橄榄CaERF109和CaABR1基因的克隆及其表达特性分析
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摘要
橄榄(Canarium album)是我国热带亚热带地区特色名贵果树,其抗寒能力弱,常常遭受低温冻害,APETALA2/乙烯响应因子(APETALA2/ethylene responsive factor, AP2/ERF)与植物生长发育和逆境胁迫响应密切相关。为了解橄榄AP2/ERF转录因子的调控功能,本研究以福榄1号橄榄为材料,采用反转录PCR(reverse transcription PCR, RT-PCR)技术克隆了2个AP2/ERF超家族成员,命名为CaERF109(GenBank登录号:MH670905)和CaABR1(abscisic acid repressor 1, GenBank登录号:MH670906),并对其进行生物信息学和qRT-PCR表达分析。结果发现,CaERF109和CaABR1开放阅读框分别为888和1 473 bp,预测可分别编码295和490个氨基酸。生物信息学分析表明,CaERF109和CaABR1密码子偏好性较弱,均编码不稳定疏水碱性蛋白,具有AP2/ERF家族转录因子中ERF亚族的典型特征,预测分别定位于线粒体基质和过氧化物酶体。qRT-PCR分析表明,CaERF109和CaABR1在不同器官中呈特异性表达,其中表达量最高的分别是根和叶;低温胁迫过程中,随着温度降低,CaERF109和CaABR1均极显著上调表达(P<0.01)。研究结果表明,CaERF109和CaABR1可能在橄榄器官发育过程中存在功能差异,并且可能参与橄榄低温胁迫响应过程。本研究结果可为橄榄低温胁迫响应的分子机制研究和抗寒育种提供理论依据。
Canarium album is one of the characteristic and precious fruit in tropical and subtropical regions of China. Generally, C. album is poor in cold resistance, thus, often suffers from low temperature freezing damage. Recently, accumulated evidences have revealed that APETALA2/ethylene responsive factor(AP2/ERF) is closely related to growth and stress response of many plants. To investigate the regulatory functions of AP2/ERF transcription factors in C. album, two AP2/ERF gene superfamily members, named CaERF109(GenBank No. MH670905) and CaABR1(abscisic acid repressor 1, GenBank No. MH670906) were cloned from C album cv. Fulan-1 tree, and their bioinformatics and qRT-PCR expression patterns were performed inthis study. The results showed that the ORF of CaERF109 and CaABR1 were 888 bp and 1 473 bp respectively, and predicted to encode 295 and 490 amino acids. Bioinformatics analysis showed that the codon biases level of CaERF109 and CaABR1 were low, and both of them encoded unstable hydrophobic basic proteins, contained ERF typical features that belonged to AP2/ERF transcription factor superfamily.Subcellular location prediction showed that CaERF109 and CaABR1 located in mitochondrial matrix and peroxisomes respectively. According to q RT-PCR analysis, both CaERF109 and CaABR1 showed organspecific expression patterns, and of which was highly expressed in root and leaf respectively. In addition, both genes were extremely significantly up-regulated with the decrease of temperature during cold stress(P<0.01).This research revealed that both CaERF109 and CaABR1 may play different regulation functions during development of organs, and involve in low temperature stress responses of C. album. This study could provide the theoretical foundation for molecular mechanism research of low temperature stress and cold resistance breeding in C. album.
Canarium album is one of the characteristic and precious fruit in tropical and subtropical regions of China. Generally, C. album is poor in cold resistance, thus, often suffers from low temperature freezing damage. Recently, accumulated evidences have revealed that APETALA2/ethylene responsive factor(AP2/ERF) is closely related to growth and stress response of many plants. To investigate the regulatory functions of AP2/ERF transcription factors in C. album, two AP2/ERF gene superfamily members, named CaERF109(GenBank No. MH670905) and CaABR1(abscisic acid repressor 1, GenBank No. MH670906) were cloned from C album cv. Fulan-1 tree, and their bioinformatics and qRT-PCR expression patterns were performed inthis study. The results showed that the ORF of CaERF109 and CaABR1 were 888 bp and 1 473 bp respectively, and predicted to encode 295 and 490 amino acids. Bioinformatics analysis showed that the codon biases level of CaERF109 and CaABR1 were low, and both of them encoded unstable hydrophobic basic proteins, contained ERF typical features that belonged to AP2/ERF transcription factor superfamily.Subcellular location prediction showed that CaERF109 and CaABR1 located in mitochondrial matrix and peroxisomes respectively. According to q RT-PCR analysis, both CaERF109 and CaABR1 showed organspecific expression patterns, and of which was highly expressed in root and leaf respectively. In addition, both genes were extremely significantly up-regulated with the decrease of temperature during cold stress(P<0.01).This research revealed that both CaERF109 and CaABR1 may play different regulation functions during development of organs, and involve in low temperature stress responses of C. album. This study could provide the theoretical foundation for molecular mechanism research of low temperature stress and cold resistance breeding in C. album.
引文
蔡文华,陈惠,李文,等.2006.2004/2005年冬季连江县低温考察和橄榄树冻害指标初探[J].中国农业气象,27(3):200-203.(Cai W H,Chen H,Li W,et al.2006.Investigation of low air temperature and preliminary study of indexes of Olive freeze harm in the winter of 2004/2005in Lianjiang county[J].Chinese Journal of Agrometeorology,27(3):200-203.)
曹诣斌,石瑞,陈文荣,等.2011.低温胁迫下佛手和枳乙烯应答因子6(ERF6)表达变化的比较分析[J].园艺学报,38(10):1873-1882.(Cao Y B,Shi R,Chen W R,et al.2011.Comparative analysis of expression changes of ethylene response factor6(ERF6)in fingered citron and trifoliate orange under cold stress[J].Acta Horticulturae Sinica,38(10):1873-1882.)
高敏霞,冯新,赖瑞联,等.2018.米良1号猕猴桃漆酶基因的克隆与表达分析[J].农业生物技术学报,26(1):65-76.(Gao M X,Feng X,Lai R L,et al.2018.Cloning and expression analysis of the laccase genes from kiwifruit(Actinidia chinensis var.deliciosa'Miliang-1')[J].Chinese Journal of Agricultural Biotechnology,26(1):65-76.)
黄敏杰.2017.橄榄(Canarium album(Lour.)Raeusch.)金丝桃苷的测定及生物合成相关基因的克隆与表达分析[D].福建农林大学,导师:陈清西,pp.12-24.(Huang M J.2017.Measurement of hyperoside content,cloning and expression of the synthesis genes in Canarium album[D].Fujian Agriculture and Forestry University,Supervisor:Chen Q X,pp.12-24.)
赖瑞联,陈瑾,冯新,等.2017.中国橄榄种质资源评价与抗寒性研究进展[J].热带作物学报,38(11):2188-2194.(Lai R L,Chen J,Feng X,et al.2017.Advances on germplasm resource evaluation and cold resistance in Chinese olive of China[J].Chinese Journal of Tropical Crops,38(11):2188-2194.)
赖钟雄,蔡英卿,许伟东,等.2007.橄榄优质栽培与综合利用[M].北京:中国三峡出版社,pp.14-17.(Lai Z X,Cai Y Q,Xu W D,et al.2007.Quality Cultivating and Integrated Utilization of Chinese Olive[M].Beijing:Chinese Sanxia Press,pp.14-17.)
杨德鑫.2012.水稻ERF蛋白OsERF109的特性及功能分析[D].中国农业科学院,导师:黄荣峰,pp.23.(Yang DX.2012.The characterization and function analyses of rice ERF protein OsERF109[D].Chinese Academy of Agricultural Sciences,Supervisor:Huang R F,pp.23.)
于晓惠.2013.巴西橡胶ERF转录因子基因的克隆与功能分析[D].海南大学,导师:张治礼,pp.34-35.(Yu X H2013.Cloning and functional analysis of the HbERFfrom Hevea brasiliensis[D].Hainan University,Suprevisor:Zhang Z L,pp.34-35.)
翟莹,张军,赵艳,等.2016.大豆ERF转录因子基因GmERF8的克隆与表达分析[J].植物遗传资源学报,17(6):1036-1040.(Zhai Y,Zhang J,Zhao Y,et al.2016Cloning and expression analysis of ERF transcription factor GmERF8 in soybean(Glycine max L.)[J].Journa of Plant Genetic Resources,17(6):1036-1040.)
Cai X T,Xu P,Zhao P X,et al.2014.Arabidopsis ERF109 mediates cross-talk between jasmonic acid and auxin biosynthesis during lateral root formation[J].Nature Communications,5(5):5833.
Charfeddine M,Sa?di M N,Charfeddine S,et al.2015.Genome-wide analysis and expression profiling of the ERFtranscription factor family in potato(Solanum tuberosum L.)[J].Molecular Biotechnology,57(4):348-358.
Fujimoto S Y,Ohta M,Usui A,et al.2000.Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[J].Plant Cell,12(3):393-404.
Gao C,Li P,Song A,et al.2015.Isolation and characterization of six AP2/ERF transcription factor genes in Chrysanthemum nankingense[J].International Journal of Molecular Sciences,16(1):2052-2065.
Kenneth J L,Thomas D S.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-??Ctmethod[J].Methods,25:402-408.
Lu X,Zhang L,Zhang F,et al.2013.AaORA,a trichome-specific AP2/ERF transcription factor of Artemisia annua,is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea[J].New Phytologist,198(4):1191-1202.
Ma Y,Zhang L,Zhang J,et al.2014.Expressing a Citrus ortholog of Arabidopsis ERF1 enhanced cold-tolerance in tobacco[J].Scientia Horticulturae,174(1):65-76.
Maki H,Sato M,Ogawa K,et al.2011.Cloning and expression profile of an ERF gene isolated from cold-stressed poplar cells(Populus nigra)[J].Cytologia,76(1):11-18.
Mizoi J,Shinozaki K,Yamaguchi-Shinozaki K.2012.AP2/ERF family transcription factors in plant abiotic stress responses[J].Biochimica et Biophysica Acta-Gene Regulatory Mechanisms,1819(2):86-96.
Nakano T,Suzuki K,Fujimura T,et al.2006.Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiology,140(2):411.
Sears M T,Zhang H,Rushton P J,et al.2014.NtERF32:Anon-NIC2 locus AP2/ERF transcription factor required in jasmonate-inducible nicotine biosynthesis in tobacco[J].Plant Molecular Biology,84(1-2):49-66
Stockinger E J.1997.Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Proceedings of the National Academy of Sciences of the USA,94(3):1035-1040.
Wang S,Yao W,Zhou B,et al.2016.Structure analysis and expression pattern of the ERF transcription factor family in poplar[J].Acta Physiology Plant,38(10):239.
Yan H W,Hong L,Zhou Y Q,et al.2013.A genome-wide analysis of the ERF gene family in sorghum[J].Genetics and Molecular Research,12(2):2038-2055.
Yin X R,Allan A C,Xu Q,et al.2012.Differential expression of kiwifruit ERF genes in response to postharvest abiotic stress[J].Postharvest Biology and Technology,66(2):1-7.
Zhang W,Zou A,Miao J,et al.2011.LeERF-1,a novel AP2/ERF family gene within the B3 subcluster,is down-regulated by light signals in Lithospermum erythrorhizon[J].Plant Biology(Stuttgart,Germany),13(2):343-348
Zhang Z,Huang R.2010.Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis[J].Plant Molecular Biology,73(3):241-249.
Zhuo C,Liang L,Zhao Y,et al.2018.A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover,antioxidant protection,and proline accumulation[J].Plant Cell Environment,41(9):2021-2032.
曹诣斌,石瑞,陈文荣,等.2011.低温胁迫下佛手和枳乙烯应答因子6(ERF6)表达变化的比较分析[J].园艺学报,38(10):1873-1882.(Cao Y B,Shi R,Chen W R,et al.2011.Comparative analysis of expression changes of ethylene response factor6(ERF6)in fingered citron and trifoliate orange under cold stress[J].Acta Horticulturae Sinica,38(10):1873-1882.)
高敏霞,冯新,赖瑞联,等.2018.米良1号猕猴桃漆酶基因的克隆与表达分析[J].农业生物技术学报,26(1):65-76.(Gao M X,Feng X,Lai R L,et al.2018.Cloning and expression analysis of the laccase genes from kiwifruit(Actinidia chinensis var.deliciosa'Miliang-1')[J].Chinese Journal of Agricultural Biotechnology,26(1):65-76.)
黄敏杰.2017.橄榄(Canarium album(Lour.)Raeusch.)金丝桃苷的测定及生物合成相关基因的克隆与表达分析[D].福建农林大学,导师:陈清西,pp.12-24.(Huang M J.2017.Measurement of hyperoside content,cloning and expression of the synthesis genes in Canarium album[D].Fujian Agriculture and Forestry University,Supervisor:Chen Q X,pp.12-24.)
赖瑞联,陈瑾,冯新,等.2017.中国橄榄种质资源评价与抗寒性研究进展[J].热带作物学报,38(11):2188-2194.(Lai R L,Chen J,Feng X,et al.2017.Advances on germplasm resource evaluation and cold resistance in Chinese olive of China[J].Chinese Journal of Tropical Crops,38(11):2188-2194.)
赖钟雄,蔡英卿,许伟东,等.2007.橄榄优质栽培与综合利用[M].北京:中国三峡出版社,pp.14-17.(Lai Z X,Cai Y Q,Xu W D,et al.2007.Quality Cultivating and Integrated Utilization of Chinese Olive[M].Beijing:Chinese Sanxia Press,pp.14-17.)
杨德鑫.2012.水稻ERF蛋白OsERF109的特性及功能分析[D].中国农业科学院,导师:黄荣峰,pp.23.(Yang DX.2012.The characterization and function analyses of rice ERF protein OsERF109[D].Chinese Academy of Agricultural Sciences,Supervisor:Huang R F,pp.23.)
于晓惠.2013.巴西橡胶ERF转录因子基因的克隆与功能分析[D].海南大学,导师:张治礼,pp.34-35.(Yu X H2013.Cloning and functional analysis of the HbERFfrom Hevea brasiliensis[D].Hainan University,Suprevisor:Zhang Z L,pp.34-35.)
翟莹,张军,赵艳,等.2016.大豆ERF转录因子基因GmERF8的克隆与表达分析[J].植物遗传资源学报,17(6):1036-1040.(Zhai Y,Zhang J,Zhao Y,et al.2016Cloning and expression analysis of ERF transcription factor GmERF8 in soybean(Glycine max L.)[J].Journa of Plant Genetic Resources,17(6):1036-1040.)
Cai X T,Xu P,Zhao P X,et al.2014.Arabidopsis ERF109 mediates cross-talk between jasmonic acid and auxin biosynthesis during lateral root formation[J].Nature Communications,5(5):5833.
Charfeddine M,Sa?di M N,Charfeddine S,et al.2015.Genome-wide analysis and expression profiling of the ERFtranscription factor family in potato(Solanum tuberosum L.)[J].Molecular Biotechnology,57(4):348-358.
Fujimoto S Y,Ohta M,Usui A,et al.2000.Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[J].Plant Cell,12(3):393-404.
Gao C,Li P,Song A,et al.2015.Isolation and characterization of six AP2/ERF transcription factor genes in Chrysanthemum nankingense[J].International Journal of Molecular Sciences,16(1):2052-2065.
Kenneth J L,Thomas D S.2001.Analysis of relative gene expression data using real-time quantitative PCR and the2-??Ctmethod[J].Methods,25:402-408.
Lu X,Zhang L,Zhang F,et al.2013.AaORA,a trichome-specific AP2/ERF transcription factor of Artemisia annua,is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea[J].New Phytologist,198(4):1191-1202.
Ma Y,Zhang L,Zhang J,et al.2014.Expressing a Citrus ortholog of Arabidopsis ERF1 enhanced cold-tolerance in tobacco[J].Scientia Horticulturae,174(1):65-76.
Maki H,Sato M,Ogawa K,et al.2011.Cloning and expression profile of an ERF gene isolated from cold-stressed poplar cells(Populus nigra)[J].Cytologia,76(1):11-18.
Mizoi J,Shinozaki K,Yamaguchi-Shinozaki K.2012.AP2/ERF family transcription factors in plant abiotic stress responses[J].Biochimica et Biophysica Acta-Gene Regulatory Mechanisms,1819(2):86-96.
Nakano T,Suzuki K,Fujimura T,et al.2006.Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiology,140(2):411.
Sears M T,Zhang H,Rushton P J,et al.2014.NtERF32:Anon-NIC2 locus AP2/ERF transcription factor required in jasmonate-inducible nicotine biosynthesis in tobacco[J].Plant Molecular Biology,84(1-2):49-66
Stockinger E J.1997.Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE,a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Proceedings of the National Academy of Sciences of the USA,94(3):1035-1040.
Wang S,Yao W,Zhou B,et al.2016.Structure analysis and expression pattern of the ERF transcription factor family in poplar[J].Acta Physiology Plant,38(10):239.
Yan H W,Hong L,Zhou Y Q,et al.2013.A genome-wide analysis of the ERF gene family in sorghum[J].Genetics and Molecular Research,12(2):2038-2055.
Yin X R,Allan A C,Xu Q,et al.2012.Differential expression of kiwifruit ERF genes in response to postharvest abiotic stress[J].Postharvest Biology and Technology,66(2):1-7.
Zhang W,Zou A,Miao J,et al.2011.LeERF-1,a novel AP2/ERF family gene within the B3 subcluster,is down-regulated by light signals in Lithospermum erythrorhizon[J].Plant Biology(Stuttgart,Germany),13(2):343-348
Zhang Z,Huang R.2010.Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis[J].Plant Molecular Biology,73(3):241-249.
Zhuo C,Liang L,Zhao Y,et al.2018.A cold responsive ethylene responsive factor from Medicago falcata confers cold tolerance by up-regulation of polyamine turnover,antioxidant protection,and proline accumulation[J].Plant Cell Environment,41(9):2021-2032.