棉花WRKY22基因分离及其对黄萎病的抗性分析

GhWRKY22 Isolation and Function Analysis in Cotton Resistance to Verticillium wilt

为了克隆棉花抗黄萎病相关基因,利用生物信息学、病毒诱导基因沉默技术(VIGS)、实时定量PCR(q PCR)和接菌分析来研究棉花WRKY基因对大丽轮枝菌的诱导响应和抗性。结果从棉花中筛选出8个与拟南芥直系同源的棉花抗病相关WRKY基因,这些直系同源WRKY基因表达受到大丽轮枝菌浸染诱导响应。其中有6个GhWRKY基因的表达均受到大丽轮枝菌诱导上调,而GhWRKY70和GhWRKY48的表达量随着不同的时间点呈现上调或下调。GhWRKY22等5个基因表达量在24,72 h分别表现为上调,呈现出双峰曲线。对GhWRKY22表达特征进一步分析,结果显示,GhWRKY22基因在茎里优势表达,同时表达受到大丽轮枝菌、水杨酸和茉莉酸激素的诱导。GhWRKY22基因沉默植株对大丽轮枝菌的敏感性增加,抗病标志基因(PR1、PR3、PR4、PR5、PAL和PDF1. 2)表达量也显著降低,揭示GhWRKY22是通过SA和JA信号途径参与棉花对大丽轮枝菌的响应过程。总之,棉花中8个WRKY基因参与棉花对黄萎病抗性调控,其中GhWRKY22基因正调控棉花的抗性,可作为棉花抗病育种的候选基因。

The development of plant disease-resistant cultivars by disease-resistant gene mining becomes a most available measure.The bioinformation,virus induced gene silencing,qPCR and fungus inoculation were employed to analyze cotton WRKY resistance to Verticillium dahliae.Eight cotton orthologous WRKY genes corresponding to8Arabidopsis defense-related WRKYs was selected by phylogenetic tree analysis.And their expression was induced by V.dahliae infection,indicating that they participate in cotton defense to this fungus.Among these genes,six GhWRKYs showed up-regulation expression,and expression levels of GhWRKY70and GhWRKY48exhibited up-or down-regulation over time.Five cotton WRKY genes including showed two expression peaks at24,72h after pathogen inoculation.GhWRKY22gene was chosen for further charactering its function in cotton resistance to V.dahliae infestation.GhWRKY22expressed predominantly in stem,and its expression level was obviously increased upon pathogen infestation and responded positively to the defense signaling molecules such as salicylic acid and jasmonic acid hormones.Knockdown of GhWRKY22gene increased susceptibility of plants to V.dahliae infection.Additionally,the expression level of defense marker genes,including PR1,PR3,PR4,PR5,PAL and PDF1.2,significantly decreased in GhWRKY22-silenced plants compared to the control plants,which suggesed that GhWRKY22participates in cotton defense against Verticillium wilt through SA and JA signaling pathways.Taken together,these results showed that8GhWRKYs were identified and characterized in regulation of resistance to V.dahliae.Among them,GhWRKY22was a positive regulation component in cotton defense,which could be regarded as a candidate gene to breed plant defense cultivars.