Analysis of gene expression and protein accumulation of an E3 ligase mcCPN1 in ice plant
| Autor: | Yu-Chan Chen, 陳玉嬋 |
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| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 96 McCOPINE1 (mcCPN1), a RING-type copine, was identified from halophyte ice plant (Mesembryanthemum crystallinum L.) and known to interact with salt stress-induced proteins mcSKD1 (suppressor of K+ transport defect 1) and mcSNF1 (sucrose non-fermenting 1). McSKD1 is a salt-induced gene and involves in intracellular protein trafficking, whereas mcSNF1 is predicted to be a protein kinase and possibly involves in salt-stress signaling. Our previous study showed that mcCPN1 contains two conserved domains, N-terminal copine A domain and C-terminal RING (really interesting new gene)-finger domain. RING-finger proteins usually possess E3 ligase catalytic activity executing the last step of ubiquitination in recognizing and ubiquitinating substrate proteins. Thus, the main focus of this thesis is to examine the expression and accumulation of mcCPN1 under salt stress, along with the analysis of its E3 ligase activity in order to access its function in salt-tolerant mechanism of ice plant. To understand the role of mcCPN1 in salt-tolerant mechanism, gene expression and protein accumulation of mcCPN1 were examined at both cellular and intact plant levels. RT-PCR and Northern blotting were used to analyze mcCPN1 expression and anti-mcCPN1 antibody was used to detect mcCPN1 accumulation. At cellular level, gene expression of mcCPN1 was constitutive after addition of salt, whereas a significant change was found in the accumulation of mcCPN1. Long-term salt stress induced mcCPN1 accumulation; however, short-term salt stress suppressed it. Temperal accumulation of mcCPN1 indicated that cell proliferation has a profound influence on mcCPN1 accumulation. At intact plant level, constitutive expression of mcCPN1 was found in salt-stressed roots and leaves, whereas protein accumulation of mcCPN1 was salt-induced suggesting that mcCPN1 might participate in the salt stress response of ice plant. Due to the reason that the changes in the accumulation mcCPN1 were found at cellular and intact plant level, the catalytic activity of mcCPN1 was further examined. The activity of mcCPN1 was demonstrated by the in vitro ubiquitination assay. When using mcCPN1 interacting protein mcSKD1 or mcSNF1 as the in vitro substrate, mcSKD1 can be ubiquitinated but mcSNF1 can not. Furthermore, mcSNF1 reduced the amount of ubiquitinated mcSKD1 in the in vitro system. These results suggested that mcSKD1 is one of the substrate proteins for E3 ligase mcCPN1 and mcSNF1 might play a regulatory role in mcCPN1-mediated mcSKD1 ubiquitination. Taken together, these results indicated that the E3 ligase mcCPN1 participates in the cell proliferation and salt stress response in ice plant and the accumulation of mcCPN1 was regulated at translational and/or post-translational level. The involvement of mcCPN1 in salt-tolerant mechanism of ice plant is possibly by way of recognizing and ubiquitinated salt stress-related protein mcSKD1. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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