香菇GPX基因克隆及其在采后贮藏中的表达分析

Cloning and Expression Analysis of Lentinus edodes GPX Gene during Postharvest Storage

以香菇(Lentinula edodes)为材料,对谷胱甘肽过氧化物酶(Glutathione peroxidase,GPX)进行基因的鉴定、编码序列克隆、蛋白生物信息学分析以及基因表达模式分析。结果表明:成功鉴定并克隆1个LeGPX基因,该基因开放阅读框(Open reading frame,ORF)621 bp,所编码蛋白包含206个氨基酸残基;系统发育分析显示LeGPX蛋白与小皮伞(Marasmius fiardii)和灰色果腐菌(Moniliophthora roreri)的GPX亲缘关系较近,同源比对结果显示LeGPX蛋白包含GPX特征序列和催化位点。生物信息学分析表明:LeGPX为亲水性不稳定蛋白,蛋白分子质量22.81 kD,理论等电点8.83;二级结构以无规则卷曲为主,不含信号肽和跨膜结构。通过原核表达和纯化成功获得了LeGPX可溶性蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-polyacrylamide gel electrophoresis,SDS-PAGE)显示蛋白大小符合预期。荧光定量PCR分析表明,香菇褐变发生过程中LeGPX基因表达量快速上升,并且与菌盖相比,LeGPX在香菇菌柄部位表达更为活跃。

In this study,the glutathione peroxidase(GPX)of Lentinula edodes(LeGPX)was identified and cloned via coding sequence,and the protein bioinformation and gene expression pattern of LeGPX were analyzed.The results showed that a LeGPX gene was successfully identified and cloned,the open reading frame(ORF)of LeGPX gene was 621 bp,and the encoding protein contained 206 amino acid residues.The phylogenetic analysis showed that LeGPX protein was closely related to GPX in Marasmius fiardii and Moniliophthora roreri,and homology comparison showed that LeGPX protein contained characteristic sequence and catalytic site of GPX.Bioinformatic analysis showed that LeGPX was a hydrophilic unstable protein with molecular weight of 22.81 kD and theoretical isoelectric point of 8.83.The protein presented mainly random curly secondary structure without signal peptide and transmembrane structure.LeGPX soluble protein was obtained by prokaryotic expression and purification,and SDS-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the protein size was in accordance with expectations.Fluorescence quantitative PCR analysis showed that the expression level of LeGPX gene was increased rapidly during L.edodes browning,and the LeGPX gene expression in mushroom stipe was more active compared with that in the cap.