【作者】 金婷婷；

【导师】 刘鹏；

【作者基本信息】 浙江师范大学 ， 植物学， 2008， 硕士

【摘要】 本研究首先对19个大豆品种进行耐铝性筛选,选择浙春2号和浙春3号作为实验材料,主要采用水培法,结合砂培法和小盆钵土培法,比较研究了铝胁迫下大豆根系分泌物的主要成分变化和分泌特征;综合分析了铝胁迫下大豆根系分泌物对土壤P和K的活化作用、对大豆种子萌发和幼苗生长的化感作用、与铝的络合作用和清除自由基作用,以及对根际土壤微生物区系的影响、微生物的物质转化功能和土壤酶活性的影响;初步探讨了大豆根系分泌的有机酸在大豆体内与铝的结合和运输,并深入研究了铝胁迫协同柠檬酸分泌抑制剂作用下大豆根尖相关酶活性变化,揭示了铝对大豆根系分泌物的诱导机制。此外,还研究了模拟主要根系分泌物（外源柠檬酸）加入条件下,二大豆品种根系形态、根尖铝积累的荧光染色以及根尖呼吸代谢等生理特性。现将主要研究成果总结如下:1.耐酸铝筛选结果表明,浙春2号的相对根长为127%,明显高于其他品种,表明耐性最强;而2DD06097和浙春3号的相对根长较短,是敏感品种。从品种推广的角度,我们选择优质品种浙春2号和浙春3号作为实验材料。2.柠檬酸是铝胁迫下大豆根系分泌物的特征性物质,而氨基酸,化感类物质以及可溶性糖等成分也可以检测出来。2h铝处理后大豆根系就能分泌一些特征性物质如柠檬酸,以响应铝胁迫;而根系脱离铝处理液6h后,大豆根系基本上不能继续维持正常铝胁迫下的分泌特性,各组分分泌的增长速度减缓。3.大豆根系分泌物对土壤P、K有明显的活化效应,活化效应随分泌物浓度和植株培养时间的增长而缓缓增强。铝胁迫下根系分泌物的活化能力又比正常根系分泌物有所增强。此外,根系分泌物具有一定的螯合Al³⁺的能力和清除DPPH自由基的功能。化感作用结果显示,催芽3d时,大豆根系分泌物显著抑制种子的萌发率和胚根增长,具有明显的自毒作用;而7d时,大豆根系分泌物更多表现出促进效应。4.土壤铝含量较低条件下(0.2,0.4 g kg^-1),大豆根系分泌物的应激分泌促使土壤微生物数量增多、微生物物质转化能力增强和土壤酶活性增大;土壤铝含量较高(0.8 g kg^-1)时,根系分泌物的分泌相对受抑制,土壤微生物活性和土壤酶活性相应地受到抑制;外源根系分泌物也能影响土壤微生态,显示出一定的缓解铝毒能力。5.随铝浓度的增加,浙春2号根际土壤相对于非根际土壤而言,Al³⁺、（AlOH）²⁺、Al（OH）₂⁺等形态铝含量均有所降低,单聚体羟基铝[（AlOH）²⁺、Al（OH）₂⁺]含量的降低尤为显著,而腐殖酸铝（HA-Al）含量有所升高;根系分泌物对根系土壤具有一定的酸化作用,而铝的形态与pH值有直接的关系,所以根系分泌物对铝形态还存在一定的间接影响。6.铝在大豆根部以铝—苹果酸（1:3）复合物和铝—柠檬酸（1:1）复合物二种铝-有机酸复合物形态存在,其化学位移在11.16ppm和-0.38ppm左右,而在茎和叶中,没有检测到铝-有机酸复合物。大豆根茎叶中可溶性铝主要以[AlO₄Al₁₂（OH）₂₄（H₂O）₁₂]形态存在,其化学位移在63.2ppm左右。茎和叶铝的含量比根少,表明大豆抗铝毒作用主要在根部,根部通过分泌有机酸及在根内部形成铝-有机酸复合物来排斥铝和解毒。同时说明大多数铝都不能通过根进入地上部分,一旦铝运输到大豆地上部分,就不能形成铝-有机酸复合物来解毒,而植物体内铝水合物中的铝对植物的毒性较大。7.铝胁迫下大豆根尖细胞质膜H⁺-ATP酶与根系柠檬酸分泌的关系密切,同时柠檬酸合成酶和酸性磷酸酶活性与大豆根系柠檬酸分泌也有关,而苹果酸脱氢酶、磷酸烯醇式丙酮酸羧化酶、NAD-柠檬酸脱氢酶的活性几乎不受影响。柠檬酸分泌抑制剂A9C和柠檬酸载体抑制剂Mersal对柠檬酸分泌的显著抑制作用。8.大豆根尖细胞细胞壁积累铝的现象十分明显,且随着铝处理浓度的增加和处理时间的延长,细胞壁固定的铝逐渐增多;外源柠檬酸存在下,细胞壁铝积累明显减少,浙春3号尤为显著。浙春3号和浙春2号铝积累量随着铝浓度的增大和铝处理时间的延长而呈不同的变化趋势。从铝形态结果分析,可以判断Al_a和Al_b活性铝的含量变化趋于相同,外源柠檬酸作用下,各浓度梯度的Al_a和Al_b活性铝含量均较单铝胁迫时有所降低,而Al_c反而增加。9.水溶性蛋白表达结果表现为低浓度铝利于其表达,而高浓度和较长时间处理则起抑制作用;浙春3号在铝处理12h和24h,蛋白表达量比相应处理下浙春2号明显要低。随铝浓度和处理时间的增加,浙春2号和3号根尖呼吸速率均有不同程度地降低;在外源柠檬酸作用下,呼吸速率较相应浓度对照组有所上升,但在无铝(0μM/L Al³⁺)状态及铝胁迫初期（1h）,浙春2号反而降低。线粒体H⁺-PPase活性变化与呼吸作用相似,随铝浓度和处理时间的上升呈一定下降趋势;而外源柠檬酸作用下,浙春3号的增强比例大于浙春2号。更多还原

【Abstract】 In these experiments,19 high quality varieties and lines of soybean（Glycine max L.）from East and nabe were tested for their tolerance to aluminum toxicity. With solution culture,sand culture and pot culture,two varieties of soybean （Zhechun No.2 and Zhechun No.3）were employed as the main material for the research.The important contents were comparatively study of nutritional components and exudating characteristics of soybean root exudations under aluminum;Synthetically analysis of the effect of soybean root exudates on mobilization of soil sparingly soluble phosphorus and potassium,chelation of Al³⁺,scavenging activity against DPPH free radical,and allelopathy of soybean seedings,as well as microorganism population;biochemical function and soil enzyme;Primarily probe into chelation with almuimun and transportation of important organic acids in soybean and lucubrating the inducing system of aluminum on root exudation of soybean.Further more,the dynamic changes of root morphology,accumulation of Al and respiratory metabolism in root tips were studied.The main research were as follows:1.19 cultivars were analysed and found to differ considerably in aluminum （Al）resistance,it shows that relative root length of Zhechun No.2 was 127%which was universally higher than others for strongest resistance of Al. By contraries,2DD06097 and Zhechun No.3 were most sensitive to Al.So Zhechun No.2（Al-resistant）and Zhechun No.3（Al-sensitive）were selected for further analysis,considering the facts.2.Citric acid was the idiocratic substance in soybean exudation under Al,but amino acid,isoflavone,phenolic acid,carbohydrate and so on were also detected with dose and time matter.The effects of Al stress on soybean exudation had beguin after 2-h induration period,yet Al had a continuous effect on all these components when roots was removed from Al treatment solution within 6-h.But the effect palled on after 6-h.3.The result shows that soybean root exudates has remarkable mobilization ability of soil sparingly soluble phosphorus and potassium;The mobilization effect enhanced slowly with the concentration of root exudate and culture time of seedings,and the activation ability is stronger of root exudates under Al treatment than that of the CK.Furthermore,it has the efficacity of chelating Al and scavenging activity against DPPH free radical by soybean root exudates.The result of allelopathy reveals that it restrains the seed germination and radicle length of soybean as be considered the notable autotoxicity for 3 days of breeding;But it showed promotion effect for 7days of breeding.4.Results indicate that in treatments low in soil Al(0.2 and 0.4 g kg^-1),irritated secretion from the soybean roots increased the population of soil microbes, strengthened their capability of material transformation and stimulated the activity of soil enzymes,whereas in treatments high in soil Al(0.8g kg^-1), root exudation was restrained comparatively,resulting in inhibition of the activity of microorganisms and soil enzymes.The results also demonstrated that exogenous root exudation could also influence the microbial ecosystem.5.Result indicates that exchangeable aluminum[Al³⁺]and[（AlOH）²⁺, Al（OH）₂⁺]of Zhechun NO.2 decreased,while[HA-Al]increased compare to non-rhizosphere soils.It reveals that the root exudates had something to do with the acidification of soil,and the form of aluminium had a direct relationship with pH,so the root secretion also had a certain impact on the form of aluminum indirectly.6.The NMR results show that Al in soybean roots was mainly chelated to citrate and malate in Al-citrate（1:3）and Al-malate（1:1）stoichiometry with the corresponding chemical shifts at 11.16ppm and-0.38ppm respectively, but no Al-organic acid complexes were found in soybean stems or leaves. Dissolubility Al in soybean root,stems or leaves exists in [AlO₄Al₁₃（OH）₂₄（H₂O）₁₂]form with the chemical shift at63.2ppm.It make clear that it is mainly in roots that some important organic acids chelate with almuimun and form complexes with little toxicity which can be transport to shoots.7.The experiment also showed that the activity of H⁺-ATPase had relationship with citrate acids efflux of soybean root,as well as the activity of CS and AP. But MDH,NADP-ICDH and PEPCase associate little with citrate acids efflux.Moreover,citrate carrier inhibitor A9Cand anion channel inhibitor Mersal restrain availably the exudation of citrate acids.8.Results showed it was significant that aluminum accumulated in the cell wall of soybean root tip cells,and it became more and more remarkable with dose and time increased;in the present of exogenous citric acid,the content of aluminum accumulated in the cell wall decreased,especially in Zhechun No.3.The change of comparative Al content in root tips of Zhechun No.3 and Zhechun No.2 did not play the same pattern in dose-and time-dependent manner.From the analysis of aluminum speciation,it could be seen that the change pattern of the content of Al_a and Al_b are parallel;the content of the two form decreased but Al_c increased with exogenous organic acids.9.Results of water-soluble protein’s expression showed that lower aluminum concentration can promote water-soluble protein’ expression,but higher aluminum concentration and longer treatment time inhibited its expression; the degree of water-soluble protein’s expressions in Zhechun NO.3 were obviously lower than that in Zhechun NO.2 after 12 h and 24 h treatments in the same aluminum concentration.With increasing of aluminum concentration and treatment time,the respiratory rate of root tips fell to different extent in both varieties;with the treatment of extraneous source citric acid.The change of Mitochondria’ H⁺-PPase activity was similar to the respiration’s,with increasing of aluminum concentration and treatment time,activity in both varieties fell; with the treatment of extraneous source citric acid,H⁺-PPase activity in Zhechun NO.3 is higher than that in Zhechun NO.2.更多还原