大豆GmHsp 17.6-L热激启动子的克隆及功能分析毕业论文

 2021-04-21 11:04

摘 要

随着植物转基因技术的发展,如何对转入的外源基因的表达进行精确调控,使其能够在植物体内适时、适量和有效地表达,成为人们关注的焦点问题。目前,可应用的植物基因表达调控系统主要有乙醇调控系统、糖皮质激素调控系统、铜调控系统、IPTG 调控系统和四环素调控系统等。这些系统均利用了外源诱导物,而维持外源基因的高水平持续表达需要不断供给或多次施用诱导物,这在实际应用中存在难度且受环境因素的影响较大;同时,有些诱导物如四环素容易被光分解,铜、银离子等超过一定浓度时会对植物造成生理伤害。热激调控系统是利用热激启动子调控目的基因,只受温度控制,克服了利用诱导物对植物产生毒性的缺点,不存在药物残留,不会带来人畜伤害和潜在的环境污染问题,经济投入少且便于在试验条件下进行精确调控。

本论文从大豆基因组DNA中扩增了热激启动子GmHsp 17.6-L,成功构建了该启动子调控的GUS报告基因表达载体,并利用浸花法对以拟南芥进行了遗传转化,获得了T0代转基因种子,截止论文成稿时,尚处于筛选阶段。下一步将开展大豆GmHsp 17.6-L热激表达的严谨性进行检测。

关键词:热激启动子;GUS;表达载体;拟南芥;浸花法转化

Cloning and functional analysis of Soybean heat shock promoter GmHsp 17.6-L from soybean

Abstract

With the development of plant transgenic technology, there is an active demand to regulate the expression of the transferred foreign gene accurately, so that it can be expressed in plant timely, appropriate and effectively. Nowadays, the application of plant gene expression regulation system includes alcohol control system, glucocorticoid regulation system, copper regulatory system, recombinant vector control system and tetracycline control system, etc. These systems use exogenous inducers to maintain the high expression level of exogenous genes, which is difficult and influenced by environmental factors in practical application. In addition, some inducers, such as tetracycline and silver ions, are easily decomposed or may cause physiological damage for plants. Heat shock control system utilise the heat shock promoter to control the target gene, which is only controlled by the temperature and overcome the disadvantages of the use of elicitor to plant toxicity.

In this study, we amplified the heat shock promoter from the soybean genomic DNA, and successfully constructed an expression vector composing of the GUS reporter gene under the control of the promoter. T0 generation transgenic seeds of Arabidopsis thaliana were obtained by using the floral-dip method. The seeds are still under antibiotic screening when this thesis is finished. The next step will be to detect the stringency of the heat shock promoter GmHsp 17.6-L.

Keywords: heat shock promoter; GUS; expression vector; Arabidopsis thaliana; floral-dip method

目录

摘要 I

1.1 前言 1

1.1热激蛋白研究进展 1

1.2热激启动子研究进展 1

1.3基因的表达调控 2

1.4本研究的目的和意义 3

2 材料与方法 4

2.1试验材料 4

2.2试验方法 4

2.2.1大豆基因组DNA提取 4

2.2.2引物设计原则 5

2.2.3 PCR反应成分及用量 6

2.2.4凝胶回收 6

3 实验结果与分析 10

3.1大豆基因组DNA提取 10

3.3 GmHsp 17.6-L的PCR扩增 10

3.4 载体构建 11

3.5 拟南芥种子筛选 11

4 问题与讨论 13

4.1 影响PCR结果的探讨 13

4.2拟南芥种植的探讨 13

5 结论及展望 15

致谢 16

参考文献 17

1.1 前言

1.1热激蛋白研究进展

HSR是动植物细胞或器官对热刺激的一种保护性反应。过度的热激会导致细胞损害甚至死亡,而耐热水平的提高是由于亚致死强度的热激条件会诱导细胞发生HSR,使细胞或机体免受热胁迫的伤害从而表现出来的。在分子生物学水平上,HSR实质上是在正常蛋白质合成受阻的同时产生热激蛋白(heat shockprotein, HSP)的一种细胞生理活动[1]

在高于生物正常生长温度8~12℃的条件下,生物体内合成一些新的蛋白质称为热激蛋白(HSP)。热激蛋白是高温诱导生物体合成的一类蛋白质[2]。植物热激蛋白(HeatShockProtein, HSP)是指在高于植物正常生长温度刺激下诱导合成的应激蛋白,与多种逆境生理密切相关[3]。1974年,Tisseres从热激果蝇幼虫的唾液腺等部位分离到了6种新的蛋白质,即HSPs[4]。研究者们期望通过调节HSP的表达水平来提高植物的抗逆性,而HSP积累量的变化主要与启动子的活性有关[5]。HSP具有保守性、短时性、多样性的特点[6]

1.2热激启动子研究进展

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