山葡萄VaERF095基因表达及其启动子功能分析

10Gene expression of VaERF095 and functional analysis of its promoter from Vitis amurensis

【目的】克隆山葡萄乙烯响应因子基因VaERF095及其启动子序列,分析在低温和外源激素诱导下该基因的表达模式和启动子活性,为深入探究VaERF095基因参与低温胁迫响应机理提供理论依据。【方法】采用同源克隆方法从山葡萄品种左山-1中获得VaERF095基因及其启动子,并通过实时荧光定量PCR检测VaERF095基因在低温(4℃)胁迫和外源激素诱导下及不同组织中的表达情况。同时构建VaERF095基因启动子融合GUS蛋白表达载体,瞬时转化葡萄叶片,通过β-葡萄糖苷酸酶(GUS)活性定量分析VaERF095基因启动子受低温胁迫和外源激素诱导的表达情况。【结果】从山葡萄克隆获得VaERF095基因,编码区(CDS)全长393 bp,编码130个氨基酸残基,该蛋白含有1个保守的AP2/ERF结构域,与欧洲葡萄和河岸葡萄的亲缘关系较近,氨基酸序列相似性分别为100.00%和93.08%。Va ERF095基因可响应低温和外源激素乙烯(ET)、水杨酸(SA)、茉莉酸甲酯(MeJA)和脱落酸(ABA)的诱导,呈现出不同的表达模式。VaERF095基因在老叶、茎、花序、卷须和果实均有表达,其中在卷须中的表达量最高,在幼叶中的表达量极低或几乎不表达。克隆获得长度为1360 bp的VaERF095基因启动子,含有2个低温响应元件(LTR)、1个ET响应元件(ERE)、1个SA响应元件(TCA-element)、2个MeJA响应元件(CGTCA-motif和TGACG-motif)、1个ABA响应元件(ABRE)、1个GA响应元件(P-box)和1个防御和胁迫响应元件(TC-rich repeats)。在低温胁迫及外源激素(ET、SA和ABA)诱导下,VaERF095基因启动子活性较对照极显著升高(P<0.01),在MeJA诱导下,VaERF095基因启动子活性显著增强(P<0.05)。【结论】VaERF095基因及其启动子正向响应低温(4℃)及外源激素(ET、SA、ABA和MeJA)的诱导,具有明显的组织表达特异性。

【Objective】In this study,the transcription factor gene VaERF095 and its promoter were cloned from Chi-nese 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 hor-mones and in different tissues were analyzed by real-time fluorescence quantitative PCR.The GUS protein expression vec-tor inserted with VaERF095 gene promoter was constructed and transiently transformed into grape leaves.Theβ-glucuroni-dase(GUS)activity assay was performed to analyze the expression of VaERF095 gene promoter induced by low tempera-ture and exogenous hormones.【Result】The VaERF095 gene was cloned from V.amurensis and the total length of the co-ding sequence(CDS)was 393 bp,encoding 130 amino acid residues.The protein contained a conserved AP2/ERF do-main,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 induc-tion 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 ten-dril 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 ele-ment(LTR),one ET responsive element(ERE),one SA responsive element(TCA-element),two MeJA responsive ele-ments(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 VaERF095 gene promoter was extremely enhanced in response to low temperature and exogenous hormones ET,SA and ABA(P<0.01).Meanwhile,the activity of VaERF095 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.