本文選題：GAAP1 + 互作因子��； 參考：《華東師范大學(xué)》2015年碩士論文

【摘要】：內(nèi)質(zhì)網(wǎng)是蛋白質(zhì)加工的主要場(chǎng)所之一。當(dāng)植物體受到脅迫條件時(shí),就可能導(dǎo)致內(nèi)質(zhì)網(wǎng)中未折疊或者錯(cuò)誤折疊蛋白的積累,即內(nèi)質(zhì)網(wǎng)脅迫(ER脅迫)。細(xì)胞啟動(dòng)包括未折疊蛋白響應(yīng)(UPR)途徑在內(nèi)的保護(hù)途徑來(lái)緩解內(nèi)質(zhì)網(wǎng)脅迫,當(dāng)ER脅迫比較嚴(yán)重時(shí),UPR也會(huì)啟動(dòng)程序性細(xì)胞死亡程序。Bax-inhibitorl(BI-1)類似蛋白是真核生物中保守的抗細(xì)胞凋亡的因子,在哺乳動(dòng)物中參與調(diào)控UPR途徑。GAAP是BI-1的亞家族蛋白,在植物中的功能還未見(jiàn)報(bào)道。在擬南芥中有五個(gè)GAAP同源基因。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),GAAP1能夠幫助擬南芥抵抗鹽脅迫和ER脅迫,但其機(jī)制不清楚,為此,本文對(duì)其抗ER的機(jī)理進(jìn)行了探究,主要結(jié)果如下：1. GAAP1的轉(zhuǎn)錄表達(dá)受ER脅迫調(diào)控。對(duì)啟動(dòng)子與GUS的融合(PGAAP1::GUS)表達(dá)轉(zhuǎn)化子進(jìn)行GUS染色分析,發(fā)現(xiàn)在營(yíng)養(yǎng)生長(zhǎng)時(shí)期,GAAP1主要在根系中表達(dá),并受到TM誘導(dǎo)后表達(dá)增強(qiáng)；在生殖生長(zhǎng)時(shí)期,GAAP1主要在雌蕊及柱頭、花絲、未成熟的種子的珠柄處表達(dá)。2.GAAP1緩解TM誘導(dǎo)的ER脅迫。通過(guò)3,3'-diaminobenzidine(DAB)染色以及mondansylcadverin (MDC)染色檢測(cè)gaap1、Col、GAAP1-OX中活性氧及自噬體的發(fā)生情況,結(jié)果表明GAAP1過(guò)量表達(dá)能夠減少擬南芥中TM對(duì)H202和自噬體的誘導(dǎo)產(chǎn)生。3. GAAP1通過(guò)負(fù)調(diào)控UPR參與ER脅迫。通過(guò)QRT-PCR的技術(shù)手段檢測(cè)GAAP1過(guò)量表達(dá)或突變對(duì)UPR的影響,結(jié)果表明,在急性脅迫條件下,GAAP1的缺失或者過(guò)量對(duì)UPR通路的基因表達(dá)沒(méi)有明顯的影響；在ER脅迫的早期及持續(xù)一段時(shí)間后,GAAP1過(guò)量表達(dá)都抑制或減弱了保護(hù)性UPR基因的上調(diào)；gaap1缺失突變體在ER脅迫早期對(duì)UPR影響不大,而在持續(xù)的ER脅迫條件下能夠減弱UPR的上調(diào)。由此推測(cè)GAAP1可能參與持續(xù)ER脅迫條件下對(duì)UPR的減弱作用。通過(guò)分析急性脅迫后恢復(fù)生長(zhǎng)不同時(shí)間,野生型、gaap1gaap3雙突變體和GAAP1過(guò)量表達(dá)株系中UPR通路的表達(dá)情況,進(jìn)一步的驗(yàn)證了GAAP1抑制UPR的推測(cè)。為了探究GAAP1抑制UPR通路的分子機(jī)制,采用Western Blot的技術(shù)手段檢測(cè)了TM處理前后gaap1、Col、 gaaplgaap3雙突變體以及GAAP1-OX中BIP蛋白的表達(dá)量。結(jié)果表明GAAP1可能至少是通過(guò)上調(diào)BIP蛋白的表達(dá)量來(lái)減弱UPR通路。4. GAAP1與其互作因子共定位在細(xì)胞膜上并且受到脅迫后其定位發(fā)生變化。分別構(gòu)建基因與YFP和CFP的融合表達(dá)載體,通過(guò)煙草葉片瞬時(shí)轉(zhuǎn)化體系分析發(fā)現(xiàn),GAAP1定位于細(xì)胞膜,并且與互作因子在細(xì)胞膜上有共同的定位；構(gòu)建擬南芥35S::eYFP-GAAP1穩(wěn)定轉(zhuǎn)化子,觀察TM處理前后GAAP1的定位變化,結(jié)果表明,在正常生長(zhǎng)條件下,GAAP1定位于細(xì)胞膜、保衛(wèi)細(xì)胞膜及細(xì)胞骨架和葉綠體外膜上；TM脅迫處理?xiàng)l件下,GAAP1定位于細(xì)胞膜、保衛(wèi)細(xì)胞膜上。對(duì)于脅迫處理前后,保衛(wèi)細(xì)胞定位的變化與其功能的關(guān)系需要進(jìn)一步的研究。5. GAAP1與互作因子在在植物體內(nèi)互作。分別構(gòu)建GAAP1與FLAG及互作因子與TAP的融合表達(dá)載體,通過(guò)煙草葉片瞬時(shí)轉(zhuǎn)化體系,并利用Co-Immunoprecipitation (CO-IP)的技術(shù)手段研究發(fā)現(xiàn),GAAP1與互作因子在植物體內(nèi)互作。6.構(gòu)建了gaap1和互作因子突變體的雙突變體。為進(jìn)一步探究gaap1與互作因子抗ER脅迫的功能及gaapl與互作因子的作用機(jī)理,通過(guò)PCR的手段篩選互作因子以及GAAP1純合子突變體并通過(guò)遺傳雜交方法構(gòu)建雙突變體,為以后的深入探究奠定一定的基礎(chǔ)。綜上所述,GAAP1影響ER脅迫條件下的活性氧及自噬體的誘導(dǎo),在脅迫早期和中后期,對(duì)UPR都起削弱作用,GAAP1能上調(diào)BIP蛋白水平,對(duì)ER脅迫的調(diào)控作用是多層面的。進(jìn)一步分析GAAP1與其互作因子的相互作用在ER脅迫中的作用,有可能對(duì)GAAP1的功能提供更多細(xì)節(jié),為GAAP家族在植物中的作用機(jī)理奠定一定的基礎(chǔ)。
[Abstract]:Endoplasmic reticulum (endoplasmic reticulum) is one of the main sites for protein processing. When plant body is stressed, it may lead to unfolded or misfolded protein accumulation in the endoplasmic reticulum, that is, endoplasmic reticulum stress (ER stress). Cells start to protect the endoplasmic reticulum stress, including unfolded protein response (UPR) pathway, when ER stress is strict. When heavy, UPR also initiates a programmed cell death program,.Bax-inhibitorl (BI-1) similar protein, a conservative anti apoptotic factor in eukaryotes, and participates in the regulation of UPR pathway.GAAP as a subfamily of BI-1 in mammals, and has not been reported in plants. There are five GAAP homologous genes in Arabidopsis thaliana. The study found that GAAP1 could help Arabidopsis resistance to salt stress and ER stress, but the mechanism was not clear. Therefore, the mechanism of its anti ER was explored in this paper. The main results are as follows: 1. GAAP1 is regulated by ER stress. GUS staining analysis is used for the fusion of promoter and GUS (PGAAP1:: GUS) expression transformant, and it is found in nutritive students. In the long period, GAAP1 was mainly expressed in the root system and enhanced by TM induction. In the period of reproductive growth, GAAP1 was mainly expressed in the pistil and stigma, the silk and the immature seeds of the Pearl stem to express the ER stress induced by TM. 3,3'-diaminobenzidine (DAB) staining and mondansylcadverin (MDC) staining were used to detect gaap1, Col, and Col. The occurrence of active oxygen and autophago in OX showed that excessive expression of GAAP1 could reduce the induction of H202 and autophagic in Arabidopsis by TM, and.3. GAAP1 was involved in ER stress through negative regulatory UPR. The effect of GAAP1 overexpression or mutation on UPR was detected by QRT-PCR technique. The results showed that GAAP1 was deficient under acute stress. The loss or overdose had no significant effect on the gene expression in the UPR pathway; overexpression of GAAP1 inhibited or weakened the up regulation of the protective UPR gene in the early and continuous periods of ER stress, and the gaap1 deletion mutant had little effect on UPR in the early stage of ER stress, and could weaken the up-regulation of UPR under the persistent ER stress. It is speculated that GAAP1 may be involved in the weakening effect of UPR under continuous ER stress. By analyzing the expression of UPR pathway in different periods of growth, wild type, gaap1gaap3 double mutants and GAAP1 overexpressed strains after acute stress, the GAAP1 inhibition of UPR is further verified. In order to explore the molecular mechanism of GAAP1 inhibition of UPR pathway, Western Blot was used to detect the expression of gaap1, Col, gaaplgaap3 double mutants and BIP protein in GAAP1-OX before and after TM treatment. The results showed that GAAP1 may at least weaken the UPR pathway and its interaction factor in the cell membrane by up regulation of the expression of BIP protein. The fusion expression vector of YFP and CFP was constructed respectively. Through the analysis of the transient transformation system of tobacco leaves, it was found that GAAP1 was located in the cell membrane, and there was a common location with the interaction factor on the cell membrane, and the construction of Arabidopsis 35S:: eYFP-GAAP1 stable transformant was observed, and the localization of GAAP1 was observed before and after TM treatment. The results showed that it was normal. Under the condition of growth, GAAP1 is located in the cell membrane, defending the cell membrane and the cytoskeleton and the outer membrane of the chloroplast. Under the condition of TM stress, GAAP1 is located in the cell membrane and defending the cell membrane. The relationship between the changes of the location of the guard cells and the function of the guard cells before and after stress treatment requires a further study of the.5. GAAP1 and interaction factors in the plant body. Internal interaction. The fusion expression vector of GAAP1 and FLAG and interaction factors and TAP were constructed respectively. Through the transient transformation system of tobacco leaves, and using the technique of Co-Immunoprecipitation (CO-IP), the two mutants of gaap1 and mutual factor mutants were constructed by GAAP1 and interaction factors in the plants of the plant, and to further explore GA. The function of AP1 and interaction factors against ER stress and the mechanism of interaction between gaapl and interaction factors, screening interaction factors and GAAP1 homozygous mutants by means of PCR and constructing double mutants through genetic hybridization, laying a certain foundation for further exploration. To sum up, GAAP1 affects active oxygen and autophago under ER stress. The induction, in both early and middle and late stages of stress, weakens the UPR, and GAAP1 can increase the level of BIP protein. The regulation of ER stress is multifaceted. Further analysis of the interaction of GAAP1 and its interaction factor in ER stress may provide more details on the function of GAAP1 and the mechanism of GAAP family in plants. Lay a certain foundation.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：Q943.2

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