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学术型硕士学位论文

外源亚精胺对高温胁迫下番茄幼苗叶片蛋白质组和抗氧化系统

的影响

桑勤勤

指导教师郭世荣教授

学科门类农学

专业名称蔬菜学

研究方向设施园艺与无土栽培

答辩日期二〇一七年五月

分类号 S641.2

学号 2014104053

EFFECTS OF EXOGENOUS SPERMIDINE ON

LEAVE PROTEOMICS AND ANTIOXIDANT SYSTEM

IN TOMATO SEEDLINGS UNDER HIGH

TEMPERATURE STRESS

By

SANG QINQIN

A Thesis

Presented to Nanjing Agricultural University

in Partial Fulfillment of the Requirements

for the Degree of

Master of Agronomy

in

Olericulture

Supervised by

Professor GUO SHIRONG

College of Horticulture

Nanjing Agricultural University

Nanjing, China

May, 2017

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目录

i

目录

摘要 ..........................................................................................................................................................I

ABSTRACT .............................................................................................................................................. III

缩略词表 .................................................................................................................................................... V

前言 ..................................................................................................................................................... VII

第一章文献综述 ..................................................................................................................................... 1

第一节高温对植物的影响以及植物的耐热机理 ......................................................................... 1

1 高温对植物的影响 ...................................................................................................................... 1

2 植物的耐热机理 .......................................................................................................................... 1

2.1 细胞膜的耐热性 ................................................................................................................. 1

2.2 抗氧化防御 ......................................................................................................................... 2

2.3 热激蛋白 ............................................................................................................................. 3

2.4 渗透调节物质 ..................................................................................................................... 4

2.5 植物激素 ............................................................................................................................. 4

第二节多胺与信号转导 ................................................................................................................ 5

1 多胺与温度胁迫响应 .................................................................................................................. 5

1.1 多胺与高温胁迫 ............................................................................................................... 5

1.2 多胺与低温胁迫 ............................................................................................................... 5

2 多胺与信号转导 .......................................................................................................................... 6

2.1 多胺与活性氧 ................................................................................................................... 7

2.2 多胺与脱落酸 ................................................................................................................... 7

2.3 多胺与一氧化氮 ............................................................................................................... 8

2.4 多胺与离子转运 ............................................................................................................... 9

2.5 多胺与 MAPK ................................................................................................................ 10

第三节蛋白质组学研究进展 ...................................................................................................... 11

1 蛋白质组学的研究技术 ............................................................................................................ 11

2 蛋白质组学在非生物胁迫中的应用......................................................................................... 11

2.1 高温胁迫 ......................................................................................................................... 11

2.2 低温胁迫 ......................................................................................................................... 12

2.3 干旱胁迫 ......................................................................................................................... 13

2.4 盐胁迫 ............................................................................................................................. 14

第二章外源亚精胺对高温胁迫下番茄幼苗叶片蛋白质组的影响 ................................................... 17

1 材料与方法 ................................................................................................................................ 18

1.1 材料与处理 ..................................................................................................................... 18

外源亚精胺对高温胁迫下番茄幼苗叶片蛋白质组和抗氧化系统的影响

ii

1.2 测定方法 ......................................................................................................................... 18

2 结果与分析 ................................................................................................................................ 19

2.1 外源 Spd 对高温胁迫下番茄幼苗生长的影响 ........................................................... 19

2.2 差异蛋白的功能分类 ..................................................................................................... 20

2.3 差异蛋白的聚类分析 ..................................................................................................... 26

2.4 差异蛋白互作网络 ......................................................................................................... 27

3 讨论 ............................................................................................................................................ 28

3.1 光合作用 ......................................................................................................................... 28

3.2 抗氧化和防御响应 ......................................................................................................... 29

3.3 蛋白合成、折叠和降解 ................................................................................................. 30

3.4 能量代谢 ......................................................................................................................... 31

3.5 氨基酸代谢 ..................................................................................................................... 32

3.6 信号转导 ......................................................................................................................... 32

第三章外源亚精胺对高温胁迫下番茄幼苗叶片抗氧化系统的影响 ............................................... 35

1 材料与方法 ................................................................................................................................ 36

1.1 材料与处理 ..................................................................................................................... 36

1.2 测定方法 ......................................................................................................................... 36

2 结果与分析 ................................................................................................................................ 38

2.1 外源 Spd 对高温胁迫下番茄幼苗 ROS 产生的影响 ................................................. 38

2.2 外源 Spd 对高温胁迫下番茄幼苗膜脂伤害的影响 ................................................... 38

2.3 外源 Spd 对高温胁迫下番茄幼苗抗氧化酶活性的影响 ........................................... 39

2.4 外源 Spd 对高温胁迫下番茄幼苗非酶抗氧化剂含量的影响 ................................... 40

2.5 外源 Spd 对高温胁迫下番茄幼苗抗氧化酶相关基因表达的影响 ........................... 42

3 讨论 ............................................................................................................................................ 42

第四章外源亚精胺诱导 MAPK 级联途径参与高温胁迫下的抗氧化防御 ..................................... 45

1 材料与方法 ................................................................................................................................ 46

1.1 材料与处理 ..................................................................................................................... 46

1.2 测定方法 ......................................................................................................................... 46

2 结果与分析 ................................................................................................................................ 48

2.1 外源 Spd 和 PD98059 对高温胁迫下番茄幼苗叶片 MDA 含量和 O2·-产生速率的影响

......................................................................................................................................................... 48

2.2 外源 Spd 和 PD98059 对高温胁迫下番茄幼苗叶片抗氧化酶活性的影响 ................ 48

2.3 外源 Spd 和 PD98059 对高温胁迫下番茄幼苗叶片抗氧化酶相关基因表达水平的影

响 ..................................................................................................................................................... 49

2.4 外源 Spd 和 PD98059 对高温胁迫下番茄幼苗叶片 H2O2含量及其相关基因的影响

......................................................................................................................................................... 51

目录

iii

2.5 外源 Spd 和 PD98059 对高温胁迫下番茄幼苗叶片基因 MAPK3 和 MAPK6 表达水平

的影响 ............................................................................................................................................. 52

3 讨论 ............................................................................................................................................ 52

全文结论 ................................................................................................................................................... 55

参考文献 ................................................................................................................................................... 57

攻读硕士学位期间发表的论文 ............................................................................................................... 73

致谢 ....................................................................................................................................................... 75

摘要

I

外源亚精胺对高温胁迫下番茄幼苗叶片蛋白质组和抗氧

化系统的影响

摘要

高温是影响植物生育的一种非生物胁迫，直接影响植株的生理生化过程，间接改

变植物的生育模式，影响幼苗的形态建成。番茄（Lycopersicon esculentum Miller）是

一种主要的设施栽培蔬菜作物，在春末、夏秋季的温室生产中，高温胁迫已成为限制

生产的重要因子。多胺（Polyamines, PAs）是植物生长的一种调节物质，是多聚阳离

子的低分子量脂肪族含氮化合物，包括腐胺（putrescine, Put）、亚精胺（spermidine, Spd）

和精胺（spermine, Spm）等，调控植物的生育过程，参与植物对非生物胁迫的响应。

本研究采用高温敏感型番茄品种‘浦红 968’为试验材料，运用基质栽培方式，

通过叶面喷施外源亚精胺（1mM Spd），研究外源 Spd 对高温胁迫下番茄幼苗叶片蛋

白质组和抗氧化防御系统的影响，探究外源 Spd 缓解高温胁迫的相关机制。主要研

究结果如下：

1．外源 Spd 显著缓解了高温胁迫对番茄幼苗生长的抑制。双向电泳（2-DE）结

合串联飞行时间质谱技术（MALDI-TOF/TOF-MS），鉴定得到 67 个差异蛋白点响应

高温胁迫和/或外源 Spd，主要涉及光合作用、抗氧化和防御响应、氨基酸代谢、蛋白

合成折叠和降解、能量和代谢、信号转导；其中大多数蛋白参与光合作用、细胞防御

响应和能量代谢，且大部分被外源 Spd 诱导上调，说明 Spd 促进高温胁迫下番茄叶

片的光合作用、抗氧化防御和能量代谢。通过蛋白质组研究发现，Spd 通过调节高温

胁迫下番茄叶片蛋白质的表达水平，增强番茄植株的高温耐性。

2. 高温胁迫下，番茄幼苗叶片 ROS（O2.–产生速率和 H2O2含量）显著升高，膜

脂过氧化损伤加重。外源 Spd 处理不同程度地提高了高温胁迫下番茄幼苗叶片的抗

氧化酶活性，上调抗氧化酶相关基因的表达水平；同时，外源 Spd 处理显著增加番

茄幼苗叶片中的抗氧化剂（AsA 和 GSH）含量，提高还原型 AsA 和 GSH 所占的

比例，维持细胞内的氧化还原平衡。外源 Spd 可以通过激活抗氧化酶和非酶抗氧化

剂这两大抗氧化系统，显著降低 O2.– 产生速率和 H2O2、MDA 含量，缓解高温胁迫

对番茄幼苗造成的氧化伤害，降低膜脂过氧化程度。

3. 为进一步探究高温胁迫下外源 Spd 诱导产生的抗氧化耐性，研究高温胁迫下

外源 Spd 诱导促分裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）级联


II

途径参与高温胁迫下的抗氧化防御。施用 MAPK 级联途径抑制剂 PD98059 研究

MAPK 级联途径在 Spd 诱导产生的抗氧化系统中的作用。结果显示，外源 Spd 可

以通过提高抗氧化酶活性及相关基因表达来缓解高温胁迫造成的氧化伤害。利用

MAPK 抑制剂 PD98059（10 μ mol·L-1）处理显著加重高温胁迫造成的氧化损伤，同

时，也在处理的较短时间内明显抑制了 Spd 诱导的抗氧化酶活性及相关基因表达的

增加。上述结果表明，MAPK 参与外源 Spd 诱导的抗氧化防护过程，可能与外源 Spd

诱导产生的高温耐性有关。

关键词：番茄；高温胁迫；亚精胺；蛋白质组；抗氧化系统；MAPK 级联途径

ABSTRACT

III

FFECTS OF EXOGENOUS SPERMIDINE ON LEAVE

PROTEOMICS AND ANTIOXIDANT SYSTEM IN

TOMATO SEEDLINGS UNDER HIGH

TEMPERATURE STRESS

ABSTRACT

High temperature is one of the major abiotic stresses, affecting the physiological and

biochemical processes of plants directly, altering the growth patterns of plants indirectly,

and affecting seedlings morphogenesis. Tomato (Lycopersicon esculentum Miller) is one of

the main type of vegetable crops in protected cultivation, which is vulnerable to high

temperature stress, especially in spring, summer and autumn greenhouse planting.

Polyamines (PAs) are kinds of low molecular weight and ubiquitous nitrogenous

compounds, mainly including putrescine (Put), spermidine (Spd) and spermine (Spm),

participating in the regulation of plant growth and responsing to abiotic stress.

In the present study, we used heat-sensitive tomato cultivar (cv. Puhong 968) as the

test material, to study the effects of exogenous spermidine (1 mM Spd) on proteomics and

antioxidant system in tomato seedlings under the high temperature stress (38/28℃ ,

day/night), to investigate the physiological and molecular mechanism of how exogenous

Spd improved the high temperature resitance of tomato seedlings. The main results are as

follows:

1. Exogenous Spd significantly alleviated the inhibition of growth induced by the high

temperature stress. A two-dimensional gel electrophoresis (2-DE) coupled with

matrix-assisted laser desorption/ ionization tandem time of flight mass spectrometry

(MALDI-TOF/TOF-MS) were performed. A total of 67 differentially accumulated protein

spots were confidently identified in response to high temperature stress and/or exogenous

Spd. The resulting proteins involved in photosynthesis, cell rescue and defense, amino acid

metabolism, protein synthesis, folding and degradation, energy and metabolism, and signal

transduction. Exogenous Spd up-regulated most identified proteins involved in

photosynthesis, rescue defense and energy metabolism, implying an enhancement in


IV

photosynthetic and antioxidant defense capacity. These results suggested that exogenous

Spd can regulate the protein expression level of tomato leaves under high temperature stress,

then enhance the high temperature tolerance, and promote the growth of tomato seedlings.

2. Under high temperature stress, the levels of ROS (including O2.– and H2O2) in

tomato leaves were significantly increased, causing membrane lipid peroxidation damage.

Application of exogenous 1 mM Spd significantly increased the activities of antioxidant

enzymes and regulated the gene expression of the antioxidant enzymes. Moreover,

exogenous Spd also increased the content of non-enzymatic antioxidants (AsA and GSH),

maintaining the intracellular redox balance, alleviating the lipid peroxidation induced by

the high temperature stress. These results suggested that exogenous Spd can significantly

activate the antioxidant enzymes and non-antioxidant antioxidants, reduce O2.–generation

rate and the content of H2O2 and MDA, alleviate the oxidative damage of tomato seedlings

caused by high temperature stress and decrease membrane lipid peroxidation.

3. In order to further explore the antioxidant tolerance induced by exogenous Spd

under high temperature stress, the effects of mitogen-activated protein kinase (MAPK)

cascade pathway induced by exogenous Spd were studied under high temperature stress.

The MAPK cascade pathway inhibitor PD98059 was used to study the role of the MAPK

cascade pathway in Spd induced antioxidant system. The results showed that exogenous

Spd could alleviate the oxidative damage caused by high temperature stress through

improving the activity of antioxidant enzymes and the expression of the related genes. The

use of MAPK inhibitor PD98059 (10 μ mol·L-1) significantly increased the oxidative

damage, and inhibited Spd-induced antioxidant enzyme activity and related gene expression.

These results indicated that MAPK was involved in exogenous Spd - induced antioxidant

protection process, which may be related to the high temperature tolerance induced by

exogenous Spd.

KEY WORDS: Tomato; High temperature stress; Spermidine; Proteome; Antioxidant

system; MAPK cascade pathway

缩略词表

V

缩略词表

Abbreviations

缩写

Abbreviation

英文全称

English Name

中文全称

Chinese Name •OH Hydroxyl radical 羟基自由基

2-DE Two-dimensional gel electrophoresis 双向凝胶电泳

ABA Abscisic acid 脱落酸

APX Ascorbate peroxidase 抗坏血酸过氧化物酶

AsA Ascorbate 抗坏血酸

CAT Catalase 过氧化氢酶

CBB Coomassie Brilliant Blue 考马斯亮蓝

CS Cysteine synthase 半胱氨酸合成酶

DAO Diamine oxidase 二胺氧化酶

DHA Dehydroascorbate 脱氢抗坏血酸

DHAR Dehydroascorbate reductase 脱氢抗坏血酸还原酶

DTT DL-Dithiothreitol 二硫苏糖醇

GR Glutathione reductase 谷胱甘肽还原酶

GSH Reduced glutathione 还原型谷胱甘肽

GSSG Oxidized glutathione 氧化型谷胱甘肽

H2O2 Hydrogen peroxide 过氧化氢

IEF Isoelectric focusing 等电聚焦

MALDI-TOF/

TOF-MS

Matrix-assisted laser desorption/

ionization tandem time of flight mass

spectrometry

基质辅助激光解析电离串

联飞行时间质谱

MAPK Mitogen-activated protein kinases 促分裂原活化蛋白激酶

MDA Malondialdehyde 丙二醛

MDH Malate dehydrogenase 苹果酸脱氢酶

MDHAR Monodehydroascorbate reductase 单脱氢抗坏血酸还原酶

NO Nitric oxide 一氧化氮

O2•- Superoxide anion radical 超氧阴离子

PAs Polyamines 多胺


VI

续表（Continue）：

PAO Polyamine oxidase 多胺氧化酶

PMSF Phenylmethyl sulfanyl fluoride 苯甲基璜酰氟

POD Peroxidase 过氧化物酶

Put Putrescine 腐胺

qRT -PCR Real-time polymerase chain reaction 实时定量聚合链式反应

ROS Reactive oxygen species 活性氧

SAMDC S-adenosylmethionine decarboxylase S-腺苷蛋氨酸脱羧酶

SDS Sodium dodecyl sulfate 十二烷基硫酸钠

SOD Superoxide dismutase 超氧化物歧化酶

Spd Spermidine 亚精胺

SPDS Spermidine synthase 亚精胺合成酶

Spm Spermine 精胺

TCA Tricarboxylic acid 三羧酸循环

TK Transketolase 转酮酶

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