猕猴桃AcWRKY70基因序列克隆及其对溃疡病病原菌和激素处理表达模式分析

Cloning and Expression Analysis of AcWRKY70 Gene Sequence under Psa and Hormone Treatment in Kiwifruit

为探究猕猴桃AcWRKY70转录因子在不同抗病性品种中响应猕猴桃溃疡病胁迫的作用机制,以抗性病品种徐香和高感病品种红阳猕猴桃叶片cDNA为模板克隆AcWRKY70基因,并对红阳AcWRKY70基因序列特征进行分析,对编码蛋白进行系统进化分析及亚细胞定位预测。通过RT-qPCR分析AcWRKY70基因的组织特异性,不同抗性猕猴桃品种对丁香假单胞杆菌猕猴桃致病变种(Psa)和水杨酸(SA)、茉莉酸甲酯(MeJA)处理下表达模式的差异。结果显示,红阳AcWRKY70基因序列总长906 bp(GenBank登录号:MW881147),开放阅读框885 bp,编码294个氨基酸;含有典型的WRKYGQK保守结构域,锌指结构为C2-HC,属于WRKY家族第Ⅲ类组,亚细胞定位预测表明,其定位于细胞核,系统进化树分析表明,其与茶树亲缘关系最近。红阳和徐香AcWRKY70基因均在叶片中表达量最高。在Psa处理下,12 h徐香品种AcWRKY70基因相对表达量迅速上升至对照的80.58倍;而在红阳中48 h该基因相对表达水平上升至最高。在SA与Psa(SA+Psa)和MeJA与Psa(MeJA+Psa)共同处理下,12 h徐香AcWRKY70基因相对表达量达到最高;而红阳AcWRKY70基因分别在72,24 h表达量达到最大。AcWRKY70基因在猕猴桃抗病胁迫方面具有一定作用,不同抗性的猕猴桃品种中响应病原菌的抗性机制可能存在较大差异。

In order to explore the mechanism of AcWRKY70 transcription factor in response to kiwifruit canker stress in different resistant varieties,AcWRKY70 gene was cloned from the leaf cDNA of resistant variety Xuxiang and highly susceptible variety Hongyang kiwifruit.The sequence structure characteristics of Hongyang AcWRKY70 gene,subcellular localization and evolutionary relationship of Hongyang AcWRKY70 protein were analyzed.The expression patterns of AcWRKY70 gene,including the tissue expression and different expression in resistant kiwifruit varietie and susceptible variety under Pseudomonas syringae pv.Actinidiae(Psa),salicylic acid(SA)and methyl jasmonate(MeJA)treatment were analyzed by RT-qPCR.Results showed that the full length of the Hongyang AcWRKY70 gene was 906 bp(GenBank accession number was MW881147).AcWRKY70 gene contained 885 bp of open reading frame(ORF)and encoded 294 amino acids.AcWRKY70 protein had typical WRKYGQK domain and the zinc finger structure was C2-HC.It belonged to classⅢgroup of WRKY family and islocated in the nucleus.Phylogenetic tree analysis showed that it had the closest genetic relationship with tea plant.Both Hongyang and Xuxiang AcWRKY70 genes had the highest expression level in leaves.Under Psa treatment,the highest expression of AcWRKY70 gene reached at 12 h in Xuxiang,the expression of Hongyang reached the maximum at 48 h.Under the co-treatment of salicylic acid and Psa(SA+Psa),Methyl jasmonate and Psa(MeJA+Psa),the highest expression of AcWRKY70 gene reached at 12 h in Xuxiang.Under SA+Psa and MeJA+Psa treatment,the expression of Hongyang reached the maximum at 72,24 h,respectively.AcWRKY70 gene played a certain role in resistance stress of kiwifruit,and the response mechanism to pathogens in different resistance kiwifruit varieties may vary considerably.