植物中磷脂酶D介导的转移磷脂酰基反应的功能研究 
贾艳霞 
学位类型博士
导师李唯奇 
2010-11
学位授予单位中国科学院研究生院
学位专业植物学
摘要转移磷脂酰基反应(transphosphatidylation)是指,在磷脂酶D(phospholipaseD,PLD)的催化作用下,甘油磷脂的磷酸二酯键发生断裂,并把磷脂的磷脂酰基转移到伯位醇上或者水上,从而生成磷脂酰醇或者磷脂酸的反应。该反应在动物、植物和微生物的生理过程中起到非常重要作用,但是其参与的很多生理过程及其机制尚且未知或不清楚。转移磷脂酰基反应中影响其功能的有底物、酶和产物三个因素。本论文以模式植物拟南芥为研究对象,通过改变转移磷脂酰基反应发生所必须的两个条件——受体底物类型和催化酶PLD的类型——来深入研究其发生的机制和产生的功能。论文的研究包括三个方面,通过改变PLD介导的转移磷脂酰基反应的受体底物,研究醇类结构对其生理学作用的影响及其意义;通过遗传操作改变介导转移磷脂酰基反应的PLD以研究其在植物衰老过程的调控作用;用正丁醇抑制绝大部分PLD来源的磷脂酸(phosphatidic acid,PA)之后植物对磷饥饿的响应。主要结果如下:1. 小分子醇类物质对植物生长发育的影响机制的初探。在本课题中,我们主要研究了小分子醇类物质对种子萌发、幼苗生长、膜脂分子组成的影响,并通过基因芯片来分析正丁醇对植物影响的信号通路和代谢途径,发现醇类的极性与其毒性呈正相关;醇类对植物的伤害可能与丁酸基和丙酸基的代谢有关而并不完全是由于阻止了磷脂酸的生成而造成的。另外,我们还筛选得到了5株正丁醇不敏感突变体,目前我们已经确定了这5株突变体的突变基因,也已初步观测到,在相同浓度正丁醇胁迫下,这5株突变体无论是生理生化水平还是细胞水平都显著好于野生型拟南芥,突变体的获得对于我们揭示醇类对植物毒害的机制提供了更直接的证据。2. 激素诱导的拟南芥离体叶片衰老过程中膜脂分子组成的变化以及磷脂酶Dα1 (phospholipaseDα1,PLDα1)和磷脂Dδ(phospholipaseDδ,PLDδ)在此过程中的调控作用。PLDα1反义抑制突变体和PLDδ敲除突变体之所以能延缓激素促进的衰老过程可能与PLDα1 和PLDδ基因可以响应外源脱落酸和乙烯信号有关;而且PLDα1-和PLDδ-来源的PA还可能有利于衰老叶片中活性氧(reactive oxygen species,ROS)的积累;PLDα1反义抑制突变体和 PLDδ敲除突变体内源的吲哚乙酸(indole-3-acrtic acid,IAA)、玉米素核苷(zeatin riboside,ZR)的含量要明显高于其对应野生型Col和WS野生型拟南芥,而内源脱落酸(abscisic acid,ABA)和茉莉酸(jasmonic acid,JA)的含量又偏低,这可能有利于其延缓激素诱导的离体叶片的衰老。3. 叔丁醇提高了低磷情况下植物对磷的吸收和利用。正丁醇的加入对正常生长和磷饥饿下生长的幼苗都造成了严重胁迫,而叔丁醇却可以在一定程度上减弱植物的缺磷症状,主要表现在减少了花青素的积累,增加了植物体内无机磷含量,增加了植株的果荚数目等。这可能是因为叔丁醇可以提高有限磷元素的吸收利用率,但是我们需要进一步实验来证明其机制。
资助项目The reaction of transphosphatidylation is which phospholipase D catalyzes hydrolytic cleavage of the terminal phosphate diester bond of glycerophosphatides, transfer the phosphatidyl moiety of a phospholipids to a primary alcohol or water, producing phosphatidyl alcohol or phosphatidic acid (PtdOH). Although this reaction plays important role in regulating physical process in plants and anminals, the pathway involved in and detailed regulation mechanism are still unknown or not clear. There are three factors which can affect transphosphatidylation, substrates, enzymes and products. This paper intended to uncover the physical effect of the reaction in detail by changing two indispensable factors, the type of substrate and the PLDs. This paper consist of three researches, the first one, changing the accept substrate of the reaction aims to study the relationship between the structure of alcohols and the physical effect, to reveal the significance of the reaction; secondly, using genetic methods to regulate the expression of PLDs mediating transphosphatidylation intends to study its roles in regulating senescence; thirdly, reducing mostly PA derived from PLDs by addition 1-Butanol try to study the effect of PLD-derived PA on phosphate starvation. 1. Initial research of the effect of alcohols on the palnt growth. In this study, we study on the effect of alcohols on Arabidopsis seed germination, seeding growth and membrane lipids molecules, the metabolic procees and signaling pathway invoved in plant responding to 1-Butanol. According to the results we observed, the toxicity of alcohols and its polarity is positively correlated; the toxicity of alcohols to plants may be related with the butanoate and propanoate metabolic pathway rather than transphosphatidylation, the increase of free IAA is contributed to the resistance to 1-Butanol for WS ecotype Arabidopsis thaliana. Futhermore, five 1-Butanol insensitive mutants have obtained and the mutant sites has identified. Under the same concentration 1-Butanol stress, the five mutants are in better condition no matter physical and chemical level or cellular levels compared to wild-type Arabidopsis thaliana. It is believed that the five mutants definitely provide direct evidence for us to uncover the mechanism of the toxicity of alcohols to plants. 2. The change pattern of the membrane lipid molecules responding to the phytohormones-promoted detached Arabidopsis leaves and the role of PLDα1 and PLDδ in regulating this process. The reason for PLDα1-antisense mutant and PLDδ-knockout mutant delaying phytohormone-promoted senescence may be PLDα1 and PLDδ are response to the ABA and ethylene sinaling; PLDα1-or PLDδ-derived PA contributes to reactive oxygen species (ROS) accumulation; PLDα1-antisense mutant and PLDδ-knockout mutant have higher level of indole-3-acrtic acid (IAA) and zeatin riboside (ZR) level but lower level of abscisic acid (ABA) and jasmonic acid (JA) compared to wild-type Col and wild-type WS, respectively, which is favorable to retard the phytohormone-promoted detached leaves senescence. 3. Application tert-butanol increase the efficiency of phosphate utilization and absord when the phosphate-limited. The seedinges phosphate-starved or not exposed to 1-Butanol was subjected to lethal stress. However, in low phosphate condition, application of tert-butanol lower anthocyanin accumulation, increase the phosphate levels and the siliques number, in a word, tert-butanol attenuates the symptom of phosphate-starved induced. It is suggested that tert-Butanol can increase the utilization and absord efficiency of the limited phosphate, however, more evidences are needed to prove the mechanism.
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/16082
专题昆明植物所硕博研究生毕业学位论文
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贾艳霞 . 植物中磷脂酶D介导的转移磷脂酰基反应的功能研究 [D]. 中国科学院研究生院,2010.
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