灰葡萄孢菌及其抗短梗霉素突变体AUR1基因序列及其酶活性的分析

Analysis of AURI gene sequence and enzymatic activity in Botrytis cinerea and its mutants with aureobasidin A-resistance

摘要:

【目的】探讨灰葡萄孢菌及其抗AbA突变体AUR1基因序列与IPC合成酶活性的关系。【方法】通过分子生物学方法测定野生型及突变体的AUR1的基因序列，高效液相荧光色谱法测定IPC合成酶活力，苯甲酰化法测定神经酰胺含量。【结果】AUR1基因序列和IPC合成酶活性测定表明4株不同的突变体均产生了对IPC合成酶抑制剂AbA的抗性，它们的突变类型为：(1) AUR1序列中缺失内含子；(2) AUR1序列中缺失内含子和P155S氨基酸突变；(3) AUR1序列中缺失内含子和V33A的氨基酸突变；(4) AUR1序列中缺失内含子和P155S、S177P、F237L的氨基酸突变。AUR1缺失内含子和既缺失内含子又伴随P155S氨基酸突变的突变体的AbA抗性较强。神经酰胺含量测定表明野生型IPC合成酶被抑制，导致神经酰胺积累，而突变体则能抵抗AbA对IPC合成酶的抑制作用。【结论】AUR1基因中的内含子对IPC合成酶的调控起重要的作用。AbA通过抑制IPC合成酶引起神经酰胺积累，IPC合成酶是鞘脂代谢的关键酶。

Abstract:

[Objective] To explore the relationship between AURI gene sequences in Botrytis cinerea and its mutants with aureobasidium A-resistance and the activity of Inositol phosphorylceramide (IPC) synthase. [Methods] Molecular-biology methods were adopted to determine the AUR1 gene sequences of the wild type and the mutants. The activity of IPC synthase was analysed by HPLC-FLD. The content of ceramide was used the method of benzene formylation. [Results] The results showed that 4 different mutant strains resisted to inhibitor AbA of IPC synthase. Their mutant types were: (1) AUR1 gene lost an intron; (2) AUR1 gene lost an intron and one amino acid mutated (P155S); (3) AUR1 gene lost an intron and one amino acid mutated (V33A); (4) AUR1 gene lost an intron and three amino acids mutated (F237L, S177P, and P155S). Both the mutant with the AUR1 gene lack of an intron and the mutant with the AUR1 gene lack of an intron and the mutant of P155S had stronger resistance to AbA. The determination of ceramide content showed that IPC synthase of wild type was inhibited to cause ceramide accumulation, and the mutants could resist to AbA inhibition on IPC synthase. [Conclusion] The intron of AUR1 gene plays an important role in the regulation of IPC synthase. AbA inhibites IPC synthase from inducing ceramide accumulation, IPC synthase is a key enzyme in sphingolipid metabolism.