关键词: 巨桉, NAC基因, 非生物逆境, 基因表达

Abstract: [Objective] NAC(NAM,ATAF and CUC2 proteins) is a family of special transcriptional factors in plants. The members in this family play important roles in life process of plants such as growth, development, signal transduction of hormones and stress responses.In order to provide more clues for further studies of the functions of EgrNAC1(Eucgr.I00058) gene, which possibly plays a very important role in Eucalyptus grandis, EgrNAC protein sequence, subcellular localization, gene expression under low temperature, high temperature, drought, salinity, ABA and MeJA were analyzed.[Method] Firstly, sequences of gene, promoter, and protein of EgrNAC1 were downloaded from genome database of Eucalyptus grandis. Then, bioinformatics software of SMART, MatInspector, and MEGA were applied to analyze the coding protein structure and cis-elements in promoter of EgrNAC1. EgrNAC1∷sGFP fused expression vector was also constructed with enzyme cutting method in the primary vector of pCAMBIA1300. And, the plasmid of EgrNAC1∷sGFP was transformed into onion epidermal cells via gene gun bombardment to identify subcellular localization of EgrNAC1 protein. Secondly, with the DGE (digital gene expression) data from the treatment of different time course under 4℃ with 3-month-old seedlings, genes co-expression with EgrNAC1 were analyzed with WGCNA software. Finally, to get more information of expression pattern of EgrNAC1 under different abiotic stress conditions, expression of EgrNAC1 under different temperatures (-8, -4, 0, 4, 8℃), time courses(0,2,6,12,24,48 h)at 4℃, high temperature (42℃), drought, salinity, ABA and MeJA were evaluated by real time fluorescence quantitative RT-PCR method.[Result] The NAC domain in EgrNAC1 contains 5 classical sub-domains A, B, C, D and E, including 2 α-helix and 5 β-sheets. There was also one nuclear localization sequence but no transmembrane region was found in the protein sequence. Phylogenetic analysis result showed that EgrNAC1 was classified into ATAF group in subfamilyⅠof NAC. In the NACs that were involved in stress responses, it belongs to SNAC-A subclass. ABRE, MBS, MCS, DREB and other cis-elements were found in the promoter of EgrNAC1 and most of them are related with abiotic stresses.Result of EgrNAC1 merged protein with GFP in onion cells showed the EgrNAC1 mainly expressed in the nuclear. In EgrNAC1 co-expression analysis under treatment of different time courses(0,2,6,12,24,48 h)at 4℃, among the 20 genes with the highest correlation coefficients,most were involved in the stress responses. EgrNAC1 was induced under the low temperature. The expression level of it increased with the time course under 4℃ treatment. For different low temperature treatments, the improvement of EgrNAC1 expression was higher under 4, 8℃ compared to the other low temperature. The induction of EgrNAC1 were also found under treatment of drought, high salinity (200 mmol·L^-1NaCl), ABA(100 μmol·L^-1) and MeJA(100 μmol·L^-1). After 1-day treatment of drought stress, EgrNAC1 expression level increased, followed by a slow decrease. It only needs 2 hours to stimulate EgrNAC1 expression under MeJA treatment, but for the induction by ABA, it needs 24 hours.[Conclusion] EgrNAC1 is an important NAC gene which is not only involved in responses to abiotic stresses such as low temperature, drought, high salinity, but also possibly have interactions with ABA and MeJA hormone signal transduction in these stress responses.

Key words: Eucalyptus grandis, NAC gene, abiotic stress, gene expression