CN116406668A - 油菜素内酯在提高水稻樱花素含量上的应用 - Google Patents
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Abstract
本发明公开油菜素内酯在提高水稻樱花素含量上的应用。本发明首次发现BL可用于提高水稻樱花素含量，并对其机理进行推测，认为BL处理诱导樱花素合成酶OsNOMT的表达，从而提高樱花素的含量。本发明实施简单、效果显著，为提高水稻樱花素含量、改善水稻免疫能力提供强有力的参考。
Description
技术领域
本发明属于生物技术领域，尤其是涉及油菜素内酯在提高水稻樱花素含量上的应用。
背景技术
油菜素甾醇(brassinosteroids，BRs)是植物内源合成的一类重要的生长促进型甾醇类激素。BR途径除了参与植物生长发育和生理相关过程，在寄主抵御细菌、真菌及病毒等病原物侵染过程中也发挥重要作用。前人研究表明，BR途径在调控植物次生代谢物质的代谢合成方面也发挥重要的作用，特别是类黄酮化合物。类黄酮化合物是由C6-C3-C6构成的一系列化合物的总称，可分为黄酮、黄酮醇、黄烷酮、异黄酮和花青素等。植物类黄酮化合物除了调节植物本身生命活动方面以外，在医药方面,类黄酮化合物具有较强的生理活性,有止咳平喘、抗菌消炎、抗病毒、抗肿瘤的功能以及对心血管系统、中枢神经系统疗效显著；食品工业领域,作为食品抗氧化剂的类黄酮化合物具有高效、低毒、廉价、易得等特点而日益受到人们重视。油菜素内酯(Brassinolide，BL)是目前发现的生物活性最高的BRs。外源性BL处理抑制拟南芥和苹果的类黄酮合成。然而，在黄瓜和葡萄中，喷施BL则诱导类黄酮积累。也就是说，BL处理对不同植物的类黄酮途径的调控作用不同。
樱花素(sakurenatin)是水稻在感染稻瘟病菌后诱导的16种植保素中唯一的类黄酮物质，在体内外具有很强的抗稻瘟病菌活性和去毒活性。同时，在延缓衰老，抵抗病毒，治疗糖尿病，预防癌症等方面发挥非常积极的作用。正常生长条件下，水稻中的樱花素含量很低，但是在受到胁迫比如紫外照射、CuCl2及茉莉酸处理时，樱花素的合成被显著诱导。樱花素是类黄酮合成途径的黄烷酮分支产物，柚皮素-7-O-甲基转移酶(naringenin 7-O-methyltransferase，NOMT)是合成樱花素的关键性合成酶。至今尚未有文献公开报道BR诱导樱花素合成的报道。
发明内容
本发明的目的是针对现有技术的不足，提供一种油菜素内酯在提高水稻樱花素含量上的应用，是利用BL提高水稻樱花素含量的方法，具体是一种在水稻种植过程中，通过BL喷施水稻幼苗，建立快速有效提高水稻樱花素碱含量的方法。
第一方面，提供油菜素内酯在提高水稻樱花素含量上的应用。
第一方面，提供一种提高水稻樱花素含量的方法，是通过外源喷施BL提高水稻叶片樱花素含量。
作为优选，将BL喷施在水稻叶片上；
作为优选，所述的水稻大小为2-3叶龄期；
作为优选，所述的BL的浓度为1μM；
作为优选，所述的喷施周期为2天处理一次；
作为优选，所述的喷施容量为浸湿整个叶片，即叶片上开始滴水为止；
作为优选，BL诱导樱花素合成酶OsNOMT的表达，进而提高樱花素的含量。
本发明的有益效果：BL处理水稻幼苗经后，诱导水稻樱花素合成基因OsNOMT的表达，提高水稻樱花素的含量。本发明首次发现BL可用于提高水稻樱花素含量，并对其机理进行推测，认为BL处理诱导樱花素合成酶OsNOMT的表达，从而提高樱花素的含量。本发明实施简单、效果显著，为提高水稻樱花素含量、改善水稻免疫能力提供强有力的参考。
附图说明
图1是BL处理后樱花素合成酶基因OsNOMT的表达。
图2是标准品及样品HPLC/MS色谱图。
图3是BL处理后水稻叶片樱花素含量检测结果。
具体实施方式
下面结合具体实施方案对本发明做进一步的说明，这些实施例应被理解为仅为本发明的最佳实例，而非以任何方式限制本发明，凡在分发明原则之内所做的任何改进、修饰和等同替换，均应包含在本发明的范围之内。
实施例1：BL喷施水稻叶片处理
1、水稻栽培：日本晴水稻种子浸泡在带水的锥形瓶中，置于37℃恒温培养箱中浸泡2天。每天早晚各换一次水，直到露白为止。1L烧杯底部装200mL的营养土,将露白种子播于烧杯中，每杯20株，温室(28℃，12h光照每天)内培养，待用。
2、BL工作液配制：BL首先用无水乙醇溶解配制母液，再用0.1％Triton X-100配制工作液。0.1％Triton X-100加入等体积无水乙醇作为对照组(CK)。
3、BL喷施水稻：2-3叶大小日本晴水稻分别用喷壶喷施1μM BL和0.1％Triton X-100(CK)，处理一次。
4、喷施容量为浸湿整个叶片，即叶片上开始滴水为止。
实施例2：荧光定量PCR检测樱花素合成酶基因OsNOMT的表达量
BL和CK处理0、1、2、4、6、9、12、24小时，Trizol法分提取水稻地上部叶片的RNA，按照HiScript 1st Strand cDNA Synthesis Kit的方法将上述RNA进行逆转录(20μl逆转录体系加入1μg的Total RNA)得到cDNA。按照下列组分配制10μL反应体系如下:
扩增程序如下：94℃5min；94℃10s，60℃20s，72℃20s，40Cycles；72℃收集荧光，以OsUBQ5为内参。
由图1可知，BL处理水稻后2h时，樱花素合成酶基因OsNOMT显著性上调表达，一直持续到24h，12h时达到顶峰，说明OsNOMT在BL诱导樱花素合成过程中发挥重要作用。
用于上述定量检测OsNOMT基因表达量的引物序列为如下：
RT-OsNOMT-F：CTAGCCGGATGCATGAAAGT，如SEQ ID No.1所示；
RT-OsNOMT-R：TGCACGTATAGGCACACACA，如SEQ ID No.2所示；
RT-OsUBQ5-F：ACCACTTCGACCGCCACTACT，如SEQ ID No.3所示；
RT-OsUBQ5-R：ACGCCTAAGCCTGCTGGTT，如SEQ ID No.4所示；
实施例3：樱花素含量的检测
1、配制樱花素提取液：乙醇：水：乙腈：乙酸＝79:13.99:7:0.01(体积比)。
2、樱花素提取：准确称取BL处理48h的水稻地上部(茎和叶)粉末，加入2ml提取液，剧烈震荡后在4℃旋转(20rpm)孵育24h。4℃，16000g离心15min，收集上清于新离心管，4℃，16000g再次离心15min，收集上清。采用LC-MS/MS方法检测樱花素。
3、标准品配制：准备称取28.63mg樱花素标准品，溶解与1ml樱花素提取液中，配制10mg/ml母液。按梯度稀释，配制100,25,6.25,1.56,0.39,0.09ng/ml梯度，用于制作标注曲线计算样本樱花素浓度。如图2所示，BL处理组的信号强度显著性高于CK对照。樱花素浓度计算标注曲线为y＝6981.15279x+49.09438(r2＝0.99980)。
4、色谱质谱采集条件：
采用SCIEX超高效液相三重四极杆串联质谱仪Triple QuadTM LC-MS/MS 5500+系统进行采集，包括超高效液相色谱(Ultra Performance Liquid Chromatography UPLC，ExionLCTM AD)和三重四极杆串联质谱(Triple QuadTM LC-MS/MS 5500+)。
色谱柱：ZORBAX Eclipse Plus C18柱(1.8μm，3.0mm*100mm；流动相A：超纯水(加入0.01％甲酸，2mM甲酸铵)；流动相B：甲醇(加入0.01％甲酸，2mM甲酸铵)。流速0.4mL/min，柱温40℃，进样量1μL。采用15min梯度洗脱方法，洗脱梯度设置详细下表：
表：液相洗脱梯度
| 时间 | 流动相成分(v/v) |
| 0～1.0min | 25％B |
| 1.0～11.0min | 25％B |
| 11.0～13.0min | 95％B |
| 13.0～13.1min | 95％B |
| 13.1～15min | 25％B |
质谱条件：利用软件Analyst 1.7.1采集数据，离子源为电喷雾离子源(Electrospray Ionization，ESI)；检测方式为多反应监测(MRM)；喷雾电压(IonSprayVoltage，IS)负离子模式下为-4500V；离子源温度(Temperature，TEM)为500℃；雾化气(IonSource Gas1，Gas1)压力为50psi；辅助气(Ion Source Gas2，Gas2)压力为55psi；气帘气(Curtain Gas，CUR)压力为35psi。碰撞气(Collision Gas，CAD)为8。根据优化的去簇电压(Declustering Potential，DP)和碰撞能(Collision Energy，CE)，对离子对进行扫描检测。
如图3，1μM BL处理水稻48h后，水稻叶片中樱花素含量是对照组的28.5倍，受到显著诱导。这些结果表明，BL处理诱导樱花素的合成。
上述实施例并非是对于本发明的限制，本发明并非仅限于上述实施例，只要符合本发明要求，均属于本发明的保护范围。
Claims (8)
1.油菜素内酯在提高水稻樱花素含量上的应用。
2.根据权利要求1所述应用，其特征在于油菜素内酯诱导樱花素合成酶OsNOMT的表达，进而提高樱花素的含量。
3.一种提高水稻樱花素含量的方法，其特征在于通过外源喷施油菜素内酯提高水稻叶片樱花素含量。
4.根据权利要求3所述方法，其特征在于将油菜素内酯喷施在水稻叶片上。
5.根据权利要求3所述方法，其特征在于所述的水稻大小为2-3叶龄期。
6.根据权利要求3或4所述方法，其特征在于所述的油菜素内酯的浓度为1μM。
7.根据权利要求3所述方法，其特征在于油菜素内酯喷施周期为2天处理一次。
8.根据权利要求3或4所述方法，其特征在于油菜素内酯喷施容量为浸湿整个叶片。
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| CN116406613B (zh) * | 2023-03-22 | 2024-04-30 | 浙江省农业科学院 | 通过缺磷提高樱花素含量增强水稻对稻瘟病抗性的方法和应用 |
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