作 者 ：赵曾强,韩泽刚,李会会,李潇玲,张 析,张 薇*
期 刊 ：西北植物学报 2015年 1期 页码：10~15
关键词：棉花；WRKY转录因子；枯萎病菌；基因克隆；表达分析；
Keywords:cotton, WRKY transcription factors, Fusarium oxysporum f.sp.vasinfectum, gene clone, expression analysis,
摘 要 ：该研究以枯萎病菌诱导棉花根部基因表达谱中筛选得到的WRKY基因片段为探针,通过电子克隆结合RT-PCR技术,从高抗枯萎病品种‘中棉所12’中克隆到1个与抗枯萎病相关的WRKY转录因子基因GhWRKY44(GenBank登录号KJ801807)。序列分析表明,GhWRKY44基因开放阅读框1 197 bp,编码398个氨基酸,含有2个WRKY结构域及C2H2型锌指结构,属于棉花WRKY转录因子家族第Ⅰ类;系统进化分析显示,GhWRKY44基因与拟南芥AtWRKY44亲缘关系最近。实时荧光定量PCR(qRT-PCR)检测该基因表达特性,结果发现枯萎病菌诱导后,GhWRKY44基因在抗病品种中优势表达,随处理后时间的推移,其表达量呈先增加后降低再增加的变化趋势,处理后3 h时GhWRKY44基因表达量达到最大;而在感病品种中,GhWRKY44基因表达水平明显低于抗病品种,对病原菌响应时间也晚于抗病品种,处理后6 h时GhWRKY44基因表达量才达到最大。水杨酸和茉莉酸均能诱导GhWRKY44基因的表达,水杨酸诱导后,GhWRKY44基因表达量迅速增加,且其表达量一直维持在一个较高水平;而茉莉酸诱导后,GhWRKY44基因表达量呈先增加后降低的变化趋势,且其表达水平明显低于水杨酸诱导。研究表明,GhWRKY44基因可能参与了棉花对病原菌和激素胁迫的应答反应。
Abstract:In this study,the WRKY gene fragment from a digital expression profiling of cotton root tissues infected by Fusarium oxysporum f.sp.vasinfectum (Fov) was used as a probe,a WRKY transcription factor gene was cloned from the roots of ‘Zhongmiansuo 12’ through the in silico cloning and RT-PCR,named as GhWRKY44(Genebank:KJ801807).Sequence analysis showed that the ORF of GhWRKY44 was 1 197 bp,encoding 398 amino acids.GhWRKY44 contained two conserved WRKY domains and one C2H2 zinc finger structure,and belonged to group I of cotton WRKY transcription factor.Phylogenetic analysis showed that GhWRKY44 closed to AtWRKY44.The GhWRKY44 expression was detected by Real-time Quantitative PCR(qRT-PCR) and the results showed that GhWRKY44 gene was predominantly expressed in resistant variety.With the duration of the treatment of Fov,the expression of GhWRKY44 increased firstly and decreased,then increased again.When the treatment time was over 3 hours,the level of GhWRKY44 expression reached the maximum in the resistant variety.However,the relative expression of GhWRKY44 was significantly lower and the response was later in the susceptible variety than that in resistant variety and the level of GhWRKY44 expression reached the maximum when the treatment time was over 6 hours.Both salicylic acid and jasmonic acid could induce the expression of GhWRKY44.After the treatment of salicylic acid,the level of GhWRKY44 expression increased rapidly and maintained at a high level.However,the expression of GhWRKY44 firstly increased and then decreased when induced by jasmonic acid.Compared with salicylic acid,the induced expression levels of GhWRKY44 were significantly lower than that of under jasmonic acid.The results demonstrated that GhWRKY44 may be response to pathogen and hormone stress in cotton.