本文選題：類受體激酶CRPK1 + 磷酸化��； 參考：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：低溫作為一種重要的環(huán)境因子,對植物的生長、發(fā)育、代謝和地理分布有重要的影響。為了適應(yīng)環(huán)境,植物進化出了復(fù)雜的應(yīng)對機制來抵抗低溫脅迫。首先,植物遭受低溫脅迫時,細胞膜可能是作為初級信號感受器,細胞膜流動性變化使植物率先感受到低溫信號。但是感受信號的精確機制以及信號傳遞機制還不清楚。其次,在低溫信號轉(zhuǎn)導(dǎo)途徑研究中,目前主要是圍繞轉(zhuǎn)錄因子CBF/DREB1(C-repeat-bindingfactor/drought responsive element binding protein 1)展開的。低溫迅速誘導(dǎo)CBF基因大量表達,高量的CBF進一步激活其下游冷響應(yīng)基因COR(cold responsive)的大量表達,從而使植物抵抗低溫脅迫。但是研究多集中在CBBF基因的轉(zhuǎn)錄水平,發(fā)現(xiàn)了一系列調(diào)控CBF基因的轉(zhuǎn)錄因子,而在CBF蛋白水平調(diào)控機制研究方面則是空白。此外,由于CBF基因過量表達抑制植物生長,植物如何精細地調(diào)控低溫應(yīng)答和生長發(fā)育的平衡也不清楚。本研究通過凍處理實驗篩選類受體激酶突變體,克隆了一個新的類受體激酶CRPK1(cold-responsive protein kinase 1),其缺失突變體具有明顯的抗凍表型,突變體中CBFs受冷誘導(dǎo)水平微弱地高于野生型,而冷響應(yīng)基因受冷誘導(dǎo)水平顯著高于野生型。并且,野生型的CRPK1基因能夠回復(fù)突變體的抗凍表型,CBF及冷響應(yīng)基因表達水平也回復(fù)到野生型水平。證明CRPK1參與了低溫調(diào)控,并且調(diào)控過程依賴于CBF信號途徑。通過顯微鏡觀察CRPK1-GFP定位和組分分離實驗證明CRPK1定位在細胞膜上。另一方面,體內(nèi)體外的磷酸化實驗證明CRPK1具有激酶活性,并且低溫處理能夠激活其激酶活性。將CRPK1關(guān)鍵的保守位點K69突變后,激酶活性完全消失,并且將突變型的CRPK1K69E轉(zhuǎn)入crpk1-1突變體中,無法回復(fù)突變體的抗凍表型,證明CRPK1激酶活性對于其執(zhí)行低溫應(yīng)答功能至關(guān)重要。進一步通過酵母雙雜交實驗,篩選到CRPK1互作蛋白14-3-3蛋白。體內(nèi)體外的磷酸化實驗均證明,在低溫脅迫下14-3-3λ能夠被CRPK1磷酸化,并且證明S70、S112、S193和T214是14-3-3λ上主要的磷酸化位點,當(dāng)這四個位點突變成丙氨酸后,CRPK1磷酸化14-3-3λ的信號幾乎喪失。并且14-3-3kλ雙突變體同樣具有抗凍表型,野生型的14-3-3λ能夠回復(fù)雙突變體的表型,而突變型的14-3-3λ4A不能回復(fù)雙突變體的表型。證明14-3-3s也參與了低溫調(diào)控,并且調(diào)控過程依賴于CRPK1對其磷酸化。一方面,研究發(fā)現(xiàn)低溫處理導(dǎo)致細胞質(zhì)中14-3-3λ被CRPK1磷酸化,被磷酸化的14-3-3λ進入細胞核中。另一方面,植物體內(nèi)體外的實驗證明14-3-3λ與CBF蛋白互作,并且互作后會促進CBF1和CBF3蛋白的降解。進一步研究發(fā)現(xiàn),14-3-3s入核過程依賴于CRPK1對其磷酸化,并且突變成非磷酸化形式的14-3-34A不能促進CBF1和CBF3蛋白降解。遺傳實驗進一步證明,CRPK1和14-3-3s參與低溫調(diào)控依賴于CBF途徑。CRPK1和14-3-3s精細調(diào)控植物體內(nèi)CBF的蛋白水平,影響CBF下游冷響應(yīng)基因的表達。綜合上述結(jié)果,新的細胞膜類受體激酶CRPK1在低溫信號轉(zhuǎn)導(dǎo)通路中是一個負調(diào)控因子。低溫脅迫激活CRPK1激酶活性,磷酸化14-3-3蛋白,細胞質(zhì)中被磷酸化的14-3-3s進入細胞核中,促進細胞核中的CBF1/CBF3蛋白的降解,從而影響CBF下游冷響應(yīng)基因的表達,調(diào)控植物對低溫脅迫的響應(yīng)。CRPK1介導(dǎo)的負調(diào)控途徑作為一個"剎車"精細地調(diào)控植物冷響應(yīng)的強度,避免過度冷響應(yīng)造成的生長抑制,從而平衡植物生長發(fā)育和抵抗逆境間的關(guān)系。上述研究不僅揭示了低溫信號從細胞膜傳遞至細胞核中的負調(diào)控機制,也闡釋了 CBF蛋白翻譯后修飾的調(diào)控機理,為闡明植物低溫信號轉(zhuǎn)導(dǎo)途徑提供了重要的信息。
[Abstract]:As an important environmental factor, low temperature has an important influence on the growth, development, metabolism and geographical distribution of plants. In order to adapt to the environment, plants have evolved a complex coping mechanism to resist low temperature stress. First, when plants suffer from low temperature stress, the cell membrane may be used as a primary signal receptor and the cell membrane fluidity changes It is the first to feel the low temperature signal. But the precise mechanism of the sensory signal and the mechanism of signal transmission are not clear. Secondly, in the study of the low temperature signal transduction pathway, the current mainly focuses on the transcription factor CBF/DREB1 (C-repeat-bindingfactor/drought responsive element binding protein 1). The low temperature rapidly induces a large number of CBF genes. The high amount of CBF further activates the heavy expression of the downstream cold response gene COR (cold responsive), which makes plants resist low temperature stress. However, the study focused on the transcriptional level of the CBBF gene, and found a series of transcriptional factors regulating the CBF gene, while the study on the regulation mechanism of the CBF protein level is blank. In addition, C is due to C. BF gene overexpression inhibits plant growth, and it is not clear how plants regulate the balance of low temperature response and growth and development. In this study, a new receptor kinase CRPK1 (cold-responsive protein kinase 1) was cloned through the freeze treatment experiments, and the missing mutant had obvious antifreeze phenotype. The cold induced level of CBFs in the mutant was slightly higher than that in the wild type, while the cold response gene was significantly higher than that of the wild type. And the wild type CRPK1 gene could revert to the antifreeze phenotype of the mutant, and the expression level of CBF and the cold response gene reverted to the wild type. It was confirmed that CRPK1 was involved in low temperature regulation, and the regulation process was dependent on the control process. Depending on the CBF signal pathway, the CRPK1-GFP localization and component separation experiments show that CRPK1 is located on the cell membrane. On the other hand, the phosphorylation experiment in vitro and in vivo shows that CRPK1 has kinase activity, and the low temperature treatment can activate its kinase activity. The kinase activity completely disappeared after the mutation of the key conserving site K69 of CRPK1. Moreover, the mutant CRPK1K69E was transferred into the crpk1-1 mutant and could not recover the antifreeze phenotype of the mutant. It was proved that the activity of CRPK1 kinase was crucial to its low temperature response function. Further, the yeast two hybrid experiment was used to screen the CRPK1 interaction protein 14-3-3 protein. The phosphorylation experiment in the body outside the body proved that 14- was under low temperature stress. 3-3 lambda can be phosphorylated by CRPK1, and it is proved that S70, S112, S193 and T214 are the main phosphorylation sites on 14-3-3 lambda. When these four loci become alanine, the signal of CRPK1 phosphorylated 14-3-3 lambda is almost lost. And the 14-3-3k lambda double mutant has the same antifreeze phenotype, and the wild type 14-3-3 lambda can respond to the phenotypes of the double mutants, and the mutations are mutated. The type of 14-3-3 lambda 4A does not respond to the phenotype of the double mutants. It is proved that 14-3-3s is also involved in low temperature regulation, and the regulation process depends on the phosphorylation of CRPK1. On the one hand, the study found that low temperature treatment leads to CRPK1 phosphorylation in the cytoplasm and the phosphorylated 14-3-3 lambda into the cell nucleus. On the other hand, the experimental evidence in vitro and in vivo is found in the plant. The interaction between the 14-3-3 lambda and CBF protein and the interaction between CBF1 and CBF3 proteins will promote the degradation of CBF1 and CBF3 proteins. Further studies have found that the 14-3-3s nucleation process relies on the phosphorylation of CRPK1 to them, and that 14-3-34A in the form of a non phosphorylated form does not promote the degradation of CBF1 and CBF3 proteins. Genetic experiments show that CRPK1 and 14-3-3s are involved in the low temperature regulation dependence. The CBF pathway.CRPK1 and 14-3-3s regulate the protein level of CBF in the body of the plant and affect the expression of cold response genes in the downstream of CBF. The results show that the new cell membrane receptor kinase CRPK1 is a negative regulator in the low temperature signal transduction pathway. The activity of CRPK1 kinase, phosphorylated 14-3-3 protein, and phosphoric acid in the cytoplasm are activated by low temperature stress. The transformed 14-3-3s enters the nucleus and promotes the degradation of the CBF1/CBF3 protein in the nucleus, thus affecting the expression of the cold response genes in the downstream CBF and regulating the negative regulation pathway mediated by the response of the plant to low temperature stress as a "brake" to regulate the intensity of the cold response of the plant and avoid the growth inhibition caused by the excessive cold response. To balance the relationship between plant growth and resistance to stress, the above studies not only reveal the negative regulation mechanism of the transmission of low temperature signals from the cell membrane to the nucleus, but also explain the regulatory mechanism of the posttranslational modification of CBF protein, which provides important information for clarifying the pathway of plant low temperature signal transduction.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

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1 劉子妍;類受體激酶CRPK1調(diào)控擬南芥低溫應(yīng)答的分子機制研究[D];中國農(nóng)業(yè)大學(xué);2017年

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