Gene expression of VaERF095 and functional analysis of its promoter from Vitis amurensis

【Objective】In this study, the transcription factor gene VaERF095 and its promoter were cloned from Chinese Vitis amurensis. The gene expression and promoter activity under the induction of low temperature and exogenous hormones were analyzed, so as to provide theoretical basis for the regulation mechanism of VaERF095 gene in response to low temperature stress. 【Method】The VaERF095 gene from V. amurensis variety Zuoshan-1 and its promoter were cloned by homology-based cloning. The expressions of VaERF095 gene induced by low temperature(4 ℃) and exogenous hormones and in different tissues were analyzed by real-time fluorescence quantitative PCR. The GUS protein expression vector inserted with VaERF095 gene promoter was constructed and transiently transformed into grape leaves. The β-glucuronidase(GUS) activity assay was performed to analyze the expression of VaERF095 gene promoter induced by low temperature and exogenous hormones. 【Result】The VaERF095 gene was cloned from V. amurensis and the total length of the coding sequence(CDS) was 393 bp, encoding 130 amino acid residues. The protein contained a conserved AP2/ERF domain, which was closely related to V. vinifera L. and V. riparia, with amino acid sequence similarity of 100.00% and 93.08%, respectively. The VaERF095 gene showed different expression patterns in response to low temperature and induction of exogenous hormones ethylene(ET), salicylic acid(SA), methyl jasmonate(MeJA) and abscisic acid(ABA).VaERF095 gene was expressed in mature leaf, stem, inflorescence, tendril and berry, with the highest expression in tendril and very low or almost no expression in young leaf. The VaERF095 gene promoter with a length of 1360 bp was cloned. The sequence analysis indicated that VaERF095 gene promoter containing two low temperature responsive element(LTR), one ET responsive element(ERE), one SA responsive element(TCA-element), two MeJA responsive elements(CGTCA-motif and TGACG-motif), one ABA responsive element(ABRE), one GA responsive element(P-box) and one defense and stress responsive element(TC-rich repeats). The activity of Va ERF095 gene promoter was extremely enhanced in response to low temperature and exogenous hormones ET, SA and ABA(P<0.01). Meanwhile, the activity of Va ERF095 gene promoter was significantly enhanced in response to MeJA induction(P<0.05). 【Conclusion】VaERF095 gene and its promoter positively respond to the induction of low temperature(4 ℃) and exogenous hormones such as ET, SA, ABA and MeJA, and has obvious tissue expression specificity.