The Ethylene Response Factor (ERF) was used to screen members of the ERF transcription factor family in the transcriptome of ‘Xiangling’ walnut. Following homology alignments, open reading frame (ORF) confirmation, sequence basic characteristics analyses, and PCR-based vector cloning, an ERF termed JrERF2?2 was selected for further analyses.

Using the ExPASy bioinformatics portal, prediction analyses suggested a 906 bp ORF of the JrERF2?2 gene that encodes a protein consisting of 262 amino acids; the mole?cular weight of this protein is 74.43 ku, and the theoretical isoelectric point is 5.07. CD?Search showed an AP2 domain in the JrERF2?2 protein. BLASTP and MEGA analyses suggested considerable similarity of the JrERF2?2 protein with ERF proteins of Betula platyphylla and Quercus suber, indicating potentially similar functions of JrERF2?2 and its homologs. Moreover, to investigate potential adaption to adverse stimuli, a 1 455 bp upstream promoter was identified in the walnut genome, and cis-acting elements were predicted using New PLACE analyses. The results showed that this promoter segment included a variety of stress-related cis-acting elements such as the drought early response element (ACGTATERD1), heat stress response element (CCAATBOX1), and low temperature response element (LTRE1HVBLT49), suggesting that this promoter may regulate JrERF2?2 expression during stress responses. Therefore, to further investigate the role of JrERF2?2 in stress response mechanisms, 2-year-old grafted ‘Xiangling’ walnut seedlings were subjected to salt, drought and abscisic acid (ABA) stress. JrERF2?2 expression was quantified using reverse-transcription quantitative PCR (qRT-PCR) analysis. Salt treatment was applied using 0.3 mol/L sodium chloride (NaCl) for 0, 3, 12 and 24 h; drought stress was elicited using 100 g/kg PEG₆₀₀₀ for 0, 3, 4 and 5 days, and the ABA treatment was applied using 0.1 mmol/L ABA for 0, 3, 24 and 48 h. Total RNA was isolated from leaf samples using a cetyltrimethylammonium bromide (CTAB) method. RNA was then reverse-transcribed to cDNA which was diluted 10-fold for use as a qRT-PCR template. Reverse transcription was performed using a PrimeScript^TM RT Reagent Kit (CWBIO, Kangwei Century, China), and the reaction was incubated at 42 ℃ for 60 min and at 85 ℃ for 5 s. A SYBR Green Real-time PCR Master Mix and the PCR primers JrERF2-2-F (5′-TGTCACCGAAGTTCCGGAT-3′) and JrERF2-2-R (5′-GATGCAGCTTCTCTAGTC-3′) were used for qRT-PCR. Walnut 18S rRNA (HE574850) was used as an internal reference. Relative expression le?vels were recorded using a 2^-ΔΔCt method. The qRT-PCR results showed that JrERF2?2 expression was induced by the NaCl, PEG₆₀₀₀ and ABA treatments, at different transcription levels. Under NaCl stress, JrERF2?2 expression levels increased over time. Relative expression at 12 and 24 h was 2.68- and 6.70-fold higher, respectively, than that at 3 h. Under exposure to PEG₆₀₀₀, JrERF2?2 expression also increased continuously over time with up to 4.12-fold increased expression compared to the control. In the ABA treatment, JrERF2?2 expression was similar to that under NaCl stress, and maximum values observed at 48 h were 6.57-fold higher than those at 3 h. NaCl, drought and ABA treatment results indicated that JrERF2?2 expression can be induced by these stressors.

Thus, JrERF2?2 is an important member of the ERF family and may have similar functions to homologs in other species. Its upstream promoter contains many cis-elements associated with stress responses, suggesting that this promoter can effectively regulate JrERF2?2 expression in response to stress. JrERF2?2 expression can be induced by salt, drought and ABA treatments, to varying degrees, and the expression patterns shared certain similarities, indicating that JrERF2?2 can respond to drought- and salt-induced osmotic stress. Moreover, JrERF2?2 may be involved in the ABA signaling pathway. Taken together, JrERF2?2 is an important candidate gene for revealing the mechanism of walnut adaptation to adverse conditions.