尖镰孢5-氧脯氨酸酶基因的鉴定及功能分析

Identification and Functional Analysis of the 5-Oxoprolinase Genes in Fusarium oxysporum

【目的】5-氧脯氨酸酶(5-oxoprolinase,OXP)是γ-谷氨酰循环中6种核心酶之一。鉴定尖镰孢(Fusariumoxysporum)5-氧脯氨酸酶基因,明确其与尖镰孢致病力的关系,为解析尖镰孢致病分子机制以及棉花枯萎病防治提供理论依据。【方法】利用生物信息学方法从尖镰孢基因组中鉴定FoOXP,分析其基因结构、编码蛋白的结构域、染色体定位及进化关系。通过基因敲除突变株和回补菌株分析FoOXP2突变后尖镰孢的表型,并检测突变株和野生型菌株对棉花幼苗的致病力差异。利用寄主诱导的基因沉默(host-inducedgenesilencing,HIGS)技术调查转化棉花的病级率及病情指数。利用实时荧光定量PCR(qRT-PCR)技术检测棉花中真菌生物量和转化FoOXP2干扰片段的棉花茎中FoOXP2的表达量。【结果】在尖镰孢基因组中共鉴定出两个FoOXP(FoOXP1和FoOXP2),其编码序列长度分别为4080和3921 bp,分别编码1359和1306个氨基酸,蛋白质分子量分别为14.90和14.07kDa,理论等电点分别为5.73和5.30。FoOXP1蛋白定位于线粒体,FoOXP2蛋白定位于细胞骨架。FoOXP1位于染色体JH657921,FoOXP2位于染色体JH657938,未形成基因簇,其序列相似性为52.00%。系统发育分析发现,FoOXP1和FoOXP2分属于两个亚群。与野生型菌株相比,FoOXP2敲除突变株产孢量和孢子萌发率显著降低,穿透能力丧失,对刚果红和山梨醇的耐受力有所增强,但对细胞壁胁迫(SDS和CFW)、氧化胁迫(H_(2)O_(2))和渗透胁迫(NaCl和KCl)更敏感。同时,对棉花的致病力显著下降。HIGS结果表明,接菌14和21 d后,FoOXP2基因沉默后的棉花植株发病明显减轻,其病情指数(17.3和40.2)显著低于对照(28.2和77.1),FoOXP2表达量和真菌生物量显著低于对照。【结论】FoOXP2正向调控尖镰孢的致病力,推测其在宿主-病原体互作过程中发挥重要作用。

【Objective】5-Oxoprolinase(OXP)is one of the six core enzymes in theγ-glutamine cycle.The objective of this study is to characterize the OXP gene of Fusarium oxysporum,clarify its relationship with the pathogenicity of F.oxysporum,and to provide a theoretical basis for the analysis of the molecular mechanism of F.oxysporum pathogenicity and the prevention and control of cotton wilt.【Method】Bioinformatics methods were used to identify the FoOXPs from the genome of F.oxysporum and analyze their gene structure,domains of the encoded proteins,chromosomal localization and evolutionary relationships.The phenotypes of F.oxysporum after FoOXP2 mutation were analyzed by using knockout mutant and complementary strain and the pathogenicity differences between the mutant and wild-type strains on cotton seedlings were detected.Furthermore,the disease grade rate and disease index on cotton were investigated by using host-induced gene silencing(HIGS)technology.The fungal biomass in cotton and expression of FoOXP2 in cotton stems transformed with interfering fragments of the FoOXP2 were examined by real-time quantitative PCR(qRT-PCR).【Result】A total of two FoOXPs(FoOXP1 and FoOXP2)were identified in the F.oxysporum genome with coding sequence lengths of 4080 and 3921 bp,encoding 1359 and 1306 amino acids,respectively.Their protein molecular weights are 14.90 and 14.07 kDa,and theoretical isoelectric points are 5.73 and 5.30,respectively.The FoOXP1 protein is located in the mitochondria and the FoOXP2 protein is located in the cytoskeleton.FoOXP1 and FoOXP2 locate on chromosome JH657921 and chromosome JH657938,do not form gene clusters,with a sequence similarity of 52.00%.Phylogenetic analysis revealed that FoOXP1 and FoOXP2 belonged to two subgroups.Compared with the wild-type strain,the FoOXP2 knockout mutant strain showed significantly lower spore production and spore germination,loss of penetration ability,and increased tolerance to CR and sorbitol,but was more sensitive to cell wall stress(SDS and CFW),oxidative stress(H_(2)O_(2)),and osmotic stress(NaCl and KCl).Moreover,the pathogenicity on cotton was significantly reduced.The results of the HIGS assay illuminated that after 14 and 21 d of inoculation,the incidence of FoOXP2 silenced cotton plants was significantly reduced,whose disease indexes(17.3 and 40.2)were significantly lower than those of the control(28.2 and 77.1).The expression of FoOXP2 and fungal biomass were significantly lower than those of the control.【Conclusion】The FoOXP2 positively regulates the pathogenicity of F.oxysporum and may play an important role in host-pathogen interactions.