关键词: 巨桉, EgrCR, 低温胁迫, 基因表达, 基因功能

Abstract: [Objective] The protein structure, subcellular localization and expression with treatment of abiotic stresses for EgrCR(Eucgr.B02857), a cold responsive gene in Eucalyptus grandis, were characterized. And, the phenotype of Arabidopsis thaliana lines which over-expressed EgrCR were also analyzed to elucidate the roles it played in response to low temperature and other abiotic stresses in E. grandis. [Method] The characterization of EgrCR protein, cis-elements in promoter sequence of the gene and construction of phylogenetic tree of homologous proteins of EgrCR in different plants were analyzed with Protparam, PSIPRED, TMHMM, MatInspector and MEGA softwares. Subcellular localization of EgrCR was characterized with the method of introducing EgrCR-GFP fused genes into onion epidermal cells via gene gun bombardment. Gene expression analysis under treatments of abiotic stresses and circadian rhythm were carried on by semi-quantitative and quantitative RT-PCR methods respectively. The 35S::EgrCR over-expression vector was constructed and transformed into Arabidopsis thaliana, and the phenotypes of transgenic plants under different low temperatures (0, 4℃) were analyzed to elucidate the function of EgrCR under the treatment of low temperature. [Result] The protein encoded by EgrCR contains 144 amino acids and there were 4 α helixes and 3 β sheets. No domain and trans-membrane region were found in the protein. The phylogenetic tree based on homology comparison showed that it was closed to its homologous protein in Populus trichocarpa, sharing a 68% protein similarity for them. To the promoter sequence of EgrCR, Some cis-elements related with plant stresses response were found in it. Nuclear localization for EgrCR merged protein with GFP implied EgrCR was located in the nucleus. Under normal condition, EgrCR were mainly expressed in stems and leaves, and qRT-PCR result of EgrCR under 0, 2, 4, 6, 8℃ and time course (2, 6, 12, 24, 48 h) treatments at 4℃revealed that it was induced strongly by low temperature. In addition, EgrCR expression was not influenced by 100 μmol·L^-1 ABA. However, under the salt stress (200 mmol·L^-1), it was inhibited firstly and then induced. Circadian rhythm also regulates the EgrCR expression, and the transcription level of it was promoted under light and hampered in the darkness. When Arabidopsis thaliana transgenic lines of EgrCR overexpression were treated at 4℃, the accumulation of anthocyanin was reduced obviously. And, with one week recovery after 3 days treatment at 0℃, the transgenic lines can return to grow quickly, showing cold resistance compared to the wild type.[Conclusion] The EgrCR was involved in responses to cold and salt stresses in Eucalyptus grandis. Circadian rhythm also had an effect on its expression.

Key words: Eucalyptus grandis, EgrCR, cold stress, gene expression, gene function