本文選題：擬南芥　切入點(diǎn)：AtureG　出處：《東北林業(yè)大學(xué)》2016年碩士論文

【摘要】：鹽脅迫導(dǎo)致農(nóng)作物產(chǎn)量降低的現(xiàn)象普遍存在。鹽脅迫在種子萌發(fā)時(shí)期對(duì)于種子來說尤為敏感。脲酶(urease)是一個(gè)依賴鎳離子的金屬酶,在大多數(shù)植物和微生物體內(nèi)用以分解尿素。在尿素代謝中,脲酶水解尿素生成氨和二氧化碳。在擬南芥(Arabidopsis thaliana)中,催化脲酶生物活性需要鎳離子和三個(gè)輔助蛋白(ureD、ureF、ureG)的參與。三個(gè)脲酶輔助蛋白基因的表達(dá)特性分析結(jié)果表明,三個(gè)脲酶輔助蛋白基因在擬南芥的各個(gè)器官中均有表達(dá),且在果夾中表達(dá)量最大；在種子萌發(fā)時(shí)期,AtureD和AtureF在吸脹過程中表達(dá)量都明顯下調(diào)表達(dá),而AtureG基因則上調(diào)表達(dá)；在種子萌發(fā)后期,NaCl脅迫和NH4Cl處理下,三個(gè)脲酶輔助蛋白均受到誘導(dǎo)表達(dá),但在表達(dá)譜上AtureD和AtureF基因的表達(dá)特性有著高度的相似性,AtureG在表達(dá)譜上不同于其他兩個(gè)基因,所以本研究中以AtureG為研究對(duì)象進(jìn)行以下研究。利用PCR、Northern blot等手段對(duì)AtureG基因缺失突變體在DNA和RNA水平進(jìn)行鑒定并得到了分別為外顯子插入(atureG-1、cs352398)和內(nèi)含子插入(atureG-2、 cs352350)的兩個(gè)純合突變體。以野生型擬南芥和脲酶基因缺失突變體(aturease)作為對(duì)照,脲酶活性分析的結(jié)果顯示所得到的兩個(gè)純合突變體(atureG-1、atureG-2)均未檢測(cè)到脲酶的活性。對(duì)野生型擬南芥、atureG-1和atureG-2在鹽等逆境處理下的表型分析結(jié)果顯示,在NaCl脅迫處理下,atureG-1和atureG-2可提高對(duì)鹽脅迫的抗性,根長(zhǎng)和鮮重都顯著性高于野生型；在過量的NH4Cl處理下,atureG-1、atureG-2與野生型相比突變體在鮮重上略有優(yōu)勢(shì)。進(jìn)一步對(duì)擬南芥野生型、atureG-1和atureG-2植株體內(nèi)的尿素含量和NH4+含量進(jìn)行了測(cè)定。研究結(jié)果顯示：鹽脅迫處理下,atureG-1和atureG-2體內(nèi)的尿素含量高于野生型,而NH4+含量則低于野生型。最后,為了進(jìn)一步了解AtureG基因在擬南芥植株體內(nèi)的作用機(jī)制,我們構(gòu)建了pGEX-6p-3-AtureG載體,并對(duì)其蛋白進(jìn)行了誘導(dǎo)和純化。結(jié)果得到了純化后的AtureG蛋白。為今后在蛋白水平上的研究打下堅(jiān)實(shí)基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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