【作者】 周强；

【导师】 於丙军；

【作者基本信息】 南京农业大学 ， 植物学， 2011， 博士

【摘要】 盐分和干旱胁迫是威胁全球农业生产的主要因素,对植物产生广泛而深远的影响。一般认为,渗透调节、多胺代谢和水孔蛋白调控被认为是植物耐受盐分和干旱胁迫的重要生理机制。香根草(Vetiveria zizanioides)是禾本科香根草属多年生C。类草本植物,具有很强的忍耐盐分和干旱等水分逆境环境的能力,有着广泛的利用价值。目前对香根草的科学研究,主要集中在重金属土壤修复和水土保持等生态环境保护方面,而对其抗逆机理的研究很少,特别是在分子机理方面基本处于空白状态。本文着重从香根草的基本生物学特性、适应盐分和干旱胁迫的主要生理和分子机制等方面进行了深入研究。研究结果表明：1、香根草的光合能力强,其Pn最高可达15.3±1.77μmol C02 m-2 s-1；生长快速,最高生长速率出现在6月,达42.1±5.1 cm month-1;地上部分产量为6634.24±889.69 Kg hm-2；香根草叶片矿质元素含量较低；其纤维素、半纤维素、木质素分别约占生物质干重的32.7±0.58%0、37.48±0.94%、15.08±1.22%；叶片水解后,主要成分葡萄糖和木糖的含量分别约为36.41±1.93%和22.50±2.59%。无论是净光合速率、产量、抗逆性还是有效化学成分、水解产物组分,均与已广泛报道的能源植物柳枝稷接近,并且香根草耐虫、病、杂草的危害,自身不会扩散为杂草,并有广泛的种植基础。所以,香根草可认为是一种潜在的优良纤维素能源植物。2、在100,200,300 mmol L-1 NaCl处理9d后,香根草根、叶中无机离子(K+、Na+、ca2+、Mg2+、Cl-、NO3-、S,P)和有机溶质(可溶性糖、有机酸、游离氨基酸)的含量随着盐胁迫强度的增加而增加,无机离子(主要是Na+、K+和C1-)对渗透调节的贡献(71.50%-80.56%)远高于有机溶质的贡献(19.43%-28.50%),在根中无机离子在渗透调节中的贡献随着盐胁迫的增强而增加,而在叶中的变化则相反。但渗透调节作用仅在香根草处于中度盐胁迫(≤200 mmol L-1 NaCl)下才有明显的积极作用。3、在中度盐胁迫(100、200 mmol L-1 NaCl)9天时,香根草基本能够正常生长,但在重度盐胁迫(300 mmol L-1 NaCl)下,其生长受到严重抑制。在NaCl处理下,香根草根和叶中游离态腐胺(Put)、尸胺(Cad)、精胺(Spd)、亚精胺(Spm)和游离态多胺(PAs)总量明显下降,在重度盐胁迫(300 mmol L-1 NaCl)下降幅更大；结合态Put、Cad、Spd、Spm和结合态PAs总量显著上升,但在重度盐胁迫下升幅较小或与对照相当;束缚态Put、Cad和束缚态PAs总量均减少,而束缚态Spd和Spm含量在叶中是下降的,在根中则增加,且在中度盐胁迫(≤200 mmol L-1 NaCl)下更明显。对根和叶片而言,除游离态(Spd+Spm)/Put比值在重度盐胁迫下较对照显著下降外,其它游离态、结合态、束缚态和总的(Spd+Spm)/Put比值在不同盐胁迫下均上升,在中度盐胁迫下更明显。这表明,维持体内多胺总量的稳态和较高的(Spd+Spm)/Put比值是香根草适应中度盐胁迫的一个重要机制。4、当香根草遭受中度干旱胁迫(20%,40%PEG-6000溶液)和重度干旱胁迫(60%PEG-6000溶液)处理6d后,植株的伤害程度(以细胞完整性、生长率、含水量和光合作用等参数评价)增加,游离态和结合态Put含量随PEG处理浓度的增加而降低,在中度干旱胁迫下,香根草通过降低渗透势,增加游离态和结合态Spd和Spm含量以维持生存；复水后,渗透平衡得到重建,大部分生理参数完全或部分恢复到对照水平,而在重度干旱胁迫后复水,其生理参数没有得到恢复。这些结果表明,香根草能够通过渗透调节和维持游离态、结合态和束缚态三种形态多胺总量的稳态等策略以应对中度干旱胁迫。5、利用简并引物克隆了香根草水孔蛋白基因片段,并采用生物信息软件进行了相似性分析。结果表明,克隆到的基因片段VZPIP1和VZPIP2与拟南芥,水稻,玉米等植物的水孔蛋白具有极高的相似性,VZPIP1属于质膜膜内蛋白PIP1类；VZPIP2属于质膜膜内蛋白PIP2类。在干旱胁迫下香根草PIPl和PIP2基因在根部的表达量均显著增加,大幅提高了根系静水压导水率和渗透压导水率,提高了根系导水能力；PIP1基因在香根草叶片中的表达量显著增加,减小了气孔导度和叶片导水率,降低了蒸腾作用,减少了水分散失。通过这一机制,在干旱胁迫下,香根草有效地维持了植株体内的水分平衡,减轻了胁迫伤害。更多还原

【Abstract】 Salinity and drought stresses were the most deleterious abiotic stresses, which adversely influence plant growth, development, and were also the important factores in limiting agricultural productivity. Generally, osmolytes, polyamines and aquaporins (AQPs) were considered to be the important mechanism in plant to drought and saline environments. Vetiver grass (Vetiveria zizanioides), is a kind of sterile and perennial grass with multi-tolerance to lots of abiotic stresses, such as drought, salinity, et al., has high photosynthesis and yield. At present, vetiver grass is being used worldwidely as means of rehabilitation of heavy metal mines, soil and water conservation, and so on. However, up to date, its tolerance mechanisms were still undefined, and especially on the molecullar biological mechanisms. In this study, the basic biological characteristics, the mechanisms of osmolytes, polyamines and aquaporins (AQPs) in the adaptation of vetiver grass to saline and drought environments were investigated. These results showed as follows:Vetiver grass (Vetiveria zizanioides) has fast growth, high net photosynthesis rate and dry yield, and low content of mineral elements. The max Pn was 15.3±1.77μmol CO2 m-2 s-1, the highest growth rate was 42.1±5.1 cm month-1 in June; its dry yield up to 6634.24±889.69 Kg hm-2. Vetiver grass leaf compositions were evaluated with regard to cellulose (32.7±0.58%), hemicelluloses (37.48±0.94%), and lignin (15.08±1.22%). And their hydrolysis products were determined by HPLC, which including glucose (36.41±1.93%), xylan (22.50±2.59%). In brief, the photosynthesis, yield, stress tolerance and leaf compositions and its hydrolysis products of vetiver grass are very near to switchgrass. In addition, vetiver grass can withstand pests, pathogen and weeds; and it can not be spread to the weed, and is planted in many countries. So, we think that vetiver grass can be considered as a potential lignocellulosic energy plant.The accumulation of inorganic and organic osmolytes and their role in osmotic adjustment were investigated in roots and leaves of vetiver grass (Vetiveria zizanioides) seedlings stressed with 100,200,300 mmol L-1 NaCl for 9 days. although the contents of inorganic (K+, Na+, Ca2+, Mg2+, Cl-, NO3-, S, P) and organic (soluble sugar, organic acids and free amino acids) osmolytes all increased with NaCl concentration in vetiver grass seedlings, the contribution of inorganic ions (mainly Na+, K+ and Cl-) to osmotic adjustment was hingher (71.50%-80.56% of total) than that of organic solutes (19.43%-28.50%). The contribution of inorganic ions increased and that of organic solutes decreased in roots with the enhanced NaCl concentration, whereas the case in leaves was opposite. On the other hand, the osmotic adjustment is only effective for vetiver grass seedlings under moderate saline stress (less than 200 mmol L-1 NaCl).Under moderate salt stresses (100 and 200 mmol L-1 NaCl) for 9 days, vetiver grass grew at similar vigor when compared with seedlings under normal growth conditions. However, growth was severely arrested when plants were treated with severe salt stress (300 mmol L-1 NaCl). Under the above 3 different concentrations of NaCl stress, free Put, Cad, Spd, Spm and total free PA contents substantially decreased in roots and leaves, more severe losses of free PAs was observed under higher NaCl concentration. Conjugated Put, Cad, Spd, Spm, and total conjugated PAs remarkably increased, the extent of the increase after 300 mmol/L NaCl treatment was smaller than those after 100 or 200 mmol/L NaCl treatments. Bound Put, Cad and total bound PAs decreased in both roots and leaves under salt stress, bound Spd and Spm decreased in leaves but increased in roots in response to salt treatments, the more obvious rise was displayed under moderate salt stresses. With the exception of the significant decreases of free (Spd+Spm)/Put ratio were observed in roots and leaves after 300 mmol/L NaCl treatment, the other ratios of free, conjugated, bound and the total (Spd+Spm)/Put increased in roots and leaves of vetiver grass seedlings after different salt treatments, especially under moderate salt stresses. These results indicate that maintaining homeostasis of total PAs content and high (Spd+Spm)/Put ratios could be an adaptation mechanism in vetiver grass to moderate saline environment.When vetiver grass was exposed to the moderate (20% and 40% PEG-6000 solutions) and severe (60% PEG solution) water deficit for 6 days, the plant injury degree (expressed as the parameters of plant growth, cell membrane integrity, water relations and photosynthesis) increased and contents of free and conjugated Put decreased with the rise of PEG concentration. Under the moderate water deficit, the plants could survive by the reduced osmotic potential (ψs), increased free and conjugated Spd and Spm in leaves. After subsequent rewatering, the osmotic balance was re-established; most of the above investigated physiological parameters were fully or partly recovered to the control levels. However, it was not the case for the severely-stressed and rewatering plants. It indicates that, vetiver grass can cope well with the moderate water deficit/drought stress by using the strategies of osmotic adjustment and maintenance of total contents of free, conjugated and bound PAs in leaves.PIP1 and PIP2 gene segment sequences of aquaporins were successfully cloned from vetiver grass, and the similarity between them and cDNA sequences of other plant aquaporins was analyzed by bioinformatics software. The results showed that VzPIPl and VzPIP2 sequence have highly similarity with other known aquaporins, such as ZmPIP1;1 and ZmPIP2;1, and VzPIPl was belonging to the PIP1 class, and the VzPIP2 belonging to the PIP2 class. And when vetiver grass was exposed to the moderate water deficit (25% PEG solution) for 3 days, the expression levels of PIP1, PIP2 significantly increased in roots, which improved the hydrostatic conductance (Lph) and osmotic conductance (Lpos), and enhanced the root hydraulic conductance. The expression levels of PIP1 significantly increased in leaves, which decreased the stomatal conductance, leaf hydraulic conductance, and reduced the transpiration and loss of water. So, by the above strategies, vetiver grass can maintain the water balance, mitigate the stress injury and cope well with the drought stress.更多还原