灰葡萄孢菌AUR1基因内含子对肌醇磷脂酰神经酰胺合成酶表达的影响及致病性

Effect of AUR1 gene intron on the expression of inositol phosphorylceramide synthase and pathogenicity in Botrytis cinerea

摘要:

摘要:【目的】AUR1编码的肌醇磷脂酰神经酰胺(IPC)合成酶是真菌鞘脂代谢的关键酶，在转录水平和翻译水平研究AUR1内含子对其基因表达的影响，以及AUR1内含子对相关致病因子的影响，为内含子调控基因表达的分子机制提供理论依据。【方法】实时定量PCR测定野生型灰葡萄孢菌(BcAUR1)和AUR1缺失115 bp内含子突变体(BcAUR1a)的mRNA表达量，高效液相层析测定IPC合成酶活性，分别采用辣根过氧化物酶法、邻苯三酚自氧化法、愈创木酚法和紫外分光光度法测定单位菌体的H2O2含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的酶活力。【结果】突变体BcAUR1a的IPC合成酶基因cDNA测序结果表明，IPC合成酶无氨基酸突变。实时定量PCR和高效液相层析的结果表明BcAUR1a的AUR1基因mRNA表达量和IPC合成酶活力比野生型BcAUR1分别增加了50.2%和14.16%。短梗霉素A(AbA)显著刺激BcAUR1 H2O2、SOD、POD和CAT的分泌，但对BcAUR1a的这几种物质的分泌无显著影响。【结论】突变体BcAUR1a的AUR1基因在转录和翻译水平上表达上调，AbA显著增强野生型灰葡萄孢菌致病力，但对突变体影响较小。突变体产生了对AbA的抗性，推测AUR1基因内含子在AUR1基因表达调控中起转录抑制子的作用。

Abstract:

Abstract:［Objective］AUＲ1 encoding inositol phosphorylceramide (IPC) synthase is the key enzyme for the sphingolipid metabolism in fungi. In this study，we explored the mechanism of AUR1 intron on the regulation of AUR1 gene expression at transcriptional and translational levels in Botrytis cinerea，as well as the influence of AUR1 intron on the pathogenicity．［Methods］AUR1 mRNA expression of wild-type B．cinerea (BcAUR1 ) and the mutant with deletion of 115 bp intron (BcAUR1a) was detected by Real-time quantitative PCR．The activity of IPC synthase from BcAUR1 and BcAUR1a was measured through high-efficiency liquid fluorescent chromatogram．In addition，H2O2 concentration and activities of superoxide dismutase (SOD)，peroxidase (POD) and catalase (CAT) per unit fungus were determined by horseradish peroxidase，pyrogallol oxidation，guaiacol and ultraviolet spectrophotometric，respectively.［Results］IPC synthase had no amino acid mutation in mutant BcAUR1a．The expression of AUR1 gene at mRNA level and the activity of IPC synthase in BcAUR1a increased by 50.2% and 14.16% compared to those in BcAUR1．The secretion of H2O2，SOD，POD and CAT in BcAUR1 was significantly stimulated by Aureobasidin A (AbA) treatment，in contrast，no significant influence was detected upon the secretion of these substances in BcAUR1a via AbA treatment.［Conclusion］The expression of AUR1 in BcAUＲ1a is significantly up-regulated at transcriptional and translational levels．AbA treatment can significantly enhance the pathogenicity of BcAUR1，but has a minor influence on the BcAUR1a． BcAUR1a is AbA-resistant．The results suggest that AUR1 gene intron regulate the expression of AUR1 as a transcriptional repressor．