【作者】 黄志；

【导师】 邹志荣；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2010， 博士

【摘要】 甜瓜作为一种重要的园艺作物,在日光温室中栽培生产中,尤其是在干旱少雨的西北地区,常常面临严重的水分亏缺问题。研究包括菌根真菌在内的各种改善甜瓜抗旱性的措施和方法具有重要的现实价值和应用前景。本研究以甜瓜品种‘中蜜3号’为试材,在筛选水分胁迫条件下适合甜瓜栽培的菌种基础上,对接种丛枝菌根真菌(AMF)提高甜瓜抗旱性的生理及分子机制进行了系统的研究,主要结果如下:1.在水分胁迫条件下接种三种不同丛枝菌根真菌(Glomus mosseae, G. versiforme, G. intraradices)均不同程度的促进了甜瓜幼苗的生长和光合效率。在两种水分条件下,接种AMF都显著提高了甜瓜幼苗的株高、根系长度、抗氧化酶活性、可溶性糖含量、净光合速率和水分利用率。但是,不同的AMF的作用不尽相同,其中对甜瓜接种效应最好的丛枝菌根真菌是G. mosseae。水分胁迫条件下,AMF通过改善寄主植株抗氧化酶系统活性、加速CO2同化物质的双向传输和提高光合能力等方式促进了植株的生长,提高甜瓜的抗旱性。2.对水分胁迫条件下接种丛枝菌根真菌(AMF)影响甜瓜幼苗根系形态建成和营养吸收的研究结果显示:(1)接种AMF可以促进甜瓜幼苗根系的生长,增加了甜瓜幼苗根系活跃吸收面积,为植株吸收和利用营养元素提供了良好条件;(2)正常水分条件下,接种AMF可以显著提高甜瓜幼苗叶片N、P、K等元素的含量,在水分胁迫条件下,这种效应更为突出,其中水分胁迫下接菌幼苗根系中的P含量在各处理中最高,较未接菌对照增加了36.7%,也显著高于其他处理。接种AMF提高了甜瓜幼苗N、P的积累,增强了对N、P的吸收。在水分胁迫下对P元素的富集在植株根系中尤为突出。研究表明,接种AMF显著提高了寄主植株根系吸收面积,同时形成的功能强大的菌丝网进一步增加了水分胁迫条件下植株的根系生长和对营养元素的吸收,促进了植株的生长,提高了甜瓜的抗旱能力。3.对水分胁迫条件下接种丛枝菌根真菌(AMF)影响甜瓜幼苗叶片脯氨酸(Pro)积累、AsA-GSH循环关键酶和内源激素的研究结果显示:(1)接种AMF可以调节水分胁迫条件下甜瓜幼苗叶片的Pro积累量,增强抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)活性,保护甜瓜幼苗AsA-GSH循环,使菌根化甜瓜幼苗在活性氧代谢中处于有利地位;(2)正常水分条件下,接种AMF可以显著提高甜瓜幼苗叶片和根系IAA、ZR、GA的含量,减少ABA的积累。随着水分胁迫时间的延长,除了甜瓜幼苗叶片ZR含量逐渐下降以外其他处理甜瓜幼苗的IAA、ZR、GA和ABA含量大部分呈现先上升后下降的趋势,其中接菌处理的IAA、ZR、GA显著高于未接菌处理,ABA积累量则显著低于未接菌处理。研究表明,AM真菌能够减轻水分胁迫对甜瓜AsA-GSH循环造成的损伤,增加Pro的积累以及IAA、ZR、GA的含量,减少ABA积累,保持活性氧(reactive oxygen species,ROS)代谢和内源激素相对平衡,促进植株的生长,提高甜瓜的抗旱能力。4.对水分胁迫条件下接种丛枝菌根真菌(AMF)影响甜瓜幼苗叶片光响应、CO2响应以及光合日变化的研究结果显示:(1)接种AMF可以提高不同水分条件下甜瓜幼苗叶片光补偿点(LCP)、光饱和点(LSP)、羧化效率(CE)和最大潜在同化效率(Amax.)等指标;(2)水分胁迫显著降低了甜瓜幼苗叶片的净光合速率(Pn)、气孔导度(Gs)、蒸腾速率(Tr)和羧化效率(CE)。在水分胁迫下,Pn、Gs和Tr均出现较大的日下降变化。在水分胁迫中胞间CO2浓度变化与气孔导度及净光合速率的变化趋势相反,这表明水分胁迫对菌根化甜瓜幼苗叶片光合作用的影响主要表现为非气孔因素的影响。5.对接种丛枝菌根真菌(AMF)影响甜瓜植株水分胁迫(10 d)及复水过程中幼苗叶片光能吸收和叶绿素荧光的研究结果显示:(1)水分胁迫在一定程度上抑制了AM真菌的侵染,水分胁迫下菌根甜瓜的侵染率、侵入点和丛枝数较正常水分条件显著下降。(2)随水分胁迫时间的延长,接种AMF和未接菌处理的甜瓜幼苗叶片相对含水量、叶片生长指数(PI)、光合速率和各叶绿素荧光参数逐渐下降,但接种AMF的甜瓜幼苗下降幅度显著小于未接菌植株;胁迫解除后,只有接种AMF的甜瓜幼苗叶片相对含水量、生长指数和光合荧光系统可以迅速恢复到正常水分条件下幼苗的水平。研究表明,接种AMF能有效提高甜瓜幼苗叶片水分胁迫下的光能吸收、电子传递能力,促进幼苗的光合作用,缓解水分胁迫对甜瓜幼苗的损害。6.利用GenBank登录的抗旱相关基因序列进行比对,在保守区域设计一对引物,利用RT-PCR获得了一个甜瓜抗旱表达差异基因,命名为MeP5CS。生物信息学分析表明,该基因全长1000 bp,开放阅读框(ORF)753bp,编码250个氨基酸;MeP5CS蛋白大小约82.18 kD,理论Pi值为4.90;MeP5CS编码蛋白与桐花树、猕猴桃和葡萄同源性较高,分别为94%、81%和73%。MeP5CS编码蛋白是疏水性蛋白,有2个跨膜螺旋结构和13个磷酸化位点。半定量RT-PCR表明,MeP5CS在甜瓜根系中表达最高,茎中次之,叶片中表达较低。试验结果表明AMF可以诱导甜瓜在水分胁迫下MeP5CS基因的表达,增强甜瓜的抗旱能力;组织表达差异可能和水分胁迫处理时间有关。更多还原

【Abstract】 Melon (Cucumis melo L.)-an important horticultural crop that is often cultivated in simply-equipped solar greenhouses in northwestern regions of China, usually suffers under poor water management. Arbuscular mycorrhizal (AM) symbiosis can play a great role in enhancing drought tolerance. In this thesis, bases on the selecting of arrbuscular mycorrhizal fungi (AMF) to drought in melon(Cucumis melo L. cv.‘Zhongmi 3’), a series of experiments had been performed on physiological responses and molecular mechanisms of drought tolerance improved by arbuscular mycorrhizal fungi. The main results were as follows:1. The plant growth, physiological, and photosynthetic responses of melons inoculated with three Glomus species under two water conditions were investigated. Results show that inoculation with Glomus elevates the physiological and photosynthetic parameters of several seedlings compared with non-AM seedlings. Regardless of water conditions, plant height, root length, biomass production, antioxidant enzyme activity, soluble sugar content, net photosynthetic rate, and photosynthetic water use efficiency are elevated in AM seedlings compared to non-AM seedlings. Each Glomus species manifests unique effects under the two water conditions. We posit that arbuscular mycorrhizal symbiosis can protect melon plants against water deficiencies by improving their reactive oxygen activity, bi-directional transport, and photosynthetic capacity. In addition, regardless of water conditions, the most efficient fungus for melon (Cucumis melo L.) is Glomus mosseae.2. The root growth and nutrient acquisition of melons inoculated with Glomus mosseae under two water conditions were investigated. The results showed that: (1) inoculated with AMF can promote the melon seedling root growth, increased melon root active absorption area; (2) under well-watered conditions, inoculated with AMF can significantly improve melon leaves N, P, K and other elements of content. Under water stress conditions, this effect is highly efficient, which inoculated seedlings roots in the highest P content in comparison with non-AMF treatment increase 36.7%, significantly higher than other treatments. Melon seedlings inoculated with AMF increased N, P accumulation, increase of N, P absorption. Concentration of P content in the plant roots especially higher under water stress. The results showed that AMF inoculation significantly increased the host plant root absorption area, while the AM hyphae links formed a powerful highway further increase root growth and nutrient absorption, promote plant growth, improve the drought resistance of melon.3.The proline content, Ascorbat-glutahione cycle and endogenous hormones of melons inoculated with Glomus mosseae under two water conditions are investigated. The results showed that: (1) melon seedlings inoculated with AMF can adjust the Pro accumulation, increased APX and GR activities, to protect melon seedlings AsA-GSH cycle, to avoid injured from Reactive oxygen species; (2) under well-watered conditions, inoculated AMF can significantly improve the melon leaves and roots IAA, ZR, GA content, reducing the accumulation of ABA. Under water stress, except the ZR content of melon leaves decreased processing, melon seedlings IAA, ZR, GA and ABA content first increased and then decreased generally, which as IAA, ZR, GA content significantly higher in AM seedlings than in non-AM control, same as ABA accumulation was significantly lower. The results showed that, AM fungi can reduce injury on AsA-GSH cycle of melon caused by water stress, increased Pro accumulation and IAA, ZR, GA content, reduced ABA accumulation, to maintain antioxidant system activities and the balance of endogenous hormones relatively, to promote plant growth and improve the capacity of melon to drought.4. The light response, A/Ci curve analysis, daily variation of net photosynthetic rate (Pn), stomatal conductance(Gs), transpiration rate (Tr), intercellular CO2 concentration(Ci), carboxylation efficiency (CE) and assimilation at saturating CO2 (Amax.)were compared between plants inoculated with Glomus mosseae (AM plants)and those not infected (Non-AM plants) under different water treatments.The results showed that the Pn, Gs, Tr, CE of plants decreased significantly under drought stress. Inoculated AMF can significantly improve melon leaves Pn, Gs, Tr, CE and Amax. under water stress conditions; (2) The diurnal change of the Pn, Gs, Tr showed larger decline under the drought stress conditions. Drought stress could improve intercellular CO2 concentration (Ci), suggesting that although both Pn and Gs decreased under the stress condition, Gs was not a limiting factor for the decline of photosynthesis. The results show that AMF improved the Gs, CE and Amax of plants to compensate for the injury caused by drought.5. The mycorrhizal development, leaf plastochron index (PI), leaf relative water content (RWC), gas exchange characteristics, chlorophyll fluorescence parameters, and photosynthetic electron transport rate (ETR) of melons inoculated with Glomus mosseae under two water conditions were investigated. The results showed that: (1) the colonization of plants by arbuscular mycorrhizal fungi (AMF) was reduced under water stress. (2) under water stress, leaf relative water content (RWC), leaf growth index (PI), photosynthetic rate(Pn) and chlorophyll fluorescence decreased both in AM seedlings and non-AM control, but the down amplitude of AM seedlings was significantly smaller than non-AM control; only RWC, PI and Photosynthetic system of seedlings inoculated with AMF showed higher recovery capacity than non-AM control upon returning to normal conditions. Study shows that inoculation of AMF can improve the melon seedlings photosynthesis absorption and electron transport capacity under water stress, promote photosynthesis, mitigation damage from water stress on melon seedlings.6. Using designed primers based on the conserved amino acid sequences of known drought-related genes to amplify cDNA fragments from melon (Cucumis melo L.) by RT-PCR, a drought-related gene named MeP5CS was obtained. Bioinformatics analysis indicated that the full-length of cDNA sequence was 1000 bp, which contained an open reading frame of 753 bp and encoded a protein of 250 amino acid residues with a calculated molecular weight of 82.18 kD and isoelectric point of 4.90. The MeP5CS protein showed 94%, 82% and 73% similarity to the P5CS from Aegiceras corniculatum, Actinidia deliciosa and Vitis vinifera. The protein includeα-helix (40.2%),β-turn (25.2%), random coil(34.6%) and a cleavage site between nineteen and twenty amino acid residues. The protein is a hydrophobic protein and there is two transmembrane helix and thirteen Phosphorylation sites. The result of RT-PCR analysis indicated that MeP5CS expression levels were different in roots、stems and leaves, and it was highest in roots and middle in stems, lower in leaves . The expression of MeP5CS is induced by AMF under water stress, but is closely related to the processing time, increasing nitrogen fixation ability of host plants by AMF and signal transduction mechanisms in different tissues. The results show that the AMF can induce MeP5CS gene expression in melon under water stress, and enhance the drought resistance of melon; expression differences in tissues may be related to the duration of water stress.更多还原