CN112314613A - 血根碱激发水稻抗性及其抗稻瘟病的应用 - Google Patents
血根碱激发水稻抗性及其抗稻瘟病的应用 Download PDFInfo
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Abstract
本发明提供了一种化合物血根碱及其抗稻瘟病功能的应用，该化合物存在于罂粟科植物血根草的根茎部，是具有抗菌、消炎、抗氧化性能的天然生物碱，同时还具抑制肿瘤细胞增殖和促凋亡作用。本发明借助体外添加血根碱化合物检测其对稻瘟病菌菌丝生长、孢子萌发、附着胞发育和致病的影响，结果显示，外源添加一定浓度血根碱能有效激活水稻抗病相关基因的表达，从而显著抑制稻瘟病菌的致病力。本发明提出了血根碱对稻瘟病菌致病的抑制作用，表明该化合物可用于防治稻瘟病。
Description
技术领域
本发明属于生物技术领域，具体涉及外源添加一种化合物血根碱在水稻抗稻瘟病中的应用。
背景技术
病原真菌引起的作物病害是全球粮食生产及食品安全的严重威胁之一。真菌病害是植物病害中最大的一类，约占植物病害的70～80%，许多分布广、危害重的植物病害都是真菌引起的。水稻是我国最重要的粮食作物，全国近60%的人口以稻米为主食，可见水稻的生产安全涉及到我国的国计民生。稻瘟病菌(Magnaporthe oryzae)引起的稻瘟病是全球水稻生产上的毁灭性病害之一，在水稻整个发育时期内均可发生，其在适温高湿的外界条件下利于发病，极易爆发成灾，每年平均造成10%~30%的产量损失，严重威胁全球粮食生产安全。此外，稻瘟病菌除危害水稻外，还可以引起其他禾本科植物发病，如稷(Eleusine coracana)、大麦(Hordeum vulgare)和小麦(Triticum aestivum)。生产上防治稻瘟病的主要办法是选用对区域稻瘟病菌群体抗病的水稻品种，同时还要辅以每年若干次的化学农药防治。但是，稻瘟病菌田间小种多且易产生变异，单一的化学农药频繁使用极易导致病菌产生耐药性。因此，迫切需要研发新的杀菌剂防治稻瘟病。
植物可以通过多种方式来抵御病原菌的侵染，如活性氧爆发、植物抗毒素的合成或超敏反应等。血根碱是一种含氮的次生代谢产物，也是一种有效的植物抗毒素，主要存在于多种植物（如血根草的根部、罂粟科植物的种子等）的提取物中，具有广泛的药理作用。例如，因其具有抗菌和消炎作用，被广泛用于牙科产品中治疗牙龈发炎等；另外，因其具有类似于白屈菜红碱的结构，而被用于治疗奶牛的乳突炎。
先前的研究发现，血根碱可诱导人体恶性神经胶质瘤细胞产生活性氧依赖的ERK1/ 2激活和自噬细胞死亡。另外，血根碱还可通过抑制AKT / mTOR和NF-kB（核因子-kB）信号途径来阻断上皮性卵巢癌的发展。也有研究发现，血根碱可显著抑制白色念珠菌的生物膜形成、粘附和菌丝伸长，同时可以通过破坏膜的完整性，从而导致耐甲氧西林的病原体金黄色葡萄球菌的细胞裂解。近来在稻瘟病菌的研究中表明，血根碱可以抑制孢子萌发、促进活性氧的产生、改变细胞核的形态以及线粒体的膜电位，但其在稻瘟病菌侵染及水稻防御过程中的作用机制还不清楚，目前并没有关于血根碱在水稻抗稻瘟病应用的报道。利用生物技术手段，研发出以化合物血根碱为主要成分的杀菌剂，外源施用诱导水稻抗性反应并抑制稻瘟病菌生长，在实际应用中具有重要价值。因此，可通过利用血根碱改良或创制新型杀菌剂，达到防治稻瘟病的目的。
发明内容
本发明的目的在于提供一种化合物血根碱在防治水稻稻瘟病中的应用，可以利用该化合物进行杀菌剂研发，创造出抗稻瘟病的新型杀菌剂。
为实现上述目的，本发明采用以下技术方案：
一种血根碱在防治水稻稻瘟病中的应用，所述防治稻瘟病包括抑制稻瘟病生长或诱导水稻抗稻瘟病。
所述血根碱的结构如下：
所述应用的具体操作方法为：将血根碱外源添加到培养基或孢子悬浮液中处理稻瘟病菌。
外源添加一定浓度的血根碱到培养基中能够显著抑制稻瘟病菌的菌丝生长。同时，将血根碱与孢子悬浮液混合后有效抑制了稻瘟病菌在疏水玻片上附着胞形成率。定量PCR结果显示，添加血根碱激活了水稻抗病相关基因的表达；将添加血根碱的孢子液喷雾接种水稻叶片后，发现血根碱能有效抑制稻瘟病菌的致病力。
本发明具有以下有益效果：
本发明中外源添加血根碱可有效提高水稻抗病相关基因的表达，抑制稻瘟病菌的致病过程，显著提高了水稻抗稻瘟的能力，该化合物可用于创制新型杀菌剂。
附图说明
图1 血根碱的结构图。
图2 血根碱对稻瘟病菌菌丝生长的影响。
图3 血根碱对稻瘟病菌附着胞形成的影响。
图4 血根碱对水稻抗性相关基因表达的影响。
图5 血根碱对稻瘟病菌致病力的影响。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细说明。
实施例
本发明通过外源添加一定浓度血根碱至离体培养基中，观察血根碱对病菌在培养基上的菌丝生长的作用，同时添加一定浓度血根碱至稻瘟病菌野生型菌株Guy11的分生孢子悬浮液中，观察病菌在疏水玻片上的附着胞形成、致病过程以及水稻抗性反应基因表达的影响，再通过接种含有血根碱的稻瘟病菌分生孢子悬浮液测定稻瘟病菌致病能力。
具体包括如下步骤：
外源添加血根碱对稻瘟病菌菌丝生长的作用：将血根碱用甲醇溶解至母液浓度为3.8mg/mL，加入到基本培养基中，配制得到含终浓度2.0 μM 、4.0 μM和8.0 μM血根碱的基本培养基，所述基本培养基成分如下：硝酸钠6 g、氯化钾0.52 g、七水硫酸镁0.152 g、磷酸二氢钾1.52 g、维生素Ｂ1 0.01 g、1000×微量元素溶液1 mL、葡萄糖10 g、琼脂粉15 g，溶解于1000 mL超纯水；再以加入等量甲醇或不加任何物质的培养基作对照。用打孔器切取稻瘟病菌菌落边缘上的菌丝块，接种于上述培养基的平板中央，28°C黑暗培养10 d后测量菌落直径并拍照。试验重复3次，每次设3个重复，结果取平均值。结果如图2所示，与对照相比，稻瘟病菌在添加了4.0 μM和8.0 μM血根碱的平板上菌丝生长显著受到抑制。
外源添加血根碱对稻瘟病菌附着胞形成的影响：收集稻瘟病菌野生型菌株Guy11的分生孢子悬浮液，调整浓度至5×104个/mL，分别添加不同浓度的血根碱后（0.5 μM和1.0μM），吸取20 μL滴至疏水盖玻片上于28°C黑暗诱导附着胞形成，并在4、8、12、24 h进行附着胞形成率统计分析。结果如图3，血根碱显著抑制了稻瘟病菌的附着胞形成。
血根碱对水稻抗性相关基因表达的影响：在稻瘟病菌野生型菌株Guy11分生孢子悬浮液（浓度5×104个/mL）中分别添加1 %无菌水、1 %甲醇和8.0 µM血根碱（稀释比例为：添加77 µL血根碱母液至100 mL无菌水中）后，对水稻进行喷雾接种，48 h后提取水稻叶片RNA，反转录成cDNA，以OsACTIN作为内参基因，检测水稻中抗性相关基因OsPR1A、OsPR1B、OsAOS2、OsPBZ的表达水平（引物序列见表1），使用SYBR Premix ExTaq（Perfect RealTime）试剂盒（Takara, Dalian）试剂盒在ABI 7500 Fast Real-Time System进行实时荧光定量PCR。反应程序为：95 °C 30 s，95 °C 5 s，60 °C 34 s，40个循环，95 °C 15 s，60 °C1 min，95 °C 15 s。试验重复三次，每次设三个重复。结果如图4显示，相比添加水或甲醇的对照，外源加入血根碱显著提高了上述抗性基因的表达水平。这说明血根碱可激活水稻抗性相关基因的表达。
表1 引物序列
血根碱对稻瘟病菌致病力的影响：在稻瘟病菌分生孢子悬浮液（浓度5×104个/mL）分别添加终浓度1.5、2.5、4.0、8.0、12.0、16.0、20.0 µM血根碱后，对水稻进行喷雾接种，6天后观察结果，如图5所示，以不添加血根碱作对照，加入血根碱后稻瘟病菌病斑数量显著减少，病斑扩展能力显著受限，表明血根碱显著抑制了稻瘟病菌的致病过程。
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。
SEQUENCE LISTING
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Claims (4)
1.一种血根碱在防治水稻稻瘟病中的应用。
2.根据权利要求1所述的应用，其特征在于，所述防治稻瘟病包括抑制稻瘟病生长或诱导水稻抗稻瘟病。
3.根据权利要求1所述的应用，其特征在于，所述血根碱的结构如下：
4.根据权利要求1或2所述的应用，其特征在于，将血根碱外源添加到培养基或孢子悬浮液中处理稻瘟病菌。
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|---|---|---|---|---|
| CN101240346A (zh) * | 2008-03-13 | 2008-08-13 | 云南农业大学 | 一种分析水稻抗稻瘟病基因的方法 |
| CN101352180A (zh) * | 2008-09-12 | 2009-01-28 | 中国农业大学 | 用于抑制植物病原菌的博落回生物碱及其制备方法 |
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| CN101352180A (zh) * | 2008-09-12 | 2009-01-28 | 中国农业大学 | 用于抑制植物病原菌的博落回生物碱及其制备方法 |
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| GULDBRANDSEN, NIELS ET.AL: "" Screening of panamanian plant extracts for pesticidal properties and HPLC-based identification of active compounds"", 《 SCIENTIA PHARMACEUTICA 》 * |
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