【作者】 李雪莹；

【导师】 卜媛媛；

【作者基本信息】 东北林业大学 ， 生物学， 2022， 硕士

【摘要】 拟南芥尿素转运蛋白AtDUR3能以高亲和力协同转运尿素,在植物的氮素代谢过程中有着至关重要的作用。本课题组前期研究发现,与野生型植株相比,AtDUR3基因缺失突变体植株能够明显提高对高盐胁迫的耐受性,氮素代谢在种子萌发期响应盐胁迫中发挥了重要的作用。鉴于此,本课题将进一步筛选AtDUR3相互作用蛋白并分析其在响应盐碱胁迫中发挥的功能。主要研究结果如下:1、拟南芥尿素转运蛋白AtDUR3基因主要在干种子和叶中表达,在花中表达量最低。AtDUR3基因受盐胁迫诱导下调表达,pro AtDUR3:GUS转基因材料GUS分析结果也表明,Na Cl胁迫抑制AtDUR3基因在种子萌发期时期的表达。而AtDUR3基因在Na₂CO₃和Na HCO₃胁迫处理下则被诱导上调表达;在氮饥饿（-N）、NH₄⁺与Urea分别作为唯一氮源时,AtDUR3在叶和根中整体呈现上调表达趋势。2、将诱饵蛋白p GBKT7-AtDUR3与拟南芥c DNA文库融合筛选相互作用蛋白,共得到11个阳性克隆,经酵母共转化验证及双分子荧光互补实验进一步表明AtDUR3与AtMYBR1之间存在相互作用。3、拟南芥MYB结构域蛋白AtMYBR1基因表达特性分析表明,AtMYBR1基因在果荚中表达量最高,在根中表达量最低,受Na Cl、Na₂CO₃和Na HCO₃胁迫诱导上调表达;在氮饥饿（-N）、NH₄⁺与Urea分别作为唯一氮源时,AtMYBR1在叶和根中整体呈上调表达趋势。但是在加入NH₄⁺后,拟南芥根中AtMYBR1基因的表达呈先上调后下调的趋势。4、经分子水平鉴定获得AtMYBR1基因缺失突变体atmybr1（SALK＿008606C和SALK＿039074）。在Na Cl处理下,atdur3缺失突变体的长势优于WT,而atmybr1缺失突变体的长势均弱于WT;AtDUR3基因的缺失可明显提高植物对盐胁迫的耐受性,增强对碱胁迫的敏感性;AtMYBR1基因的缺失则明显降低植物对盐碱胁迫的耐受性。更多还原

【Abstract】 Arabidopsis urea transporter AtDUR3 cooperatively transports urea and protons with high affinity.It plays an important role in the process of plant nitrogen metabolism.The previous research of our research group found that compared with wild-type plants,AtDUR3 gene deletion mutant plants can significantly improve the tolerance to high salt stress,and nitrogen metabolism plays an important role in responding to salt stress during seed germination.In view of this,this project will further screen the AtDUR3 interacting protein and analysis its function in response to saline alkali stress.The main results are as follows:1.Arabidopsis urea transporter AtDUR3 is mainly expressed in dry seeds and leaves,and the lowest expression in flowers.The expression of AtDUR3 was down regulated by salt stress.The results of GUS analysis of pro AtDUR3:GUS transgenic materials also showed that Na Cl stress inhibited the expression of AtDUR3 during seed germination.AtDUR3 was up-regulated under Na₂CO₃ and Na HCO₃ stress;When nitrogen starvation（-N）or NH₄⁺and urea were the only nitrogen sources,AtDUR3 was up-regulated in leaves and roots as a whole.2.The bait protein PGBKT7-AtDUR3 was screened with the Arabidopsis c DNA library,and 11 positive clones were obtained,The yeast Co-transformation verification and bimolecular fluorescence experiment further showed that there was an interaction between AtDUR3 and AtMYBR1.3.The analysis of expression characteristics of Arabidopsis MYB domain protein AtMYBR1 gene showed that the expression of atmybr1 gene was the highest in fruit pods and the lowest in roots.It was up-regulated by Na Cl,Na₂CO₃ and Na HCO₃ stress;When nitrogen starvation（-N）,NH₄⁺and urea were the only nitrogen sources,AtMYBR1 was up-regulated in leaves and roots.However,after the addition of NH₄⁺,the expression of AtMYBR1 gene in Arabidopsis roots showed a trend of first upregulation and then downward regulation.4.AtMYBR1 gene deletion mutants atmybr1（SALK＿008606C and SALK＿039074）were obtained by molecular level identification.Under Na Cl treatment,the growth of atdur3deletion mutants was better than WT,while the growth of atmybr1 deletion mutants was weaker than WT;The deletion of AtDUR3 gene can significantly improve the tolerance of plants to salt stress and enhance the sensitivity to alkali stress;The deletion of AtMYBR1 gene significantly reduced the tolerance of plants to saline alkali stress.更多还原