【作者】 董守坤；

【导师】 祖伟；

【作者基本信息】 东北农业大学 ， 作物栽培学与耕作学， 2008， 博士

【摘要】 本试验于2006-2007年进行,采用框栽、砂培以及组培相结合的方法进行。框栽试验,应用¹⁵N示踪技术,选用高蛋白型、高油型、丰产型、饲料型大豆和不结瘤大豆,以其中一个品种为材料,设置高氮、中氮和低氮三个施氨水平,研究大豆氮素积累与来源规律,同时测定谷氨酰胺合成酶和硝酸还原酶活性:砂培试验,以¹⁵N标记的氮肥为氮源,测定大豆氮素来源与构成及谷氨酰胺合成酶、硝酸还原酶活性,并测定硝态氮诱导下的硝酸还原酶mRNA含量;组培试验,以¹⁵N标记硫酸铵或硝酸钾以及两者混合为氮源培养大豆,测定铵态氮和硝态氮吸收比例。运用以上方法,对大豆吸收利用氮素规律及相关酶活性进行了研究,结果表明:大豆氮素吸收表现为明显的时期性,而栽培品种与秣食豆（饲料大豆）有明显差异。（1）无机氮营养期,大豆主要依靠土壤氮和肥料氮。栽培品种的无机氮营养期为出苗—R₁期,在R₁期土壤氮所占比例为83.8%～85.2%,肥料氮所占比例为12.7%～15.0%,根瘤固氮所占比例在3.0%以下;而秣食豆无机氮营养期为出苗—V₄期,在V₄期土壤氮所占比例为75.4%,肥料氮所占比例为15.7%,根瘤固氮所占比例为8.9%。（2）根瘤固氮快速增长期,大豆主要依靠土壤氮和根瘤固氮,而根瘤固氮快速增加。栽培品种表现在R₁—R₅期,根瘤固氮量和所占比例大幅度提高,土壤氮和肥料氮的积累量都有所增加,但其所占比例均下降;栽培品种在R₅期土壤氮所占比例为60.0%～67.9%,根瘤固氮所占比例达到28.3%～36.3%,肥料氮所占比例低于4.0%;而秣食豆表现在V₄—R₅期,在R₅期时根瘤固氮所占的比例达到69.1%,土壤氮所占比例为29.7%,而肥料氮所占比例仅为1.2%。（3）土壤氮和根瘤固氮同步吸收期,既依靠土壤氮,又依靠根瘤固氮。栽培大豆表现在R₅—R₈期,土壤氮和根瘤固氮仍有大量积累,肥料氮积累不明显,而土壤氮和根瘤固氮积累接近;栽培品种R₈期土壤氮所占比例在39.3%～60.5%,根瘤固氮所占比例为37.5%～59.2%,肥料氮所占比例均低于2.0%;秣食豆在这一时期主要依靠根瘤固氮,土壤氮和肥料氮的积累量较少,R₈期根瘤固氮所占的比例高达71.4%。施氮水平对大豆氮的吸收积累有明显影响。（1）在框栽条件下,东农47的氮素积累量大小顺序为:低氮处理＞中氮处理＞高氮处理,差异达到显著水平;根瘤固氮积累量的大小顺序为:低氮处理＞中氮处理＞高氮处理,低氮处理显著高于中氮处理和高氮处理,中氮和高氮处理差异不显著。（2）在砂培条件下,东农47随氮素水平增加,大豆植株中肥料氮所占比例增大,根瘤固氮所占比例减小;在出苗—V₃期、出苗—R₁、出苗—R₂期给予高氮处理,而V₃—R₈期、R₁—R₈期、R₂—R₈期变为低氮处理时,随高氮处理的时间延长氮素积累量减少,其中根瘤固氮积累量随高氮处理的时间延长明显减少,而肥料氮积累量差异不大;从出苗—R₄期、出苗—R₅期给予低氮处理,R₄—R₈期、R₅—R₈期给予高氮处理时,与全生育期给予低氮的处理相比氮素积累量明显增加,其中R₅—R₈期给予高氮的处理肥料氮积累量显著大于R₄—R₈期给予高氮的处理,两个处理根瘤氮积累量差异不显著。施氮水平对大豆产量影响很大。（1）在框栽条件下,东农47的产量大小顺序为:低氮处理＞中氮处理＞高氮处理,低氮处理和中氮处理的产量显著大于高氮处理,而低氮和中氮处理的产量差异不显著。（2）在砂培条件下,低氮处理不能满足大豆对氮的需求,产量较低,而高氮处理因抑制根瘤的生长,也不能获得较高的子粒产量。从出苗—V₃期、出苗—R₁期、出苗—R₂期给予高氮处理,而V3—R₈期、R₁—R₈期、R₂—R₈期变为低氮处理时,随高氮处理的时间延长产量降低;从出苗—R₄期、出苗—R₅期给予低氮处理,R₄—R₈期、R₅—R₈期给予高氮处理时,与全生育期给予低氮的处理相比产量显著增加,但两个处理间差异不显著。由此可见,当外源氮水平较低时,于R₄期或R₅期提高外源氮水平可以增加大豆产量。对大豆产量与子粒中肥料氮所占比例、大豆产量与根瘤固氮所占比例分别作回归分析,回归方程分别为y=-0.017x²+1.329x+25.294、y=-0.017x²+2.091x-12.828,经测验两个回归方程拟合效果很好,经求导分析得出大豆子粒中肥料氮所占比例为39.1%、根瘤固氮所占比例为61.5%时大豆产量最高。由此推断,当外源氮对子粒的贡献率为40%左右、根瘤固氮贡献率为60%左右时,能够同时满足大豆对根瘤固氮和外源氮的需求,有利于大豆产量的形成。铵态氮和硝态氮对大豆的营养作用不同,大豆吸收铵态氮比例大于硝态氮。在无菌条件利用组培方法下培养大豆,当培养基中NH₄⁺-N与NO₃^--N比例为1:1时,大豆吸收NH₄⁺-N与NO₃^--N的比例接近1.5:1,其中叶片、茎、根吸收NH₄⁺-N与NO₃^--N的比例分别为1.6:1、1.4:1和1.6:1。大豆上部幼嫩叶片硝酸还原酶活性较高,中部成熟叶片和下部老化叶片活性较低,品种间表现一致,而大豆叶片硝酸还原酶活性动态品种间存在一定差异。NO₃^-能够诱导iNR的基因表达,经NO₃^-诱导iNR1和iNR2两种同工酶mRNA含量明显增加。大豆上部幼嫩叶片和中部成熟叶片谷氨酰胺合成酶（GS）活性较高,下部老化叶片活性较低,品种间表现一致。各部位谷氨酰胺合成酶（GS）活性随生育进程总的变化趋势一致,但年份间差别较大。更多还原

【Abstract】 The experiment was conducted with frame tests（pot without bottom）,sand culture and tissue culture during the period from 2006 to 2007 year.Labeled ¹⁵N was used in the frame test and the experiment design was as following:genotype and nitrogen were chosen as the main factors,and five genotypes were chosen（high protein,high oil,high yield,forage and root noduleless soybean）, and three nitrogen levels,low,mid and high,were chosen.One genotype was used to study the nitrogen source and accumulation in soybean,determine the activity of nitrate reductase and glutamine synthetase;Labeled ¹⁵N fertilizer was used in sand culture,the nitrogen source and composition,the activity of glutamine synthetase and nitrate reductase,and the mRNA content induced by nitrate nitrogen were mensurated;In the tissue culture experiment,Labeled ¹⁵N ammonium sulfate or potassium nitrate and mixture of them were used as nitrogen source to mensurate the ratio of NH₄⁺-N:NO₃^--N which were absorbed by plants.The results were shown as following:Nitrogen uptake of soybean could be divided into there periods,there was evidence difference between cultivated and forage soybean:（1） Inorganic nitrogen period,when the plants absorbed mainly soil-N and fertilizer-N.The period of cultivated soybean began from seedling to beginning bloom（R₁）.The proportion of soil-N and fertilizer-N was between 83.8%～85.2%and 12.7%～15.0%respectively in R₁,and the nodulation-N was below 3.0%at this stage;The period of MSD began from seedling to V₄ period,the proportion of soil-N,fertilizer-N and nodulation-N was 75.4%、15.7%and 8.9%in V₄.（2） Nodulation-N fast increasing period,when the plants absorbs mainly soil-N and nodulation-N,nodulation-N increased fast.During the period from R₁ to R₅, nodulation-N accumulated fast and the proportion increased in cultivars,both soil-N and fertilizer-N accumulations still increased,but their proportions in plant decreased;The proportions of soil-N and nodulation-N in cultivation soybean were between 60.0%～67.9%and 28.3%～36.3%respectively in R₅,while that of fertilizer-N was less than 4.0%;The period of MSD was from V₄ to R₅,the proportion of nodulation-N already achieved 69.1%in R₅,soil-N was 29.7%, fertilizer-N was only 1.2%.（3） soil-N and nodulation-N synchronization absorption period,the plants absorbed both soil-N and nodulation-N,cultivation soybean began from beginning size stage to full maturity stage（R₅—R₈）,when the nodulation-N and soil-N still had the massive accumulations,and the accumulation of fertilizer-N stagnated;The proportions of soil-N and nodulation-N were between 39.3%～60.5%and 37.5%～59.2%respectively in R₈,and fertilizer-N was less than 2.0%;In this period,MSD absorbed mainly nodulation-N,the accumulation of soil-N and fertilizer-N were few.The proportion ofnodulation-N already reached as high as 71.4%in R₈.N-level used had a significant effect on N-absorption and N-accumulation of soybean. （1）Under frame conditions,the order of nitrogen accumulative amount of DN47 is low-N＞mid-N＞high-N,the difference reached significant level;the order of nodulation-N accumulative amount is low-N＞mid-N＞high-N,and the nodulation-N accumulative amount of low-N was significantly higher than that of mid-N and high-N,but there was no significant difference between mid-N and high-N.（2）Under sand culture,the proportion of fertilizer-N increased as nitrogen level heightened, but that of nodulation-N was on the contrary;Applying higher N level during these periods:from seedling to V₃,seedling to R₁,and seedling to R₂,then applied lower nitrogen level during the periods from V₃ to R₈,R₁ to R₈,and R₂ to R₈,accumulative nodulation-N amount decreased significantly as the higher nitrogen level period prolonged,but that of fertilizer-N had little difference;Applying lower nitrogen level during the periods:from seedling to R₄,and seedling to R₅,then applied higher nitrogen level during the periods from R₄—R₈,and R₅—R₈,accumulative nitrogen amount were higher than that of lower-N treatment in whole period,the accumulative nitrogen amount of the treatment that applying higher nitrogen level during the period from R₅—R₈ was significantly higher than that of the period from R₄—R₈,but there was no difference between accumulative nodulation-N amount of the two treatments.Yield of soybean was affected by N levels（2） Under frame condition,the order of yield of DN47 is low-N＞mid-N＞high-N,and the yield of low-N and mid-N was significantly higher than that of high-N,but there was no significant difference between low-N and mid-N.（2） Under sand culture,the yield of low-N was lower because nitrogen requirement could not be fulfilled,higher-N could not reach high yield became of the inhibition of higher nitrogen on the growth of nodules. Applying higher N level during these periods:from seedling to V₃,seedling to R₁,and seedling to R₂,then applied lower nitrogen level during the periods from V₃ to R₈,R₁ to R₈,and R₂ to R₈,the yield decreased as the higher nitrogen level period prolonged;applying lower nitrogen level during the periods:from seedling to R₄,and seedling to R₅,then applied higher nitrogen level during the periods from R₄—R₈,and R₅—R₈,the yield was higher than that of lower-N in whole period,but there was no significant difference,which indicated that heightened exogenous nitrogen level between R₄ and R₅ and lower nitrogen level in prophase could increase yield of soybean.The regression of yield and proportion of fertilizer-N in seed,yield and proportion of nodulation-N in seed were analyzed,and regression equations were y=-0.017x²+1.329x+25.294 and y=-0.017x²+2.091x-12.828 respectively.The two regression equation provided a better fit,and the result of derivation analysis shown that highest yield could be obtained when the proportion of soil-N and nodulation-N was 39.1%and 61.5%respectively.We could deduce:when the contribution rate of exogenous nitrogen and nodulation-N were about 40%and 60%in seeds respectively,these could satisfy the requirement of soybean and benefit to soybean yield formation. There were a difference between ammonium and nitrate nutrition,and more ammonium was absorbed by soybean than nitrate.Tissue culture in sterile condition was conducted and the result shown that when the ratio of NH₄⁺-N:NO₃^--N in culture medium was 1:1,the rates of NH₄⁺-N: NO₃^--N absorbed by soybean were approximately 1.5:1,and the ratios of NH₄⁺-N and NO₃^--N absorbed by leaf,stem and root were 1.6:1,1.4:1 and 1.6:1.The activity of nitrate reductase（NR） exhibited similarly among different varieties,higher in tender leaf and lower in old leaf,but there were some difference of the activity dynamic of nitrate reductase.Gene of iNR could be induced by NO₃^-,accordingly mRNA of iNR1and iNR2 isoenzymes could be enhanced.The activity of glutamine synthetase（GS） exhibited similarly among different varieties,higher in tender and middle leaves and lower in low leaves.The change trends of activity of glutarnine synthetase in different parts were similar,but there was a great difference among different years.更多还原