【作者】 金维环；

【导师】 陈耀锋；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2010， 硕士

【摘要】 氢离子焦磷酸化酶(H+-pyrophosphatase,H+-PPase)水解无机焦磷酸(PPi)产生自由能并与H+跨膜转运相耦联形成质子梯度,建立的跨膜质子梯度为各种溶质(如阳离子、阴离子、氨基酸和糖类等)的跨膜的次级主动运输提供驱动力。H+-PPase作为一种有效的质子泵,在植物对水分和盐分胁迫的响应中扮演着重要的角色。H+-PPase与液泡膜H+-ATPase一起,既能维持细胞离子平衡和渗透平衡,又能为减轻一些无机离子(如Na+)对细胞质的毒害起重要的作用。本研究的前期工作已从披碱草(Elymus dahuricus)中克隆了新的氢离子焦磷酸化酶基因(EdHP1)。基因功能研究证明EdHP1能够显著提高转基因烟草对磷和钾的吸收。然而,对于EdHP1促进营养吸收的生理机制和调控机理还不十分清楚。本研究通过比较转EdHP1基因烟草及野生型烟草的各项生理指标,初步分析了EdHP1促进转基因烟草吸收磷和钾的生理机制,增加对植物磷和钾生理代谢过程的了解。同时,本研究对EdHP1的抗旱性及信号调控途径也进行了初步探索:1.以转EdHP1基因烟草为实验材料,在低营养条件下,比较了转基因植株和野生型烟草在根系发育,植株形态,体内生长素含量,叶绿素含量,体内钾、磷含量等方面的差异,结果显示,在低钾条件下(0.05 mmolL-1),转基因烟草相对于野生型烟草根系明显发达,转基因烟草体内钾含量及生长素含量明显高于野生型烟草。低钾胁迫主要影响植物细根发育,转基因烟草的细根明显比野生型烟草发达,而中等根和粗根二者差别不大;在低钾条件下,采用非损伤探测技术研究了转基因烟草和非转基因烟草在根尖,伸长区,根毛区钾离子和氢离子的流速值。结果发现,在低钾条件下,钾离子外排速率转基因株系低于非转基因烟草,氢离子外排速率则高于非转基因烟草。在低磷条件下(0.016 mmolL-1),实验结果与低钾条件下的结果相似,转基因烟草根系比野生型烟草发达,体内的磷含量,生长素含量比野生型烟草高;根据以上实验结果推测,EdHP1基因的作用机制可能是提高转基因烟草根部细胞的质子梯度,质子梯度提高一方面使根部细胞酸化,提高IAA等生长素在根部的分布,促进了根系发育,另一方面可以促进细胞质膜上的磷和钾转运体的活性,促进了根部磷和钾的主动运输,最终两方面的生理过程综合作用提高了植物根部对钾、磷的吸收。2.以转EdHP1基因烟草为实验材料,在干旱条件下(2% PEG)对转基因株系和野生型烟草进行抗旱性鉴定,结果证明转基因烟草的发芽率明显高于野生型烟草,转基因烟草的根系相对于野生型烟草发达,EdHP1基因的过表达可以提高转基因植物的抗旱性。3.为了研究氢离子焦磷酸化酶基因在植物中的调控机制。以拟南芥的氢离子焦磷酸化酶(AtVP1)为诱饵,采用酵母双杂交系统筛选拟南芥的cDNA文库,初步筛选到一些阳性克隆。同时,为了研究AtVP1是否参与了两个重要的与植物营养吸收有关的信号传导途径,选择在这两个传导途径中重要的调控蛋白(LKS和SOS2),鉴定AtVP1是否和LKS、SOS2可以互作,结果显示AtVP1不与LKS、SOS2互作,证明AtVP1不受这两个重要蛋白调控。EdHP1作为H+-PPase家族中的一个重要成员既可以促进植物的营养吸收又可以提高植物的抗逆性,具有重要的应用价值。EdHP1生理机制的剖析不仅为利用这个基因改良作物的抗逆性提供理论依据,而且可以增加我们对植物吸收营养元素生理过程的了解,具有重要的理论价值。更多还原

【Abstract】 Pyrophosphate (PPi) is hydrolyzed by pyrophosphorylase (H+-PPase) and releases free energy, and then produces proton gradient, which provide driving power for secondary active trans-membrane transportation of solutes such as positive ion, negative ion, ammonium acid, sugar, and etc. As efficient H +pump, H+-PPase plays a key role in responding to salinity and drought stresses in plants. Working together with vacuole-membrane-located H+-ATPase, H+-PPase not only can maintain the balance of ion and osmotic of the cell, but also can reduce the toxic effect to the cytoplasm causing by inorganic ion such as Na+. In pervious work, an H+-PPase gene, EdHP1 had been cloned from Elymus dahuricus. Result of gene functional analysis indicated that the EdHP1 gene could improve the Na+ and K+ uptaking in transgenic tobacco plants. However, the physiological and regulation mechanisms of nutritional absorption of EdHP1 transgenic plants still remain unknown. In the present study, by comparing each physiology index between EdHP1 transgenic and non-transgenic tobacco, we studied the physiological mechanism of P +and K+ uptaking controlled by EdHP1 gene, and those results led to better understanding of respective regulation mechanism. Meanwhile, we studied signaling regulation pathway involving EdHP1underlying drought tolerance. And the results are demonstrated below:1. Using the EdHP1 transgenic tobaccos as the studying materials, under low nutritional condition of low K+ and P+ concentration (0.05 level and 0.016 respectively), we compared parameters difference between transgenic tobacco and wide-type (WT) tobacco in terms of root system development, plant morphology, growth hormone levels in vivo, chlorophyll content, K+ and P+ content of plants. The results indicated that, under low content of K+ (0.05 mmolL-1) condition, the root systems of transgenic tobacco were more healthy than that of WT’s, and similarly, the K+ content in vivo was higher than that of WT’s. In general, low content of K+ effect on thin root developing specifically, and in this research, thin root of transgenic tobacco were more healthy than wild type tobacco, whereas there were no differences between transgenic and WT tobaccos in medium-thick and thick roots. Facilitated by the Non-Invasive Detection Method, the H+ and K+ flux in the root tip, elongation zone, and hair zone of both transgenic and WT tobaccos were detected under the low potassium condition. The results indicated that there were less K+ efflux and more H+ efflux in transgenic tobaccos compared to WT ones. Similar with above, the result coming from low phosphorus experiment (0.016 mmolL-1) was demonstrated as follows: Root system of transgenic tobaccos were better developed than of WT tobaccos’, and growth hormone content of transgenic tobaccos was higher than that of WT tobaccos’. According the result above, it was suggested that the mechanism of EdHP1 gene might increase the H+ gradient in transgenic tobacco’s root cells, which can resulted in cells acidic, increasing the IAA distribution among roots, and in turn, improving the roots development. On the other hand, higher H+ gradient can also active the transporter of the potassium and phosphorus on the membrane, which can improve the active transportation in roots. Both two effects resulted in higher the potassium and phosphorus up taking in root eventually.2. Result of drought tolerance experiment (2% PEG) indicated that the seed germination of EdHP1 transgenic tobacco were higher than that of WT plants’. The root systems of transgenic tobaccos were better-developed than that of WT ones’, as suggested that overexpression of EdHP1 can confer higher drought tolerance to plants.3. To reveal the regulation mechanisms of EdHP1 in plants, Yeast Two-Hybrid system was used to screen Arabidopsis cDNA library, and AtVP1 from Arabidopsis was used as the bait protein, and finally some positive clones were obtained. On other hand, two key regulation proteins, LKS and SOS2 that are two key regulation proteins involved in signaling pathway underlying nutrition uptaking, were used for this experiment to identify whether they interacted with AtVP1. As a result, AtVP1 didn’t interacted with none of them in other words, suggested that AtVP1 was not controlled by LKS or SOS2.The an member of H+-PPase family, EdHP1 can not only increase nutrition up taking, but also improve the tolerance to abiotic stress in plants, which were important for improving crop stress tolerance. Dissection of physiological mechanism provided a theoretical basis to improve the tolerance to abiotic stress in crops, and give better understand on the process of nutrient absorption.更多还原