草菇芳香醇氧化酶基因vvaao1的序列特征与差异表达

doi:10.13560/j.cnki.biotech.bull.1985.2018-0166

摘要/Abstract

摘要： 对芳香醇氧化酶编码基因vvaao1序列特征及差异表达进行分析,以期为研究vvaao1在草菇基质降解与子实体发育中的生物学功能提供依据。应用ZOOM软件分析基因结构与序列准确性;通过生物信息分析网站预测氨基酸序列的信号肽、亚细胞定位及三级结构;采用MEGA 5.1构建系统发育树;结合数字基因表达谱（DGE）、荧光定量PCR（RT-qPCR）、蛋白质组（iTRAQ）分析进行差异表达分析。结果显示,vvaao1全长3 091 bp,含有9个外显子、8个内含子,开放阅读框长1 803 bp,编码600个氨基酸;生物信息分析结果显示：VvAAO1具备信号肽,为分泌蛋白;其三级结构由20个α-螺旋和19个β-折叠组成,与模式蛋白3FIM的一致性（Identity）达到48%。进化树结果显示,VvAAO1属于AAO家族,与侧耳属菌株Pleurotus. eryngii和P. pulmonarius的AAO序列最为接近。DGE、RT-qPCR及iTRAQ的分析结果表明,vvaao1在可孕异核体菌丝上的表达量分别是两个同核体菌株之和的51.0、49.8和2.13倍;进一步对不同发育阶段的子实体样品进行RT-qPCR检测,结果显示vvaao1在原基时期的表达量均显著高于其他时期的样品。VvAAO1具备信号肽,为分泌蛋白,其编码基因在异核体菌株H1521的表达水平显著高于两个同核体菌株之和,存在协同增效表达,且在原基时期表达量最高,vvaao1的高表达可能有利于草菇子实体的形成。

关键词: 褐腐菌, 原基形成, 芳香醇氧化酶, 基因结构, 协同增效

Abstract: A gene encoding aryl alcohol oxidase named vvaao1 was obtained from straw mushroom Volvariella volvacea. To reveal the role of vvaao1 during substrate degradation and fruiting-body development in V. volvacea,the structure,sequence characters and expression profile of vvaao1 were analyzed. ZOOM software was used to confirm the accuracy of gene sequence and analyze the gene structure. The protein sequence was submitted to Expasy ProtParam for physicochemical properties prediction,SignalP server for signal peptide prediction,TargetP server for sub-cellular localization prediction,and the Phyre 2 server for three dimensional（3D）structure analysis. The MEGA 5.1 was used for multiple sequence alignment and phylogenetic tree analysis. Digital gene expression profiling（DGE）,real time fluorescent quantitative PCR（RT-qPCR）and iTRAQ methods were used to determine the expression pattern of vvaao1. Results showed that the full sequence of vvaao1 covered 3 091 bp,containing 9 exons and 8 introns,and the open reading frame length was 1803bp,encoded a protein with 600 amino acids. Bioinformatic analysis showed that the VvAAO1 contained a secretory signal peptide,and located in secretory pathway. The 3D structures predicted showed that VvAAO1 consisted with 20 α-helices and 19 β-strands,and had 48% identity with model protein 3FIM. The phylogenetic tree showed that VvAAO1 of V. volvacea belonged to the AAO class of GMC superfamily,and had the closest relationship with AAO in Pleurotus eryngii and P. pulmonarius. The analysis of DGE,RT-qPCR and iTRAQ results showed that the expression value of vvaao1 in H1521 were 51.0,49.8 and 2.13 times as much as the sum of two homokaryons,respectively. In addition,the gene expression of vvaao1 in primordia showed significant higher than other fruiting-body samples. VvAAO1 is a secretory protein,and its high level expression may be beneficial for fruiting-body formation.

Key words: brown-rot fungi, primordium formation, aromatic alcohol oxidase, gene structure, synergistic gene expression effect

严俊杰, 仝宗军, 刘媛媛, 张磊, 张云, 谢宝贵. 草菇芳香醇氧化酶基因vvaao1的序列特征与差异表达[J]. 生物技术通报, 2018, 34(4): 107-114.

YAN Jun-jie, TONG Zong-jun, LIU Yuan-yuan, ZHANG Lei, ZHANG Yun, XIE Bao-gui. Sequence Characterization and Differential Expression Analysis of a Aryl Alcohol Oxidase Gene vvaao1 from Volvariella volvacea[J]. Biotechnology Bulletin, 2018, 34(4): 107-114.

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