猪苓菌核自噬相关基因 ATG8的克隆及表达分析

摘要

目的：克隆分离猪苓自噬相关基因 ATG8，并探讨其在猪苓防御蜜环菌入侵过程中的作用。方法以猪苓菌核为材料，采用 RT-PCR 方法得到自噬相关基因的开放读码框全长，并利用荧光定量 PCR 检测该基因在不同猪苓菌核部位的表达量。结果该基因开放读码框为387 bp，编码128个氨基酸；该蛋白分子量为14.838 kD，理论等电点为5.85。氨基酸序列多重比对及系统发育树结果显示 PuATG8与蚁巢伞属（termitomyces sp.）亲缘关系最近，与皱革菌（Punctularia strigosozonata）、密褐褶孔菌（Gloeophyllum trabeum）、立枯丝核菌（Rhizoctonia solani）有较高的同源性。PuATG8为ATG 家族同源基因。荧光定量 PCR 结果表明，PuATG8在未被蜜环菌侵染的猪苓菌核及被蜜环菌侵染的菌核中都有表达，其中被侵染部分的表达量显著上调。结论 PuATG8可能参与了猪苓菌核的防御反应。%Objective To clone and isolate the autophagy-related protein 8 (ATG8) gene and investigate the function of this gene in the defense of Polyporus umbellatus. Methods A new ATG8 gene, designated as PuATG8, was isolated from the Polyporus umbellatus sclerotia using RT-PCR. The expression analysis of PuATG8 in different parts of Polyporus umbellatus sclerotia was carried out by quantitative RT-PCR assay. Results The full-length of open reading frame was 387 bp, encoding a putative protein of 129 amino acids with a molecular weight of 14.838 kD and a theoretical pI of 5.85. Phylogenetic tree analysis indicated that PuATG8 had the highest similarity with ATG8 from termitomyces sp., and was highly homologous to Punctularia strigosozonata, Gloeophyllum trabeum and Rhizoctonia solani. Quantitative RT-PCR showed that PuATG8 was expressed in P. umbellatus sclerotia irrespective of Armillaria infection. Meanwhile, the expression of PuATG8 in the sclerotia with A. mellea infection was significantly up-regulated. Conclusion This PuATG8 gene may be involved in the defense response of Polyporus umbellatus sclerotia.