Foc4 2个谷胱甘肽S-转移酶基因的克隆、鉴定及其在外源氧化胁迫下的表达

Cloning and characterization of two glutathione S-transferases cDNAs in Foc4 and expression under exogenous oxidative stress

摘要:

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。

Abstract:

In order to identify two putative glutathione S-transferase (GSTs) genes in Fusarium oxysporum f. sp. cubense race 4 (Foc4), cDNA sequences of the entire coding regions of the two genes were cloned from Foc4 using RT-PCR method. Subsequently, the two genes were named Fogst1 and Fogst2 respectively. The length of open reading frame of Fogst1 was 609 bp and encoded a protein including 202 amino acid residues, Fogst2 possessed an open reading frame with 693 bp which encoded a 230-amino acid protein. Phylogenetic analysis showed that Fogst1 belonged to sigma (σ) subtype members of the GSTs superfamily, and Fogst2 was a new member of an unknown subfamily in the GSTs superfamily. To verify the expression of Fogst1 and Fogst2, the recombinant prokaryotic expression vector pET28a-Fogst1 and pET28a-Fogst2 were constructed and transformed into Escherichia coli expression strain BL21(DE3). The soluble recombinant proteins Fogst1 and Fogst2 were obtained after being induced by IPTG. GSTs activity assays showed that both of the two recombinant proteins had specific activity with CDNB. For real time RT- PCR analysis, the mycelium samples of Foc4 were collected after treatment by H2O2 for 1, 5, 12, 24 hours. The results showed that the expression of Fogst1 and Fogst2 were significantly up-regulated in the first 5 hours, and then decreased and returned to normal level. These results suggested that Fogst1 and Fogst2 may be involved in the process of Foc4 resistance to exogenous oxidative stress.