苹果U-box型E3泛素连接酶MdPUB24的耐盐性和ABA敏感性鉴定

从‘皇家嘎拉’苹果(Malus×domestica Borkh.)中克隆了一个E3泛素连接酶基因(序列号:MDP0000199588)。基因序列测序发现,该基因包含长为1 212 bp完整的开放阅读框,编码404个氨基酸。系统进化树分析表明,这一E3泛素连接酶与拟南芥At PUB24同源序列相似性最高,因此将其命名为MdPUB24。利用Plant Care数据库进行启动子顺式作用元件预测分析表明,MdPUB24启动子序列中含有与脱落酸、光、茉莉酸及干旱信号相关的顺式作用元件。荧光定量PCR分析表明,MdPUB24在‘皇家嘎拉’苹果的不同组织中均有表达,且在叶片中最高;外源ABA、NaCl和低温胁迫处理能够抑制‘皇家嘎拉’苹果组培苗中MdPUB24的表达。MdPUB24过量表达的‘王林’苹果愈伤组织和异位表达的拟南芥幼苗在盐胁迫条件下,生长势与野生型相比明显变弱,表明MdPUB24负调控盐胁迫。相反,在外源ABA处理条件下,MdPUB24过量表达苹果愈伤和异位表达拟南芥与对照相比,生长势明显增强,表明MdPUB24对ABA不敏感。

Functional Identification of Salt Tolerance and ABA Sensitivity of Apple U-box E3 Ubiquitin Ligase MdPUB24

An E3 ubiquitin ligase gene（Gene ID：MDP0000199588）was cloned from‘Royal Gala’ apple（Malus × domestica Borkh.）. Sequence analysis showed that the length of MdPUB24 gene was 1 212 bp，which encoded 404 amino acids. A phylogenetic tree analysis indicated that our cloned apple E3 ubiquitin ligase exhibited the highest sequence similarity to Arabidopsis AtPUB24，so named MdPUB24. In silico analysis suggested that the promoter sequence of MdPUB24 contained several typical cis-acting elements，including abscisic acid-，light-，MeJA- and drought-responsive elements using PlantCare Databases. qPCR analysis showed that MdPUB24 widely expressed in different tissues of‘Royal Gala’ apple，and highly expressed in apple leaves. The expression of MdPUB24 was significantly inhibited by exogenous ABA and NaCl and low temperature. The growing potential of MdPUB24-expressing apple calli of‘Orin’cultivar and Arabidopsis were much weaker than that of control under salt stress condition，suggesting that MdPUB24 negatively regulated salt tolerance. In contrast，the growing potential of MdPUB24-expressing apple calli of‘Orin’and Arabidopsis were much stronger than that of control in the presence of exogenous ABA，suggesting that MdPUB24 was insensitive to exogenous ABA.