巨桉EgrCBF3基因的克隆与逆境响应表达分析

Isolation and expression analysis under stress of EgrCBF3 in Eucalyptus grandis

【目的】克隆巨桉(Eucalyptus grandis)抗逆相关基因EgrCBF3(GenBank登录号:JQ068829)的全长cDNA序列,分析EgrCBF3在低温、干旱、脱落酸(ABA)处理和高盐条件下的表达。【方法】利用RT-PCR结合RACE技术,克隆巨桉抗逆相关基因EgrCBF3全长cDNA序列;分析正常条件下,巨桉根、茎和叶中EgrCBF3的表达情况;同时采用qRT-PCR,分析不同低温(0,2,4,6,8℃)和4℃处理不同时间(2,4,8,24和48h),以及干旱、ABA和高盐等逆境条件下EgrCBF3的响应表达情况。【结果】EgrCBF3cDNA全长1 161bp,含有1个675bp的ORF,编码224个氨基酸,包含1个AP2结构域。该基因编码的蛋白与蓝桉中的CBF蛋白同源性最高,达97%。EgrCBF3在巨桉的叶、茎和根中均有表达,但表达量无明显差异。对不同低温和4℃不同处理时间条件下EgrCBF3表达的qRT-PCR分析表明,EgrCBF3基因受低温诱导,在2℃条件下诱导表达量最强;在4℃条件下随低温时间的延长,其诱导表达特性不变,仅略有波动。在100μmol/L ABA、200mmol/L NaCl和干旱处理条件下,EgrCBF3的表达不受ABA和盐胁迫的影响,但在干旱胁迫下被诱导。【结论】EgrCBF3响应低温胁迫诱导,同时可能也参与了干旱胁迫的逆境响应机制。

【Objective】 The study aimed to isolate EgrCBF3（GenBank No.JQ068829） from Eucalyptus grandis and analyze its expression under stresses of low temperature,drought,abscisic acid（ABA） and high salt.【Method】 Using RT-PCR and RACE methods,EgrCBF3 was cloned,and its expression in roots,stems,and leaves under normal growth condition was studied.qRT-PCR method was used to analyze the expression change of EgrCBF3 under different low temperatures（0,2,4,6 and 8 ℃）,different times（2,4,8,24 and 48 h） at 4 ℃,ABA,drought and salinity.【Result】 The EgrCBF3 cDNA was 1 161 bp encoding a protein of 224 amino acids and contained an AP2 domain.The EgrCBF3 protein shared a 97% homology with EgCBF1.Result of expression showed that EgrCBF3 expressed in leaf,stem and root with no significant difference.And,the EgrCBF3 transcripts were induced by different low temperatures,its expression reached the highest level at 2 ℃.At the treatments of different times at 4 ℃,EgrCBF3 was induced at different time points,and slightly fluctuated.Changes of expression of EgrCBF3 under 100 μmol/L ABA,200 mmol/L NaCl and drought implied that EgrCBF3 was insensitive to ABA and salt treatment but induced under drought stress.【Conclusion】 EgrCBF3 was induced by cold stress and possibly involved in drought stress.