【作者】 聂兆君；

【导师】 胡承孝；

【作者基本信息】 华中农业大学 ， 植物营养学， 2014， 博士

【摘要】 钼是植物必需的微量元素之一。不同作物对钼的需要量和吸收能力不同,因此植物钼营养存在基因型差异。钼高效品种97003和钼低效品种97014在干物质量、产量、钼含量及累积量、含钼酶及其调控过程等方面均存在显著差异。本文在已有研究的基础上,通过盆栽、根箱及营养液培养实验,应用生理生化研究技术,深入研究了两个冬小麦品种对钼吸收利用的差异及其调控机制。主要结果如下：1.研究了两个冬小麦品种根际过程对土壤钼生物有效性影响的差异。结果表明：缺钼条件下,两个冬小麦品种根区土壤有效钼含量显著高于不种植物的对照,且显著高于非根区土壤；施钼条件下,两个冬小麦品种根区土壤有效钼含量显著低于不种植物的对照,且显著低于非根区土壤。说明在缺钼条件下,冬小麦能够通过根系活化土壤钼以适应缺钼胁迫。缺钼条件下,两个冬小麦品种根区土壤pH显著高于不种植物的对照,且显著高于非根区土壤。说明冬小麦根际过程能提高根区土壤pH进而提高土壤钼的有效性。两个冬小麦品种根系N03-的吸收和NH4+的外排导致了H+的内流,说明H+内流是冬小麦根系提高根区土壤pH的原因。两个冬小麦品种根系活化土壤钼存在差异。缺钼条件下,97003根区土壤有效钼提高的幅度高于97014；施钼条件下,97003根区土壤有效钼降低的幅度低于97014,说明97003根系活化土壤钼的作用强于97014。缺钼条件下,97003外排较少的NH4+和吸收较少的N03-导致了较低的H+内流,进而导致了较低的根区pH。但是,97003根系较强的活化土壤钼的能力可能与较低的H+内流导致较低的根区土壤pH无关。2.研究了两个冬小麦品种钼吸收、转运及同化相关蛋白基因表达的差异。结果表明：两个冬小麦品种根系TaSultr5.1、TaSultr5.2和TaCnxl基因表达与钼吸收量密切相关。根系TaSultr5.2基因表达与钼吸收速率密切相关。当供钼水平低于1μmol、L时,97003具有较强的钼吸收能力,这与97003根系较高的TaSultr5.1、 TaSultr5.2和TaCnxl基因表达有关。相反,当供钼水平高于5μmol、L时,97003根系TaSultr5.2和TaCnx1基因的下调表达导致了其较弱的钼吸收能力。说明TaSultr5.1、TaSultr5.2和TaCnx1基因的不同表达模式可能是两个冬小麦品种不同供钼水平下钼吸收能力差异的原因。3.研究了竞争离子对两个冬小麦品种钼吸收转运相关蛋白基因表达的影响。结果表明：硫和磷的缺乏分别提高了两个冬小麦品种地上部以及根系钼含量、钼迁移系数及单株钼累积量,表明缺硫和缺磷均能促进冬小麦钼的吸收和迁移。同时,硫和磷的缺乏分别降低了两个钼水平下根系TaSultr1.1和TaPht1.1基因表达,提高了TaSultr5.1和TaSultr5.2基因表达。说明缺硫和缺磷提高TaSultr5.1和TaSultr5.2基因表达可能是缺硫和缺磷促进小麦钼吸收和迁移的原因。两个冬小麦品种钼吸收转运相关蛋白的基因表达对缺硫和缺磷的响应程度不同。4.研究了两个冬小麦品种钼辅因子合成相关蛋白基因表达的差异。结果表明：缺钼条件下,97003叶片或根系NR和SO活性高于97014,叶片AO和XDH活性低于97014；施钼条件下,97003叶片SO活性同样高于97014,根系SO活性低于97014；叶片AO和XDH活性低于97014,根系AO和XDH活性高于97014。缺钼条件下,97003叶片NR基因表达显著低于97014、叶片及根系AO和XDH基因表达显著高于97014；施钼条件下,97003叶片及根系AO和XDH基因表达显著低于97014,说明两个冬小麦品种含钼酶活性和基因表达在不同钼水平下表现出不同的差异。缺钼和施钼条件下,97003叶片和根系TaCnx2和TaCnx5基因表达显著低于97014,说明97003体内有较少的环状蝶呤单磷酸盐(cPMP)和钼喋呤(MPT)的累积。97003叶片和根系TaCnxl基因表达显著高于97014,说明97003体内有更多的钼辅因子(Moco)合成。97014根系具有较高的TaAba3基因表达,可能与钼辅因子相对缺乏诱导TaAba3基因表达有关。综上所述,两个冬小麦品种吸收利用土壤钼差异的可能机制是：缺钼条件下,钼高效品种97003根区土壤有效钼含量高于钼低效品种97014,说明97003具有较强的根系活化土壤钼的能力；97003根内钼吸收转运蛋白TaSultr5.2和TaSultr5.1基因表达显著高于97014,说明97003具有较强的钼吸收转运能力；97003体内钼同化过程关键蛋白TaCnx1基因表达显著高于97014,说明97003具有较强的钼辅因子生物合成能力。因此,冬小麦钼高效品种高效吸收利用土壤钼在于其在缺钼条件下具有较强的活化土壤钼的能力、钼吸收转运能力以及钼辅因子生物合成能力。其中,钼吸收转运能力的差异是两个冬小麦品种吸收利用土壤钼差异的主要原因。更多还原

【Abstract】 Molybdenum (Mo) is an essential trace element for higher plants. Plants can have genotypic differences in Mo uptake and use. There were significient differences in dry matter (DM), grain yield, Mo concentration, Mo accumulation, Mo enzyme activities and their regulating processes between Mo-efficient winter wheat cultivar97003and Mo-inefficient winter wheat cultivar97014. Based on the results of earlier studies, soil and solution culture experiments were conducted to investigate the differences in Mo uptake, utilization and its regulation mechanisms between the two winter wheat cultivars. The main results were as follows.1. Rhizobox and hydroponic culture experiments were conducted to investigate the difference in rhizosphere precesses affecting availability of Mo between the two cultivars.The results showed that the available Mo concentration in the rhizosphere soil of the two cultivars was significantly inceased relative to the control without plants, and Mo concentration in the rhizosphere soil of the two cultivars was significantly higher than that in the non-rhizosphere soil in the-Mo treatment. In the+Mo treatment, the available Mo concentration in the rhizosphere soil of the two cultivars was significantly lower relative to the control without plants, and that in the rhizosphere soil of the two cultivars was significantly lower than that in the non-rhizosphere soil. This suggests that winter wheat can adapt to Mo deficiency stress through root activation of soil Mo. Under Mo-deficient conditions, the pH in the rhizosphere soil of the two cultivars was significantly inceased relative to the control without plants, and in the rhizosphere soil of the two cultivars was significantly higher than that in the non-rhizosphere soil. This indcates that rhizosphere processes of winter wheat can increase pH of the rhizosphere soil and thus increase the availability of Mo in the rhizosphere soil. The two cultivars absorbtion of NO3-and excretion of NH4+induced an influx of proton (H+), suggesting that H+influx into the roots of winter wheat might be the reason for increased pH of the rhizosphere soil. The two cultivars had differential abilities for activating soil Mo via rhizosphere processes. The available Mo in the rhizosphere soil of97003was higher than that in the rhizosphere soil of97014both in the-Mo and+Mo treatments. This suggests that97003has a greater ability for activating soil Mo. The pH of the rhizosphere soil of97003was lower than that of97014, which might be due to a lower net and mean H+influx into97003under Mo-deficient conditions.97003absorbed less NO3-and excreted less NH4+than97014, and thus induced a lower H+influx. However, the differences in the pH of the rhizosphere soil between the two cultivars might be nothing to do with the difference in the available Mo in the rhizosphere soils.2. A hydroponic culture experiment was conducted to investigate the difference in the expression of gene related to Mo uptake, transport and assimilation between the two cultivars.The results indicated a close relationship between Mo uptake and TaSultr5.1, TaSultr5.2and TaCnxl expression, according to a stepwise regression analysis of the time course of Mo uptake in the two cultivars. Meanwhile, expression of TaSultr5.2in roots also showed a positive relationship with Mo uptake rates.97003had stronger Mo uptake than97014at low Mo-application rates (less than1μmol Mo L-1) due to the higher expression of TaSultr5.2, TaSultr5.1and TaCnxl in roots. In contrast, Mo uptake of97003was weaker than97014at high Mo application rates (ranging from5to20μmol Mo L-1), which was related to significant down-regulation of TaSultr5.2and TaCnx1genes in roots of97003compared to97014. This indicates that the differential-expression intensities of TaSultr5.2, TaSultr5.1and TaCnxl could be the cause of the difference in Mo uptake between the two cultivars at low and high Mo application levels.3. A hydroponic culture experiment was conducted to investigate the influence of competitive ions on the expression of gene related to Mo uptake and transport in the two cultivars. The results showed that S and P deficiency increased Mo concentrations in shoots and roots, Mo translocation coefficients, and accumulation per plant, respectively. This shows that S and P deficiency can enhance Mo uptake and translocation in winter wheat, respectively. Meanwhile, S and P deficiency decreased the expression of TaSultr1.1and TaPht1.1but increased expression of TaSullr5.1and TaSultr5.2in roots of the two cultivars. This shows that S and P deficiency can enhance Mo uptake and translocation, which might be related to the increased TaSultr5.1and TaSultr5.2expression. There were different responses to S and P deficiency in Mo uptake, Mo translocation and expression of gene relted to Mo uptake and translocation between the two cultivars.4. A hydroponic culture experiment was conducted to investigate the difference in the expression of gene related to Moco biosynthesis between the two cultivars. The results showed that the activities of nitrate reductase (NR) and sulfite oxidase (SO) in leaves or roots of97003were higher than those of97014, but the activities of aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) in leaves of97003were lower than those of97014under Mo-deficient conditions. Under Mo-sufficient conditions,97003had higher SO activity in leaves, AO and XDH activities in roots but lower SO activity in roots, and AO and XDH activities in leaves than97014. The expression of NR in leaves of97003was lower than that of97014, but the expression of AO and XDH in leaves and roots of97003were lower that those of97014in the-Mo treatment.97003showed lower expression of AO and XDH in leaves and roots than97014in the+Mo treatment. This suggests that the difference in Mo enzyme activities and their gene expression between the two cultivars was different under Mo-deficient and Mo-suficient conditions respectively.97003had lower expression of TaCnx2and TaCnx5in leaves and roots than97014, which might result in the lower accumulation of cyclic pyranopterin monophosphate (cPMP) and molybdopterin (MPT) in97003. The expression of TaCnxl in leaves and roots of97003was significantly higher than that of97014, indicating more Moco formation in97003.97014had higher expression of TaAba3in roots than97003, which might be related to the increase of TaAba3expression incuded by the Moco shortage.Possible mechanisms of differences in the Mo uptake and utilization between two cultivars are summarized. Under Mo-deficient conditions, the available Mo concentration in the rhizosphere soil of97003was higher than that in the rhizosphere soil of97014, which suggested that97003had a stronger ability of activating soil Mo via rhizosphere processes; The expression of TaSultr5.2and TaSultr5.1in roots of97003were higher than those of97014, which suggested that97003had a stronger ability of Mo uptake and transport; The expression of TaCnxl in leaves and roots of97003were higher than those of97014, which suggested that97003had a stronger ability of Moco biosynthesis. Therefore, under Mo-deficient conditions, Mo-efficient cultivar using soil Mo efficiently was due to the stronger ability of activating soil Mo, Mo uptake and transport, and Moco biosynthesis. The difference in the ability of Mo uptake and transport might be the most important reason for the difference in the Mo uptake and utilization between the two winter wheat cultivars.更多还原