论文总字数：26689字

摘 要

植物激素指的是一类在植物体内自然产生的微量的，能参与植物自身生理过程调节的有机分子或物质^[1]。这些植物激素对植物生长发育各有其独特作用，同时相互间亦存在着促进或抑制的作用。它们对一些如砷、低氮、高温、低温等胁迫环境下植物的生长也有着重要的作用。植物激素的高准确度和高通量定量是揭示植物激素代谢、转运和分子机制的前沿研究的关键。因此，本课题以水稻为研究对象，建立了一种简便的，能同时广泛地定量多种植物激素的高分辨液质联用检测方法，并对方法进行验证。具体结果如下：

（1）植物激素液质条件的优化。本文从轰击能量和流动相选择两方面入手。通过质谱多反应监测（multiple reaction monitoring，MRM）)，确定特征离子及轰击能量；确定流动相：A:0.1%甲酸水溶液；B:纯乙腈。梯度洗脱程序为：0~3min，10%B，3~8min，10%~65%B；8~15min，65%~100%B；15~18min，100%B；18~18.1min，100%~10%B；18.1~22min，10%B。根据植物激素物化性质，色谱柱选用C18反相柱。

（2）水稻样品前处理方法的优化。本文选择水稻地上部分（Shoot）、地下部分（Root）和培养液为三种基质，考察基质用量、提取试剂、富集纯化方法等方面，并通过添加回收实验优化前处理方法。具体为：称取液氮研磨后样本量，地上部分25mg，地下部分50mg，1mL乙腈提取三次，首次提取需过夜12h，30℃氮吹后用1 mL 10%乙腈水溶液复溶，复溶液过SPE HLB小柱浓缩净化，用2 mL洗脱液（80:19:1=乙腈：水：甲酸）洗脱，收集洗脱液，过0.22μm膜后上机检测，培养液取20mL直接过SPE HLB小柱浓缩净化，过膜上机检测。该方法地上部分回收率为51.95%~89.81%，地下部分回收率为62.66%~91%，培养液回收率为65.72%~104.88%，由于基质效应的影响，地上部分与地下部分回收率较低；由于水杨酸本底含量较高，而标准样添加量较少，水杨酸的回收率测定结果不够准确，故整体的回收率波动范围较大。

关键词：多种植物激素 高分辨 液质联用 水稻 基质效应

THE STUDY ON THE SYNCHRONOUS DETECTION METHOD OF MULTIPLE PLANT HORMONES BY HIGH RESOLUTION LIQUID CHROMATOGRAPHY MASS SPECTROMETRY

Abstract

Plant hormones, a trace substance in plant tissues, were known to participate in the regulation of the physiological processes. They played a unique role in plant growth and development, as well as making positive or negative effects on each other. Besides that, some of them are also essential to the growth of plants under stressed conditions such as arsenic, low nitrogen concentration, and unusual temperature. High accuracy and high-throughput quantification of phytohormones are the key to reveal the frontier research of phytohormone metabolism, transport and molecular mechanism. Therefore, a simple high-resolution liquid chromatography-mass spectrometry method for simultaneous and extensive quantification of various plant hormones was established and validated. The specific results are as follows:

(1) Optimizing the conditions of liquid chromatography-mass spectrometry (LC-MS) method for phytohormones. This paper starts with the selection of collision energy and mobile phase. We used multiple reaction monitoring (MRM) to find characteristic ions and suitable collision energies. The mobile phases were determined as follows: A: 0.1% formic acid aqueous solution; B: pure acetonitrile. The gradient elution procedure was 0-3 min, 10% B, 3-8 min, 10%-65% B, 8-15 min, 65%-100% B, 15-18 min, 100% B, 18-18.1 min, 100%-10% B, 18.1-22 min, 10% B. According to the physicochemical properties of phytohormones, C18 reversed-phase column was selected for chromatographic column.

(2) Optimizing the pretreatment methods of rice samples. The aboveground part (Shoot), underground part (Root) and culture medium of rice were selected as three substrates. The size of substrates, extraction reagents, enrichment and purification ways were investigated. The pretreatment methods were determined by adding recovery experiments. Specifically: weigh the sample after liquid nitrogen grinding, 25mg aboveground part, 50mg underground part, and 1mL acetonitrile to extract three times, first extraction for 12h, 30°C nitrogen blowing and reconstitute with 1mL acetonitrile solution (acetonitrile content 10%，v/v).The complex solution was purified by SPE HLB column and eluted with 2 mL of eluent (80:19:1=acetonitrile : water : formic acid，v/v). The eluate was collected and passed through a 0.22 μm membrane and then detected by machine. 20 mL of the culture solution was directly subjected to solid phase extraction, and detected by a machine after passing through the membrane. The recovery rate of aboveground part is 51.95%~89.81%, the recovery rate of underground part is 62.66%~91%, and the recovery rate of culture liquid is 65.72%~104.88%. Due to the influence of matrix effect, the recovery rate of aboveground part and underground part is lower. Because the background content of salicylic acid is higher, and the standard solution is less, the determination result of salicylic acid recovery rate is not accurate enough, so the overall recovery rate fluctuates widely.

Key words：Multiple plant hormones；High-resolution；Liquid chromatography-mass spectrometry；Rice；Matrix effect

目 录

摘要………………………………………………………………………………………………I

ABSTRACT…………………………………………………………………………………… II

第一章 文献综述 1

1.1植物激素概况 1

1.2 七种植物激素 1

1.2.1 6-苄氨基嘌呤 1

1.2.2 赤霉素 2

1.2.3 独脚金内酯 2

1.2.4 茉莉酸类化合物 2

1.2.5 水杨酸 3

1.2.6 脱落酸 3

1.2.7 芸苔素内酯 3

1.3 植物激素的分析方法 4

1.3.1 气相色谱-质谱联用（GC-MS） 4

1.3.2 高效液相色谱（high performance liquid chromatography，HPLC） 4

1.3.3 液相色谱-质谱联用（LC-MS） 4

1.3.4 其他方法 5

1.4 研究与目的 5

第二章 实验材料及方法 6

2.1 材料 6

2.2 主要仪器 7

2.3实验方法 7

2.3.1水稻种子萌发及幼苗的培养和处理 7

2.3.2混标植物激素母液的配制 9

2.3.3液相色谱与质谱条件 9

2.3.4添加回收实验 9

第三章 结果与分析 11

3.1 多种植物激素的液质检测方法确定 11

3.1.1 上机溶剂的选择 11

3.1.2 色谱洗脱条件 11

3.1.3 轰击能量选择 12

3.1.4 标准曲线的绘制和检出限的计算 14

3.1.5样品前处理方法的优化 15

3.1.6不同浓度、不同介质的添加回收实验结果 20

3.2分析及展望 24

参考文献 26

致谢 29

第一章 文献综述

1.1植物激素概况

自1935年，Went发现第一种植物激素——生长素（auxin）^[2]至2008年Gomez-Roldan等人发现目前最新的植物激素——独脚金内酯（strigolactones，SLs）^[3]。共有九类已知的植物激素，分别为生长素、细胞分裂素（cytokinins，CK）、赤霉素（gibberellins，GAs）、脱落酸（abscisic acid，ABA）、乙烯（ethylene，ETH）、芸苔素内酯（brassinosteroids，BRs）、水杨酸（salicylic acid，SA）、茉莉酸类（jasmonates，JAs）、独脚金内酯^[4]。

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