拟南芥GAAP1对内质网胁迫的调控作用

发布时间：2018-05-10 12:09

本文选题：GAAP1 + 互作因子　；参考：《华东师范大学》2015年硕士论文

【摘要】：内质网是蛋白质加工的主要场所之一。当植物体受到胁迫条件时,就可能导致内质网中未折叠或者错误折叠蛋白的积累,即内质网胁迫(ER胁迫)。细胞启动包括未折叠蛋白响应(UPR)途径在内的保护途径来缓解内质网胁迫,当ER胁迫比较严重时,UPR也会启动程序性细胞死亡程序。Bax-inhibitorl(BI-1)类似蛋白是真核生物中保守的抗细胞凋亡的因子,在哺乳动物中参与调控UPR途径。GAAP是BI-1的亚家族蛋白,在植物中的功能还未见报道。在拟南芥中有五个GAAP同源基因。本实验室前期研究发现,GAAP1能够帮助拟南芥抵抗盐胁迫和ER胁迫,但其机制不清楚,为此,本文对其抗ER的机理进行了探究,主要结果如下：1. GAAP1的转录表达受ER胁迫调控。对启动子与GUS的融合(PGAAP1::GUS)表达转化子进行GUS染色分析,发现在营养生长时期,GAAP1主要在根系中表达,并受到TM诱导后表达增强；在生殖生长时期,GAAP1主要在雌蕊及柱头、花丝、未成熟的种子的珠柄处表达。2.GAAP1缓解TM诱导的ER胁迫。通过3,3'-diaminobenzidine(DAB)染色以及mondansylcadverin (MDC)染色检测gaap1、Col、GAAP1-OX中活性氧及自噬体的发生情况,结果表明GAAP1过量表达能够减少拟南芥中TM对H202和自噬体的诱导产生。3. GAAP1通过负调控UPR参与ER胁迫。通过QRT-PCR的技术手段检测GAAP1过量表达或突变对UPR的影响,结果表明,在急性胁迫条件下,GAAP1的缺失或者过量对UPR通路的基因表达没有明显的影响；在ER胁迫的早期及持续一段时间后,GAAP1过量表达都抑制或减弱了保护性UPR基因的上调；gaap1缺失突变体在ER胁迫早期对UPR影响不大,而在持续的ER胁迫条件下能够减弱UPR的上调。由此推测GAAP1可能参与持续ER胁迫条件下对UPR的减弱作用。通过分析急性胁迫后恢复生长不同时间,野生型、gaap1gaap3双突变体和GAAP1过量表达株系中UPR通路的表达情况,进一步的验证了GAAP1抑制UPR的推测。为了探究GAAP1抑制UPR通路的分子机制,采用Western Blot的技术手段检测了TM处理前后gaap1、Col、 gaaplgaap3双突变体以及GAAP1-OX中BIP蛋白的表达量。结果表明GAAP1可能至少是通过上调BIP蛋白的表达量来减弱UPR通路。4. GAAP1与其互作因子共定位在细胞膜上并且受到胁迫后其定位发生变化。分别构建基因与YFP和CFP的融合表达载体,通过烟草叶片瞬时转化体系分析发现,GAAP1定位于细胞膜,并且与互作因子在细胞膜上有共同的定位；构建拟南芥35S::eYFP-GAAP1稳定转化子,观察TM处理前后GAAP1的定位变化,结果表明,在正常生长条件下,GAAP1定位于细胞膜、保卫细胞膜及细胞骨架和叶绿体外膜上；TM胁迫处理条件下,GAAP1定位于细胞膜、保卫细胞膜上。对于胁迫处理前后,保卫细胞定位的变化与其功能的关系需要进一步的研究。5. GAAP1与互作因子在在植物体内互作。分别构建GAAP1与FLAG及互作因子与TAP的融合表达载体,通过烟草叶片瞬时转化体系,并利用Co-Immunoprecipitation (CO-IP)的技术手段研究发现,GAAP1与互作因子在植物体内互作。6.构建了gaap1和互作因子突变体的双突变体。为进一步探究gaap1与互作因子抗ER胁迫的功能及gaapl与互作因子的作用机理,通过PCR的手段筛选互作因子以及GAAP1纯合子突变体并通过遗传杂交方法构建双突变体,为以后的深入探究奠定一定的基础。综上所述,GAAP1影响ER胁迫条件下的活性氧及自噬体的诱导,在胁迫早期和中后期,对UPR都起削弱作用,GAAP1能上调BIP蛋白水平,对ER胁迫的调控作用是多层面的。进一步分析GAAP1与其互作因子的相互作用在ER胁迫中的作用,有可能对GAAP1的功能提供更多细节,为GAAP家族在植物中的作用机理奠定一定的基础。
[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.

【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q943.2

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