摘 要

近年来，全球气候变化，使旱涝灾害的发生更为频繁，干旱导致木本植物死亡也时有发生。干旱胁迫抑制植物生长发育速度，加速植物衰老进程，降低作物产量。其根源为干旱胁迫下，绿色植物的光合作用受到抑制。

银杏为中生代孑遗雌雄异株的裸子植物，具有一定代表性且有较高的经济和生态效益。银杏植株体内所特有的生物活性物质具有药用价值。其独特的扇形叶片和秋季金黄的叶色具有观赏价值，使其成为城市行道树的选择之一。

干旱胁迫对于银杏的光合作用的影响可从器官、细胞及分子三个层次进行分析：从器官水平上分析，由于植物叶片失水，气孔关闭导致二氧化碳供应不足；从细胞水平分析，植物叶绿体受损，细胞内生理生化过程发生改变；从分子水平上分析，干旱使其叶绿体蛋白的表达情况发生改变。

本次实验以两年生银杏幼苗为实验材料，以雌雄性及是否干旱胁迫进行分组，实验组进行0 d, 20 d, 40 d, 80 d的干旱处理。在对干旱胁迫下银杏雌雄植株的叶绿体超微结构和生理生化研究的基础上，从蛋白质水平研究银杏雌雄植株的抗旱差异。通过改良银杏叶片叶绿体蛋白的方法，提高所制备的蛋白质样品纯度。利用iTRAQ技术进行蛋白质组学分析，发现在干旱胁迫下，雌雄叶片中叶绿体差异蛋白121个，已知功能蛋白67个。并运用qRT-PCR进行基因检验，经验证，编码蛋白的mRNA与在蛋白水平上的表达量变化趋势相似，iTRAQ结果有效。

关键词：银杏；干旱胁迫；叶绿体蛋白；雌雄差异

Difference comparison of chloroplast protein which response the drought stress between the male and female Ginkgo

ABSTRACT

In the recent years, due to the climate variation around the world, drought and flood disasters frequently occur, and because of that, many arbors die from time to time. The drought stress inhibits the plant growth and accelerates the senescence of plant, in addition, leads to the reduction of crop yield. The source of the consequences above is that the drought impacts the plants’ photosynthesis.

Ginkgo is an ancient dioecious gymnosperm in Mesozoic, which has high economic and ecological value. The specific bio-active substance in ginkgo have medical value. Its typical fan-shaped and yellow leaves make more cities use ginkgo to be the roadsides tree.

Three levels of the consequences that the ginkgo suffers under the drought disaster are as followed. One side, in organ level, the dehydration of leaf leads to the stomata closing. The stomatal closure results in inadequate supplies of carbon dioxide. Then, in cellular level, the long-time water insufficient injures the chloroplast of plant and vary the physiological and biological reaction inside. In molecular level, the drought affect ginkgo chloroplast protein expression.

The materials of this experience were the 400 two-year-old ginkgo seedlings. Divided them into four groups：male control group; male drought group; female control group and female drought group. The ginkgo in drought groups suffered dry treatment during 0 d, 20 d, 40 d, 80 d. This experience analyzed the difference between the male and female ginkgo in protein level under the drought stress by proteomics analysis. Improved the purity of protein samples by modifying the method of extracting the chloroplast protein. Under the drought stress, 121 differential proteins were found and the function of 67 proteins in them were known by proteomics analysis with iTRAQ technique. Genetic testing by qRT-PCR indicated that the expression quantity of mRNA and protein had the similar variation tendency which means the result of iTRAQ was effective.

Key words：ginkgo；drought stress；chloroplast protein；gender difference

目 录

1 前言………………………………………………………………………………………1

1.1 实验研究背景………………………………………………………………1

1.1.1 银杏研究价值及生长习性…………………………………………………1

1.1.2 干旱胁迫对植物的影响…………………………………………………1

1.2 目前研究进展…………………………………………………………………………2

1.2.1 干旱下银杏光合及生理生化特性的相关研究……………………………………2

1.2.2 干旱下银杏性别差异的相关研究…………………………………………………3

1.2.3 与干旱相关的蛋白质组学研究…………………………………………………3

1.2.4 蛋白质组学研究方法（iTRAQ技术）…………………………………………4

1.3 实验内容及研究路线…………………………………………………………………………4

1.3.1 雌雄银杏抗旱性差异研究整体路线………………………………………………4

1.3.2 实验研究内容与目标…………………………………………………………………5

1.4 本研究的意义与创新…………………………………………………………………………6

2 银杏叶绿体蛋白提取方法改良…………………………………………………………7

2.1 实验准备……………………………………………………………………7

2.1.1 实验材料…………………………………………………………………………7

2.1.2 实验试剂…………………………………………………………………………7

2.1.3 实验仪器…………………………………………………………………………8

2.2 实验步骤…………………………………………………………………………8

2.2.1 银杏叶绿体分离……………………………………………………………8

2.2.2 叶绿体蛋白提取—TCA-丙酮沉降法………………………………………9

2.2.3 叶绿体蛋白提取—Tris-平衡酚抽提法……………………………………9

2.3 结果分析…………………………………………………………………………9

2.3.1 叶绿体完整率检测……………………………………………………………9

2.3.2 单向电泳分析…………………………………………………………………10

2.3.3 双向电泳分析…………………………………………………………………11

3 干旱胁迫下雌雄银杏叶绿体蛋白质组学研究…………………………………………………………15

3.1 实验准备……………………………………………………………………15

3.1.1 实验材料…………………………………………………………………………15

3.1.2 技术路线…………………………………………………………………………16

3.2 实验步骤……………………………………………………………………16

3.2.1 样品处理…………………………………………………………………………16

3.2.2 FASP酶解…………………………………………………………………………17

3.2.3 肽段标记…………………………………………………………………………17