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为鉴定香蕉枯萎病菌(尖孢镰刀菌古巴专化型4号生理小种,Fusarium oxysporum f.sp.cubense race 4,Foc4)中的2个假想谷胱甘肽S转移酶(GSTs),采用RT-PCR方法克隆了这2个GSTs基因cDNA编码序列,随后分别将2个基因定名为Fogst1和Fogst2。其中,Fogst1的开放阅读框长609 bp,编码202个氨基酸残基,Fogst2的开放阅读框长693 bp,编码230个氨基酸残基。进化树分析表明:Fogst1属于GSTs超家族的sigma(σ)亚型成员,Fogst2属于GSTs超家族中目前未知的亚家族成员。为了验证Fogst1和Fogst2的表达,分别构建了Fogst1和Fogst2的原核表达重组载体pET28a-Fogst1和pET28a-Fogst2,并将pET28a-Fogst1和pET28a-Fogst2转化到大肠杆菌表达菌株BL21,经IPTG诱导后获得以可溶形式表达的重组蛋白Fogst1和Fogst2。GSTs活性分析表明,以CDNB为底物检测,2个重组蛋白均具有GSTs酶活性。分别取外源氧化胁迫处理后1、5、12、24 h菌丝样品进行相对荧光定量PCR分析,结果表明:Fogst1和Fogst2在前5 h表达量均大幅上调,表达量随后下调并恢复正常水平。这些结果均暗示Fogst1和Fogst2可能参与了Foc4抗外源氧化胁迫过程。
In order to identify two hypothetical glutathione S-transferases (GSTs) in Fusarium oxysporum f.sp.cubense race 4 (Foci sp. Fusarium solanacearum 4, Foc4), Fusarium oxysporum f. Sp. -PCR method to clone these two GSTs gene cDNA coding sequence, followed by the two genes were named Fogst1 and Fogst2. The open reading frame of Fogst1 was 609 bp in length and encoded 202 amino acid residues. The open reading frame of Fogst2 was 693 bp in length and encoded 230 amino acid residues. Phylogenetic tree analysis showed that Fogst1 belongs to the sigma (σ) subtype of the GSTs superfamily, and Fogst2 belongs to the currently unknown subfamily members of the GSTs superfamily. In order to verify the expression of Fogst1 and Fogst2, the prokaryotic expression recombinant vectors pET28a-Fogst1 and pET28a-Fogst2 of Fogst1 and Fogst2 were constructed respectively, and pET28a-Fogst1 and pET28a-Fogst2 were transformed into E. coli BL21. After induced by IPTG, Soluble forms of expressed recombinant proteins Fogst1 and Fogst2. GSTs activity analysis showed that CDNB as substrate detection, two recombinant proteins have GSTs enzyme activity. The relative fluorescence quantitative PCR analysis of mycelia at 1, 5, 12, 24 h after exogenous oxidative stress treatment showed that the expression of Fogst1 and Fogst2 both increased significantly in the first 5 h, and then down-regulated and returned to normal levels . These results suggest that Fogst1 and Fogst2 may be involved in Foc4 against exogenous oxidative stress.