CN110301454B - 一种油菜短肽BnPEP5的应用 - Google Patents
一种油菜短肽BnPEP5的应用 Download PDFInfo
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- CN110301454B CN110301454B CN201910518850.3A CN201910518850A CN110301454B CN 110301454 B CN110301454 B CN 110301454B CN 201910518850 A CN201910518850 A CN 201910518850A CN 110301454 B CN110301454 B CN 110301454B
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Abstract
本发明提供一个油菜（Brassica napus）短肽BnPEP5激发十字花科植物产生对核盘菌（Sclerotinia sclerotiorum）抗性和应用。本发明应用的油菜短肽BnPEP5由27个氨基酸组成，是全长蛋白的C端部分。本发明首次通过人工合成短肽BnPEP5，阐明了该短肽对核盘菌抗性的激发作用，并提供该短肽通过浸润和喷雾处理十字花科植物油菜和芥菜（Brassica juncea）激发其产生对核盘菌抗性、从而防控油菜和芥菜菌核病的应用。本发明提供的BnPEP5短肽是植物自身合成的产物，利用BnPEP5短肽激发免疫从而防治菌核病具有天然绿色、不污染环境、对人畜无害、不会诱导病原物产生抗药性等优点。此外，BnPEP5短肽易于人工合成，获取简便，抗病激发作用强烈，所需浓度低，是一种新型生态和环境友好型作物病害防控制剂。
Description
技术领域
本发明属生物技术领域，涉及一种油菜短肽BnPEP5的免疫激发功能及其在菌核病防控中的应用。
背景技术
1、植物免疫激发技术
植物免疫是植物通过受体识别病原物分子从而激活的防卫反应。植物免疫***有多个层次，其中第一层次为植物细胞表面的跨膜分子模式识别受体(patternrecognitionreceptors，PRRs)识别源自病原物及植物自身的保守的分子模式产生的免疫，称为模式触发性免疫(pattern-triggered immunity，PTI)。通俗地说，植物能感知病原物侵染等引起的对植物细胞完整性的破坏，并产生伤害相关分子模式(damage-associatedmolecularpatterns,DAMPs)，这些DAMPs通过PRR受体识别激发植物产生PTI免疫反应，包括活性氧迸发、激酶级联的活化、防卫相关基因的表达等，最后表现对病原物的抗性。PTI在防止非适应性微生物侵染植物的非寄主抗性(nonhost resistance)和限制适应性病原物侵染感病寄主植物的基础抗性(basal resistance)中起重要作用。DAMP激发的PTI免疫具有广谱、强烈、稳定等优点。此外，由于DAMP是植物自身的天然产物，因此利用DAMP激发免疫从而达到防治病害的目的是一种环境生态友好型绿色病害防控新方法。
2、菌核病防控技术
菌核病由核盘菌(Sclerotinia sclerotiorum)侵染所致。核盘菌是坏死营养型(necrotroph)病原真菌，寄主防卫极广，是油料作物以及蔬菜作物的主要病害。每年造成巨大经济损失。由于缺乏高抗品种，化学防治仍然是重要手段。由于一些农药存在生态污染、人畜毒害、以及易使病原物产生抗药性等问题，新型绿色防治制剂亟待研发。
发明内容
本发明的目的是提供一种油菜(Brassica napus)短肽BnPEP5激发十字花科植物产生对核盘菌(Sclerotinia sclerotiorum)抗性的应用。本发明应用的油菜短肽BnPEP5序列如SEQ ID NO：1所示，共由27个氨基酸组成，是全长蛋白(序列如SEQ ID NO：2所示)的C端部分。在本发明之前，短肽BnPEP5的功能没有任何公开报告。本发明首次通过人工合成短肽BnPEP5，阐明了该短肽对核盘菌抗性的激发作用，并提供该短肽通过处理十字花科植物激发其产生对核盘菌抗性、从而防控菌核病的应用。通过以下步骤实现：
(1)BnPEP5短肽的人工合成或生物表达获取
可以委托专业多肽合成公司合成。也可通过生物表达获取短肽BnPEP5：将BnPEP5相应核苷酸序列克隆入表达载体、转化至真核或原核微生物、微生物扩繁、提取和纯化目的短肽。
(2)BnPEP5短肽的植物处理
以合适浓度的短肽BnPEP5水溶液浸润或喷雾处理植物叶片，激发植物免疫。浸润和喷雾所需短肽BnPEP5水溶液浓度以及处理规模不同。浸润处理所需BnPEP5浓度较低，一般在100nM～200nM范围内，适合小规模植物处理，而喷雾处理所需BnPEP5浓度较高，一般在200nM～1000nM范围内，适合大规模植物处理。
(3)BnPEP5短肽激发植物产生对核盘菌的抗性
短肽BnPEP5处理后迅速激发植物产生对核盘菌的免疫反应。通过病原物接种分析可检测激发作用的强度。植物产生对核盘菌的免疫反应表现为短肽BnPEP5处理使接种后产生的坏死病斑比水对照处理时更小。从作用机制角度看，BnPEP5对该免疫反应的激发与其诱发活性氧的迅速大量积累有关。
本发明的优点：(1)本发明提供的BnPEP5短肽是植物自身合成的产物，利用BnPEP5短肽激发免疫从而防治菌核病具有天然绿色、不污染环境、对人畜无害、不会诱导病原物产生抗药性等优点。(2)DAMP激发植物免疫是最新理论研究成果。BnPEP5作为一种油菜DAMP应用于十字花科作物免疫激发和病害防控有充分的理论依据，也是理论指导实践应用的范例。(3)BnPEP5短肽只有27个氨基酸，易于人工合成，获取简便易行。(4)BnPEP5短肽的抗病性激发作用强烈，所需浓度低，因此病害防治成本较低。
附图说明
图1显示BnPEP5短肽水溶液浸润或喷雾油菜(Brassica napus)叶片激发油菜产生对核盘菌的抗性。以浓度为100nM的BnPEP5短肽水溶液通过无菌一次性针筒浸润油菜植株叶片(b)，或以浓度为200nM的BnPEP5短肽水溶液通过喷枪喷雾到油菜叶片(d)，1d后以核盘菌菌丝块接种叶片，图示为接种后2d的病斑大小。结果表明，与无菌双蒸水处理对照相比(a,无菌双蒸水针筒浸润；c,无菌双蒸水喷枪喷雾)，BnPEP5处理叶片形成的病斑明显更小。表明BnPEP5处理激发油菜产生了对核盘菌的抗性。
图2显示BnPEP5短肽激发油菜叶片迅速积累活性氧。浓度为100nM的BnPEP5短肽迅速诱发油菜叶片小圆片大量积累活性氧。无菌水处理作为对照。活性氧采用基于鲁米诺的方法通过化学发光检测仪进行检测。
图3显示BnPEP5短肽水溶液浸润或喷雾芥菜(Brassica juncea)叶片激发芥菜产生对核盘菌的抗性。以浓度为100nM的BnPEP5短肽水溶液通过无菌一次性针筒浸润芥菜植株叶片(b)，或以浓度为200nM的BnPEP5短肽水溶液通过喷枪喷雾到芥菜叶片(d)，1d后以核盘菌菌丝块接种叶片，图示为接种后2d的病斑大小。结果表明，与无菌双蒸水处理对照相比(a,无菌双蒸水针筒浸润；c,无菌双蒸水喷枪喷雾)，BnPEP5处理叶片形成的病斑明显更小。表明BnPEP5处理激发芥菜产生了对核盘菌的抗性。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1B nPEP5短肽在油菜(Brassica napus)抗菌核病中的应用
本发明实施例利用人工合成的BnPEP5短肽，通过浸润或喷雾法处理油菜，激发其产生对核盘菌的抗性，从而用于油菜菌核病的控制。主要步骤包括：
1)BnPEP5短肽的人工合成
本发明提供的BnPEP5短肽序列如SEQ ID NO：1所示，共由27个氨基酸组成，是全长蛋白(序列如SEQ ID NO：2所示)的C端部分。在本发明之前，短肽BnPEP5的功能没有任何公开报告。
本实施例委托专业多肽合成公司(强耀生物科技有限公司)合成BnPEP5短肽，用于油菜抗病激发和菌核病防控功能的分析和应用。
2)BnPEP5短肽对油菜的处理
浸润处理：以浓度为100nM的BnPEP5短肽水溶液通过无菌一次性针筒浸润油菜植株叶片；
喷雾处理：以浓度为200nM的BnPEP5短肽水溶液通过喷枪喷雾到油菜植株叶片。
3)BnPEP5短肽激发油菜产生对核盘菌的抗性
BnPEP5短肽水溶液浸润或喷雾油菜叶片均能激发油菜产生对核盘菌的抗性。核盘菌接种分析结果显示，以BnPEP5短肽(100nM)浸润或喷雾(200nM)处理1d后，核盘菌菌丝块接种叶片的病斑显著小于无菌双蒸水处理对照(图1)。表明BnPEP5浸润或喷雾处理均能迅速激发油菜产生对核盘菌的抗性。
活性氧定量检测分析结果显示，BnPEP5短肽(100nM)诱发油菜叶圆片迅速强烈积累活性氧(图2)。表明，BnPEP5短肽通过诱发活性氧迸发激发油菜产生对核盘菌的抗性。
实施例2BnPEP5短肽在芥菜(Brassica juncea)抗菌核病中的应用
本发明实施例利用人工合成的BnPEP5短肽，通过浸润或喷雾法处理芥菜，激发其产生对核盘菌的抗性，从而用于芥菜菌核病的控制。主要步骤包括：
1)BnPEP5短肽的人工合成
本发明提供的BnPEP5短肽序列如SEQ ID NO：1所示，共由27个氨基酸组成，是全长蛋白(序列如SEQ ID NO：2所示)的C端部分。本实施例委托专业多肽合成公司(强耀生物科技有限公司)合成BnPEP5短肽，用于芥菜抗病激发和菌核病防控功能的分析和应用。
2)BnPEP5短肽对芥菜的处理
BnPEP5短肽的植物处理方式包括浸润和喷雾。两者所需短肽BnPEP5水溶液浓度以及处理规模不同。浸润处理所需BnPEP5浓度较低，适合小规模植物处理，而喷雾处理所需BnPEP5浓度较高，适合大规模植物处理。
浸润处理：以浓度为100nM的BnPEP5短肽水溶液通过无菌一次性针筒浸润芥菜植株叶片；
喷雾处理：以浓度为200nM的BnPEP5短肽水溶液通过喷枪喷雾到芥菜植株叶片。
3)BnPEP5短肽激发芥菜产生对核盘菌的抗性
BnPEP5短肽水溶液浸润或喷雾芥菜叶片均能激发芥菜产生对核盘菌的抗性。核盘菌接种分析结果显示，以BnPEP5短肽(100nM)浸润或喷雾(200nM)处理1d后，核盘菌菌丝块接种叶片的病斑显著小于无菌双蒸水处理对照(图3)。表明BnPEP5浸润或喷雾处理均能迅速激发芥菜产生对核盘菌的抗性。
通过以上实施例，本领域技术人员可以确定，短肽BnPEP5可以有效激发十字花科植物产生对核盘菌(Sclerotinia sclerotiorum)抗性，应用于菌核病的防控。
序列表
<110> 浙江大学
<120> 一种油菜短肽BnPEP5的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> PRT
<213> 甘蓝型油菜(Brassica napus)
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Gly Lys Arg Gly Gly Val Asn Asn His Asp Met
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<210> 2
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<213> 甘蓝型油菜(Brassica napus)
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Claims (1)
1.一种油菜Brassica napus短肽BnPEP5激发十字花科作物产生对核盘菌Sclerotinia sclerotiorum抗性的应用，其特征在于，该油菜短肽BnPEP5氨基酸序列如SEQ ID NO：1所示，所述应用是将短肽BnPEP5水溶液浸润或喷雾处理十字花科作物叶片，激发植物免疫，所述十字花科作物为油菜、芥菜Brassica juncea。
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