Characterization and functional analysis of an E3 ubiquitin ligase gene in the glucose-insensitive mutant in Arabidopsis thaliana

• Main Authors: Li-Chen Hsieh, 謝禮臣
• Other Authors: Co-Shine, Wang
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/88383204382217024590

碩士 === 國立中興大學 === 生物科技學研究所 === 103 === Upon abiotic stresses, plants may initiate a series of complicate processes of ubiquitination in which E3 ubiquitin ligase can specifically degrade its target proteins. We have screened to identify an Arabidopsis T-DNA insertion mutant, ul, that exhibited glucose-insensitivity in the presence of high glucose conditions. AtUL gene encodes an E3 ubiquitin ligase that has 411 amino acids with a calculated molecular mass of 45.4 kDa. The protein contains a C4HC3 RING-variant domain and a region of four transmembrane domains at the C-terminus. Subcellular localization analysis demonstrated that protein fused with EGFP was located to the plasma membrane. The gene can be significantly induced by glucose, salt, abscisic acid (ABA) and dehydration. The two mutant lines 216 and 480 displayed germination and cotyledon greening percentages higher than wild-type (WT) under various concentrations of ABA, salt and mannitol, suggesting that they are ABA-insensitive. Salt resistance has also demonstrated that ul mutants exhibited significantly resistance to salt. Transpiration analysis of the two mutant leaves displayed higher water loss than WT, suggesting of the insensitivity of the mutant. The stomatal openness in the two mutant lines was markedly higher than that in WT when leaves of 3-week-old plants were either treated with low concentrations of ABA or dehydrated for 12 days. The survival rate of the two mutant lines was lower than that of WT when plants were recovered after 15 days of dehydration. The ABA content in the ul seedling increased 1.3 fold which is lower than that in the WT after 4 hour dehydration also suggesting of the insensitive nature of two mutant lines. At the molecular level, quantitative real-time polymerase chain reaction analysis revealed that most transcripts of ABA/stress responsive genes were repressed in the two mutant lines compared to WT. We have demonstrated that AtUL acts in a signaling network upstream of HXK1 and ABI4 and plays a positive role in the regulation of glucose mediated ABA signaling pathway.