番茄ShARPC5抗白粉病功能分析

【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-induc...

Functional Analysis of Gene ShARPC5 Involved in Tomato Resistance to Powdery Mildew

【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.