【作者】 查倩；

【导师】 张新忠； 韩振海； 王忆；

【作者基本信息】 中国农业大学 ， 果树学， 2014， 博士

【摘要】 低铁胁迫对于植物来说是一种很普遍的非生物胁迫,机理植物应对低铁逆境存在两种途径：一种是加强根系铁吸收,即低铁胁迫导致植物根际酸化,铁吸收相关基因上调表达；还有另一种是根质外体或细胞器等处中铁元素再利用的加强,表现为低铁胁迫下植物的各种铁载体合成和转运基因上调表达。本研究的试验材料为低铁敏感的苹果砧木山定子(Malus baccata)和耐低铁的苹果砧木小金海棠(Malus xiaojinensis),通过检测40μM Fe正常供铁和4μM Fe低铁处理下生理生化指标和相关基因表达变化,发现：(1)正常供铁条件下小金海棠根吸收相关基因FRO2、IRT1和NRMAP1铁转运相关基因CS1和FRD3表达量较山定子显著高,小金海棠叶绿素含量和根、叶活性铁含量均显著高于山定子,山定子遭遇低铁胁迫后第3d叶活性铁含量降低到84.3mg/kg,表现明显黄化,而小金海棠遭遇低铁胁迫后第9d叶活性铁含量降低到75mg/kg,表现轻微黄化。(2)低铁胁迫发生第1-3d小金海棠根系Fe铁吸收相关基因HA7、FRO2和IRT1和柠檬酸铁转运相关基因CS1和FRD3持续显著升高,低铁胁迫第3d,小金海棠的根际pH显著降低,根际三价铁还原酶活性从第3d开始迅速升高,根中的柠檬酸含量持续显著增加,最终低铁胁迫发生后第1-3d叶中活性铁每日降率(DRPD值)低于山定子(DRPD值),而山定子中铁吸收不加强。(3)低铁胁迫下铁转运表现为成熟叶柠檬酸含量和尼克酰胺含量显著增多,基因CS1、FRD3、NAS1和叶细胞体内二价铁转运相关基因NRAMP3上调表达。山定子在低铁处理第1d,叶活性铁含量达到103.67mg/kg左右即出现上述变化,而小金海棠在低铁处理第6d,叶活性铁降低到107mg/kg时加强铁转运。低铁胁迫下,植物感受低铁信号介导其下游的低铁适应性反应。通过对小金海棠进行低铁胁迫下的转录组测序,发现一系列与信号物质相关的合成基因和信号通路基因在根系上调表达,有乙烯、NO, IAA和H202。本试验通过检测小金海棠根系在不同处理下生理生化指标和基因表达变化,发现：(1)低铁胁迫发生后,小金海棠的根系积极响应低铁逆境：NO含量在24h显著增多,ROS含量在36h显著减少,乙烯含量在36h显著升高；低铁胁迫诱导HA2基因和IRT1基因在24h显著上调表达；(2)分根低铁处理的低铁侧24hROS产量开始降低,NO作为抑制ROS产生的信号,促进基因HA2表达,此过程中植物体内ROS的变化可以改变氧化还原作用,抑制IRT1基因表达。(3)介质酸化和HA2基因上调表达在分根低铁处理两侧均有。发现乙烯被抑制IRT1基因也被抑制表达,而乙烯在分根低铁处理的低铁侧产生被隔绝,不能移动到供铁侧,所以在低铁处理和分根低铁处理的低铁侧,乙烯可以优先调控IRT1的上调表达。所以低铁胁迫下小金海棠介质酸化和IRT1基因加强表达是受不同信号调控的。细胞内金属离子浓度对维持细胞平衡作用重大,NRAMP蛋白是一个负责细胞水平上金属离子转运的蛋白。本研究从小金海棠上克隆到两个基因MxNRAMP1和MxNRAMP3,通过进化树分析和跨膜区分析进一步确定这两个基因；利用共聚焦荧光显微镜观察到,MxNRAMP1定位在质膜上；MxNRAMP3定位在液泡膜上。并发现MxNRAMP1和MxNRAMP3基因表达与铁、镉、锰、锌胁迫相关。更多还原

【Abstract】 Iron deficiency is one of the most hazardous abiotic stresses for plants. For strategy I plants, one mechanism of iron deficiency tolerance is enhanced iron uptake and distribution in the roots. Another potential mechanism is the remobilisation of iron stored in organdlles under iron-deficient conditions. To better understand the mechanism of low-iron stress tolerance in Malus xiaojinensis, the differences in physiological parameters and iron uptake and transport-related gene expression between an iron deficiency-sensitive species, M. baccata, and an iron deficiency-tolerant species, M. xiaojinensis were investigated under low-iron (4μM Fe) conditions.(1) Under iron sufficient conditions, the expressions of iron uptake-and transport-related genes, i.e. FIT1, IRT1, CS1, FRD3and NRMAP1, and the immanent leaf and root active iron contents were higher in M. xiaojinensis than those in M. baccata. On day3of low iron stress, the leaf active iron contents of M. baccata decreased to84.3mg/kg and the leaf appeared chlorosis; on day9of low iron stess, the leaf active iron contents of M. xiaojinensis decreased to75mg/kg and the leaf apprared slightly chlorosis (2) However, on the first three days of low iron stress, the rhizospheric pH decreased and the root ferric chelate reductase (FCR) activity and the expression of ferrous uptake-and iron transport-related genes in the roots increased significantly only in M. xiaojinensis. Leaf chlorosis occurred on the3rd and the9th day after low-iron treatment in M. baccata and M. xiaojinensis, respectively. On the first3days of low iron stress, the DRPD of M. xiaojinensis lower than the DRPD of M. baccata. However, the iron uptake system did not strengthene in M. baccata.(3) The expression of iron relocalization-related genes, such as NAS1, FRD3and NRMAP3, increased after the5th or6th day of low iron stress in leaves of M. xiaojinensis, whereas the expression of NAS1, FRD3and NRMAP3in the leaves of M. baccata increased immediately after the onset of low iron treatment. On the day1of low iron stess, the leaf iron contents of M. baccata became to about103.67mg/kg, the iron relocalization strengthened; on On the day1of low iron stess, the leaf iron contents of M. xiaojinensis became to107mg/kg, the iron relocalization strengthened.Under low iron stress, plant porduced some signals to downstream the iron deficiency response. Through the transcriptome sequencing of M. xiaojinensis under low iron stress, found the expression of a series of related signal substance synthesis genes and signal pathway genes in root increased, including ethylene, NO, auxin and hydrogen peroxide. By detecting different physiological parameters and gene expression in the roots of M. xiaojinensis under treatments, we found:(1) ROS increased at the fisrt time of low iron stress, and then NO occurred low iron stress to clean ROS, so ROS decreased on the36h of low iron stess. ETH increased on the36h of low iron stress. The gene expression of HA2and IRT1increased on the24hr of low iron stress.(2) On the24h of Low iron stress, ROS began to decrease, NO as the signal to inhibit the production of ROS, promoted the gene expression of HA2, and changed the ROS in the plants. This process can change the redox to inhibit the expression of IRT1gene.(3) inhibition of ETH declined the expression of IRT1gene. And ETH in the-Fe side of split-root treatment had to isolated and cannot move to the other side. So in the low iron stress and the-Fe side of split-root treatment ETH can eliminate the inhibitory effects of NO and ROS on IRT1expression.The intracellular metal ion concentration of major to maintain cell balance effect. NRAMP protein take response to intracellular transport of metal ions. We isolated two genes MxNRAMPl and MxNRAMP3from Malus xiaojinensis, and identify the role of those genes through phylogenetic analysis and further analysis of transmembrane region. we used confocal fluorescence microscopy to identify that:MxNRAMPl located on the plasma membrane; and MxNRAMP3positioned in the vacuole membrane. Gene expression of MxNRAMP1and MxNRAMP3have relationship with Fe, Cd, Mn, Zn stress.更多还原