【作者】 欧红梅；

【导师】 张自立；

【作者基本信息】 安徽农业大学 ， 植物营养学， 2006， 硕士

【摘要】 稀土元素（Rare Eareth Elements）的农业应用日趋广泛,特别是随着稀土化肥的推广,加速了土壤与环境中稀土元素的富集和积累。由于稀土是重金属,稀土进入土壤和环境后对生态环境和人类健康的影响,已引起全社会的广泛关注。目前关于农用稀土在土壤中累积后对生态环境产生的影响研究已取得了一定成果,但稀土对植物的作用机理研究尚不系统。拟南芥是一种模式植物,生物学的不同领域都对这一遗传资源进行了大力开发和利用。拟南芥的许多突变体已被广泛用于植物发育和代谢途径的研究。拟南芥突变体的筛选已成为研究许多重要理论问题的前提。在稀土农用研究中利用拟南芥的工作还很少,目前还没有关于拟南芥抗稀土元素突变体筛选的研究。本试验试图筛选出拟南芥抗La突变体,为La抗性基因定位提供基础材料。拟南芥抗La突变体的研究将从基因角度阐明稀土积累的遗传效应,填补稀土研究的空白,完善稀土农用的环境安全性评价体系。由于La³⁺很容易和其它阴离子发生络合沉淀,降低了La在培养基中的有效浓度,同时也使得La分布不均匀,不利于突变体的筛选,因此进行了筛选条件的摸索。在pH5.8、0.8%琼脂条件下,在1/2MS+20 mg/kg及以上La浓度培养基中出现白色沉淀,在50 mg/kg La³⁺浓度水平,植株也能生长。为避免了离子的沉淀,以1/2MS（-P）为基础培养基和纯稀土培养基试验,20 mg/kg La³⁺完全抑制幼苗主根的生长,而且两者试验结果相同,确立了以纯稀土培养基为筛选培养基。为保证稀土培养基的均一性,避免空气中CO2造成La³⁺沉淀,将pH调至5.5。筛选方法试验中,双层培养试验根生长至50mg/kg La³⁺浓度稀土培养基中仍能继续生长。由于拟南芥幼苗茎和叶片处于非胁迫条件,植物的根吸收的La向上部转运很少,导致幼苗可以忍耐更高浓度的胁迫。考虑到双层培养基每皿的接种量有限,种子发芽不一致,根生长条件很难控制,因而选用了根向地性弯曲生长为突变指标的筛选方法。筛选压力试验,在6、6.5mg/kg La³⁺培养基上,拟南芥根弯曲与对照一致;7、7.5 mg/kg La³⁺培养基上,拟南芥能生长,但比对照长得慢,根弯曲较短;8mg/kg及以上,拟南芥不生长。含La³⁺的培养基较对照软,而且稀土含量越高,培养基越软。由于琼脂用量和培养基的pH值在改善培养基凝固效果方面有互补作用。为得到在pH5.5下合适的凝固效果,提高琼脂浓度至1.0%。发现拟南芥在7-13mg/kg La³⁺水平培养基上生长处于抑制状态,仍能生长;在14 mg/kg及以上浓度基本不生长。在pH5.5,1%琼脂浓度条件下,确立了野生型拟南芥完全不能生长的14mg/kgLa³⁺浓度为最适宜筛选压力。对所获得的5120个T2代株系约50,000粒种子,用上述确定的筛选浓度和筛选方法进行筛选。共筛选到63株可能突变体,在营养土中培养,分单株收获。在培养过程中发现一株晚熟株,其抽薹比对照晚15天。对获得的63株可能突变体用上述方法进一步鉴定,尚未发现稳定遗传的抗La突变株系。更多还原

【Abstract】 Rare earth elements（REEs） has been applied widely in agriculture ,especially application of REEs chemical fertilizer quicken the enrichment and accumulation of rare earth in the soil and environment.Because RE are heavy metal,the effects of REEs on the entironment and human body have aroused whole society attention. At the present time, study of REEs’effect on the entironment after accumulated in the soil has obtained a few fruit, but study of REEs’effect mechanism on the plant has no system.Arabidopsis is a mode plant. Its heredity resource has been exploited and utilized in the different field of biology. Arabidopsis’s many mutants have been used for the study of plant growth and metabolize pass. Screening mutants of Arabidopsis have been precondition of many important theory question solved. But utilization of Arabidopsis in REE used in agriculture is few, the study of screening REE-resistance mutants of Arabidopsis is absence at presence.This study tries to screen La-resistance mutants of Arabidopsis. The mutants will provide base material for gene mapping of La-resistance. The study of La-resistance mutants of Arabidopsis will clarify heredity effect of REE accumulate from the angle of gene , fill up the blank of REE study,perfect environment security evaluate system of REEs’application in agriculture.Due to La³⁺ occuring deposition with other negative ion, it reduces the efficiency concentration of La in the medium, also cause La distributing asymmetry at the same time, and go against mutants’screening, so the condition of screening is explored. At the condition of pH5.8、0.8% agar, white deposition appears in the medium containing 1/2MS+20 mg/kg La ,plant are also able to grow at the 50 mg/kg La³⁺.In order to avoid ionic deposition, base medium containing 1/2MS（-P）and medium containing pure La are tested. 20 mg/kg La³⁺ restrains entirely growth of seeding taproot, furthermore both examinations’result are same,so the medium containing pure La is established for screening medium. In order to the uniformity of La in the medium, and avoid CO2 of air getting deposition, pH is brought to 5.5.Through two-layer solid-medium of screening method experiment,roots could grow through the upper layer and penetrate the lower layer containing 50mg/kg La³⁺.Stem and leaf are under the non-intimidate ,and La absorbed by roots of the plant is transfer few to the upside,so seeding can endure even more higher concentration. In view of inoculating quantity in per vessel being finity, and seed germination being variance, and growth condition of root being very hard to control, the screening method using the root-bending assay is selected.Among the screening stress experiment,root-bending is consistent to CK in the medium containing 6 and 6.5mg/kg La³⁺;in the medium containing 7 and 7.5 mg/kg La³⁺ the plant can grow ,but it grow slower than CK,and the root-bending length is shorter. It cannot grow in the medium concaining 8mg/kg and up La³⁺.The medium containing La³⁺ is softer than CK,and that concentration of La³⁺ higher ,medium softer .Because agar dosage and pH of medium have complementary effect in improving solidification , fine solidification is gained when agar concentration increasing to 1.0% at pH5.5. Arabidopsis is restrained at La³⁺ from 7mg/kg to 13mg/kg, but still can grow, and it cannot at and up 14 mg/kg La³⁺. 14mg/kg La³⁺ concertration of Col-0 Arabidopsis be entirely incapable of growing is selected for the best screening pressure at pH5.5 and 1.0% agar.Using the above selected screening concentration and screening method the 5120 T2 series （about 50,000 seeds） are screened. 63 putative mutants are obtained, and cultured in nutritional soil, harvested disparting individual plant. In the process of culture a later ripe plant of later 15 days than CK is discovered.63 putative mutants are identified using the previous method, La-resistance mutant of stable heredity is not yet discovered.更多还原