棉花抗枯萎病相关转录因子基因GhERFB101的克隆与表达分析

摘要

为了探究ERF转录因子家族与棉花枯萎病抗性之间的关系，也为海岛抗枯萎病品种的选育工作提供新的基因资源，从Solexa高通量测序技术建立的棉花基因表达谱中筛选探针序列，通过电子克隆结合RT-PCR技术从高抗枯萎病的棉花品种中棉所12中克隆到一个新的 ERF-B1亚组转录因子基因，命名为 GhERFB101（ GenBank：KF850521）。序列分析表明，该基因开放阅读框738 bp，编码245个氨基酸，含有一个保守的AP2／ERF结构域，在进化上与拟南芥AtERF11的亲缘关系最近。实时荧光定量PCR分析显示，枯萎病菌诱导后，GhERFB101基因在抗病品种根中的表达量呈现下降趋势；而在感病品种中，该基因呈上调表达，随着病菌处理后时间延长，其表达量呈现先增加后降低的变化趋势，在病菌处理后12 h表达量达到最大，在48 h下降到最低。乙烯和茉莉酸诱导后，该基因表达量均呈现明显的先增加后降低的变化趋势，在乙烯处理后2h基因的表达量达到最大；茉莉酸则在处理后1h基因的表达量达到最大；而水杨酸诱导后基因的变化幅度不大；推测该基因可能通过茉莉酸、乙烯信号传导途径参与对枯萎病菌的防御反应。%In order to explore the relationship between ERF transcription factor family and cotton resistance to Fusarium wilt,and provide new gene resources for breeding work of sea island cotton resistance to Fusarium wilt. Before this study ,we constructed a digital expression profiling of cotton root tissues infected by Fusarium oxysporum f.sp.vasinfectum(Fov)in different time,then we found a query sequence in it ,through the method of silico cloning and RT-PCR, we cloned a new ERF-B1 subgroup transcription factor gene , named GhERFB101 ( GenBank:KF850521 ) from the roots of Zhongmiansuo 12 which was a high resistant cultivar to the infection of Fov.,sequence analysis indicated that the ORF of GhERFB101 was 738 bp encoding 245 amino acids and a conserved AP 2/ERF domain ,and closed to Arabidopsis AtERF11 in the phylogenetic relationship .Real-time RT-PCR analysis indicated that,the relative expression of GhERFB101 decreased in resistant cultivar after the treatment of Fov.while in the susceptible cultivar the gene expression was up regulation .With the treatment time going on ,the expression level in-creased first and decreased then .It reached the highest after 12 h and the lowest after 48 h.After the treatment of ethylene and jasmonic acid ,the expression also showed the same trend like before ,the expression level reached the maximum at 2 h after the treatment of ethylene ,and it reached the maximum at 1 h after the treatment of jasmonic acid.While the changes of salicylic acid treatment was not significant;we speculated that GhERFB101 may involve in jasmonic acid and ethylene signal pathway to defense Fov.