中国农业科学

• 植物保护 • 上一篇    

转CiNPR4基因柑橘抗溃疡病的机制解析

张婧芸,刘语诺,王兆昊,彭爱红,陈善春,何永睿   

  1. 西南大学柑桔研究所,重庆 400712
  • 发布日期:2021-05-12

Analysis of resistance mechanism of CiNPR4 transgenic plants to citrus canker

ZHANG Jingyun, LIU Yunuo, WANG Zhaohao, PENG Aihong, CHEN Shanchun, HE Yongrui   

  1. Citrus Research Institute, Southwest University, Chongqing 400712
  • Online:2021-05-12

摘要: 【目的】诱导抗性在植物防御病原菌的感染过程中起着重要作用。病程相关基因非表达子1(nonexpressor of pathogenesis-related gene 1,NPR1)是水杨酸(salicylic acid,SA)介导的系统获得性抗性信号转导途径中一个重要的转录因子,在调节植物的抗病方面发挥着关键性的作用。研究耐黄龙病的‘Jackson’葡萄柚(Citrus paradisi)中的NPR1-like基因CiNPR4对柑橘溃疡病的抗性,解析过表达CiNPR4的转基因晚锦橙(C. sinensis)抗柑橘溃疡病的机理。【方法】选取黄龙病抗性增强的转CiNPR4基因柑橘,采取完全成熟的叶片利用针刺法进行柑橘溃疡病的离体抗性评价分析;在此基础上,对溃疡病抗性增强的转CiNPR4基因植株利用针刺法离体接种溃疡病菌(Xanthomonas citri pv. citri,Xcc),统计接种Xcc后0、1、3、5、7和9 d的细菌数量;利用注射法对溃疡病抗性增强的转CiNPR4基因植株叶片接种Xcc,接种后0、3和5 d收集接种部位的叶片,测定叶片中SA和茉莉酸(jasmonic acid,JA)含量;同时,收集接种Xcc后0、3和5 d接种部位的叶片,利用实时荧光定量 PCR分析SA和JA分别介导的防御反应相关基因CsPR1和CsPDF1.2在Xcc诱导下表达水平的变化;根据CiNPR4蛋白与TGA转录因子相互作用网络预测CiNPR4互作蛋白为Ciclev10005080m和Ciclev10001081m,以甜橙为参考基因组,将这两个基因在https://www.citrusgenomedb.org/网站上进行blastx分析,预测CiNPR4在甜橙基因组中互作的候选蛋白,克隆CiNPR4和候选蛋白基因的cDNA,利用同源重组法分别构建诱饵载体和猎物载体,并将诱饵质粒和猎物质粒共转入酵母菌株Y2HGold中,进行点对点酵母双杂交分析。【结果】CiNPR4的超量表达减轻了转基因柑橘叶片上溃疡病的症状,柑橘溃疡病抗性增强的转CiNPR4基因植株叶片上Xcc的生长比较缓慢,在接种Xcc后9 d,转基因植株含有显著低于野生型(wild type,WT)晚锦橙的Xcc数量;Xcc诱导0 d,溃疡病抗性增强的转CiNPR4基因植株含有与WT植株无差异的SA含量,随着Xcc诱导时间的延长,转CiNPR4基因植株中SA的含量显著增加;而Xcc处理0 d时,WT植株含有显著高于转CiNPR4基因植株的JA含量,随后,JA含量在转CiNPR4基因植株中逐步上升,Xcc处理5 d时,达到显著高于WT植株的水平;而WT植株在整个处理期间SA和JA的含量基本保持不变;溃疡病抗性增强的转基因植株受Xcc诱导3 d时CsPR1的表达水平迅速上升,达到与WT植株差异显著的水平,而CsPDF1.2的表达水平在Xcc诱导5 d时上升;WT植株受Xcc诱导后CsPR1的表达水平没有发生显著变化,而CsPDF1.2的表达水平在Xcc诱导5 d时上升,且显著高于转CiNPR4基因植株中CsPDF1.2的表达水平;酵母双杂交分析证实,CiNPR4与甜橙基因组中的CsTGA2转录因子互作。【结论】CiNPR4与CsTGA2转录因子互作,通过促进SA而抑制JA介导的防御反应,调控转基因柑橘对溃疡病的抗性。

关键词: CiNPR4, 柑橘溃疡病, 水杨酸, 茉莉酸, TGA转录因子

Abstract: 【Objective】Induced resistance is an important part of plant defense response against infection of pathogens. Pathogenesis-related gene 1 (NPR1) is an important transcription factor in salicylic acid (SA)-mediated signal transduction of systemic acquired resistance and plays a key role in regulating plant disease resistance. CiNPR4, a NPR1-like gene, came from Huanglongbing-tolerant ‘Jackson’ grapefruit (Citrus paradisi). The objective of this study is to evaluate the resistance of CiNPR4 to citrus canker and preliminarily explore the resistance mechanism of CiNPR4 transgenic plants to citrus canker.【Method】The fully mature leaves from Huanglongbing-resistant CiNPR4 transgenic citrus plants were selected to challenge the Xanthomonas citri pv. citri (Xcc) pathogen via in vitro pin-prick inoculation. On this basis, the CiNPR4 transgenic plants with enhanced resistance to citrus canker were inoculated with Xcc in vitro by the pin-prick method and then counted the number of Xcc at 0, 1, 3, 5, 7, and 9 d after inoculation. The leaves of CiNPR4 transgenic plants with enhanced resistance to citrus canker were inoculated with Xcc by injection and collected at 0, 3, and 5 d after inoculation. The contents of SA and jasmonic acid (JA) in the leaves were measured, meanwhile, the expression level of defense-related genes CsPR1 and CsPDF1.2 mediated by SA and JA, respectively, were analyzed by quantitative real-time PCR. According to the interaction network of CiNPR4 protein and TGA transcription factor, it is predicted that Ciclev10005080m and Ciclev10001081m can interact with CiNPR4, respectively, these two genes were analyzed by blastx on the website https://www.citrusgenomedb.org/ to predict the candidate proteins that interact with CiNPR4 in the sweet orange genome. Then, the cDNA sequences of CiNPR4 and candidate protein genes were cloned, and inserted into the pGBKT7 and pGADT7 plasmid to construct the bait and prey vectors by homologous recombination method, respectively. After that, the bait and prey plasmids were co-transformed into yeast strain Y2HGold for point-to-point yeast two-hybridization analysis.【Result】The disease resistance evaluation in vitro showed that the CiNPR4 transgenic plants displayed reduced symptoms of citrus canker. Xcc grew slowly in the leaves of CiNPR4 transgenic plants with enhanced resistance to citrus canker during the entire observation period, and had significantly lower Xcc cells than wild-type (WT) plants at 9 d after inoculation. The SA contents among the transgenic citrus lines and WT plants were not significantly different at 0 d. However, with the prolongation of Xcc induction time, the SA content in CiNPR4 transgenic plants significantly increased. The JA content in WT plants was significantly higher than that in CiNPR4 transgenic plants at 0 d after inoculation, after that, the JA content in CiNPR4 transgenic plants gradually increased and was significantly higher than that in WT plants at 5 d after Xcc induction. However, the SA and JA contents in WT plants were not significantly changed during the entire induction period. The expression level of CsPR1 was significantly increased in the transgenic plants with increased canker resistance than that in WT plants after Xcc induction for 3 d, and the expression level of CsPDF1.2 in the above-mentioned transgenic plants increased at 5 d after inoculation. However, WT plants exhibited no significantly different expression of CsPR1 after Xcc induction, but had an increased expression level of CsPDF1.2 at 5 d after inoculation, which was significantly higher in WT plants than that in the CiNPR4 plants. The interaction between CiNPR4 and CsTGA2 was confirmed by the yeast two-hybrid analysis.【Conclusion】These results indicated that CiNPR4 interacts with CsTGA2 to regulate the resistance of transgenic plants to citrus canker by promoting SA- and inhibiting JA-mediated defense response.

Key words: CiNPR4, citrus canker, salicylic acid, jasmonic acid, TGA transcription factor