山葡萄<i>VaERF095</i>基因表达及其启动子功能分析

王岚; 许建韧

doi:10.3969/j.issn.2095-1191.2024.01.004

山葡萄VaERF095基因表达及其启动子功能分析

王岚¹,
许建韧²

1. 宁夏大学葡萄酒与园艺学院, 宁夏银川 750021;
2. 北方民族大学生物科学与工程学院, 宁夏银川 750021

基金项目:

宁夏自然科学基金项目(2021AAC03092)

宁夏重点研发计划项目(2020BEB04024)

详细信息

作者简介:
王岚(1990-),https://orcid.org/0000-0002-6496-8227,博士,主要从事葡萄抗逆分子育种研究工作,E-mail:wanglan@nxu.edu.cn

中图分类号: S663.103.6
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- 文章访问数: 65
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- PDF下载量: 8
出版历程
- 收稿日期: 2023-10-14
- 网络出版日期: 2024-05-23

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

WANG Lan¹,
XU Jian-ren²

1. College of Enology and Horticulture, Ningxia University, Yinchuan, Ningxia 750021, China;
2. College of Bioscience and Engineering, North Minzu University, Yinchuan, Ningxia 750021, China

Funds:

Ningxia Natural Science Foundation (2021AAC03092)

Ningxia Key Research and Development Plan Projec(t2020BEB04024)

摘要

摘要: 【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%。VaERF095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。
- 山葡萄 /
- ERF转录因子 /
- 基因表达 /
- 启动子 /
- 功能分析
Abstract: 【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.
- Vitis amurensis /
- ERF transcription factor /
- gene expression /
- promoter /
- functional analysis

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参考文献(30)

陈悦,陈茜,董伟峰,才晓溪,沈阳,杨珺凯,贾博为,孙明哲,孙晓丽.2022.水稻AP2/ERF转录因子基因OsERF096启动子克隆及活性分析[J].土壤与作物,11(2):159-169.[Chen Y,Chen X,Dong W F,Cai X X,Shen Y,Yang J K,Jia B W,Sun M Z,Sun X L.2022.Cloning and activity analysis of promoter of an AP2/ERF transcription factor gene OsERF096 in rice[J].Soils and Crops,11(2):159-169.]doi: 10.11689/j.issn.2095-2961.2022.02.005.

高玉迪,李可,朱友银,刘向蕾,尹亚红,王月,郭卫东.2021.中国樱桃乙烯响应因子PpcERF5基因克隆与功能分析[J].植物生理学报,57(1):59-68.[Gao Y D,Li K,Zhu YY,Liu X L,Yin Y H,Wang Y,Guo W D.2021.Cloning and functional analysis of ethylene responsive factor gene PpcERF5 from Chinese cherry[J].Plant Physiology Jour-nal,57(1):59-68.]doi: 10.13592/j.cnki.ppj.2020.0486.

贺普超,晁无疾.1982.我国葡萄属野生种质资源的抗寒性分析[J].园艺学报,9(3):17-21.[He P C,Chao W J.1982.Studies on the cold hardiness of eight species of Vitis L.growing wild in China[J].Acta Horticulturae Sinica,9(3):17-21.]

黄仁维,任迎虹,祁伟亮,曾睿,刘欣宇,邓彬艳.2022.桑树MaERF105-Like的克隆及其在干旱胁迫下的表达分析[J].园艺学报,49(11):2439-2448.[Huang R W,Ren YH,Qi W L,Zeng R,Liu X Y,Deng B Y.2022.Cloning of mulberry MaERF105-like gene and its expression under drought stress[J].Acta Horticulturae Sinica,49(11):2439-2448.]doi: 10.16420/j.issn.0513-353x.2021-0543.

王雪贺缘,郭耀祖,陈桂森,张苗苗,吴紫璇,左丁卉,谢鑫爽,贺琳.2021.玉米中低温应答ERF基因鉴定及表达分析[J/OL].分子植物育种. https://kns.cnki.net/kcms/detail/46.1068.S.20210809.1656.021.html" target="_blank"> https://kns.cnki.net/kcms/detail/46.1068.S.20210809.1656.021.html.[Wang X H Y,Guo Y Z,Chen G S,Zhang M M,Wu Z X,Zuo D H,Xie X S,He L.2021.Identification and expression analysis of low tem-perature response ERF genes in maize[J/OL].Molecular Plant Breeding. https://kns.cnki.net/kcms/detail/46.1068.S.20210809.1656.021.html" target="_blank"> https://kns.cnki.net/kcms/detail/46.1068.S.20210809.1656.021.html.]

徐伟荣.2010.中国华东葡萄抗白粉病芪合成酶基因及启动子克隆与功能分析[D].杨凌:西北农林科技大学.[Xu W R.2010.Cloning and functional analysis of stilbene synthase and its promoter from powdery mildew-resistant Chinese Vitis pseudoreticulata[D].Yangling:Northwest A&F University.]

赵龙飞,明聪,张中荣,袁玥,刘戈辉,张薇.2022.棉花AP2/ERF-B3亚组转录因子基因GhERF14的克隆与表达分析[J].分子植物育种,20(9):2804-2811.[Zhao L F,Ming C,Zhang Z R,Yuan Y,Liu G H,Zhang W.2022.Cloning and expression analysis of a transcription factor gene GhERF14 from cotton AP2/ERF-B3 subgroup[J].Molecu-lar Plant Breeding,20(9):2804-2811.]doi: 10.13271/j.mpb.020.002804.

Bolt S,Zuther E,Zintl S,Hincha D K,Schmülling T.2017.ERF105 is a transcription factor gene of Arabidopsis thaliana required for freezing tolerance and cold acclimation[J].Plant,Cell and Environment,40(1):108-120.doi: 10.1111/pce.12838.

Ding F,Wang C,Xu N,Wang M L.2022.The ethylene response Factor SlERF.B8 triggers jasmonate biosynthesis to promote cold tolerance in tomato[J].Environmental and Experimental Botany,203:105073.doi: 10.1016/j.envexp-bot.2022.105073.

Feng K,Hou X L,Xing G M,Liu J X,Duan A Q,Xu Z S,Li MY,Zhuang J,Xiong A S.2020.Advances in AP2/ERF superfamily transcription factors in plant[J].Critical Reviews in Biotechnology,40(6):750-776.doi: 10.1080/07388551.2020.1768509.

Ghorbani R,Zakipour Z,Alemzadeh A,Razi H.2020.Genomewide analysis of AP2/ERF transcription factors family in Brassica napus[J].Physiology and Molecular Biology of Plants,26:1463-1476.doi: 10.1007/s12298-020-00832-z.

Gong Z Z,Xiong L M,Shi H Z,Yang S H,Herrera-Estrella LR,Xu G H,Chao D Y,Li J R,Wang P Y,Qin F,Li J G,Ding Y L,Shi Y T,Wang Y,Yang Y Q,Guo Y,Zhu J K.2020.Plant abiotic stress response and nutrient use effi-ciency[J].Science China Life Sciences,63(5):635-674.doi: 10.1007/s11427-020-1683-x.

Han D G,Han J X,Xu T L,Li X G,Yao C Y,Li T M,Sun XH,Wang X H,Yang G H.2021.Overexpression of MbERF12,an ERF gene from Malus baccata (L.) Borkh,increases cold and salt tolerance in Arabidopsis thaliana associated with ROS scavenging through ethylene signal transduction[J].In Vitro Cellular&Developmental Biology-Plant,57(5):760-770.doi: 10.1007/s11627-021-10199-9.

Hao L D,Shi S B,Guo H B,Li M,Hu P,Wei Y D,Feng Y F.2020.Genome-wide identification and expression profiles of ERF subfamily transcription factors in Zea mays[J].PeerJ,8:e9551.doi: 10.7717/peerj.9551.

He S T,Hao X M,He S L,Hao X G,Zhang P,Chen X N.2021.Genome-wide identification,phylogeny and expres-sion analysis of AP2/ERF transcription factors family in sweet potato[J].BMC Genomics,22(1):748.doi: 10.1186/s12864-021-08043-w.

Hong Y B,Wang H,Gao Y Z,Bi Y,Xiong X H,Yan Y Q,Wang J J,Li D Y,Song F M.2022.ERF transcription fac-tor OsBIERF3 positively contributes to immunity against fungal and bacterial diseases but negatively regulates cold tolerance in rice[J].International Journal of Molecular Sciences,23(2):606.doi: 10.3390/ijms23020606.

Jofuku K D,den Boer B G,Van Montagu M,Okamuro J K.1994.Control of Arabidopsis flower and seed development by the homeotic gene APETALA2[J].The Plant Cell,6(9):1211-1225.doi: 10.1105/tpc.6.9.1211.

Li B B,Wang X H,Wang X F,Xi Z M.2023.An AP2/ERFtranscription factor VvERF63 positively regulates cold tolerance in Arabidopsis and grape leaves[J].Environmental and Experimental Botany,205:105124.doi: 10.1016/j.envexpbot.2022.105124.

Licausi F,Giorgi F M,Zenoni S,Osti F,Pezzotti M,Perata P.2010.Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[J].BMC Genomics,11:719.doi: 10.1186/1471-2164-11-719.

Licausi F,Ohme-Takagi M,Perata P.2013.APETALA/ethylene responsive factor(AP2/ERF) transcription factors:Mediators of stress responses and developmental programs[J].New Phytologist,199(3):639-649.doi: 10.1111/nph.12291.

Lv K W,Wu W Q,Wei H R,Liu G F.2021.A systems biology approach identifies a regulator,BplERF1,of cold tolerance in Betula platyphylla[J].Forestry Research,1(1):96-105.doi: 10.48130/FR-2021-0011.

Ohme-Takagi M,Shinshi H.1995.Ethylene-inducible DNAbinding proteins that interact with an ethylene responsive element[J].Plant Cell,7(2):173-182.doi: 10.2307/3869993.

Ritonga F N,Ngatia J N,Wang Y,Khoso M A,Farooq U,Chen S.2021.AP2/ERF,an important cold stress-related tran-scription factor family in plants:A review[J].Physiology and Molecular Biology of Plants,27:1953-1968.doi: 10.1007/s12298-021-01061-8.

Sun M Z,Shen Y,Chen Y,Wang Y,Cai X X,Yang J K,Jia BW,Dong W F,Chen X,Sun X L.2022.Osa-miR1320 tar-gets the ERF transcription factor OsERF096 to regulate cold tolerance via JA-mediated signaling[J].Plant Physiology,189(4):2500-2516.doi: 10.1093/plphys/kiac208.

Sun X M,Zhang L L,Wong D C J,Wang Y,Zhu Z F,Xu G Z,Wang Q F,Li S H,Liang Z C,Xin H P.2019a.The ethylene response factor VaERF092 from Amur grape regulates the transcription factor VaWRKY33,improving cold tolerance[J].Plant Journal,99(5):988-1002.doi: 10.1111/tpj.14378.

Sun X M,Zhao T T,Gan S H,Ren X D,Fang L C,Karungo SK,Wang Y,Chen L,Li S H,Xin H P.2016.Ethylene posi-tively regulates cold tolerance in grapevine by modulating the expression of ETHYLENE RESPONSE FACTOR 057[J].Scientific Reports,6:24066.doi: 10.1038/srep24066.

Sun X M,Zhu Z F,Zhang L L,Fang L C,Zhang J S,Wang QF,Li S H,Liang Z C,Xin H P.2019b.Overexpression of ethylene response factors VaERF080 and VaERF087 from Vitis amurensis enhances cold tolerance in Arabidopsis[J].Scientia Horticulturae,243:320-326.doi: 10.1016/j.scienta.2018.08.055.

Wang L,Liu W D,Wang Y J.2020.Heterologous expression of Chinese wild grapevine VqERFs in Arabidopsis thaliana enhance resistance to Pseudomonas syringae pv.tomato DC3000 and to Botrytis cinerea[J].Plant Science,293:110421.doi: 10.1016/j.plantsci.2020.110421.

Xiao X M,Si J,Wei W,Yang Y Y,Shan W,Kuang J F,Lu WJ,Chen J Y,Chen J W.2023.Banana ERF transcription factor MaERF110 is involved in ethylene-induced chilling tolerance by regulating ROS accumulation[J].Postharvest Biology and Technology,197:112218.doi: 10.1016/j.post-harvbio.2022.112218.

Zhu X L,Wei X H,Wang B Q,Wang X,Zhang M J.2020.Identification and analysis of AP2/ERF gene family in tomato under abiotic stress[J].Biocell,44(4):777-803.doi: 10.32604/biocell.2020.010153.