猕猴桃<i>AcWRKY70</i>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

doi:10.7668/hbnxb.20192987

摘要/Abstract

摘要：

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。

关键词: 猕猴桃, AcWRKY70, 表达分析, 猕猴桃溃疡病, 水杨酸, 茉莉酸甲酯

Abstract:

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 class Ⅲ group 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.

Key words: Kiwifruit, AcWRKY70, Expression analysis, Kiwifruit canker, Salicylic acid, Methyl jasmonate

曲东, 燕飞, 刘欣瑞, 康雪, 曾海涛, 张羽. 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[J]. 华北农学报, 2022, 37(5): 166-173. doi: 10.7668/hbnxb.20192987.

QU Dong, YAN Fei, LIU Xinrui, KANG Xue, ZENG Haitao, ZHANG Yu. Cloning and Expression Analysis of AcWRKY70 Gene Sequence under Psa and Hormone Treatment in Kiwifruit[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 166-173. doi: 10.7668/hbnxb.20192987.

http://www.hbnxb.net/CN/Y2022/V37/I5/166

图/表 7

图1 红阳AcWRKY70基因PCR产物(A)和 AcWRKY70转录因子的保守结构域(B) M.GL DNA Marker 2000 Ladder;1.红阳AcWRKY70基因PCR 扩增产物。

Fig.1 The PCR amplified product of AcWRKY70 gene(A)and the conserved domain of AcWRKY70 transcription factor(B) M.GL DNA Marker 2000 Ladder;1.PCR product of Hongyang AcWRKY70 gene.

图2 AcWRKY70 与不同物种WRKY 转录因子氨基酸序列多重比对分析红色框线.WRKYGQK保守结构域;红色箭头.C2-HC锌指结构域。

Fig.2 Multiple alignment of amino acid sequences of AcWRKY70 and WRKY transcription factors in different species Red box.The conserved domain of WRKYGQK;Red arrows.The zinc finger domain of C2-HC.

图3 红阳猕猴桃AcWRKY70蛋白系统进化树分析红阳猕猴桃AcWRKY70蛋白序列位于红框中。

Fig.3 Phylogenetic tree analysis of AcWRKY70 protein in Hongyang The AcWRKY70 protein sequence of kiwifruit is located in the red frame.

图4 AcWRKY70 蛋白磷酸化位点预测

Fig.4 The phosphorylation sites prediction of AcWRKY70 protein

图5 AcWRKY70蛋白三级结构预测

Fig.5 The tertiary structure prediction of AcWRKY70 protein

图6 徐香和红阳品种猕猴桃AcWRKY70基因在不同组织中表达模式不同小写字母代表不同处理存在显著差异(P<0.05)。图7同。

Fig.6 Expression difference of AcWRKY70 gene in different tissues of Xuxiang and Hongyang Different lowercase indicate significant difference in different treatments, P<0.05.The same as Fig.7.

图7 病原菌(A)、SA+Psa(B)和MeJA+Psa(C)处理下AcWRKY70基因的表达分析

Fig.7 Different expression of AcWRKY70 gene under Psa(A),SA+Psa(B)and MeJA+Psa(C)treatment

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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

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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

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