灵芝14-3-3蛋白基因的电子克隆与表达分析

In Silico Cloning and Expression Analysis of 14-3-3 Gene in Ganoderma lucidum

通过电子克隆技术,获得14-3-3基因的cDNA序列全长,并采用生物信息学方法,借助电子计算机和相关的生物信息学软件,对该基因编码蛋白从基本理化性质、疏水性/亲水性、亚细胞定位、跨膜区结构、信号肽、高级结构及系统发育树分析等进行了预测和分析。该cDNA编码蛋白由257个氨基酸组成,相对分子质量为29.014 5 kDa,亲水性和疏水性比较平衡,定位于细胞质,不存在信号肽,无跨膜螺旋区,且二级结构由6.23%无规则卷曲,66.54%α-螺旋和27.24%延伸链组成。同源比对分析显示,该酶基因编码的氨基酸序列与污叉丝孔菌、双孢菇和木耳等蘑菇类高等真菌中的14-3-3基因所编码的氨基酸序列高度同源。实时荧光定量PCR结果表明,灵芝14-3-3基因在液体静置培养过程中的表达量显著高于振荡培养。

A novel 14-3-3 gene from Ganoderma lucidum was cloned in silico based on the corresponding Gan-oderma lucidum EST sequences in NCBI database .Some characters of amino acids encoded by 14-3-3 gene,inclu-ding the composition of amino acid sequence ,physical and chemical properties ,transmembrane domain ,hydropho-bicity /hydrophilicity ,subcellular localization ,secondary and tertiary structure of protein plus the functional domain , were analyzed by bioinformatics tools .A 14-3-3 gene from Ganoderma lucidum was 1 372 bp and it contained a complete ORF which encoded 257 amino acids localized in the cytoplasm ,there was not signal peptide and trans-membrane helical regions,was not a secreted protein .The secondary structure were composed of 6.23% random coil,66.54% α-helix and the 27.24% extension chain.Homology comparison and phylogenetic analysis showed that the amino acid encoded by 14-3-3 gene in Ganoderma lucidum was highly homologous with those encoded by 14-3-3 gene in mushroom species .As the real-time PCR results showed ,the gene transcription expression level un-der the liquid static cultivation was obvious higher than that in liquid submerged cultivation .