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为了探究AP1转录因子在尖孢镰刀菌古巴专化型4号生理小种(Foc4)中是否参与香蕉枯萎病的致病过程,借助尖孢镰刀菌Fo5176菌株(GenBank序列号:AFQF01001482.1)全基因组序列,通过PCR和RT-PCR技术克隆获得了Foc4中AP1转录因子的基因组DNA和cDNA编码序列。利用PEG介导的原生质体转化法获得AP1基因敲除转化子。利用qRTPCR分析AP1可能调控的下游基因表达。利用灌根法(直接在根部浇菌)检测了AP1缺失突变体的致病能力。结果表明Foc4的AP1转录因子cDNA编码序列长1 770 bp,编码63.9 kDa(589 aa)蛋白,是一个典型的bZIP型转录因子,命名为FoAP1;FoAP1缺失突变体的气生菌丝大量减少,菌丝的入侵生长受到严重限制。对外源氧化胁迫不敏感,但致病能力减弱。
In order to explore whether AP1 transcription factor involved in pathogenicity of banana wilt disease in Fusarium oxysporum 4 Foc4, Fusarium oxysporum Fo5176 strain (GenBank accession number: AFQF01001482.1) The genomic sequence was cloned by PCR and RT-PCR to obtain the genomic DNA and cDNA coding sequence of AP1 transcription factor in Foc4. AP1 knockout transformants were obtained using PEG-mediated protoplast transformation. Downstream gene expression that AP1 might regulate was analyzed using qRTPCR. The pathogenicity of AP1-deficient mutants was tested by the method of root-filling (directly rooted at the root). The results showed that the AP1 transcription factor cDNA encoding Foc4 was 1 770 bp in length encoding a protein of 63.9 kDa (589 aa) and was a typical bZIP-type transcription factor designated as FoAP1. The number of vacuolar mycelium of FoAP1 deletion mutant was greatly reduced, Silk invasion growth is severely limited. Not sensitive to exogenous oxidative stress, but pathogenic ability weakened.