【作者】 贺忠群；

【导师】 邹志荣； 张志斌；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2007， 博士

【摘要】 本研究以“中杂9号”番茄为试材,在筛选适于番茄有机土栽培的菌种基础上,对AMF提高番茄耐盐的生理及分子机制进行了系统研究,主要结果如下:1.有机土上番茄接种6种不同的丛枝菌根真菌（Glomus versiforme,Glomus mosseae-2, Glomus intraradices, Glomus diaphanum, Glomus mosseae, Glomus etunicatum）均不同程度上促进了植株生长,并筛选出Glomus versiforme, Glomus mosseae-2是对番茄生长促进效果最好的菌种。接种Glomus versiforme, Glomus mosseae-2后,干物重分别比对照高75%和65%, Glomus versiforme, Glomus mosseae-2用于有机土栽培具有较大的生产潜力。2.用不同浓度NaCl（0.5%,1%）对接种Glomus mosseae-2和未接种番茄处理后,研究Glomus mosseae-2对番茄耐盐性的影响。结果表明,持续盐胁迫下与未接菌株相比,接菌株具有较大的叶面积、地上部茎流量和根系活力,因而表现较强的地上及地下部生长。盐胁迫虽抑制菌根的形成,但随盐浓度增加,菌根效应并未减小。在同一盐胁迫浓度下,接种AMF能提高番茄的耐盐系数,因而提高番茄的耐盐性。3.在0.5%和1%NaCl胁迫下,对接菌及未接菌番茄营养吸收平衡及离子毒害机制进行了分析。结果表明,番茄接种Glomus mosseae-2,显著提高了地上部及根系N、P、K⁺和Ca²⁺的含量,显著降低Na⁺含量,对Cl^-含量虽有减少但无显著影响。接种AMF还显著影响盐胁迫下植株的营养吸收及平衡,增加地上部及根系K⁺/ Na⁺、P/ Na⁺、Ca²⁺/ Na⁺及根系P/Cl^-值。这些比值与植株总干重均呈显著正相关,其中与K⁺/ Na⁺、P/ Na⁺、Ca²⁺/ Na⁺相关性最大。接菌番茄耐盐性提高与AMF改善植株营养状况尤其是提高K、P含量,保持较高K⁺/ Na⁺、P/ Na⁺、Ca²⁺/ Na⁺比值、降低植株Na⁺含量从而降低Na⁺对植株的毒害作用有关。4.为研究AMF降低Na⁺对番茄毒害作用的分子机理。首次用RT-PCR、Realtime-PCR技术对接种Glomus mosseae-2及未接菌株叶片及根系液泡膜Na⁺/H⁺逆向转运蛋白基因（LeNHX1）mRNA表达进行分析。结果发现,无NaCl处理时,接菌与未接菌株LeNHX1基因表达量无显著差异。盐处理后,盐胁迫诱导接菌及未接菌株LeNHX1基因的大量表达,但接菌株LeNHX1基因的表达量低于未接菌株,AMF对LeNHX1没有显著诱导作用。说明Glomus mosseae-2能提高番茄的耐盐性与LeNHX1基因关系不大,即接菌番茄降低植株Na⁺毒害作用,不是通过AMF诱导LeNHX1基因的过量表达,使Na⁺区域化至液泡中这条途径来实现的。5.不同浓度NaCl（0.5%和1%）持续胁迫40 d过程中,对接种Glomus mosseae-2番茄有机渗透调节物质含量的研究分析表明,盐胁迫下,与未接菌番茄相比,接种AMF番茄能显著促进叶片和根系可溶性糖的积累、增加叶片可溶性蛋白含量及根系脯氨酸含量,使植株耐盐能力增强。接菌株可溶性蛋白的增加,尤其可溶性糖以及根系脯氨酸的大量积累在AMF提高番茄耐盐的渗透调节机制中具有重要的作用。6.在不同浓度NaCl（0.5%和1%）持续胁迫过程中,在无NaCl处理和NaCl处理下,AMF能显著提高超氧化物歧化酶（SOD）、过氧化物酶（POD）、抗坏血酸过氧化物酶（APX）及谷胱甘肽过氧化物酶（GSH-PX）活性,过氧化氢酶（CAT）活性被AMF短暂地诱导后与其相应未接菌株无显著差异;AMF能显著减小盐胁迫下番茄细胞膜透性和膜脂过氧化,因而提高了番茄的耐盐性;接菌番茄耐盐性的提高主要与AMF增强SOD、POD、APX和GSH-PX活性,进而增强清除氧自由基的能力有关,而与CAT关系不大。7.在0.3%,0.6%,1%NaCl持续胁迫下,盐胁迫降低了番茄净光合速率和光饱和点,AMF虽然未提高番茄的光饱和点,但能提高叶片净光合速率（Pn）、蒸腾速率（Tr）、气孔导度（gs）、表观量子产量（AQY）、CO₂羧化效率（CE）,同时提高了叶绿体光合磷酸化活性,有利于维持叶绿体吸收光能的能力,因此,接种AMF能提高番茄光能转化效率及CO₂的利用效率;接菌与未接菌株在0.3-0.6%盐浓度处理时,光合作用下降主要受气孔限制,1%NaCl处理28 d后,光合作用下降主要受非气孔因素影响,而接种AMF能提高盐胁迫下番茄的光合作用,增强植株生长及耐盐性。8.在0.3%,0.6%,1%NaCl胁迫下,生长促进物质IAA、GA3和Zeatin含量下降,生长抑制物质ABA含量增加,而接种Glomus mosseae-2增加了这些激素的含量。菌根形成过程中AMF参与调节内源激素平衡,同一盐浓度下与未接菌株相比,AMF降低叶片ABA/IAA、ABA/GA3、ABA/Zeatin及ABA/ （IAA+GA3+Zeatin）的比值。通过gs与ABA/Zeatin比值的相关分析,gs和ABA/Zeatin值呈极显著负相关。同一盐浓度下,接菌株有较高gs和较低的ABA/Zeatin值,而未接菌株有较高的ABA/Zeatin值和较低的gs。与未接菌株相比,接菌株较高ABA含量并没导致较小的气孔导度,ABA和Zeatin共同调节气孔对盐胁迫的响应,维持接菌株较高的气孔导度,从而维持较高的光合作用,增强番茄的耐盐性。通过外源IAA、GA3对番茄相关耐盐指标（植株干物重、可溶性糖、可溶性蛋白等）的影响研究发现,外源IAA、GA3能增强番茄的耐盐性,由AMF诱导的内源IAA、GA3可起到类似的作用,与AMF提高番茄耐盐性密切相关。9.在0.5%、1%NaCl胁迫下,随盐浓度增加和盐胁迫持续番茄叶片相对含水量、叶片水势及叶片水分利用率、根系水导均不同程度降低,接种AMF番茄能显著提高这些指标的值。接种AMF后具有减缓番茄受盐害而失水的作用,这种作用在较高盐浓度下表现得更为明显。为进一步研究盐胁迫下AMF促进番茄水分吸收的分子机制,用RT-PCR、Realtime-PCR技术分析了盐胁迫下接菌及未接菌番茄叶片及根系中的LePIP1,LePIP2, LeTRAMP,LeAQP2（质膜水孔蛋白基因）,LeTIP（液泡膜水孔蛋白基因）5种水孔蛋白基因mRNA的表达量。结果发现,AMF及盐胁迫均在转录水平上调控了这5个基因的表达。AMF及盐胁迫对以上各基因的调控表达在叶片和根系中有很大差异即表现出组织表达差异性。盐胁迫下,与未接菌株相比,这5个基因在叶片中均过量表达,说明这些水孔蛋白基因参与了水分的跨膜转运,有利于水分在叶片中快速转运,因此利于维持接菌株较高的叶片水势。而在根部,与未接菌株相比,LePIP1、LePIP2、LeTRAMP、LeTIP基因表达下调, LeAQP2则上调,因此, LeAQP2基因在接菌株根中的过量表达与盐胁迫下AMF提高番茄根系水导有关。盐胁迫下,其它基因在接菌或未接菌株根系中的表达,由于AMF与盐胁迫共同对其基因表达的上调或下调以不同调控方式参与了渗透调节。更多还原

【Abstract】 In this thesis, on the bases of studies on selecting AMF strains to suit to organic culture, a series of studies had been conducted on physiological and molecular mechanisms of salt tolerance improved by arbuscular mycorrhizal fungi in tomato （Lycopersicon esculentum L. Var. zhongza 9）. The main results were as follows:1. when tomato seedlings inoculated with six AMF strains （ Glomus versiforme, Glomus mosseae-2, Glomus intraradices, Glomus diaphanum, Glomus mosseae, Glomus etunicatum1） under organic culture, positive effect on growth in AM plants was observed compared to CK and Glomus versiforme, and Glomus mosseae-2 were screened out as the best strains for tomato growth. Glomus versiforme, and Glomus mosseae-2 increased dry weight by 75% and 65% respectively which indicated that the two AMF strains may be potential in organic culture.2. The effects of Glomus mosseae-2 on salt tolerance of tomato were studied under 0.5% and 1% NaCl stress. The results showed that higher leaf area, root activity and stem sap flow were detected in AM tomato than corresponding non-AM plants under continuous salt stress which lead to higher shoot and root growth. Although formation of mycorrhizae was restrained by salt stress, the mycorrhizal benefit still increased with salt concentration increasing. Under the same NaCl concentration, coefficient of salt tolerance was improved , and so salt tolerance was enhanced.3. Nutrition absorbing in AM and non-AM tomato and mechanisms of ion damage to plant were studied under 0.5% and 1% NaCl stress. N, P, K +and Ca+ content improved and Na⁺ decreased significantly, but Cl^- content decreased indistinctively after AMF inoculation. Under the same salt stress, higher K⁺/ Na⁺, P/ Na⁺, Ca²⁺/ Na⁺ and P/Cl^- ratio were observed in AM plants compared to corresponding non-AM plants. These values of ratio had significantly positive relation with dry weight and K⁺/ Na⁺, P/ Na⁺, Ca²⁺/ Na⁺ were more closely related with dry weight. So enhanced salt tolerance in AM tomato were related with improved nutrition condition, especially higher K, P content, K⁺/ Na⁺、P/ Na⁺ and lower Na⁺ content which reduced Na⁺ damage to plant.4. To study mechanisms of the reduced Na⁺ damage to plant induced by AMF under salt stress, RT-PCR, Realtime-PCR were performed to assay the vacuolar Na⁺/H⁺ antiporter （LeNHX1） mRNA level in leaves and roots of AM and non-AM tomato. The results showed that expression of LeNHX1 gene in AM tomato was similar to that of non-AM plant under non- NaCl condition. Expression of LeNHX1 gene was induced by NaCl stress in AM and non-AM plants, but AMF had no significant effect on LeNHX1 mRNA level and lower expression was observed in AM tomato. So mechanisms of the reduced Na⁺ damage to plant induced by AMF was in little relation to LeNHX1 which can export Na⁺ from the cytosol to the vacuole across the tonoplast.5. The effects of AMF （Glomus mosseae-2） on osmotic adjustment matter content of tomato were studied under continuous NaCl stress （0.5% and 1%） for 40 days. The results showed that AMF-inoculation significantly promoted the accumulation of soluble sugar in leaves and roots, increased soluble protein in leaves and proline content in roots under salt stress, so salt tolerance of tomato was improved . The accumulation of soluble protein, soluble sugar and root proline induced by AMF played an important role in osmotic adjustment mechanism of enhanced salt tolerance in AM tomato.6. The effect of Glomus mosseae-2 on cell membrane damage and the antioxidants responses in leaves and roots of AM tomato and control were examined under different NaCl stress （ 0.5% and 1%） for 40 days. The results indicated that superoxide-dismutase （SOD）, ascorbate peroxidase （APX）, peroxidase （POD）, Glutathione peroxidase （GSH-PX） activity in leaves and roots of AM symbiosis were significantly higher than corresponding non-AM plants under NaCl stress or non- NaCl condition. However, CAT activity was transient significantly induced by AMF and then suppressed to a level similar with non-AM seedlings. AMF significantly reduced cell membrane osmosis and membrane peroxidation in leaves and roots in salinity. So, the salt tolerance of tomato was enhanced by AMF. This research suggested that the enhanced salt tolerance in AM symbiosis was mainly related with the elevated SOD, POD, APX and GSH-PX activity by AMF which degraded reactive oxygen species and so alleviated the cell membrane damages under salt stress.7. Under 0.3%, 0.6%, 1%NaCl stress, the net photosynthetic rate （Pn）, light saturation point （LSP） was decreased in salinity, whereas AMF improved Pn, stomatal conductance （gs）, transpiration rate （Tr）, apparent quanturn yield （AQY）, and carboxylation efficiency （CE） remarkly, although LSP in AM plants was not increased. At the same time, AMF improved photosphorylation activity in chloroplast which was favour to keeping higher ability to absorb light energy. So, AM tomato had higher capacity of CO₂ fixation and light energy exchange compared to corresponding non-AM tomato. Under 0.3-0.6% NaCl stress, the decline of Pn was caused mainly by stomatal factors, but under1% NaCl stress for 28 d, the decline of Pn was caused mainly by non-stomatal factors, whereas AMF improved photosynthesis in AM tomato and consequently improved plant growth and enhanced salt tolerance.8. Under 0.3%, 0.6%, 1%NaCl stress, the content of endogenous IAA, GA3, Zeatin in leaves and roots of AM and non-AM decreased and the content of ABA increased, whereas AMF inoculation improved the content of these endogenous hormones. AMF adjusted the balance of endogenous hormones and decreased the ratio of ABA/IAA、ABA/GA3、ABA/Zeatin and ABA/（IAA+GA3+Zeatin） compared to corresponding non-AM plants. The correlation analysis showed a significant reverse relation between gs and the ratio of ABA/Zeatin. AM tomato had higher gs and lower ratio of ABA/Zeatin than that of corresponding non-AM plants. So, higher ABA content did not led to lower gs compared to non-AM plants and ABA and Zeatin adjusted response of stoma to salt stress jointly and kept higher gs than non-AM ones which lead to higher photosynthesis. Studies on plant growth, dry weight, and other physiological item （soluble sugar, soluble protein and MDA content） when exdohormones （IAA, GA3） applied showed that IAA, GA3 improved salt tolerance of AM or non-AM tomato. Similarly, it also indicated that IAA, GA3 induced by AMF can also enhanced salt tolerance of AM tomato.9. Relative water content （RWC）, water potential and water use efficient （WUE） in leaves of AM and non-AM tomato were examined under NaCl （0.5%, 1%） stress. The results showed that RWC,water potential,WUE declined with NaCl concentration increasing under continuous salt stress, however, AMF improved the value of these parameters. Water absence due to salt stress can be alleviated in AM tomato and the positive effect of AMF on water absorb was more obvious compared to non-AM plants especially under high NaCl （1%） stress. Consequently, AM tomato had higher capability to keep the water balance in plant under salt stress. At the same time, water condunce affected by AMF and salinity jointly. Salt stress decreased Lp and higher Lp in roots of AM plants also observed.To study molecular mechanism of water absorbing enhanced by AMF deeply, RT-PCR and Realtime-PCR was firstly used to study mRNA levels of five aquaporin genes （LePIP1, LePIP2, LeTRAMP, LeAQP2 and LeTIP）in leaves and roots of AM and non-AM tomato under 1% NaCl stress. The results indicated that the five aquaporin genes were all regulated at transcriptional level by AM fungi and salt stress. The expression of the five genes adjusted by AMF or salt stress was significantly different in leaves and roots. Higher level of the five genes mRNAs were observed in leaves compared to corresponding non-AM tomato under salt stress which indicated that overexpression of the five genes facilitated water transport across biomembranes, thus AM tomato can transport water faster and keep higher leaf water potential. However, LePIP1, LePIP2, LeTRAMP, LeTIP were down-regulated in roots of AM tomato in salinity compared to that of non-AM ones, but expression of LeAQP2 gene in AM tomato roots was higher than corresponding non-AM plants. Accordingly, overexpression of LeAQP2 gene in roots may be relation to enhanced root conductivity due to AMF. Under salt stress, other genes （LePIP1, LePIP2, LeTRAMP, LeTIP） in roots of AM or non-AM tomato may involve in osmosis adjustment in different ways by up-regulating or down-regulating the expression of these genes by salt stress and AMF jointly更多还原