CmWRKY15-1通过水杨酸信号通路调控菊花白色锈病抗性

doi:10.3864/j.issn.0578-1752.2021.03.015

摘要/Abstract

摘要：

【目的】菊花白色锈病是菊花重要病害之一,严重影响其观赏品质。分析CmWRKY15-1在菊花抗白色锈病中的功能及防卫机制,为利用分子育种解决菊花产业中白色锈病病害问题提供借鉴。【方法】以菊花白色锈病抗病品种‘黄莺’为试验材料,构建CmWRKY15-1过表达载体与干扰载体,通过农杆菌介导法转化菊花,分别获得超表达与沉默植株株系,通过表型观察、病情指数统计及转基因植株内源SA含量变化、SA合成关键基因及病程相关基因的表达分析,探究CmWRKY15-1在水杨酸信号途径响应菊花白色锈病的功能。【结果】经遗传转化后获得超表达植株OE-9株系与沉默植株RNAi-4株系,接种后的表型鉴定和病情指数调查中RNAi-4株系病情指数为53.67,抗性反应表现为感病,而野生型植株接菌前后表型无变化,表明CmWRKY15-1的沉默降低了‘黄莺’对白色锈病的抗性。同时OE-9株系中内源水杨酸含量在接种前后与WT相比呈上升趋势,而RNAi-4株系中水杨酸含量降低,即CmWRKY15-1的过表达与沉默分别显著上调和下调内源SA含量,对SA的积累有正向调节作用。OE-9系、RNAi-4系和WT在接菌处理前后的SA合成基因表达情况分析结果表明,在接种处理后,OE-9株系中ICS1的表达呈先增加后降低的趋势,其最高表达量为对照的3.8倍。而在RNAi-4株系中,ICS1在接种后出现降低,且一直保持较低水平。PAL在OE-9株系中的表达相较于对照增加,其最高表达量为对照的2.3倍,而在RNAi-4株系中,该基因的表达总体呈下降趋势,这与SA含量测定的变化结果基本一致,也进一步验证CmWRKY15-1通过SA介导的信号途径响应菊花白色锈病。此外,响应SA信号的病程相关基因表达分析结果显示,随着白色锈病病原菌处理时间的延长,OE-9株系中这些基因的表达除PR5外,均在40 h时出现峰值,PR5相对延后,在48 h时出现峰值。RNAi-4株系中除PR5外,其他基因的表达量显著低于WT、OE-9中的表达量。CmWRKY15-1的过表达提高了水杨酸信号途径中PR的转录水平,与此相反,CmWRKY15-1的沉默使这些基因表达下调。【结论】CmWRKY15-1对菊花抗白色锈病具有正调控作用,且通过水杨酸介导的信号途径调控抗病。

关键词: 菊花, CmWRKY15-1, 白色锈病, 水杨酸, 功能验证

Abstract:

【Objective】Chrysanthemum White Rust is one of the most important diseases of chrysanthemum, which seriously affects its ornamental quality. This study would provide theoretical reference for the molecular mechanism of Chrysanthemum White Rust through the preliminary analysis of the function of CmWRKY15-1.【Method】In this study, the overexpression and interference vector was constructed based on CmWRKY15-1 gene sequence, which were transformed into resistant cultivar Huangying by Agrobacterium mediated method. The function of CmWRKY15-1 gene in response to Chrysanthemum White Rust in salicylic acid signal pathway was explored by phenotype observation, disease index statistics, changes of endogenous SA content in transgenic plants, expression analysis of key genes of SA synthesis, pathogenesis-related genes and defense enzyme genes. 【Result】The OE-9 and the RNAi-4 strains were obtained after transformation. The disease index of RNAi-4 strain was 53.67 in phenotype identification and disease index investigation, and the resistant response was susceptible, but the phenotype of wild type plants had no change. Meanwhile, the content of endogenous Salicylic Acid (SA) in OE-9 lines increased, compared with WT after inoculation, while in RNAi-4 lines decreased. The overexpression and silencing of CmWRKY15-1 significantly changed SA content, which had a positive regulatory effect on SA accumulation. The expression of SA synthesis genes in OE-9, RNAi-4 and WT showed that the expression of ICS1 in OE-9 upregulated first and then down-regulated, and the highest expression was 3.8 times as much as that in WT. Whereas, the expression level of ICS1 in RNAi-4 decreased after inoculation and remained at a lower level. The PAL expression indicated an increasing trend, and the highest expression was 2.6 times as much as that of the control. PAL expression in RNAi-4 was down-regulated, basically consistent with the change of SA content, which further confirmed that CmWRKY15-1 was a novel regulator mediated by SA signal pathway in respond to Chrysanthemum White Rust. In addition, the analysis of the expression of disease resistance related genes in response to SA signal showed that the expression of these genes peaked at 40 h in OE-9. However, the PR5 was relatively delayed and peaked at 48 h, and their expression in RNAi-4 was significantly lower than that in WT and OE-9 except PR5. The overexpression of CmWRKY15-1 increased the transcription level of PR gene in SA signaling pathway. On the contrary, the silencing of CmWRKY15-1 reduced the expression of these genes.【Conclusion】Therefore, CmWRKY15-1 had a positive regulatory effect on resistance of Chrysanthemum White Rust, and it might respond to Chrysanthemum White Rust through the regulation of SA signal pathway.

Key words: chrysanthemum, CmWRKY15-1 gene, Chrysanthemum White Rust, SA, functional verification

毕蒙蒙,刘迪,高歌,祝朋芳,毛洪玉. CmWRKY15-1通过水杨酸信号通路调控菊花白色锈病抗性[J]. 中国农业科学, 2021, 54(3): 619-628.

BI MengMeng,LIU Di,GAO Ge,ZHU PengFang,MAO HongYu. CmWRKY15-1 Regulates Resistance of Chrysanthemum White Rust Through Salicylic Acid Signaling Pathway[J]. Scientia Agricultura Sinica, 2021, 54(3): 619-628.

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引物Primer	序列Sequence（5′-3′）
Npt Ⅱ F	GGCTATGACTGGGCACAACA
Npt Ⅱ R	GATACCGTAAAGCACGAGGAA
Actin-F	TCCGTTGCCCTGAGGTTCT
Actin-R	GATTTCCTTGCTCATCCTGTCA
CmWRKY15-1-F	TGGTGGCTGCATCACAT
CmWRKY15-1-R	GGAAGAATCAGTGCTAATACATTAA

引物 Primer	序列 Sequence（5′-3′）
ICS1-F	TCCCTACTGAAGAGGCACGG
ICS1-R	CCAACAGCGGGTTCACTCTC
PAL-F	ATGGCACCGAAGCAAGTCACAC
PAL-R	GATACCCGAGTAACCCTGGAGGAG
NPR1-F	TGTCGAGAAGGATGGAAAGCC
NPR1-R	GGAGGCACCCATCATCAACA
PR1-F	CTCAACCAAAAGGAATAGTCGG
PR1-R	CCCTGCCAGTTTACGCTGTA
PR2-F	GGCAATGGTGGTGTTGGAAC
PR2-R	CTTCCTCCGTCAGCAGAAGG
PR5-F	CCAATGGAGTTTAGCCCCGT
PR5-R	GTCCACAACTACCACGCTCA