拟南芥脲酶辅助蛋白AtureG基因的功能解析

发布时间：2018-03-31 11:29

本文选题：拟南芥　切入点：AtureG　出处：《东北林业大学》2016年硕士论文

【摘要】：盐胁迫导致农作物产量降低的现象普遍存在。盐胁迫在种子萌发时期对于种子来说尤为敏感。脲酶(urease)是一个依赖镍离子的金属酶,在大多数植物和微生物体内用以分解尿素。在尿素代谢中,脲酶水解尿素生成氨和二氧化碳。在拟南芥(Arabidopsis thaliana)中,催化脲酶生物活性需要镍离子和三个辅助蛋白(ureD、ureF、ureG)的参与。三个脲酶辅助蛋白基因的表达特性分析结果表明,三个脲酶辅助蛋白基因在拟南芥的各个器官中均有表达,且在果夹中表达量最大；在种子萌发时期,AtureD和AtureF在吸胀过程中表达量都明显下调表达,而AtureG基因则上调表达；在种子萌发后期,NaCl胁迫和NH4Cl处理下,三个脲酶辅助蛋白均受到诱导表达,但在表达谱上AtureD和AtureF基因的表达特性有着高度的相似性,AtureG在表达谱上不同于其他两个基因,所以本研究中以AtureG为研究对象进行以下研究。利用PCR、Northern blot等手段对AtureG基因缺失突变体在DNA和RNA水平进行鉴定并得到了分别为外显子插入(atureG-1、cs352398)和内含子插入(atureG-2、 cs352350)的两个纯合突变体。以野生型拟南芥和脲酶基因缺失突变体(aturease)作为对照,脲酶活性分析的结果显示所得到的两个纯合突变体(atureG-1、atureG-2)均未检测到脲酶的活性。对野生型拟南芥、atureG-1和atureG-2在盐等逆境处理下的表型分析结果显示,在NaCl胁迫处理下,atureG-1和atureG-2可提高对盐胁迫的抗性,根长和鲜重都显著性高于野生型；在过量的NH4Cl处理下,atureG-1、atureG-2与野生型相比突变体在鲜重上略有优势。进一步对拟南芥野生型、atureG-1和atureG-2植株体内的尿素含量和NH4+含量进行了测定。研究结果显示：盐胁迫处理下,atureG-1和atureG-2体内的尿素含量高于野生型,而NH4+含量则低于野生型。最后,为了进一步了解AtureG基因在拟南芥植株体内的作用机制,我们构建了pGEX-6p-3-AtureG载体,并对其蛋白进行了诱导和纯化。结果得到了纯化后的AtureG蛋白。为今后在蛋白水平上的研究打下坚实基础。
[Abstract]:Salt stress is especially sensitive to seeds during germination. Urease is a nickel ion-dependent metalloenzyme. Used in most plants and microorganisms to break down urea. In urea metabolism, urease hydrolyzes urea to produce ammonia and carbon dioxide. In Arabidopsis thaliana, The catalytic activity of urease requires the participation of nickel ion and three auxiliary proteins, ureDureFureG. the analysis of the expression characteristics of the three urease auxin genes showed that the three urease auxin genes were expressed in all organs of Arabidopsis thaliana. The expression of AtureD and AtureF were down-regulated during seed germination, while the expression of AtureG gene was upregulated, and the expression of AtureD and AtureF were up-regulated in the later stage of seed germination. All three urease auxiliaries were induced to express, but the expression characteristics of AtureD and AtureF genes were highly similar in expression profile, and AtureG was different from the other two genes in expression profile. Therefore, in this study, AtureG was used as the research object. AtureG gene deletion mutants were identified at the DNA and RNA levels by Northern blot and two intron insertions were obtained: exon insertion cs352398) and intron insertion tautureG-2, cs352350). Homozygous mutants. Wild type Arabidopsis thaliana and urease gene deletion mutants were used as control. The results of urease activity analysis showed that the urease activity of the two homozygous mutants was not detected. The phenotypic analysis of wild type Arabidopsis thaliana atureG-1 and atureG-2 under salt and other stresses showed that the urease activity was not detected in the two homozygous mutants. The resistance to salt stress was increased under NaCl stress, and root length and fresh weight were significantly higher than that of wild type. The fresh weight of the wild type mutant was slightly superior to that of the wild type under excessive NH4Cl treatment. The urea content and NH4 content in the plants of Arabidopsis thaliana wild-type TatureG-1 and atureG-2 were further determined. The results showed that: under salt stress, the content of urea and NH4 in the wild type of Arabidopsis thaliana was determined. The urea content in Agar G-1 and atureG-2 was higher than that in wild type. Finally, in order to further understand the mechanism of AtureG gene in Arabidopsis thaliana plants, we constructed pGEX-6p-3-AtureG vector. The protein was induced and purified, and the purified AtureG protein was obtained, which laid a solid foundation for further study on protein level.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

【参考文献】