發(fā)布時(shí)間：2018-04-29 08:57

  本文選題：擬南芥 + F-box��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：ABA在植物生命周期中起著關(guān)鍵的作用,例如種子的休眠、萌發(fā)、幼苗的生長以及開花等。更重要的是,ABA使植物能夠耐受與水相關(guān)的脅迫,例如干旱和鹽度等。迄今為止,在ABA的信號(hào)傳導(dǎo)途徑中一些基本組分已經(jīng)被鑒定,比如PROTEIN PHOSPHATASES TYPE 2Cs(PP2Cs),SUCROSENON-FERME-NTING 1-RELATED PROTEIN KINASEs(Sn RK2s)和ABA-RESPONSIVE ELEM-ENTS BINDINGFACTORs(ABFs)等。在2009年,在植物細(xì)胞中ABA受體PYRABACTIN RESISTANCE 1/PYR-LIKE/REG-ULATORY COMPONENTS OF ABA RECEPTORs(PYR1/PYLs/RCARs)被證明。當(dāng)受到外源ABA誘導(dǎo)后,PYR1/PYLs/RCARs受體感知ABA并抑制PP2C的活性使Sn RK2去磷酸化,進(jìn)而調(diào)控下游基因的表達(dá)。ABA信號(hào)轉(zhuǎn)導(dǎo)通路中基本成分水平的嚴(yán)格控制對(duì)于穩(wěn)態(tài)是至關(guān)重要的。最近,許多研究已經(jīng)表明,這些中樞調(diào)節(jié)因子被泛素26S蛋白酶體系降解。在擬南芥基因組中已經(jīng)發(fā)現(xiàn)了超過1400個(gè)編碼E3連接酶的基因。這些基因可以分為兩組:單亞基型和多亞單位型。F-box蛋白作為SCF復(fù)合體最重要的亞基是由快速擴(kuò)增的真核基因家族編碼,在N端包含有40-50個(gè)氨基酸組成的保守的F-box結(jié)構(gòu)域,首先在細(xì)胞周期蛋白F中被發(fā)現(xiàn)并介導(dǎo)了與Skp1的相互作用。F-box蛋白在C端都包含一個(gè)或者多個(gè)高度可變的蛋白質(zhì)-蛋白質(zhì)相互作用結(jié)構(gòu)域。在擬南芥和水稻基因組中分別有692和779個(gè)F-box蛋白序列被發(fā)現(xiàn)。但大多數(shù)F-box家族基因的功能還未知。本研究克隆了兩個(gè)擬南芥F-box家族基因,對(duì)這兩個(gè)基因在ABA信號(hào)傳導(dǎo)途徑中的功能進(jìn)行了分析,主要研究結(jié)果如下:1.利用Infusion技術(shù),克隆了FB1和FB2基因,成功將其構(gòu)建到N-egad-LUC植物表達(dá)載體上。FB1和FB2基因組全長分別為2283 bp和1337 bp。FB1包含兩個(gè)外顯子和一個(gè)內(nèi)含子,而FB2中不包含內(nèi)含子。FB1和FB2具有典型的F-box結(jié)構(gòu)域,并與油菜中FB1和FB2親緣關(guān)系最近。2.通過農(nóng)桿菌介導(dǎo)的蘸花侵染,將重組質(zhì)粒轉(zhuǎn)化到擬南芥中,利用Basta抗性篩選,Luminometer儀瞬時(shí)檢測和Western Blot技術(shù),證實(shí)得到了FB1和FB2的過表達(dá)轉(zhuǎn)基因擬南芥,最終得到T3代純合轉(zhuǎn)基因植株。另外,從ABRC(Arabidopsis Biological Resource Center)購買到FB1和FB2的T-DNA插入突變體,利用三引物法,鑒定出純合的突變體。3.利用Gateway技術(shù)構(gòu)建了FB1-YFP融合表達(dá)載體,并瞬時(shí)轉(zhuǎn)染了擬南芥原生質(zhì)體進(jìn)行亞細(xì)胞定位分析,結(jié)果表明FB1蛋白定位于細(xì)胞質(zhì)和細(xì)胞核中。4.分別對(duì)FB1和FB2過表達(dá)擬南芥株系進(jìn)行ABA響應(yīng)表型分析,結(jié)果表明FB1和FB2過表達(dá)擬南芥種子的萌發(fā)率明顯高于野生型的,對(duì)ABA表現(xiàn)出不敏感,并隨著ABA濃度的增加表型更加顯著。5.在FB1和FB2過表達(dá)以及野生型中,分析ABA相關(guān)基因的轉(zhuǎn)錄水平,發(fā)現(xiàn)FB1和FB2過表達(dá)引起ABA信號(hào)轉(zhuǎn)導(dǎo)負(fù)向調(diào)節(jié)因子ABI1、ABI2的轉(zhuǎn)錄水平升高,正向調(diào)節(jié)因子ABI3、ABI4的表達(dá)量下降;但是如ABF轉(zhuǎn)錄因子、Sn RK2和ABI5等ABA信號(hào)正向調(diào)節(jié)因子的表達(dá)量出現(xiàn)上調(diào),推測可能存在FB1和FB2降解靶蛋白引起了蛋白水平下降而誘導(dǎo)mRNA轉(zhuǎn)錄升高的反饋。
[Abstract]:ABA plays a key role in plant life cycle, such as seed dormancy, germination, seedling growth and flowering. More importantly, ABA enables plants to withstand water-related stresses such as drought and salinity. Up to now, some basic components in the signal transduction pathway of ABA have been identified, such as PROTEIN PHOSPHATASES TYPE 2CsSU PP2CsSU SUCROSENON-FERME-NTING 1-RELATED PROTEIN KINASEs(Sn RK2s and ABA-RESPONSIVE ELEM-ENTS BINDINGFACTORs ABFs. In 2009, ABA receptor PYRABACTIN RESISTANCE 1/PYR-LIKE/REG-ULATORY COMPONENTS OF ABA RECEPTORsPYR1 / PYLs / RCARswere demonstrated in plant cells. When induced by exogenous ABA, the PYR1 / PYLsR / RCARs receptor sensed ABA and inhibited the activity of PP2C so that Sn RK2 was dephosphorylated, and then regulated the expression of downstream genes. Recently, many studies have shown that these central regulatory factors are degraded by the ubiquitin 26 S protease system. More than 1, 400 genes encoding E 3 ligase have been found in Arabidopsis genomes. These genes can be divided into two groups: single subunit type and multiple subunit type. F-box protein, as the most important subunit of SCF complex, is encoded by a rapidly amplified eukaryotic gene family and contains a conserved F-box domain consisting of 40-50 amino acids at the N-terminal. First of all, it was found and mediated the interaction with Skp1 in cyclin F. F-box protein contains one or more highly variable protein-protein interaction domains at the C-terminal. In Arabidopsis thaliana (Arabidopsis thaliana) and rice (Oryza sativa) genomes, 692 and 779 F-box protein sequences were found, respectively. But the function of most F-box family genes remains unknown. In this study, two Arabidopsis F-box family genes were cloned and their functions in ABA signaling pathway were analyzed. The main results are as follows: 1. Using Infusion technique, FB1 and FB2 genes were cloned and successfully constructed into the N-egad-LUC plant expression vector. The genome length of. FB1 and FB2 were 2283 BP and 1337 bp.FB1, respectively, containing two exons and one intron, respectively. However, FB2 does not contain introns. FB1 and FB2 have typical F-box domain, and have the closest relationship with FB1 and FB2 in rapeseed. The recombinant plasmid was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens mediated by dipping flower. The transient detection of Basta resistance sieve and Western Blot technique confirmed the overexpression of FB1 and FB2 in Arabidopsis thaliana. Finally, the T 3 fusion transgenic plants were obtained. In addition, the T-DNA insertion mutants of FB1 and FB2 were purchased from ABRC(Arabidopsis Biological Resource Center, and the homozygous mutants .3were identified by three-primer method. The fusion expression vector of FB1-YFP was constructed by Gateway technique, and the subcellular localization analysis of Arabidopsis protoplasts was carried out. The results showed that FB1 protein was located in cytoplasm and nucleus. The ABA response phenotype of Arabidopsis thaliana lines with FB1 and FB2 overexpression was analyzed. The results showed that the germination rate of FB1 and FB2 overexpression Arabidopsis seeds was significantly higher than that of wild-type Arabidopsis plants, and was insensitive to ABA, and the phenotype was more significant with the increase of ABA concentration. In the overexpression of FB1 and FB2 and wild type, the transcriptional level of ABA related genes was analyzed. It was found that the overexpression of FB1 and FB2 resulted in an increase in the transcription level of ABA signal transduction negative regulatory factor ABI1 and ABI2, and a decrease in the expression of positive regulatory factor ABI3, ABI4. However, the expression of ABA signal positive regulatory factors such as ABF transcription factors, such as Sn RK2 and ABI5, was up-regulated, which suggested that there might be feedback that the degradation of FB1 and FB2 could induce the decrease of protein level and induce the increase of mRNA transcription.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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本文編號(hào)：1819260