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  本文選題：Foc + 谷胱甘肽S轉(zhuǎn)移酶��； 參考：《生物工程學(xué)報》2017年06期

【摘要】：為鑒定香蕉枯萎病菌(尖孢鐮刀菌古巴專化型4號生理小種,Fusarium oxysporum f.sp.cubense race 4,Foc4)中的2個假想谷胱甘肽S轉(zhuǎn)移酶(GSTs),采用RT-PCR方法克隆了這2個GSTs基因cDNA編碼序列,隨后分別將2個基因定名為Fogst1和Fogst2。其中,Fogst1的開放閱讀框長609 bp,編碼202個氨基酸殘基,Fogst2的開放閱讀框長693 bp,編碼230個氨基酸殘基。進化樹分析表明:Fogst1屬于GSTs超家族的sigma(σ)亞型成員,Fogst2屬于GSTs超家族中目前未知的亞家族成員。為了驗證Fogst1和Fogst2的表達,分別構(gòu)建了Fogst1和Fogst2的原核表達重組載體pET28a-Fogst1和pET28a-Fogst2,并將pET28a-Fogst1和pET28a-Fogst2轉(zhuǎn)化到大腸桿菌表達菌株BL21,經(jīng)IPTG誘導(dǎo)后獲得以可溶形式表達的重組蛋白Fogst1和Fogst2。GSTs活性分析表明,以CDNB為底物檢測,2個重組蛋白均具有GSTs酶活性。分別取外源氧化脅迫處理后1、5、12、24 h菌絲樣品進行相對熒光定量PCR分析,結(jié)果表明:Fogst1和Fogst2在前5 h表達量均大幅上調(diào),表達量隨后下調(diào)并恢復(fù)正常水平。這些結(jié)果均暗示Fogst1和Fogst2可能參與了Foc4抗外源氧化脅迫過程。
[Abstract]:In order to identify two hypothetical glutathione S-transferase (GSTs) from Fusarium oxysporum f.sp.cubense race 4 (Fusarium oxysporum f.sp.cubense race 4 Foc4), two GSTs gene cDNA coding sequences were cloned by RT-PCR. Then the two genes were named Fogst1 and Fogst2 respectively. The open reading frame length of Fogst1 was 609 BP, and that of Fogst2 encoding 202 amino acid residues was 693 BP, encoding 230 amino acid residues. The phylogenetic tree analysis shows that the member of the sigma (蟽) subtype of the GSTs superfamily belongs to the GSTs superfamily and belongs to the unknown member of the GSTs superfamily. In order to verify the expression of Fogst1 and Fogst2, the prokaryotic expression vectors pET28a-Fogst1 and pET28a-Fogst2 of Fogst1 and Fogst2 were constructed, and the pET28a-Fogst1 and pET28a-Fogst2 were transformed into E. coli expression strain BL21. The results of Fogst1 and Fogst2.GSTs activity analysis showed that the recombinant protein was expressed in soluble form after IPTG induction. Using CDNB as substrate, the two recombinant proteins had GSTs enzyme activity. The relative fluorescence quantitative PCR analysis was performed on the mycelium samples treated with exogenous oxidative stress for 24 h. The results showed that the expression of Fogst1 and Fogst2 increased significantly in the first 5 hours, then decreased and returned to normal level. These results suggest that Fogst1 and Fogst2 may be involved in the process of Foc4 resistance to exogenous oxidative stress.
【作者單位】： 海南大學(xué)熱帶生物資源教育部重點實驗室;海南大學(xué)海洋學(xué)院;中國熱帶農(nóng)業(yè)科學(xué)院環(huán)境與植物保護研究所;
【基金】：國家自然科學(xué)基金(No.31560491) 海南大學(xué)博士科研啟動基金(No.kyqd5143)資助~~
【分類號】：S436.68

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