摘要：

为通过电子克隆得到灵芝LaeA基因序列,分析其基因序列信息,并初步探究其调控功能。采用电子克隆的方法,以已知桔青霉的LaeA蛋白序列为模板,在灵芝的EST数据库中进行序列相似性搜索比对(Blast),经序列拼接、序列验证和序列延伸等电子克隆方法获得灵芝LaeA基因的cDNA序列;采用生物信息学软件对LaeA基因编码蛋白质的基本理化性质、疏水性/亲水性、亚细胞定位、信号肽、二级结构、三级结构及进化关系等方面进行了预测和分析。结果表明,LaeA基因全长1 134 bp,编码378个氨基酸,蛋白分子质量为42.895 3 ku,存在于细胞质中,是亲水性蛋白;LaeA蛋白结构主要由47.88%无规则卷曲、33.33%α-螺旋和18.78%延伸链组成,有S-腺苷甲硫氨酸结合位点,属于AdoMet_MTases超家族蛋白;系统进化分析结果显示,LaeA蛋白与云芝、污叉丝孔菌等白腐担子菌类的亲缘关系较为亲近;qRT-PCR结果表明,LaeA基因在灵芝细胞液体静置培养过程中的表达量显著高于振荡培养方式。推测LaeA蛋白作为1种甲基转移酶蛋白,通过参与组蛋白的甲基化修饰,进而影响基因簇的表达水平。

关键词: 灵芝, LaeA, 电子克隆, 生物信息学

Abstract:

Obtaining the LaeA gene sequence of Ganoderma lucidum through electronic cloning,analyzing its gene sequence information and preliminarily exploring its regulatory function.This study adopts the method of electronic cloning,using the known LaeA protein sequence of Penicillium citrinum as a template,perform sequence similarity search and alignment(Blast)in the EST database of Ganoderma lucidum and obtain the cDNA sequence of the LaeA gene of Ganoderma lucidum through electronic cloning methods such as sequence splicing,sequence validation and sequence extension.Some characters of amino acids encoded by LaeA gene,including the physical and chemical properties,hydrophobicity/hydrophilicity,subcellular localization,secondary and tertiary structure of protein,and phylogenetic relationship were analyzed by bioinformatics tools.The length of LaeA gene from G.lucidum was 1 134 bp,encoded 378 amino acids.The protein encoded molecular weight of 42.895 3 ku.The protein was an instability protein that was present in the cytoplasm and not secreted to the extracellular.Furthermore,the structure of LaeA protein was mainly composed by 47.88% random coil,33.33%α-helix and 18.78% extension strand,contained SAM binding site,belonged to AdoMet_MTases superfamily proteins.Phylogenetic analysis showed that LaeA was closely related to white rot basidiomycetes such as Trametes versicolor and Dichomitus squalens;the Real-time Fluorescence Quantitative PCR results showed that the expression level of LaeA in Ganoderma lucidum cells during liquid static culture was significantly higher than in oscillatory culture.It is speculated that the LaeA protein as a methyltransferase protein that participates in histone methylation modification,thereby affecting the expression level of gene clusters.

Key words: Ganoderma lucidum, LaeA, Silico cloning, Bioinformatics