【作者】 王玉波；

【导师】 王庆祥；

【作者基本信息】 沈阳农业大学 ， 作物学， 2011， 博士

【摘要】 本研究于2009年、2010年在东北农业大学校内和香坊科研实习基地进行。校内试验以二倍体纯系品种甜研7号为材料,运用RT-PCR和5’/3’-RACE法首次进行了NiR基因cDNA全长的克隆；田间框栽试验以当前生产主栽品种KWS0143为材料,采用测土配方施肥项目中推荐的“3414”肥料效应田间试验设计方案,进行氮磷钾3因素4水平试验。根据试验结果得出的肥料效应函数确定甜菜适宜的肥料用量及比例；测定甜菜不同生育时期、不同器官的氮代谢关键酶活性,分析各酶间的活性变化关系及其对甜菜产质量的影响；研究各营养元素对氮代谢关键酶活性的调控规律,进而探讨甜菜氮素同化的代谢机理,为提高肥料利用率提供理论依据。研究结果如下：1.以经30mmol/L KNO3-诱导4h的甜菜幼苗叶片为材料,采用Trizol法获得的总RNA,具有清晰的28S,18S和5S三个条带,RNA完整性好、质量高,可以满足后续的分子生物学实验。根据已知的甜菜NiR基因的DNA部分序列(AF173663,638bp)和菠菜mRNA的部分序列(X07568)设计特异引物,经RT-PCR和5’-RACE及3’-RACE试剂盒扩增了cDNA 5’端序列(489bp)、cDNA3’端序列(1600bp)以及中间序列P0(423bp)全长序列,运用DNANMAN软件将所有的片段进行拼接,得到了拼接的全长cDNA。依据拼接的全长序列设计引物,扩增cDNA全长,首次得到甜菜NiRcDNA全长序列(全长为2014bp,登录号HQ224499),将所得序列进行ORF分析,其含有完整的读码框,编码599个氨基酸。2.NR与NiR的偶联调节作用：在甜菜生育期间,叶片enNRA、exNRA的变化动态基本上是一致的,呈双峰曲线变化,在苗期活性最高,叶丛形成末期至块根增长初期出现第二个高峰,峰值远小于苗期；NiRA亦呈双峰曲线变化,叶片NiRA在6月22日出现第一个高峰,叶片和块根NiRA在8月7日同步出现峰值。NiRA高峰出现时间较NRA滞后一个取样时期。各生育时期内,exNRA均高于enNRA、块根NiRA均低于叶片NiRA。各生育时期均为高氮处理的酶活性大于低氮处理的酶活性,对照酶活性均最低。NRA、NiRA与氮素处理间,除叶片NiRA与氮素处理在7月24日相关不显著外,其它时期均表现出显著或极显著正相关关系,不同氮素水平处理间的NRA、NiRA表现出很强的规律性：即随氮素水平的升高,酶活性增强。磷、钾肥与NRA均在叶丛形成未期表现出极显著相关,与NiRA均在6月22日和8月7日表现出显著正相关,与酶活性的变化动态相一致。NiR与NR分别在6月6日、6月22日、9月11日表现出显著正相关关系,在8月7日和生育末期与exNRA相关达极显著水平,NR、NiR变化极其同步,存在偶联作用。3.GS与GOGAT的协同作用：在生育期间,叶片GSA呈双峰曲线变化,苗期活性最高,8月7日出现第二个高峰,峰值小于苗期；根中GSA呈单峰曲线变化,高峰出现在块根增长初期。GOGATA呈低—高—低的变化动态,在块根增长初期达极值。在各生育时期GSA、GOGATA随氮肥水平的升高而升高,高氮处理均大于其它处理,无氮处理酶活性一直处于最低水平。各生育时期均为叶片的GSA大于块根GSA,在酶活性高峰期(8月初)表现的更为明显,且叶片酶活性高峰期持续的时间长；叶片GOGATA亦同样大于块根GOGATA.各元素与GSA、GOGATA在全生育时期内均保持着不同程度的正相关关系,与叶片GSA在苗期显著正相关,与根中GSA在8月7日(酶活性高峰期)表现显著相关；与GOGATA在块根增长期相关达到显著或极显著水平。甜菜GS和GOGAT,除在7月8日叶片GSA与根中GOGATA相关不显著外,其它各生育期一直呈显著正相关关系,表明在甜菜氮代谢中,GS和GOGAT两者是协同作用进行氨的同化。4.各氮代谢酶的相关关系：内、外源NRA间,分别在苗期、叶丛形成末期、块根增长初期和糖分积累期呈显著相关,这与甜菜各生育期NR的变化动态一致；叶片与块根NiRA间除幼苗期相关不显著外,其它时期均呈显著正相关；叶片和块根的GSA间、GOGAT间全生育期内相关显著。NRA和叶片GSA在苗期极显著相关,NRA和GSA在叶丛形成末期、糖分积累期保持着显著相关关系；GOGATA在7月8日与exNRA间、在8月25日与enNRA间相关不显著,其它时期与NRA相关均达显著水平；NiR与氨同化酶的关系,除了NiRA与根中GSA间在苗期相关不显著和叶片NiRA在7月24日与GSA间、与叶片GOGATA间相关不显著外,其它时期均呈显著或极显著相关关系,NiR与氨同化酶的关系变化比NR更同步。5.氮磷钾肥对不同取样时期的氮素代谢酶活性表现不同程度的促进作用,而这种促进作用以氮肥为最大,磷钾肥次之,且存在明显的互作效应。在不同的生育期内,氮代谢关键酶活性对甜菜块根产量、含糖率和产糖量的影响作用也不同。除了9月11日,酶活性与含糖率的回归方程不显著外,在其它各取样时期,各酶活性与产量、含糖率和产糖量的回归方程均达极显著水平。在生育前期对产量形成影响最大的是NiRA,对含糖率影响最大的是GOGATA,对产糖量影响最大的是NiRA和根中GOGATA,而到生育后期,NRA、GSA则成为影响甜菜块根产量、含糖率和产糖量的最主要因素。6.各因素对甜菜产质量的影响不同。氮、磷、钾肥的用量和配比是影响甜菜产量的重要因素,N、P、K各养分施用的最高增产率分别为56.0%、13.51%和10.64%；从养分效率看,中氮、中磷和中钾水平利用效果最好；氮、磷、钾肥之间存在一定的交互作用,配合施用能提高肥效和促进甜菜的生长。本试验条件下,最大施用量N、P205和K20分别为198.51kg·hm-2、115.97kg·hm-2和103.63kg·hm-2,三要素比例为1：0.584：0.522时,甜菜块根产量最高为65005.59kg·hm-2。氮肥用量对品质有主要影响作用,在同等肥力下,含糖率随着施氮量的增加而下降,随着磷肥的增加而升高,钾肥对含糖率影响不大。随着氮肥施用量的增加氨态氮的含量呈升高趋势,其中氮磷肥表现出相互促进作用。钾离子含量与氮肥、钾肥的施用量相关,钠离子含量与氮肥、磷肥的施用量相关。更多还原

【Abstract】 In this dissertation, the experiments were made in the campus and practice base of Xiangfang from 2009 to 2010. Tianyan 7, the diploid pure lines of sugar beet as materials, Full-length NiR gene was the first cloned by the method of RT-PCR and 5’/3’-RACE; KWS0143, the current large-scale cultivars as materials, dynamic activities of key enzymes of nitrogen assimilation of sugar beet leaf and root at different growth stages and the impact of nitrogen, phosphorus, potassium on the yield and quality of sugar beet were systematically studied by the means of the field experiments with 3414 best regression design of fertilizer effect. According to test results concluded the fertilizer effect function and further determined appropriate fertilizer application for sugar beets.Through measured nitrogen metabolism key enzymes activity of different organs at different growth stage, we comprehensively analyzed the relationship between various enzyme activity and effect of them on yield and quality; the control laws of nutrition elements on the key enzyme activity, further explored beets nitrogen assimilation mechanism. These researches will provide theoretical basis for improve fertilizer efficient utilization. The results were as follows:l.With beet seedling leaves through 30mmol/L KNO3 induced 4h for materials, we got total RNA by the Trizol method, with clear 285,18 S and 5S three bands, RNA integrity, good quality, it could satisfy the follow-up of molecular biology experiment. Specific primers was designed according to DNA partial sequencing of the known beet NiR gene (AF173663,638bp) and mRNA part of the sequence (X07568) of spinach. Telomere sequence cDNA 5’(489bp), cDNA 3 (1600bp) and full-length sequence P0 (423bp) were amplified by RT-PCR,5’-RACE and 3’-RACE kit. All the fragments were joined together Using software of DNANMAN, got the whole length of the mosaics cDNA. According to the length of the length of stitching sequence designed cDNA primers, amplification, and then sugar beets Full-length NiR gene was the first obtained (registration number, HQ224499). By ORF analysis, the series contained the complete read code box, coding 599 amino acids.2.Coupling regulatory role between NR and NiR:The dynamic change of enNRA、exNRA in leaf were basically the same during growth stage of sugar beets, they were diauxie curve changes and with the highest activity in seedling stage, the second peak appeared in telophase of leafage form to initial stage of root growth and the peak was far less than seedling stage’s. NiRA also showed diauxie curve changes, the first peak of leaf NiRA appeared on June 22, peak of leaf and root NiRA were synchronous on 7 August. NiRA peak time was more NRA lag appear a sampling period. The time of NiRA peak appearance was lag a growth period than NRA’s. Each growth stage, exNRA were all higher than the enNRA, root NiRA were below blade NiRA.Enzyme activity in high nitrogen treatment was greater than low nitrogen treatment at different growth period, and enzyme activity was lowest in the control treatment.There was significant or very significant positive correlation between NRA、NiRA and nitrogen application at all growth stage expect leaf NiRA and nitrogen application on July 24. The NRA and NiRA showed a strong regularity between different nitrogen applied:With nitrogen level increased, enzyme activity enhanced. P, k and the NRA had a very significant correlation at the end of leafage formation, a significant positive correlation with NiRA on June 22 and August 7, and also same to the changes dynamic of enzyme activity. Nitrogen and NiR, NR showed significant positive correlation respectively on June 6,June22 and September 11,had a very significant with exNRA on Augst 7 and growth late stage.On 7 August and birth late exNRA related with significantly positively, NR, NiR change extremely synchronization, exist coupling effect. The change of NR and NiR were extremely synchronization, and between them had coupling effect.3.GS and GOGAT Synergy:GSA in leaf was diauxie curve changes during growth stage of sugar beets and with the highest activity in seedling stage, the second peak of leaf GSA appeared on August 7, and the peak was lower than seedling stage’s. GSA in root was single-peak curve changes during growth stage of sugar beets and with the highest activity in root growth of early stage. GOGATA showed low-high-low dynamics, and got extreme value in root growth of early stage. With nitrogen level increased, GSA and GOGATA enhanced. High nitrogen treatment was larger than other treatments, without nitrogen treatment had been in the lowest level of enzyme activity. The GSA of blades was greater than root’s, especially in the strongest period of Enzyme activity (in early August), and Enzyme activity, and it could continues a long time. Leaves GOGATA were also greater than roots GOGATA.There was different degree of positive correlativity between nitrogen, phosphorus, potassium and GSA and GOGATA from the foliage formation period to the end stage of tuber growth. Each element had significant positive correlation with GSA of leaves at seedling, with GSA of roots on August 7 (enzyme activity strongest period); and with GOGATAGOGATA at root growth stage reached significant or very significant correlation. GS was positive related with GOGAT in all growth stage of sugar beets expect in July 8, in which GSA in leaves and GOGATA in roots hadn’t significant correlation. These indicated that GS and GOGAT were synergistic action for ammonia assimilation in nitrogen metabolism of beet.4.The relationship of nitrogen metabolic enzymes:The change tendencies of inside NRA and exogenous NRA were the same to NR. They had significant correlation at seedling stage, the end of leafage formation, at the initial of root growth and sugar accumulation period. NiRA in leaves and roots were significant correlation in all growth stage of sugar beets expect at seeding stage. GSA in leaves and roots were significant relationship in all growth stage, GOGAT either.There was a very significant correlation between NRA and GSA of leaves at seedling stage, and they were significant correlation at leafage formation stage and sugar accumulation stage. GOGATA was significant related with NRA in all growth stage, but not significant with exNRA On July 8, with enNRA on August 25. There were significant or very significant correlation between NiR and Ammonia assimilation enzyme in all growth stage in addition to NiRA and GSA of roots in seeding, NiRA and GSA on July 24, NiRA and GOGATA. The relationship changes of NiR and ammonia assimilation enzyme were more synchronized than NR.5.NPK fertilizers would increase different degree of the activity of nitrogen metabolic enzymes at different growth stage. The promoting function was the largest with nitrogen and phosphorus, potassium was the second, there existed obvious interaction effect among them. In different growth stage, activity of nitrogen metabolism key enzyme had different effect on sugar beets root yield and sugar content and sugar yield. Regression equations of enzyme activity and yield and sugar content and sugar yield production were very significant correlation in every sampling period, in addition to September 11, the regression equation of enzyme activity and sugar content was not significant correlation. In the early growth stage, NiRA was the greatest influence factor on yield formation, GOGATA was the greatest influence factor on sugary rate, NiRA and GOGATA of roots were important factors to sugar yield production; in the late growth stage, NRA and GSA were the most main affecting factor to root yield and sugar yield rate and sugar yield production of sugar beet.6.Different N,P,K factors had different effect on the quality of beet. The fertilizing amounts and proportion of nitrogen, phosphorus and potassium fertilizer are important factors which affect sugar beet yield. The highest increasing yield rate is 56.0%、13.51% and 10.64% respectively by fertilizing N、P and K. According to the nutrient efficiency, the fertilizer application of nitrogen, phosphorus, potassium at medium level has the best effect; There were interactions among nitrogen, phosphorus and potassium fertilizer, coordinated applications of nitrogen, phosphorus and potassium fertilizer improve fertilizer efficiency and promote sugar beet growth. The condition of the experiment, the best NPK application of sugar beets with maximum economical yield of 65005.59 kg·hm-2 was N 198.51 kg·hm-2, P2O5 115,97kg·hm-2 and K2O 103.63 kg·hm-2.The rate of N:P:K was 1:0.584:0.522. The amount of applied nitrogen had a major impact on quality. Under equal fertilization, the sugar content decreases with the amount of nitrogen increased, phosphorus is conducive to the increase of sugar content, potassium has little effect on sugar content. Along with the increase of nitrogen application, content of ammonia nitrogen was enhanced. Nitrogen and phosphorus could promote each other. Potassium ion content was related with the nitrogen and potash application; the sodium content of was related with the nitrogen and phosphorus application.更多还原