【作者】 杨升；

【导师】 张华新；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2014， 博士

【摘要】 沙枣（Elaeagnus angustifolia L.）作为我国荒地和盐碱地的重要先锋树种，已经广泛应用于盐碱地的改良和治理。为了对沙枣进行选优，并且系统研究沙枣的耐盐生理机制，本文以6个沙枣种源（阿拉尔、昌吉、金昌、银川、盐池和磴口）的实生苗为材料，通过温室盆栽试验，系统研究和比较了6个种源沙枣的耐盐性差异，从中选择了耐盐能力差异较大的两个种源系统研究了其在不同NaCl浓度，不同胁迫时间下的生理变化和矿质离子代谢特征；同时，以水培苗为材料，利用非损伤微测技术（NMT）研究了两个种源的电生理学和药理学特性差异，从不同方面系统研究了不同种源沙枣耐盐性差异的生理机制。主要结果如下：1.除阿拉尔种源在150mmol·L-1NaCl胁迫条件下各生长指标与对照之间无明显差异外，随着盐浓度的增加，沙枣幼苗相对株高生长量、地径、单株叶面积、叶片数、总生物积累量和相对生物量均显著减少，而根冠比、比叶面积和叶面积比率与对照之间在低NaCl浓度下差异不明显，在高NaCl浓度下，沙枣幼苗的根冠比均显著增加，比叶面积和叶面积比率显著减少，其中对银川种源沙枣幼苗的抑制作用最大。利用隶属函数法结合权重综合评价6个种源沙枣幼苗的耐盐能力差异，其排序为：阿拉尔>金昌>磴口>盐池>昌吉>银川。2. NaCl胁迫下，随着盐浓度的增加和胁迫时间的延长，沙枣生物量积累的抑制效应越来越明显，而阿拉尔种源沙枣幼苗的抑制程度要低于银川种源。在150mmol·L-1NaCl浓度下，与对照相比，阿拉尔种源随着胁迫时间的延长，生物量积累下降较小，根冠比值无明显变化。盐胁迫显著降低了沙枣幼苗叶片中的叶绿素a、叶绿素b和总叶绿素含量以及叶绿素a/b值，并且随着胁迫时间的延长，下降幅度逐渐越大，而盐胁迫对银川种源叶绿素含量的影响要大于阿拉尔种源。随着NaCl浓度的增加和胁迫时间的延长，沙枣叶片中的MDA含量逐渐增加，而银川种源的增加幅度要大于阿拉尔种源。脯氨酸含量随着NaCl浓度的增加而显著增加，但随着胁迫时间的延长，脯氨酸含量增加幅度逐渐下降。随盐浓度增加，处理7天后，沙枣叶片中可溶性糖含量下降，并且阿拉尔种源的下降幅度大于银川种源，当胁迫时间持续到30天，可溶性糖含量先降后升，NaCl胁迫60d后，呈逐渐上升趋势，并且阿拉尔种源的增加幅度要大于银川种源。3. NaCl胁迫后，沙枣幼苗各组织中Na+含量均急剧增加，并且随着胁迫时间延长根和叶中的增幅比较明显，而阿拉尔种源在根中的增幅要大于银川种源，在叶片中则正好相反。随着NaCl浓度的增加和胁迫时间延长，K+含量和Ca2+含量呈现逐渐下降的趋势，并且阿拉尔种源沙枣幼苗根和叶中的下降幅低于银川种源，而Mg2+含量变化规律随胁迫时间延长而有所改变，胁迫7天后，根系中随着盐浓度的升高而逐渐增加，而叶片中则逐渐减少；盐胁迫30天时，与对照组间无显著性差异；60天后，各组织呈逐渐下降的趋势。沙枣幼苗各组织和全株K+/Na+比值均随着NaCl浓度的增加和胁迫时间延长而呈下降趋势，叶片的下降幅度最大，其次为根，茎最小，根和叶片中K+/Na+比值在阿拉尔种源中的下降幅度要小于银川种源。在盐胁迫30天内，沙枣的K+选择性吸收系数和运输系数，随着NaCl浓度增加而增加或保持稳定，而当胁迫达到60天时，则随着盐浓度的增加逐渐降低，阿拉尔种源的K+吸收系数要略高于银川种源4. NaCl处理后，沙枣幼苗根系的稳态Na+和K+外流均显著增加，而且阿拉尔种源的Na+净流量要明显大于银川种源，而K+外流要低于银川种源；在瞬时加入NaCl刺激下，K+的外流在沙枣幼苗中表现出显著增加，并且银川种源的外流净流量要大于阿拉尔种源，而H+由内流变为外流，阿拉尔种源H+外流量要显著性高于银川种源。药理学试验发现3种抑制剂（氯化四乙铵，阿米洛利，正钒酸钠）分别处理后，NaCl诱导的Na+和K+外流均能受到不同程度的限制，阿米洛利处理后，阿拉尔种源Na+外流仍然明显大于银川种源。正钒酸钠处理后，Na+和K+外流与相应抑制剂处理间差异不大或无差异，表明Na+和K+外流与质膜质子泵活性密切相关。沙枣在盐胁迫下，通过Na+/H+逆向转运体将Na+排出体内。同时，盐胁迫会使细胞去极化程度加强，增加K+外流。由于Na+外排需要质膜两侧产生H+电化学势梯度，这样降低了细胞去极化，从而使细胞内K+外流减少，这样造成了Na+和K+的竞争性外流。基于上述结果，表明6个种源沙枣幼苗的耐盐能力差异，其排序为：阿拉尔>金昌>磴口>盐池>昌吉>银川。NaCl胁迫不仅能够抑制沙枣有机物的积累，还能够影响有机物的分配，而且对耐盐能力较弱的种源影响较大。随着NaCl浓度增加和胁迫时间延长，耐盐性较强的沙枣种源的叶绿素含量下降幅度较小，并且能够更好的维持膜系统稳定性，以此保障植物体内各种新陈代谢的顺利进行。在渗透调节物质中，脯氨酸主要是在胁迫早期调节植物体内微环境，而可溶性糖在短期胁迫下，主要是作为能量来源，长期胁迫下，则主要是作为渗透调节物质，并且耐盐能较强的沙枣种源的调节能力较强。同时，耐盐能力强的沙枣种源能够更加有效的把Na+阻隔在根部，减少叶片中Na+含量，并且具有更强的保留K+能力。耐盐能力较强沙枣种源具有更强的Na+/H+逆向转运体活性，使更多的Na+外流，从而减少了K+的外流，因此，更好的维持细胞质中K+/Na+平衡。更多还原

【Abstract】 Elaeagnus angustifolia L. is an important pioneer tree species for wasteland andsaline-akali land, which has been applied widely in saline-akali land improvement andmanagement. In order to select optimal E. angustifolia and reveal the salt-tolerance mechanism,E. angustifolia seedlings from six provenances (Alaer, Changji, Jinchang, Yinchuan, Yanchi,Dengkou) were used as experimental materials in this research. By pot experiment ingreenhouse, the salt tolerance of six E. angustifolia provenances were studied and comparedsystematically. The physiological changes and metabolic characteristics of mineral ions wereinvestigated with seedlings of salt-tolerance provenance and salt-sensitive provenance underdifferent NaCl concentrations and different stress time. Meanwhile, the electrophysiologicaland pharmacological properties were analyzed by using Non-invasive Mico-test Technique(NMT) with hydroponic seedlings of the two provenances. which systemic studied thephysiological mechanism from different aspects.The main results of this research were summarized as follow:1. The relative height growth, ground diameter, leaf area per plant, number of leaf,biomass accumulation and relative biomass were significantly reduced with the NaClconcentration increased, except the Alaer provenance under the150mmol·L-1NaCl stress. Theroot/shoot ratio, specific leaf area and leaf area ratio was not obvious difference under the150mmol·L-1NaCl, but under the300mmol·L-1NaCl concentration, the root/shoot ratio wassignificantly increased and decreased for specific leaf area and leaf area ratio. The growthindexes of Yinchuan provenance had a maximum inhibition under salt stress. Seedlingtolerance ability of six different provenances was assessed by membership function combinedwith weight, and the sequence from strong to weak was Alaer> Jinchang> Dengkou> Yanchi>Changji>Yinchuan. 2. Under the NaCl stress, the biomass accumulation of E. angustifolia seedlings wasinhibited more obviously with increasing salt concentration and stress time, and the Alaer E.angustifolia provenance had a lower inhibitory effect than Yinchuan provenance. When theseedlings of Alaer provenance were treated with150mmol·L-1NaCl, the biomass accumulationhad a little decline with the stress time prolonging, compared with the control, and theroot/shoot ratio was insignificantly change. The content of chlorophyll a, chlorophyll b, totalchlorophyll and chlorophyll a/b ratio were all remarkable reduced by salt stress, and thedecrement was gradually increased with the salt stress time extending. The salt stress hadgreater inhibited effect on chlorophyll content of Yinchuan provenance than that of Alaerprovenance. With increase of the NaCl concentration and stress time, malonaldehyde (MDA)content of E. angustifolia leaves was gradually increased, and the increased rate of MDAcontent in leaves of Yinchuan provenance was greater than that of Alaer provenance. Theproline content was significantly increased with increase of the NaCl stress, but the increasedrate of proline content decreased with prolong of the stress time. After salt treatment for7days,the soluble sugar content of E. angustifolia leaves decreased with the NaCl concentrationincreased, and the Alaer provenance had more decrement than Yinchuan provenance. Thesoluble sugar content rose after the first drop with increase of the salt concentration after saltstress30days. The soluble sugar content showed a gradual upward trend at NaCl stress60days later, and the rate of increment was greater in Alaer provenance than Yinchuanprovenance.3. After NaCl stress, the Na+content in organizations of E. angustifolia seedlingsincreased dramatically with increase of NaCl concentration, and the Na+contents in roots andleaves were more obvious increase with prolonged of stress time. The increment of roots ofAlaer provenance was greater than Yinchuan provenance, but in leaves which was opposite.With the increased of NaCl concentration and prolonged stress, K+and Ca2+content levelsshowed a gradual downward trend, but the contents of root and leaf were fall lower in Alaerprovenance than Yinchuan provenance. Variation of Mg2+content followed NaCl concentration which was changed with prolonged stress time. After salt stress for7days, the Mg2+content ofroots increased with increase of salt concentration, while the leaves were gradually reduced.There was no significant difference compared with the control group after30days, but theMg2+content showed a gradual downward trend in each tissue. K+/Na+ratio in each tissue of E.angustifolia seedlings and whole plant K+/Na+ratio were all decrease with increased of NaClconcentration and stress time, and K+/Na+ratio of the leaves dropped sharpest, followed by theroots and stem smallest. The decrement rate of roots and leaves K+/Na+ratio in Alaerprovenance was less than that of Yinchuan provenance. The K+selective absorption coefficientand transport coefficients of E. angustifolia increased or remained stable as the NaClconcentration increased in30days of salt stress, but the two coefficients were decreasedgradually with increasing salt concentration as the stress time reached60days. Meanwhile, theK+selective absorption coefficient and transport coefficients of Alaer provenance was slightlyhigher than Yinchuan provenance.4. The steady-state Na+and K+of E. angustifolia were efflux and significantly increasedafter NaCl treatment24hour. The Na+efflux of Alaer provenance was greater than Yinchuanprovenance, but the K+efflux was lower than Yinchan provenance. When the transientstimulation of NaCl was given, the K+efflux in E. angustifolia seedlings exhibited aremarkable increase, and net K+flux of Yinchuan provenance was higher than Alaerprovenance. In pharmacology experiment, Na+efflux and K+efflux which induced by NaClwere inhibited by three kinds of inhibitors (TEA, Amiloride, Vanadate). After amiloridetreatment, the Na+efflux of Alaer provenance was still significantly larger than Na+efflux ofYinchuan provenance. The Na+and K+efflux which were treated by vanadate had a littledifference or no difference compared with that of corresponding inhibitor treatment, whichindicated the Na+and K+efflux closely associated with the plasma membrane proton pumpactivity. When the E. angustifolia seedlings were under the NaCl stress, Na+of the roots wasexpelled by the Na+/H+antiporter. In the meantime, salt stress strengthened the celldepolarization and increased K+efflux. Because the Na+efflux required H+electrochemical potential gradient at the sides of the membrane which reduced the cell depolarization and K+efflux, this caused the competitive efflux of Na+and K+.Based on these results, the sequence of the salt-tolerance from strong to weak of the six E.angustifolia provenances is Alaer>Jinchang>Dengkou>Yanchi>Changji>Yinchuan. NaClstress can not only inhibit the accumulation of organic matter of E. angustifolia, but also canaffect the distribution of organic matter, and which has greater impact for salt-sensitiveprovenance. As the NaCl concentration increased and stress time prolonged, the salt-toleranceprovenance of E. angustifolia has a lesser decrement rate of chlorophyll content, and betterability to maintain the stability of the membrane system, thus all kinds of metabolisms areensured to successfully carry on. In the osmotic adjustment, the proline can regulate the vivomicroenvironment for early stress of E. angustifolia. The soluble sugar is mainly as an energysource in the short-term salt stress and primarily as an osmotic adjustment in the long-termstress, and the salt-tolerance E. angustifolia provenance has stronger regulation ability thansalt-sensitive porenance. Meanwhile, the salt-tolerance E. angustifolia provenance can be moreeffective to obstruct Na+in the roots, reducing leaf Na+content, and has a stronger ability toretain K+for the plants. In addition, the salt-tolerance E. angustifolia provenance has strongerNa+/H+antiporter activity, which increased Na+efflux and decreased K+efflux, thereby theplants can be better to maintain the cytoplasm K+/Na+balance.更多还原