褪黑素UPLC-MS/MS检测方法优化及其对干旱和盐胁迫下葡萄幼苗褪黑素含量的分析  

Optimization of UPLC-MS/MS Method for Melatonin Detection and Analysis of Melatonin Content in Grape Seedlings under the Conditions of Salt and Drought Stress with the Optimized Method

作　　者：吴艳迪 焦健[1] 姜建福[1] 张颖[1] 樊秀彩[1] 孙海生[1] 刘崇怀[1] 

机构地区：[1]中国农业科学院郑州果树研究所,郑州450009

出　　处：《西北植物学报》2017年第8期1657-1663,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：现代农业产业技术体系建设专项资金资助(CARS-30-yz-1);中国农业科学院科技创新工程专项经费项目(CAAS-ASTIP-2017-ZFRI);国家农作物种质资源平台(NICGR2016-037);农业部物种保护项目(2130135-34)

摘　　要：褪黑素是一种吲哚类激素,对植物的生理活动和抗逆性起到显著的调节作用。该研究建立了褪黑素超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法,并分别对干旱胁迫和盐胁迫条件下‘赤霞珠’葡萄幼苗根系和叶片中褪黑素含量的变化进行分析,以探讨葡萄中褪黑素的生理功能及其响应逆境胁迫的调控机制。结果显示:(1)褪黑素UPLC-MS/MS检测方法的优化条件为:采用超声波破碎法提取葡萄组织中的褪黑素;色谱条件为:色谱柱为Agilent Eclipse XDB-C18(1.8μm,3.0×50mm),流动相A为0.1%(v/v)的甲酸水溶液,流动相B为纯甲醇,梯度洗脱,柱温42℃,进样量1μL,流速0.2mL/min;质谱条件为:电喷雾正离子模式(ESI+)电离,多反应监测模式(MRM)检测,检测离子对为m/z 233→174。该方法测量结果的相对标准偏差(RSD)低于7%,最低检测限(LOD)和最低定量限(LOQ)分别为0.04ng/mL和0.12ng/mL。(2)干旱胁迫和盐胁迫下葡萄幼苗根系和叶片中褪黑素含量较对照组均显著增加,且胁迫程度越强增幅越大;用120mmol/L的NaCl溶液处理幼苗以后,幼苗根系和叶片中褪黑素含量分别达到627.25和3 220.42pg/g,大约都是相应对照组幼苗根系和叶片中褪黑素含量的7倍;10%PEG6000处理植株后其根系和叶片中褪黑素含量也远远高于对照组。研究表明,超高效液相色谱串联质谱法可作为一种准确、高效、简便易操作且具有较高的灵敏度和精确度的植物内源褪黑素含量检测方法;葡萄中褪黑素的合成是其对逆境胁迫的一种应激响应,暗示着褪黑素在缓解逆境胁迫方面具有重要作用。Melatonin is an indoleamine,which plays a significant role in regulation of the physiological activities and stress-tolerance of plants.This study employed Ultra Performance Liquid Chromatography t and em Mass Spectrometry(UPLC-MS/MS) and measured the changes of melatonin content in ‘CabernetSauvignon'grape leaves and roots under the conditions of salt and drought stress,respectively, and explored the physiological functions and regulation mechanisms of melatonin in response to stress.The results showed that:(1)the UPLC-MS/MS method for determination of melatonin was optimized, and melatonin was extracted from different grape tissues by the method of ultrasonication.The HPLC condition was as follows:The analytes were separated using an Agilent Eclipse XDB-C18(1.8μm,3.0×50mm)at 42 ℃ with linear elution gradient at 0.2mL/min flow rate.The injection volume was 1μL.Mass spectrometric conditions:The electrospray was operated under the positive ionization mode and the sample was identified by multiple reaction monitoring(MRM)mode.The ion of monitor was at m/z 233→174.The established method exhibited high specificity and sensitivity,the relative st and ard deviation(RSD)was less than 7%.In addition,the limit of detection(LOD) and the limit of quantity(LOQ)was 0.04ng/mL and 0.12ng/mL,respectively.(2)The treatments of drought and salt stress significantly increased the melatonin content in leaves and roots of grape seedlings, and the increase range of melatonin content was enlarged with the increasing degree of stress treatment.After the seedlings were treated with 120mmol/L NaCl,the melatonin content of grape roots and leaves reached 627.25 and 3 220.42pg/g,respectively,which was about 7 times higher than that of control seedlings.When treated with 10% PEG6000,the content of melatonin in roots and leaves was much higher than that in the control group.As a conclusion,UPLC-MS/MS is an accurate and efficient method for the detection of endogenous melatonin in plants.the melatonin biosynthesis in grape is a response

关 键 词：葡萄 褪黑素 超高效液相色谱-串联质谱 干旱胁迫 盐胁迫 

分 类 号：Q503[生物学—生物化学] Q946.83