因。利用生物信息学方法对其结构域及功能进行预测,包括开放阅读框分析、基本理化性质分析、保守结构域分析、信号肽预测、蛋白修饰位点分析、疏水性分析、系统进化分析、蛋白质二级结构及三级结构预测等。结果表明:经测序VaCBF3基因序列全长为854bp,编码239个氨基酸,序列提交至GenBank中(登录号为:EU672969;蛋白登录号为:ACD45468.1)。该基因属于AP2 superfamily家族,具有AP2保守结构域;蛋白相对分子量为25.9kDa,分子式为C1109H1754N332O364S11,原子总数为3570,理论等电点为7.1;为不稳定亲水蛋白,不含信号肽;共计43个磷酸化位点,1个N-糖基化位点,39个O-糖基化位点;二级结构中α-螺旋占25.94%,β-转角占4.60%,不规则卷曲链56.07%,延伸链占13.39%;三级结构含有1个α-螺旋和3个β-折叠。系统发育树分析表明,VaCBF3蛋白与Vitisamurensis聚为一支,亲缘关系最近,与V.vinifera、V.riparia亲缘关系较远。

Cloning and Bioinformatic Analysis of VaCBF3 Gene from Vitis amurensis

In order to predict the function of the VaCBF3 gene in Vitis amurensis, by using RT-PCR method, a cold resistance gene VaCBF3 was cloned from mountain grape which had been treated at low temperature. Bioinformatics method was used to predict its domain and function, including open reading frame analysis, basic physical and chemical properties analysis, conservative domain analysis, signal peptide prediction, protein modification site analysis, hydrophobicity analysis, phylogenetic analysis, protein secondary structure and tertiary structure prediction, etc. The results were as follows: the total length of VaCBF3 gene was 854 bp (GenBank accession number: EU672969), encoded 239 amino acids (GenBank accession number: ACD45468.1). VaCBF3 belonged to the AP2 superfamily family and had an AP2 conservative domain. The relative molecular weight of protein was 25.9 kDa, molecular formula was C1109H1754N332 O364S11, total number of atoms was 3 570 and theoretical isoelectric point was 7.1;Protein is unstable and hydrophilic, had no signal peptides, contained 43 phosphorylated sites, 1 N-glycosylated site and 39 O-glycosylated sites. In its secondary structure, the percentage of α-helix was 25.94 %,β-angle was 4.60 %, irregular curled chain was 56.07 % and extended chain was 13.39 %. Its tertiary structure contained one alpha-helix and three beta-folds. Phylogenetic analysis showed that the VaCBF3 protein was closely related to that of Vitis amurensis, but distantly related to that of V. vinifera and V. riparia.