猕猴桃AcNPR1基因克隆及抗病响应表达分析

Cloning of AcNPR1 Gene in Kiwifruit and Expression Analysis of Disease Resistance Response

为探究猕猴桃AcNPR1基因在抗病响应中的作用及其在不同抗性猕猴桃品种间抗性差异机制,以高感病品种‘红阳’和抗病品种‘徐香’猕猴桃叶片为材料克隆AcNPR1基因序列;利用ExPASy-ProtParam tool、MEGA7.0等对‘红阳’AcNPR1蛋白的理化性质进行分析、亚细胞定位进行预测、进化关系等进行分析;通过RT-qPCR分析AcNPR1基因在不同品种猕猴桃植株中的组织表达模式以及病原菌和水杨酸(Salcylicacid,SA)对其诱导表达模式。结果表明,AcNPR1基因开放阅读框1770bp(Genbank登录号MW881148),编码589个氨基酸,理论等电点6.27,具有典型的BTB/POZ结构域、ANK重复序列、NPR-like等NPR1蛋白保守结构域,AcNPR1蛋白预测定位于细胞核中;蛋白质的二级结构以α螺旋和无规卷曲为主;系统进化显示其与茶树的亲缘关系最为密切,相似性为83.94%。组织特异性分析表明AcNPR1基因在‘红阳’和‘徐香’品种叶片中的表达水平最高。在丁香假单胞杆菌猕猴桃致变种(Pseudomonas syringae pv.actinidiae,Psa)处理下,抗病品种‘徐香’AcNPR1基因的相对表达水平在处理0h后迅速增加,为对照的3.6倍;而高感品种‘红阳’在48h后显著增加,为对照的2.7倍。在SA+Psa处理下,‘徐香’AcNPR1基因相对表达水平在24h内达到最高水平,是对照的7.7倍;‘红阳’在72h后达到最大值,仅为对照的1.6倍。‘徐香’AcNPR1基因的抗病响应反应早于‘红阳’且更易受SA诱导表达。AcNPR1基因在猕猴桃抗病胁迫方面具有一定作用,在不同抗性的猕猴桃品种抗病机制中存在差异。

To explore the role of AcNPRI gene in disease resistance response and its tissue expression patterns and induced expression by pathogen and SAC Salicylic.acid)in kiwifruit.In this study,the AcNPRI Bene sequence was cloned from the leaves of highly susceptible"Hongyang and middle re sistant.Xuxiang".The physical and chemical properties,subcellular localiation and evolutionary re lationship of‘Hongyang'AcNPRI protein were.analyzed by ExPASY protparam tool and MEGA 7.0.The tissue expression pattern of AcNPR1 gene in kiwifruit plants and its induced expression pattern.by Psa(Pseudomonas syringae py.actinidiae),and SA were analyzed by RT qPCR.The results showed that the open reading frame of AcNPRI gene was 1770 bp(GenBank acession number MW881148).encoding 589 amino acids and a theoretical isoelectric point of 6.27,and had typical con-servative domains of NPR1 protein such as BTB/POZ domain.ANK repeat sequence and NPR1_like_C.The subcellular localization predietion results showed that AcNPR1 protein was localized in the nu-cleus.The secondary structure of protein was mainly composed of a helix and random coil Phyloge-netic evolution showed that it had the elosest genetie relationship with tea plant,and its similarity was 83.94%.Tissue specifie analysis showed that the expression level of AeNPRI Bene was the highest in the leaves of‘Hongyang'and‘Xuxiang’.Under the treatment of Pseudomonas syringae pv.actin-idiae(Psa).the relative expression level of AcNPR1 gene indisease resistant variety.Xuxiang'in-creased rapidly after 0 h of treatment,3.6 times of the control.However,the highly susceptible varie-ty"Hongyang"increased significantly after 48 hours,2.7 times of the control.Under SA+Psa treat-ment.the relative expression level of AeNPRI gene of.Xuxiang"reached the highest level within 24 hours,7.7 times of the control"Hongyang reached its maximum after 72 hours,1.6 times of the control.The disease resistance response of Xuxiang AcNPRI gene was earlier than that of"Hongyang"and was more easily induced by SA.AcNPRI gene plays an important role in kiwifruit disease resistance,and there are differences in disease resistance mechanism among different resistance ki-wifruit varieties.