关键词: 巨桉, EgrZFP6, 非生物逆境, 基因功能

Abstract: [Objective] EgrZFP6(Eucgr.A01232)is a gene which is involved in abiotic stresses response in Eucalyptus grandis. With the study of protein structure and function of EgrZFP6, the roles it possibly played in abiotic stress response in E. grandis were discussed in order to provide a basis for stress resistance breeding of Eucalyptus.[Method] The EgrZFP6 protein structure was analyzed with CDD online software. And, sequences of ZFP protein in other plant species that have high similarity with EgrZFP6 were downloaded from NCBI after Blast software was used. Multiple alignments for these sequences were finished with Clustalx and their motifs were analyzed. EgrZFP6∷sGFP fused expression vector was also constructed and transformed into onion epidermal cells via gene gun bombardment to identify subcellular localization of EgrZFP6. Transformation of 35S∷EgrZFP6 into Arabidopsis thaliana was via floral-dip method. Two homozygous lines of EgrZFP6 over-expression in A.thaliana were obtained, their phenotype under normal condition, low temperature, drought and high salinity treatments were evaluated compared to wild type (COL). Based on Yeast 2 Hybridization, EgrERF4 (Eucgr.F01164), a protein can interact with EgrZFP6, was screened from the Yeast 2 Hybridization library. The expression of EgrERF4 under low temperature, drought and salinity in E.grandis seedlings was also analyzed. Protein that can interact with EgrZFP6 were screened and verified by Yeast 2 Hybridization. Gene expression of EgrERF4 under abiotic stresses was analyzed by real time fluorescence quantitative RT-PCR method.[Result] The EgrZFP6 is a classic C2H2 type zinc finger protein. There are 2 zinc finger domains with QALGGH sequence which is specific for plants in it. An ERF (ethylene responsive element binding factor) associated amphiphilic repression (EAR) motif and a L-box motif were also found in the protein sequence.Result of subcellular localization revealed the protein EgrZFP6 encoded was localized in the nuclear. Under normal condition, the two EgrZFP6 over-expression A.thaliana lines showed primary root growth inhibition compared to wide type. However, its sensitivity to the low temperature was increased. PEG treatments can promote lateral root growth and increase the number of them and the transgenic lines showed tolerance to salinity treatment to some extent. EgrERF4, an ethylene responsive factor, can interact with EgrZFP6. For the low temperature treatments to the E. grandis seedlings, EgrERF4 expression can be induced under time course treatments (0.5,2,6,12,24,48 h) at 4℃ and different low temperature (-8,-4,0,4℃,2 h) except for -8℃. EgrERF4 expression induction was also found under high salinity (200 mmol·L^-1), but drought inhibited the EgrERF4 expression.[Conclusion] EgrZFP6 transcriptional factor was possibly involved in the low temperature, high salinity and drought stresses through interacting with EgrERF4 in Eucalyptus grandis. In plants, EgrZFP6 may negatively regulate the sensitivity to cold stress. But for drought and salinity stress responses, it possibly played a positive role via changing the root pattern.

Key words: Eucalyptus grandis, EgrZFP6, abiotic stress, gene function