红花ERF1转录因子的克隆及植物表达载体的构建
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摘要
目的克隆1条红花AP2/ERF家族转录因子编码区序列,并构建植物表达载体。方法在红花转录组测序基础上,利用RT-PCR方法克隆1条红花AP2/ERF家族转录因子编码区序列(Ct ERF1)并进行生物信息学分析,利用ClustalW 1.83软件构建系统进化树,在Ct ERF1基因序列两端引入SpeI和XbaI酶切位点,构建含有35S启动子的植物超表达载体p BASTA-Ct ERF1。结果 CtERF1基因编码297个氨基酸,且具有典型的AP2/ERF基因的功能结构域,含有1个AP2区域,为ERF亚族蛋白,亚细胞定位分析推测其定位在细胞质和细胞核上。系统进化分析表明,Ct ERF1基因编码氨基酸与其他物种氨基酸具有一定的同源性,其中与杨树和人参的亲缘关系最近。通过分子生物学方法,成功构建p BASTA-CtERF1植物表达载体。结论成功克隆了1条红花AP2/ERF家族转录因子编码区基因Ct ERF1,并构建了植物表达载体p BASTA-Ct ERF1。
Objective To clone a coding region sequence of AP2/ERF transcription factor family from Carthamus tinctorius, and construct a plant expression vector. Methods A gene(CtERF1) of AP2/ERF family transcription factor was cloned by RT-PCR based on the sequence of C. tinctorius transcription sequencing, the phylogenetic tree was constructed by ClustalW 1.83 software, Spe I and Xba I restriction sites were introduced to construct over-expression vector pBASTA-CtERF1 containing 35 S promoter. Results Ct ERF1 gene had a functional domain of a typical AP2/ERF gene encoding 297 amino acids, and contained an AP2 region speculated to be located in cytoplasm and nucleus, which was ERF subprotein. Systematic evolution analysis showed that CtERF1 gene had some homology with other plant species, among which the relationship with Populus deltoides and Panax japonicus were the closest. The p BASTA-Ct ERF1 plant expression vector was constructed successfully by molecular biology. Conclusion A Ct ERF1 gene of C.tinctorius AP2/ERF transcription factor family was cloned and the plant expression vector p BASTA-Ct ERF1 was constructed successfully.
Objective To clone a coding region sequence of AP2/ERF transcription factor family from Carthamus tinctorius, and construct a plant expression vector. Methods A gene(CtERF1) of AP2/ERF family transcription factor was cloned by RT-PCR based on the sequence of C. tinctorius transcription sequencing, the phylogenetic tree was constructed by ClustalW 1.83 software, Spe I and Xba I restriction sites were introduced to construct over-expression vector pBASTA-CtERF1 containing 35 S promoter. Results Ct ERF1 gene had a functional domain of a typical AP2/ERF gene encoding 297 amino acids, and contained an AP2 region speculated to be located in cytoplasm and nucleus, which was ERF subprotein. Systematic evolution analysis showed that CtERF1 gene had some homology with other plant species, among which the relationship with Populus deltoides and Panax japonicus were the closest. The p BASTA-Ct ERF1 plant expression vector was constructed successfully by molecular biology. Conclusion A Ct ERF1 gene of C.tinctorius AP2/ERF transcription factor family was cloned and the plant expression vector p BASTA-Ct ERF1 was constructed successfully.
引文
[1]官玲亮,侯凯,刘千,等.红花ω6脂肪酸脱氢酶基因克隆及生物信息学分析[J].中国油料作物学报,2013,35(4):372-383.
[2]扈晓佳,殷莎,袁婷婷,等.红花的化学成分及其药理活性研究进展[J].药学实践杂志,2015,31(4):161-166.
[3]Nakano T,Suzuki K,Fujimura T,et al.Genomewide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiol,2006,140(2):411-432.
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[5]吴致君,卢莉,黎星辉,等.茶树AP2/ERF-B3类转录因子基因的克隆与表达特性分析[J].南京农业大学学报,2014,37(4):67-75.
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[8]Lee S Y,Hwang E Y,Seok H Y,et al.Arabidopsis AtERF71/HRE2 functions as transcriptional activator via cis-acting GCC box or DRE/CRT element and is involved in root development through regulation of root cell expansion[J].Plant Cell Rep,2015,34(2):223-231.
[9]Chung M Y,Vrebalov J,Alba R,et al.A tomato(Solanumly copersicum)APETALA2/ERF gene,SlAP2a,is a negative regulator of fruit ripening[J].Plant J,2010,64(6):936-947.
[10]Huala E,Sussex I M.LEAFY interacts with floral homeotic genes to regulate Arabidopsis floral development[J].Plant Cell,1992,4(8):901-913.
[11]Niu X,Helentjaris T,Bate N J.Maize ABI4 binds coupling element1 in abscisic acid and sugar response genes[J].Plant Cell,2002,14(10):2565-2575.
[12]吴文燕,蒋喜红,刘春生,等.丹参ERF转录因子Sm ERF1基因的表达分析和亚细胞定位[J].中国中药杂志,2013,38(7):957-961.
[13]吴素瑞,高珂,刘璇,等.AP2/ERF转录因子调控药用植物活性成分生物合成的研究进展[J].中草药,2016,47(9):1605-1613.
[14]Gantet P,Memelink J.Transcription factors:Tools to engineer the production of pharmacologically active plant metabolites[J].Trends Pharmacol Sci,2002,23(12):563-569.
[15]Broun P.Transcription factors as tools for metabolic engineering in plants[J].Curr Opin Plant Biol,2004,7(2):202-209.
[16]刘秀明,杨文婷,陈凯,等.红花不同品种和不同花期黄色素含量分析[J].时珍国医国药,2014,2(9):2233-2235.
[17]Wang C,Xin M,Zhou X,et al.The novel ethylene-responsive factor Cs ERF025 affects the development of fruit bending in cucumber[J].Plant Mol Biol,2017,95(4/5):519-531.
[18]Sun W,Gao D,Xiong Y,et al.Hairy leaf 6,an AP2/ERFtranscription factor,interacts with OsWOX3B and regulates trichome formation in rice[J].Mol Plant,2017,10(11):1417-1433.
[19]Owji H,Hajiebrahimi A,Seradj H,et al.Identification and functional prediction of stress responsive AP2/ERFtranscription factors in Brassica napus by genome-wide analysis[J].Comput Biol Chem,2017,11(71):32-56.
[20]Yu Y,Duan X,Ding X,et al.A novel AP2/ERF family transcription factor from Glycine soja,Gs ERF71,is a DNA binding protein that positively regulates alkaline stress tolerance in Arabidopsis[J].Plant Mol Biol,2017,94(4/5):509-530.
[21]Li H,Xiao Q,Zhang C,et al.Identification and characterization of transcription factor ZmEREB94involved in starch synthesis in maize[J].J Plant Physiol,2017,216:11-16.
[22]Zhao Y,Chang X,Qi D,et al.A Novel soybean ERFtranscription factor,Gm ERF113,increases resistance to Phytophthora sojae infection in soybean[J].Front Plant Sci,2017,299(8):1-16.
[23]郭慧,金司阳,刘寒,等.甘蓝AP2/ERF转录因子的克隆和生物信息学分析[J].中国药师,2017,20(1):6-10.
[24]秦捷,王武,左开井,等.AP2基因家族的起源和棉花AP2转录因子在抗病中的作用[J].棉花学报,2005,17(6):366-370.
[25]Durán-Medina Y,Serwatowska J,Reyes-Olalde J I,et al.The AP2/ERF transcription factor DRNL modulates gynoecium development and affects its response to cytokinin[J].Front Plant Sci,2017,26(8):1841-1853.
[26]Wang C,Xin M,Zhou X,et al.The novel ethyleneresponsive factor CsERF025 affects the development of fruit bending in cucumber[J].Plant Mol Biol,2017,95(4/5):519-531.
[27]Sun W,Gao D,Xiong Y,et al.Hairy Leaf 6,an AP2/ERFtranscription factor,interacts with OsWOX3B and regulates trichome formation in rice[J].Mol Plant,2017,10(11):1417-1433.
[28]Velasco R,Zharkikh A,Affourtit J,et al.The genome of the domesticated apple(Malus×domestica Borkh.)[J].Nat Genet,2010,42(10):833-839.
[29]赵金玲,姚文静,王升级,等.杨树AP2/ERF转录因子家族生物信息学分析[J].东北林业大学学报,2015,43(10):21-29.
[30]吴思阳.非生物因素对人参皂苷生物合成途径关键酶基因表达能力的影响[D].长春:吉林大学,2007.
[2]扈晓佳,殷莎,袁婷婷,等.红花的化学成分及其药理活性研究进展[J].药学实践杂志,2015,31(4):161-166.
[3]Nakano T,Suzuki K,Fujimura T,et al.Genomewide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiol,2006,140(2):411-432.
[4]Zhou X,Tang L,Xu Y,et al.Towards a better understanding of medicinal uses of Carthamus tinctorius L.in traditional Chinese medicine:A phytochemical and pharmacological review[J].J Ethnopharm,2014,151(1):27-43.
[5]吴致君,卢莉,黎星辉,等.茶树AP2/ERF-B3类转录因子基因的克隆与表达特性分析[J].南京农业大学学报,2014,37(4):67-75.
[6]张计育,王庆菊,郭忠仁.植物AP2/ERF类转录因子研究进展[J].遗传,2012,34(7):835-837.
[7]Gao C,Li P,Song A,et al.Isolation and characterization of six AP2/ERF transcription factor genes in Chrysanthemum nankingense[J].Int J Mol Sci,2015,16(1):2052-2065.
[8]Lee S Y,Hwang E Y,Seok H Y,et al.Arabidopsis AtERF71/HRE2 functions as transcriptional activator via cis-acting GCC box or DRE/CRT element and is involved in root development through regulation of root cell expansion[J].Plant Cell Rep,2015,34(2):223-231.
[9]Chung M Y,Vrebalov J,Alba R,et al.A tomato(Solanumly copersicum)APETALA2/ERF gene,SlAP2a,is a negative regulator of fruit ripening[J].Plant J,2010,64(6):936-947.
[10]Huala E,Sussex I M.LEAFY interacts with floral homeotic genes to regulate Arabidopsis floral development[J].Plant Cell,1992,4(8):901-913.
[11]Niu X,Helentjaris T,Bate N J.Maize ABI4 binds coupling element1 in abscisic acid and sugar response genes[J].Plant Cell,2002,14(10):2565-2575.
[12]吴文燕,蒋喜红,刘春生,等.丹参ERF转录因子Sm ERF1基因的表达分析和亚细胞定位[J].中国中药杂志,2013,38(7):957-961.
[13]吴素瑞,高珂,刘璇,等.AP2/ERF转录因子调控药用植物活性成分生物合成的研究进展[J].中草药,2016,47(9):1605-1613.
[14]Gantet P,Memelink J.Transcription factors:Tools to engineer the production of pharmacologically active plant metabolites[J].Trends Pharmacol Sci,2002,23(12):563-569.
[15]Broun P.Transcription factors as tools for metabolic engineering in plants[J].Curr Opin Plant Biol,2004,7(2):202-209.
[16]刘秀明,杨文婷,陈凯,等.红花不同品种和不同花期黄色素含量分析[J].时珍国医国药,2014,2(9):2233-2235.
[17]Wang C,Xin M,Zhou X,et al.The novel ethylene-responsive factor Cs ERF025 affects the development of fruit bending in cucumber[J].Plant Mol Biol,2017,95(4/5):519-531.
[18]Sun W,Gao D,Xiong Y,et al.Hairy leaf 6,an AP2/ERFtranscription factor,interacts with OsWOX3B and regulates trichome formation in rice[J].Mol Plant,2017,10(11):1417-1433.
[19]Owji H,Hajiebrahimi A,Seradj H,et al.Identification and functional prediction of stress responsive AP2/ERFtranscription factors in Brassica napus by genome-wide analysis[J].Comput Biol Chem,2017,11(71):32-56.
[20]Yu Y,Duan X,Ding X,et al.A novel AP2/ERF family transcription factor from Glycine soja,Gs ERF71,is a DNA binding protein that positively regulates alkaline stress tolerance in Arabidopsis[J].Plant Mol Biol,2017,94(4/5):509-530.
[21]Li H,Xiao Q,Zhang C,et al.Identification and characterization of transcription factor ZmEREB94involved in starch synthesis in maize[J].J Plant Physiol,2017,216:11-16.
[22]Zhao Y,Chang X,Qi D,et al.A Novel soybean ERFtranscription factor,Gm ERF113,increases resistance to Phytophthora sojae infection in soybean[J].Front Plant Sci,2017,299(8):1-16.
[23]郭慧,金司阳,刘寒,等.甘蓝AP2/ERF转录因子的克隆和生物信息学分析[J].中国药师,2017,20(1):6-10.
[24]秦捷,王武,左开井,等.AP2基因家族的起源和棉花AP2转录因子在抗病中的作用[J].棉花学报,2005,17(6):366-370.
[25]Durán-Medina Y,Serwatowska J,Reyes-Olalde J I,et al.The AP2/ERF transcription factor DRNL modulates gynoecium development and affects its response to cytokinin[J].Front Plant Sci,2017,26(8):1841-1853.
[26]Wang C,Xin M,Zhou X,et al.The novel ethyleneresponsive factor CsERF025 affects the development of fruit bending in cucumber[J].Plant Mol Biol,2017,95(4/5):519-531.
[27]Sun W,Gao D,Xiong Y,et al.Hairy Leaf 6,an AP2/ERFtranscription factor,interacts with OsWOX3B and regulates trichome formation in rice[J].Mol Plant,2017,10(11):1417-1433.
[28]Velasco R,Zharkikh A,Affourtit J,et al.The genome of the domesticated apple(Malus×domestica Borkh.)[J].Nat Genet,2010,42(10):833-839.
[29]赵金玲,姚文静,王升级,等.杨树AP2/ERF转录因子家族生物信息学分析[J].东北林业大学学报,2015,43(10):21-29.
[30]吴思阳.非生物因素对人参皂苷生物合成途径关键酶基因表达能力的影响[D].长春:吉林大学,2007.