MdWRKY40介导提高苹果与拟南芥对轮纹病菌的免疫抗性

doi:10.3864/j.issn.0578-1752.2018.21.005

摘要/Abstract

摘要：

【目的】从‘富士’苹果中克隆MdWRKY40,研究其在水杨酸（SA）诱导条件下的表达模式及在苹果轮纹病抗病信号通路中的作用,为进一步揭示苹果的抗病机制提供理论依据。【方法】以‘富士’苹果为试材,克隆MdWRKY40的全长CDS序列,对其进行生物信息学分析,采用荧光定量PCR（qRT-PCR）分析其在苹果各组织中的表达水平,及对非生物胁迫SA的响应;研究外源SA处理对苹果叶片接种轮纹病菌（Botryosphaeria dothidea）的影响,并利用qRT-PCR检测病程相关蛋白基因的表达;将MdWRKY40在拟南芥中进行异源表达,对稳定表达的拟南芥幼苗叶片进行接菌处理,观察叶片发病程度及发病叶片数量,并采用qRT-PCR分析病程相关基因的表达;测量拟南芥幼苗的根系长度,并利用qRT-PCR检测生长素相关基因的表达。【结果】MdWRKY40包含长为858 bp完整的开放阅读框,编码286个氨基酸,预测其分子量为32.088 kD,等电点为8.15。系统进化树分析表明,MdWRKY40与白梨PbWRKY40序列相似性最高,亲缘关系最近,与拟南芥AtWRKY40在不同的分支上,亲缘关系较远,利用DANMAN软件进行MdWRKY40与AtWRKY40的多序列比对分析发现,MdWRKY40蛋白与AtWRKY40蛋白虽然都含有一个WRKYGQK保守结构域,但相似度仅为29.78%。qRT-PCR分析表明,MdWRKY40在根中的表达水平最高,在叶中的表达水平最低,并且在根、茎、叶中,SA均诱导了MdWRKY40的表达,且均呈现先升高后降低的趋势,在6 h时表达量最高;外源SA处理提高了苹果叶片对轮纹病菌的抗性,未处理的叶片发病率达92.59%,SA处理后发病率降至79.26%,并显著提高了病程相关蛋白基因MdPR2、MdPR5的表达量。与野生型相比,在拟南芥中异源过量表达MdWRKY40显著提高了拟南芥叶片对轮纹病菌的抗性,野生型拟南芥发病率达77.5%,而两个转基因拟南芥株系发病率仅为21.5%和17.4%,并显著提高了病程相关基因PR1、PR3、PR4的表达。过表达MdWRKY40的拟南芥植株根系生长受到抑制,培养7 d后转基因拟南芥主根长度分别是野生型拟南芥的39.9%和43.1%,培养10 d后主根长度分别是野生型拟南芥的58.5%和55.4%。基因表达结果显示,生长素合成相关基因AtTAA1和生长素运输相关基因AtPIN1、AtPIN2的表达水平在MdWRKY40过表达株系中显著低于野生型。【结论】MdWRKY40表达受SA和苹果轮纹病菌侵染诱导;MdWRKY40是苹果中重要的轮纹病抗病基因,该基因过表达显著提高对轮纹病菌的抗性;MdWRKY40具有调控植物根系生长发育的功能,可能通过下调生长素运输相关基因的表达影响植物根系生长发育。

关键词: 苹果, MdWRKY40, 水杨酸, 苹果轮纹病菌, 免疫抗性, 根系生长

Abstract:

【Objective】The objective of this study is to clone MdWRKY40 from ‘Fuji’ apple, research its expression pattern under salicylic acid (SA)-induced conditions and its role in the disease resistance pathway of Botryosphaeria dothidea, and to provide a theoretical basis for further revealing the disease resistance mechanism of apple. 【Method】 The full-length CDS sequence of MdWRKY40 was cloned from ‘Fuji’ apple, and its bioinformatics analysis was carried out. Fluorescence quantitative PCR (qRT-PCR) was used to analyze the expression level in different apple tissues and the response to abiotic stress SA, to study the effect of exogenous SA treatment on apple leaves inoculated with pathogenic fungi B. dothidea, and to detect the expression of pathogenesis-related protein gene by qRT-PCR. MdWRKY40 was expressed heterologous in Arabidopsis thaliana, and the stably expressed A. thaliana seedlings were treated with B. dothidea to observe the degree of disease and the number of infected leaves. The expression of disease-associated genes was analyzed by qRT-PCR. The root length of A. thaliana seedlings was measured and the expression of auxin-related genes was detected by qRT-PCR. 【Result】 MdWRKY40 contains a complete open reading frame of 858 bp in length and encodes 286 amino acids. The predicted molecular weight is 32.088 kD and the isoelectric point is 8.15. Phylogenetic tree analysis showed that MdWRKY40 has the highest similarity with the PbWRKY40 sequence, and its genetic relationship is closest. MdWRKY40 and AtWRKY40 locate in different branches, and its genetic relationship is far from that of AtWRKY40. The multiple sequence alignment analysis of MdWRKY40 and AtWRKY40 by using DANMAN software revealed that both MdWRKY40 protein and AtWRKY protein contain a WRKYGQK conserved domain, but similarity is only 29.78%. qRT-PCR analysis showed that the expression level of MdWRKY40 was the highest in root and lowest in leaf. SA induced MdWRKY40 expression in root, stem and leaf, and the expression all increased first and then decreased, reached the highest level at 6 h. Exogenous SA enhanced the resistance of apple leaves to B. dothidea, the incidence of untreated leaves reached 92.59%, and the incidence after SA treatment decreased to 79.26%, and significantly increased the expression of disease-associated protein genes MdPR2 and MdPR5. Compared with the wild type, the overexpression of MdWRKY40 in A. thaliana significantly increased the resistance of A. thaliana leaves to B. dothidea. The incidence of wild type A. thaliana reached 77.5%, while the incidence of two transgenic A. thaliana lines was only 21.5% and 17.4%, and significantly increased the expression of PR1, PR3, and PR4 genes associated with disease progression. The root growth of A. thaliana plants with overexpression of MdWRKY40 was inhibited. After 7 days of culture, the length of main root of transgenic A. thaliana was 39.9% and 43.1% respectively of that of wild type A. thaliana. After 10 days of culture, the length of main root of transgenic A. thaliana was 58.5% and 55.4% respectively of that of wild type A. thaliana. The expression level of the auxin synthesis-related gene AtTAA1 and auxin transport-related genes AtPIN1 and AtPIN2 was significantly lower in the MdWRKY40 overexpression lines than in the wild type.【Conclusion】The expression of MdWRKY40 was induced by the infection of SA and the pathogenic fungi B. dothidea. MdWRKY40 is an important disease resistance gene in apple. The overexpression of MdWRKY40 significantly increased the resistance to B. dothidea. MdWRKY40 has the function of regulating the growth and development of plant roots, which may affect the growth and development of plant roots by down-regulating the expression of auxin transport-related genes.

Key words: apple, MdWRKY40, salicylic acid (SA), Botryosphaeria dothidea, immune resistance, root growth

周茜茜,邱化荣,何晓文,王宪璞,刘秀霞,李保华,吴树敬,陈学森. MdWRKY40介导提高苹果与拟南芥对轮纹病菌的免疫抗性[J]. 中国农业科学, 2018, 51(21): 4052-4064.

QianQian ZHOU,HuaRong QIU,XiaoWen HE,XianPu WANG,XiuXia LIU,BaoHua LI,ShuJing WU,XueSen CHEN. MdWRKY40 Mediated Improvement of the Immune Resistance of Apple and Arabidopsis thaliana to Botryosphaeria dothidea[J]. Scientia Agricultura Sinica, 2018, 51(21): 4052-4064.

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基因Gene	正向引物序列 Forward primer sequence (5′-3′)	反向引物序列Reverse primer sequence (5′-3′)
MdWRKY40	GAGAATGCAACCCTAAGATTCC	ATATGCATTTGGTGGTGGTG
MdPR2	ACTCACAGTCACCATCCTCAAC	AATCGAACATCAGCTGAATAGG
MdPR5	CTCACCTTGGCCATCCTCTT	TTGGATGCTAGTTCGAAGC
AtPR1	TCATGGCTAAGTTTGCTTCC	AATACACACGATTTAGCACC
AtPR3	ACGGAAGAGGACCAATGCAA	GAGCAGTCATCCAGAACCAAATC
AtPR4	ACCACCGCGGACTACTGTTC	ACCACCGCGGACTACTGTTC
AtPIN1	CGGTGGGAACAACATAAGCA	CACACTTGTTGGTGGCATCAC
AtPIN2	CCGTGGGGCTAAGCTTCTCATCT	AGCTTTCCGTCGTCTCCTATCTCC
AtTAA1	GATGAAGAATCGGTGGGAGAAGC	CGTCCCTAGCCACGCAAACGCAGG