棘孢木霉糖苷水解酶3基因家族的生物信息学及表达模式分析

Bioinformatics and expression analyses of glycoside hydrolase 3 genes in Trichoderma asperellum

摘要:

【背景】糖苷水解酶(glycoside hydrolase,GH)3基因家族成员主要编码胞外β-葡萄糖苷酶，是纤维素降解中的关键酶。【目的】鉴定棘孢木霉GH3基因家族成员，探究其在纤维素降解过程中转录水平的表达模式。【方法】通过生物信息学方法对棘孢木霉GH3基因家族成员进行鉴定，对其基因结构、系统进化、蛋白理化性质、亚细胞定位及蛋白质三级结构进行分析，并采用荧光定量PCR技术对纤维素诱导下转录水平的表达模式进行综合分析。【结果】棘孢木霉基因组共鉴定到16个GH3基因家族成员，含有1-8个外显子，编码蛋白质长度为533-934个氨基酸，分子量为57.82-101.91kDa，大多数为胞外蛋白。系统发育表明，该基因家族成员可分为4组，与里氏木霉的相似性较高。基因表达模式分析表明，纤维素诱导下，16个GH3基因均有表达，但不同成员在转录水平的表达存在差异。其中，1个基因呈组成型表达，2个基因表达下调，13个基因表达上调。棘孢木霉的胞外β-葡萄糖苷酶活力在纤维素诱导下明显提升，与GH3基因家族成员在转录水平的整体表达模式相一致。【结论】棘孢木霉基因组共包含16个GH3基因家族成员，而且多为亲水性稳定蛋白质，有良好的商业开发潜力；绝大部分GH3基因家族成员的转录量在纤维素诱导下呈上调，与胞外β-葡萄糖苷酶活性变化相一致，说明其在纤维素降解中发挥重要作用，这为天然木质纤维原料的资源化利用和纤维素降解工程菌的构建提供了重要的理论依据。

Abstract:

[Background] The glycoside hydrolase 3 gene (GH3) family mainly encodes extracellular β-glucosidases, which are the key enzymes in cellulose degradation. [Objective] To identify the GH3 genes in Trichoderma asperellum (TaGH3 genes) and investigate their transcriptional patterns. [Methods] The bioinformatics tools were employed to identify the TaGH3 genes and analyze the gene structure, phylogenetic relationship, as well as the physicochemical properties, subcellular localization, and tertiary structure of the deduced proteins. quantitative real-time polymerase chain reaction (RT-qPCR) was employed to investigate the transcriptional patterns of TaGH3 genes in the presence of cellulose. [Results] A total of 16 TaGH3 genes were identified, which contained 1-8 exons. The deduced TaGH3 proteins had the length of 533-934 amino acid (aa) and the theoretical molecular weights of 57.82-101.91 kDa. The majority of TaGH3 proteins were predicted to be extracellular. Phylogenetic analysis demonstrated that all the TaGH3 proteins could be classified into 4 clades, which had higher homology with Trichoderma reesei. Cellulose affected the transcription of all the 16 TaGH3 genes, while the transcription level varied among genes. Specifically, cellulose induced the constitutive expression of 1 gene, down-regulated the expression of 2 genes, and up-regulated the expression of 13 genes. Moreover, cellulose enhanced the extracellular β-glucosidase activity, which was consistent to the transcriptional pattern of most TaGH3 genes. [Conclusion] The genome of T. asperellum carried 16 GH3 genes, most of which encoded hydrophilic proteins with the potential for commercial exploitation. The transcription of the majority of TaGH3 genes was up-regulated by cellulose, which was consistent with the enhanced β-glucosidase activity, indicating that these genes played a key role in cellulose degradation. This study systematically investigated the GH3 genes in T. asperellum, providing new insights for the resource utilization of lignocellulosic materials and the construction of strains with high cellulase production.