番茄ShARPC5抗白粉病功能分析

doi:10.3864/j.issn.0578-1752.2020.01.006

摘要/Abstract

摘要：

【目的】白粉病（powdery mildew）是番茄生产上的重要病害,严重影响番茄的产量。番茄基因组测序工作的完成为抗病基因挖掘提供了重要的信息资源。ARP2/3（actin-related protein 2 and 3）复合体是肌动蛋白微丝骨架动力学的主要调控因子,能够参与包括响应外界胁迫等多种细胞学过程。本研究通过对番茄ARPC5（actin-related protein C5）进行克隆和抗病功能验证,为番茄基因组信息完善、抗病机制解析和分子育种等方面打下基础。【方法】从番茄LA1777（Solanum habrochaites）cDNA中PCR扩增ShARPC5,使用DNAMAN 6.0进行多序列比对;MEGA 6.0构建系统发育树;应用在线工具ProtComp v. 9.0进行亚细胞定位预测。利用实时荧光定量PCR（qRT-PCR）比较接种白粉菌（Oidium neolycopersici,On-Lz）后高感品种Moneymaker（MM）和高抗品种LA1777中番茄ARPC5的表达特征,分析白粉菌侵染与ARPC5表达的相关性。应用病毒诱导的基因沉默（virus-induced gene silencing,VIGS）技术进一步验证该基因在番茄中的抗病功能,观察沉默株和野生型株系接种后表型变化,利用台盼蓝和DAB染色法检测植株产生过敏性坏死和H₂O₂的能力,并检测ShARPC5沉默后一些与植物抗病相关标记基因的表达变化。采用农杆菌浸花法遗传转化拟南芥过表达ShARPC5植株,观察转基因和野生型株系接种后表型变化,并统计单病斑分生孢子数。【结果】从番茄品种LA1777中克隆到ShARPC5,编码132个氨基酸残基,包含一个保守的P16-Arc结构域。与番茄MM-白粉菌亲和互作相比,非亲和互作的番茄品种LA1777在接种白粉菌后,ShARPC5显著上调表达,尤其在接种后18 h。在番茄上沉默ShARPC5能够增加植株对白粉菌On-Lz的敏感性,防卫反应基因PR1b1显著下调表达。组织学观察显示与对照植株相比,ShARPC5沉默植株接种后诱导产生过敏性坏死和活性氧减少。在烟草上瞬时过表达ShARPC5能够诱导产生坏死斑。相反,在拟南芥上过表达ShARPC5能够增加植物的抗病性。【结论】ShARPC5是番茄响应白粉菌侵染的重要基因,可减轻番茄白粉病的发病程度,在番茄抗白粉病机理研究方面具有较大的应用价值,可作为番茄抗白粉病分子育种的一个候选基因。

关键词: 番茄白粉病, 抗病性, 肌动蛋白微丝骨架, ARP2/3复合体, ARPC5

Abstract:

【Objective】Powdery mildew is an important disease in tomato production, and seriously affects the yield of tomato. Genome sequences of Solanum lycopersicum provide valuable information resources for disease-resistant gene searching. The actin- related protein 2 and 3 complex (ARP2/3 complex), a key regulator of actin cytoskeletal dynamics, has been linked to multiple cellular processes, including those associated with response to stress. In this study, tomato ARPC5, encoding a subunit protein of the ARP2/3 complex, was cloned and identified for disease resistance. The results will lay a foundation for upgrading tomato genomic information, further studying the resistance mechanism and molecular breeding.【Method】ShARPC5 was annotated from the genomic databases and cloned from tomato LA1777 (S. habrochaites). DNAMAN 6.0 was used for multiple sequence alignment and MEGA 6.0 was used for constructing phylogenetic tree. The prediction of protein subcellular localization was performed using ProtComp v. 9.0. The qRT-PCR was used to compare the expression of ARPC5 in the high-sensitive variety Moneymaker (MM) and high-resistant variety LA1777 induced by Oidium neolycopersici (On-Lz). The correlation between On-Lz infection and ARPC5 expression was analyzed. The function of ShARPC5 involving in tomato resistance to On-Lz was further verified by the technology of virus-induced gene silencing (VIGS). The phenotypic changes of ShARPC5-silenced and wild-type lines were observed. Hypersensitive response (HR) and H₂O₂ production were detected by trypan blue and DAB staining, respectively, and the expression of some marker genes related to plant disease resistance was assayed in ShARPC5-silenced plants. Additionally, genetic transformation of Arabidopsis thaliana was conducted by Agrobacterium-mediated floral-dip method. The phenotypic changes of transgenic and wild-type lines were observed, and the number of conidia per lesion was also counted.【Result】The S. habrochaites ARPC5 was identified and characterized. ShARPC5 encodes a 132-amino-acid protein possessing a conserved P16-Arc domain. Compared with the compatible interaction between tomato MM (Moneymaker) and On-Lz, the expression of ShARPC5 was significantly up-regulated in incompatible interaction between tomato LA1777 and On-Lz, especially at 18 hpi. Silencing of ShARPC5 in tomato could increase the susceptibility to the powdery mildew pathogen On-Lz. The expression of PR1b1, a maker gene related to signal regulation, was significantly down-regulated after silencing of ShARPC5. The histological observation showed that the induction of hypersensitive cell death and the generation of reactive oxygen were reduced in silenced-ShARPC5 tomato plants compared with wild types. Transient over-expression of ShARPC5 in tobacco could rapidly produce necrotic spots. Conversely, over-expression of ShARPC5 in A. thaliana, followed by inoculation with On-Lz, showed enhanced resistance.【Conclusion】ShARPC5 is an important gene which can reduce the incidence of tomato powdery mildew, and has a great application value in the mechanism study of tomato resistance to powdery mildew. At the same time, it can be used as a candidate gene for molecular breeding of tomato powdery mildew resistance.

Key words: tomato powdery mildew, resistance, actin cytoskeleton, ARP2/3 complex, ARPC5

冯婵婧,孙广正,王阳,马青. 番茄ShARPC5抗白粉病功能分析[J]. 中国农业科学, 2020, 53(1): 65-73.

ChanJing FENG,GuangZheng SUN,Yang WANG,Qing MA. Functional Analysis of Gene ShARPC5 Involved in Tomato Resistance to Powdery Mildew[J]. Scientia Agricultura Sinica, 2020, 53(1): 65-73.

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基因 Gene	引物序列Primer sequence
基因 Gene	正向引物Forward primer sequence (5′-3′)	反向引物Reverse primer sequence (5′-3′)
ShARPC5/SlARPC5	CGAAGGCATAATCACAAGA	CAGCAAGACAACGCAGTA
PR1b1	CATCCCGAGCACAAAAC	TGAAGTCACCACCACCCT
Glucanase A	CTTTTACTTGTTGGGCTTCT	ACTTCCTTTGAGGGCATT
Chitinase 3	ACGCCATCCCCTAAAGA	TGGACCCATCCCACATT
GAPDH (Control)	CTGGTGCTGACTTCGTTGTTG	GCTCTGGCTTGTATTCATTCTCG
AtARPC5	CGGAATGCTCAATGCTCT	CGGTCCCCAGTAGAAAGTC
UBQ10 (Control)	AGAAGTTCAATGTTTCGTTTCATGTAA	TTACGAATCCGAGGGAGCCATTG