【作者】 郁晓敏；

【导师】 方萍；

【作者基本信息】 浙江大学 ， 植物营养学， 2004， 硕士

【摘要】 化肥的施用对农业生产的突破性发展功不可没，但长期大量、单一地施用化肥特别是化学氮肥所带来的肥料利用率降低、农产品品质下降和环境污染问题正日益突出，已经对农业的可持续发展产生严重的负面影响。而现代育种策略通常是在养分充足的条件下进行的，这也使得蕴藏在植物资源中能在低养分胁迫环境下高效吸收和利用养分的遗传潜力未能发挥。 激活标签法（ACTIVATION TAGGING）是新近发展起来的一种用于基因分离和鉴定的方法。本论文实验利用激活突变使特定内源基因发生过量表达而产生显性功能获得型突变的方法，创建大规模的拟南芥突变体库，以这些转化植株作为突变体筛选的材料。 通过反复的比较实验，自行设计专为拟南芥水培设计的植物漂浮装置，该装置由两部分组成：浮板和容器。装有石英砂的浮板既可满足种子的直播培养，又可以进行幼苗的移栽培养。采用发芽、幼苗等实验确定筛选拟南芥低氮耐性突变体的适宜浓度，其含氮量为0.18mmol/L。在该浓度下，野生型基本受到抑制而不能表现，而突变体的优势则得到发挥。 利用上述实验确定的漂浮装置和选择压，对建好的突变体库中的种子（T₂）进行大规模、高通量的突变体筛选。培养过程中，以叶绿素SPAD值为主要筛选指标，并且辅之于其它较为明显的生物学性状，第一轮从中筛选出6株生长良好的植株（T₃）：第二轮从中筛选出7株生长较好的植株（T₄）；第三轮则从中筛选出于8株植株（T₅）。 将三轮筛选得到的其中4株可能突变体（T₅），首先进行抗除草剂特性的鉴定实验，从其后代（T₆）中分离出5株具有抗性的植株；然后分析这5株在低氮胁迫环境下的叶绿素含量、全氮磷钾含量，并与标准营养液培养下的野生型植株进行比较；结果表明两者之间没有明显的差异，说明可能突变体在低氮条件下对氮、磷、钾等主要营养元素仍然具有良好的吸收能力。 综合所有实验结果，本论文实验初步筛选得到5株拟南芥低氮耐性突变体，并且对其氮素营养生理生化特性进行分析和研究，为鉴定和分离其与耐低氮性状连锁的基因奠定了基础。更多还原

【Abstract】 Application of chemical fertilizer has promoted a great development in agricultural production. However, injudicious use of chemical fertilizer, especially chemical nitrogen fertilizer has induced a crucial negative impact to sustainable agriculture development, such as decrease of fertilizer utilization efficiency, reduction of agricultural product quality, and environmental pollution. But modern breeding is always proceeding under standard condition, so the high absorbing and using ability of plants under low nutrient stress are not exerted.Activation tagging is a new method for isolation and functional identification. It can generate dominant gain-of-function mutants by over expression of a particular endogenous gene. The mutant-isolation experiment is based on large seed pools (T2) which are mutagenized by activation tagging.A hydroponic system was designed for Arabidopsis, which is based on two units: a seed-holder and a tank. The quartz sand-containing seed-holder allows not only direct culture of seeds, but transplant of seedlings. Effects of low nitrogen stress on germination and growth of Arabidopsis thaliana were studied and the corresponding N concentrations for screening mutants were determined as 0.18mmol/L. Under this concentration, growth of wild type was restrained but mutants will be alive.By using floating system and corresponding screen pressure, T2 populations (seed pool) were screened for low nitrogen tolerant mutants with high efficiency and low maintenance. During the culture, with chlorophyll SPAD scores and other biological characters, 6 plants (T3) were selected for putative mutants at first round, 7 plants (T4) were selected at second round, and 8 plants (T5) were selected at third round.After the isolation, 4 plants (T5) were selected to have herbicide test and 5 plants (T6) resisted herbicide and were still alive. Then chlorophyll content, total N, P, K content of those 5 putative mutants (T6) under low-N stress were measured and they are similar with those of wild type under standard condition. These results show that 5 plants have good absorbing capacity of nutrients under low nitrogen stress.Due to the whole results, 5 low-nitrogen tolerant putative mutants of Arabidopsis thaliana were isolated and their physiological and biochemical characters of nitrogen nutrient were studied. It could provide the materials to identify and separate its corresponding gene.更多还原