CN106973944A - TALE蛋白AvrXa7在防治柑橘溃疡病害上的应用 - Google Patents

TALE蛋白AvrXa7在防治柑橘溃疡病害上的应用 Download PDF

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CN106973944A
CN106973944A CN201710319409.3A CN201710319409A CN106973944A CN 106973944 A CN106973944 A CN 106973944A CN 201710319409 A CN201710319409 A CN 201710319409A CN 106973944 A CN106973944 A CN 106973944A
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avrxa7
disease
citrus
vrxa7
tale
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邹华松
孙东玲
卓涛
范晓静
户勋
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Fujian Agriculture and Forestry University
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Fujian Agriculture and Forestry University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom

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  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Pest Control & Pesticides (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Agronomy & Crop Science (AREA)
  • Dentistry (AREA)
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  • Agricultural Chemicals And Associated Chemicals (AREA)
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Abstract

本发明提供TALE蛋白AvrXa7在防治柑橘溃疡病害上的应用，属生物技术领域，从稻黄单胞菌中分离的TALE蛋白AvrXa7通过干扰柑桔溃疡病菌pthA4对感病基因的激活，抑制溃疡病害的发生；在生防荧光假单胞菌Pf中表达AvrXa7，能够有效抑制溃疡症状的形成，AvrXa7是构建高效基因工程菌株的极佳基因资源，在柑橘溃疡病的生物防控中具有重要的应用价值。

Description

TALE蛋白AvrXa7在防治柑橘溃疡病害上的应用
技术领域
本专利属生物技术领域，涉及TALE蛋白AvrXa7在防治柑橘溃疡病害上的应用。AvrXa7来源于水稻白叶枯病菌，对柑橘溃疡病菌中的PthA4致病功能有抑制作用，干扰柑桔中感病基因的表达，从而有效控制柑橘溃疡病害发生。
背景技术
柑橘溃疡病是我国柑橘产区的一个重要细菌病害，为国内外检疫对象。引起溃疡病产生的病原菌是柑橘黄单菌（Xanthomonas citri subsp. citri），在所有已经测序得柑橘黄单胞菌中都含有4个avr/pthA基因家族成员pthA1、pthA2、pthA3和pthA4，其中pthA4是病原菌的主要致病基因，该基因突变后病原菌在柑橘寄主上的致病力完全丧失。在病害发生过程中，病原菌首先克服寄主植物表层结构，随后依赖pthA4激活柑橘中的感病基因表达，使得溃疡症状最终形成。从提高寄主抗性角度对柑橘溃疡病进行控制，除了选用基础抗性较强的柑橘品种，对pthA4的致病功能进行干扰也是一个有效的控制策略，主要的突破途径在于干扰pthA4对感病基因的激活。目前并未有利用水稻白叶枯病菌的TALE蛋白AvrXa7用于防治柑橘溃疡病的报道。
发明内容
本发明的目的在于提供水稻白叶枯病菌的TALE蛋白AvrXa7在柑橘溃疡病生物防治中应用。本发明利用水稻白叶枯病菌的AvrXa7干扰柑橘溃疡病菌的致病基因pthA4对柑橘感病基因的激活，从而达到防治柑橘溃疡病菌的目的。
本发明的优点在于：本发明的AvrXa7基因来源于引起水稻白叶枯病菌的稻黄单胞（Xanthomonas oryzae pv. oryzae）(Yang B, Zhu WG, Johnson LB and White FF.Thevirulence factor AvrXa7 of Xanthomonas oryzae pv. oryzae is a type IIIsecretion pathway-dependent nuclear-localized double-stranded DNA-bindingprotein. PNAS, 2000, 97:9807–9812.)，在基因序列上与柑橘溃疡病菌pthA4同源性很高，属同一个基因家族成员。在野生型柑橘溃疡病菌中异源表达AvrXa7，溃疡菌丧失了在柑橘上形成溃疡症状的能力。转录组学和Real-time PCR结果显示，与pthA4对应的感病基因在引入AvrXa7以后，不能被诱导表达，表明AvrXa7干扰了pthA4对柑橘感病基因的激活。将AvrXa7在荧光假单胞菌中表达，对溃疡病有显著的防治作用。相对柑橘溃疡病菌而言，AvrXa7虽然是来源于黄单胞属的不同种菌株，但可以干扰pthA4在致病过程中的功能，可用做高效生防菌株的分子改造，应用于柑橘溃疡病的生态物控制。
附图说明
图1、AvrXa7在柑橘溃疡病菌中抑制溃疡病害的发生。
图2、表达AvrXa7的荧光假单胞菌对柑橘溃疡的防治作用。
具体实施方式
实施例1
（1）在野生型柑橘溃疡病菌中表达AvrXa7
将avrXa7基因克隆到质粒pUFR034中，通过电转化方法进入野生型柑橘溃疡病菌Xac29-1中。在NB液体培养基中培养后，注射接种柑橘叶片。5天后观察溃疡症状的形成情况。接种野生型菌株的区域，一般3天后就可以看到溃疡症状出现，5天后注射接种区域的溃疡斑隆起；而表达AvrXa7的溃疡菌株接种后，观察不到溃疡症状的形成 （图1）。
（2）AvrXa7对柑橘溃疡病菌pthA4基因致病功能的干扰
柑橘植物接种携带AvrXa7的柑橘溃疡菌株以后，运用转录组学方法分析寄主植物的基因表达情况，发现一批与avrXa7和pthA4相关的基因，其中最为突出的是与pthA4对应的感病基因。接种野生型菌株后，与pthA4对应的感病基因诱导表达，表达水平增高100多倍，而接种表达AvrXa7的溃疡菌株后，感病基因没有诱导表达，表明AvrXa7干扰了柑橘溃疡病菌pthA4基因的致病功能。
（3）AvrXa7在柑橘溃疡病害生物防治中的应用
将AvrXa7转到生防荧光假单胞菌Pf中，借助假单胞菌的III型分泌系统，将AvrXa7分泌到寄主植物细胞。将野生型柑橘溃疡病菌和Pf在液体培养基中培养后，采用摩擦接种的方法在柑橘叶片制造伤口，用携带AvrXa7的假单胞菌液浸泡叶片30秒，然后放在室温下自然风干；2小时后，浸液接种柑橘溃疡病菌。以后每隔10天用携带AvrXa7的假单胞菌液处理一次叶片。1个月后观察溃疡病的发生情况。用水处理作为对照，1个月后，叶片出现典型的溃疡症状，在伤口的位置都会形成隆起的溃疡病斑。生防假单胞菌的伤口也会出现溃疡斑，但症状轻微，稍有凸起。携带AvrXa7的假单胞菌处理的叶片仅能看到叶片伤口的愈合，没有看到任何溃疡症状出现（图2）。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。

Claims (1)

1.TALE蛋白AvrXa7在防治柑橘溃疡病害上的应用。
CN201710319409.3A 2017-05-09 2017-05-09 TALE蛋白AvrXa7在防治柑橘溃疡病害上的应用 Pending CN106973944A (zh)

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Cited By (2)

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CN110791496A (zh) * 2018-07-17 2020-02-14 华中农业大学 水稻基因OsTPI1-1在水稻抗病性改良中的应用
CN115812716A (zh) * 2022-07-18 2023-03-21 广东省农业科学院果树研究所 一种增强柑橘对溃疡病抗性的免疫诱导剂及其应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110791496A (zh) * 2018-07-17 2020-02-14 华中农业大学 水稻基因OsTPI1-1在水稻抗病性改良中的应用
CN115812716A (zh) * 2022-07-18 2023-03-21 广东省农业科学院果树研究所 一种增强柑橘对溃疡病抗性的免疫诱导剂及其应用
CN115812716B (zh) * 2022-07-18 2024-06-07 广东省农业科学院果树研究所 一种增强柑橘对溃疡病抗性的免疫诱导剂及其应用

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Application publication date: 20170725