棉花抗枯萎病相关基因GhERF5-4D的克隆及功能分析

Cloning and Functional Analysis of GhERF5-4D Gene Related to Fusarium oxysporum Resistance in Cotton

研究陆地棉ERF(ethylene-responsive factor)转录因子基因GhERF5-4D在棉花抗枯萎病中的作用,为棉花抗病分子机制及棉花抗病育种研究奠定基础。以枯萎病菌诱导棉花根部基因表达谱中的差异表达基因为探针,结合棉花全基因组数据库获得1个ERF转录因子基因GhERF5-4D,并进一步利用实时荧光定量(qRT-PCR)技术和病毒诱导基因沉默(virus induced gene silencing,VIGS)技术研究枯萎病菌胁迫和激素处理后基因GhERF5-4D的表达特征及其在棉花抗病过程中的作用。结果表明,GhERF5-4D(GenBank:MF093148)位于陆地棉D07染色体上,开放阅读框(open reading frame,ORF)为663 bp,编码220个氨基酸,仅含1个AP2结构域且无内含子,属于ERF亚族。该基因的表达特征在棉花抗感枯萎病品种及不同激素胁迫处理中存在显著差异,其在抗病品种中为上调表达,在感病品种中为下调表达;乙烯、茉莉酸甲酯处理后,该基因上调表达,而水杨酸处理后则下调表达。GhERF5-4D基因沉默后并进行枯萎病菌接种实验,结果表明,与对照相比,沉默株系更感病。对下游抗病相关防卫基因表达特征进行分析,发现PR2、PR4、PR5、NPR1、ERF1相对表达量均低于对照,而WRKY70相对表达量高于对照。GhERF5-4D能够响应枯萎病菌胁迫,并通过参与乙烯、茉莉酸途径调控下游相关抗病防卫基因提高棉花对枯萎病抗性。

To study the role of ERF(ethylene responsive factor)transcription factor gene GhERF5-4D in cotton Fusarium oxysporum resistance may lay a foundation for the study of cotton disease resistance molecular mechanism and cotton disease resistance breeding.Firstly,the differentially expressed genes obtained from the gene expression profile in cotton root tissues induced by F.oxysporum were employed as probes,based on the whole genome database of upland cotton,the ERF transcription factor gene GhERF5-4D was selected as a candidate gene.Then,the expression characteristics of GhERF5-4D under F.oxysporum and multiple hormones treatment,and its roles in cotton disease resistance were investigated via qRT-PCR and virus induced gene silencing(VIGS)techniques.GhERF5-4D gene(GenBank accession number:MF093148),located on the chromosome D07 of upland cotton,its open reading frame(ORF)was 663 bp encoding 220 amino acids.There was only one AP2 domain in its coding regions without any introns and belonged to the ERF subfamily.Its expression patterns in resistant and susceptible cotton cultivars and inoculation by hormones were significantly distinct,which was up-regulated in resistant varieties and down-regulated in susceptible cultivars,up-regulated after ethylene and methyl jasmonate treatment and down-regulated after salicylic acid tratment.When GhERF5-4D gene was silenced by VIGS and then inoculated with F.oxysporum,the silenced strains showed more susceptible to disease than the control.Moreover,the relative expressions of PR2,PR4,PR5,NPR1 and ERF1 genes were lower than those of control groups,while,the relative expression of WRKY70 gene was higher than that of the control.Thus,GhERF5-4D gene was confirmed to respond to F.oxysporum stress and improve cotton resistance to F.oxysporum by regulating downstream defense genes through ethylene and jasmonic acid pathway.