棉花黄萎病菌鸟氨酸脱羧酶抗酶蛋白基因VdOAZ的功能分析

Functional Analysis of an Ornithine Decarboxylase Antizyme Gene VdOAZ in Verticillium dahliae Isolated from Cotton

【目的】明确棉花黄萎病菌中鸟氨酸脱羧酶抗酶蛋白(OAZ)基因(VdOAZ)的功能。【方法】以大丽轮枝菌野生型菌株V592的基因组DNA和cDNA为模板,对VdOAZ基因全长进行克隆并测序。构建针对VdOAZ基因的敲除载体和互补载体,通过农杆菌介导的遗传转化筛选VdOAZ基因敲除菌株和和互补菌株。以野生型菌株V592为对照,对VdOAZ基因敲除突变体和互补菌株的菌落生长速率、产孢量、微菌核产量及对棉花的致病力进行测定;通过实时荧光定量逆转录聚合酶链式反应测定致病相关的其他基因在VdOAZ基因敲除突变体中的表达量,及亚精胺诱导条件下,V592菌株中VdOAZ基因及致病相关的其他基因的相对表达量。【结果】从棉花黄萎病菌中克隆到VdOAZ基因的全长为1 006 bp,具有2个开放阅读框(Open reading frame,ORF),ORF2编码的蛋白具有OAZ所特有的ODC-AZ保守结构域。与野生型菌株V592和互补菌株相比,VdOAZ基因敲除突变体的菌落生长速率降低、微菌核产量及产孢量明显减少,对棉花的致病力下降,表明VdOAZ基因与大丽轮枝菌分生孢子和微菌核的产生有关,并参与大丽轮枝菌致病。在VdOAZ基因敲除突变体中,VdPKAC1、VMK1、VdNLP1、VdNLP2和VdSge1基因表达量显著上调;V592菌株经亚精胺诱导培养后,VdOAZ基因的表达量显著上调,而上述5个致病相关基因的表达量均明显下调,表明VdOAZ基因对其表达具有负调控作用。【结论】VdOAZ基因响应多胺水平改变,通过调控VdPKAC1、VMK1、VdNLP1、VdNLP2、VdSge1的表达影响大丽轮枝菌孢子产生、微菌核形成和致病过程。

[Objective] To determine the function of ornithine decarboxylase antizyme gene(VdOAZ) in Verticillium dahliae causing cotton wilt disease. [Method] The full length of VdOAZ gene was cloned and sequenced from V. dahliae strain V592 genomic DNA and cDNA. VdOAZ gene deletion plasmid was constructed and transformed into V592 strain by the Agrobacterium tumefaciens-mediated transformation to produce VdOAZ gene deletion strains, and the complementary plasmid was constructed and transformed into a VdOAZ gene deletion strain to produce complemented strains. Colony growth rate on potato dextrose agar medium, microsclerotia production and conidia production and virulence to cotton of VdOAZ gene deletion mutants and complementary strains were measured when compared to V592 strain. Relative expression of other genes involved in virulence in VdOAZ deletion mutants were measured by reverse transcription-quantitative real time PCR(RT-q PCR), and the relative expression of VdOAZ gene and other genes involved in virulence in V592 strain were also determined by RT-q PCR when V592 strain was cultured for 4 hours in the presence of spermidine. [Result] The full length of VdOAZ gene was determined to be 1 006 bp and it has two open reading frames(ORFs), VdOAZ protein encoded by ORF2 contained conserved ODC-AZ domain characterized by OAZ protein. VdOAZ gene deletion mutants displayed decreased colony growth rate, reduced microsclerotia and conidia, and drastic reduction in virulence to cotton compared with the wild type strain V592 and complementary strains, indicating that VdOAZ gene is participated in production of microsclerotia and conidia, and is involved in virulence in V. dahliae. In VdOAZ deletion mutants, transcriptional expression of 5 genes(VdPKAC1, VMK1, VdNLP1,VdNLP2 and VdSge1) in V. dahliae were strongly up-regulated. The transcriptional expression of VdOAZ gene was strongly up-regulated in the presence of spermidine, whereas VdPKAC1, VMK1, VdNLP1, VdNLP2 and VdSge1 were down-regulated,suggesting that VdOAZ gene negatively regulates transcriptional expression of VdPKAC1, VMK1, VdNLP1, VdNLP2 and VdSge1. [Conclusion] In response to changes in polyamine levels, VdOAZ gene regulates the expression of VdPKAC1, VMK1,VdNLP1, VdNLP2 and VdSge1 to participate in the process of conidium production, microsclerotium formation and pathogenesis of V. dahliae.