荔枝GRX基因启动子的克隆与瞬时表达分析
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摘要
为了研究荔枝GRX基因的调控机理,本研究利用PCR的方法克隆了该基因上游1 996 bp的启动子片段并对其功能进行了初步分析,结果表明:荔枝GRX基因启动子序列中含有大量的TATA-box和CAAT-box保守元件,以及ARE、HSE、MBS、TC-rich repeats、TGA-element等各种转录调控相关的顺式作用元件。基因枪表达分析表明,该启动子能驱动GUS基因在荔枝的花、叶、根、果皮以及果核中表达而在果肉中不表达,具有组织特异性。为进一步研究荔枝GRX基因的表达模式和调控机理提供了基础与依据。
In order to study the regulation mechanism of GRX gene from Litchi,PCR method was applied in this research to clone 1 996 bp upstream promoter fragments in this gene to preliminarily analyze its function.The results showed that the promoter sequence of GRX gene from Litchi contained larger quantities of TATA-box and CAAT-box conserved elements,and a variety of transcriptional regulation related cis acting elements,such as ARE,HSE,MBS,TC-rich repeats,and TGA-element.Analysis of particle bombardment expression indicated that this promoter could promote GUS gene to express in flower,leaf,root,pericarp and seed but not in pulp,which indicated that the promoter was tissue specific.The results of this researh could provide the basis and reference for the further study of the expression pattern and regulation mechanism of the GRX gene in Litchi.
In order to study the regulation mechanism of GRX gene from Litchi,PCR method was applied in this research to clone 1 996 bp upstream promoter fragments in this gene to preliminarily analyze its function.The results showed that the promoter sequence of GRX gene from Litchi contained larger quantities of TATA-box and CAAT-box conserved elements,and a variety of transcriptional regulation related cis acting elements,such as ARE,HSE,MBS,TC-rich repeats,and TGA-element.Analysis of particle bombardment expression indicated that this promoter could promote GUS gene to express in flower,leaf,root,pericarp and seed but not in pulp,which indicated that the promoter was tissue specific.The results of this researh could provide the basis and reference for the further study of the expression pattern and regulation mechanism of the GRX gene in Litchi.
引文
Dubey A.K.,Kumar N.,Sahu N.,Verma P.K.,Chakrabarty D.,Behera S.K.,and Mallick S.,2016,Response of two rice cultivars differing in their sensitivity towards arsenic,differs in their expr ession of glutaredoxin and glutathione S transferase genes and antioxidant usage,Ecotoxicol Environ Saf.,124:393-405
Guo S.S.,Wen S.Y.,Qiao Y.Q.,Wu Y.M.,Duan J.,and Li H.Y.,2017,Bioinformatics analysis of BAS1 genes from foxtail millet(Setaria italic(L.)Beauv.)and its expression under drought stress,Shanxi Nongye Kexue(Journal of Shanxi A-gricultural Sciences),45(2):151-155(郭莎莎,温思钰,乔亚琪,武懿茂,段杰,李红英,2017,谷子BAS1基因生物信息学分析及其在干旱胁迫下的表达分析,山西农业科学,45(2):151-155)
Guo Y.S.,Yu J.,Pu W.,Zou J.,Fu Q.,Yu S.Z.,Lin S.F.,Zhang X.L.,Zhao J.H.,and Xia F.J.,2017,Subcellular localization of Nb GRX1 and its role in plant response to drought stress,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),40(1):34-39(郭玉双,余婧,蒲伟,邹颉,付强,余世洲,林世锋,张孝廉,赵杰宏,夏范讲,2017,烟草谷氧还蛋白Nb GRX1的亚细胞定位及抗旱功能研究,南京农业大学学报,40(1):34-39)
He F.,Mei W.L.,Li H.L.,Guo D.,Peng S.Q.,and Dai H.F.,2014,Cloning and function analysis of the sesquiterpene guaiene synthase gene(As SGS)promoter in Aquilaria sinensis,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),35(4):712-717(何访,梅文莉,李辉亮,郭冬,彭世清,戴好富,2014,白木香愈创木烯合酶基因(As SGS)启动子的克隆及功能初步鉴定,热带作物学报,35(4):712-717)
Hu Y.,Wu Q.,Sprague S.A.,Park J.,Oh M.,Rajashekar C.B.,Koiwa H.,Nakata P.A.,Cheng N.,Hirschi K.D.,White F.F.,and Park S.,2015,Tomato expressing Arabidopsis glutaredoxin gene At GRXS17 confers tolerance to chilling stress via modulating cold responsive components,Horticulture Research,2:15051
Jin Q.F.,Chen Z.D.,Sun W.J.,Lin F.M.,Xue Z.H.,Huang Y.,and Tang X.H.,2016,Cloning and bioinformatical analysis of anthocyanin synthase gene and its promoter in Camellia sinensis,Chaye Kexue(Journal of Tea Science),36(2):219-228(金琦芳,陈志丹,孙威江,林馥茗,薛志慧,黄艳,唐秀华,2016,茶树Cs ANS基因及其启动子的克隆与生物信息学分析,茶叶科学,36(2):219-228)
Kang G.J.,Li Y.,Zeng R.Z.,and Nie Z.Y.,2013,Cloning and sequence analysis of Hb NTL1 promoter from Hevea brasiliensis,Shengwu Jishu Tongbao(Biotechnology Bulletin),(1):83-88(康桂娟,黎瑜,曾日中,聂智毅,2013,巴西橡胶树Hb NTL1基因启动子的克隆与序列分析,生物技术通报,(1):83-88)
Kong Y.B.,Li X.H.,and Zhang C.Y.,2017,Construction and activity analysis of the promoter of purple acid phosphatase gene Gm PAP4 in soybean,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),50(3):582-590(孔佑宾,李喜焕,张彩英,2017,大豆紫色酸性磷酸酶基因Gm PAP4启动子结构与活性分析,中国农业科学,50(3):582-590)
Lescot M.,Dehais P.,Thijs G.,Marchal K.,Moreau Y.,Peer Y.V.,Rouze P.,and Rombauts S.,2002,Plant CARE:a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences,Nucleic Acids Res.,30(1):325-327
Lian C.L.,Lu B.G.,Lai Z.X.,Feng X.,Lin Y.L.,Chen Y.K.,and Zhang X.H.,2015,Cloning and functional analysis of Lc Cu/SOD3 promoter from embryogenic callus of the ancient Litchi tree,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),35(1):16-22(练从龙,卢秉国,赖钟雄,冯新,林玉玲,陈裕坤,张梓浩,2015,荔枝古树胚性愈伤组织Lc Cu/Zn-SOD3基因启动子的克隆及功能验证,西北植物学报,35(1):16-22)
Minakuchi K.,Yabushita T.,Masumura T.,Ichihara K.,and Tanaka K.,1944,Cloning and sequence analys in of a c DNAencoding rice glutaredoxin,FEBS Lett.,337(2):157-160
Na S.H.,Wang Y.,and Liu G J.,2010,Cloning and analysis of Ps GRX gene from Polygonum sibiricum laxm,and its expression in response to Na HCO3,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),46(4):341-346(那守海,王垠,刘关君,2010,西伯利亚蓼Ps GRX基因的克隆及其在Na HCO3胁迫下的表达,植物生理学通讯,46(4):341-346)
Nie L.N.,Xia L.Q.,Yao M.S.,Gao D.Y.,Li L.,Yu Z.,Chen M.,Li L.,CH.,and Ma Y.Z.,2008,Progress on cloning and functional study of plant gene promoters,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),9(3):385-391(聂丽娜,夏兰琴,徐兆师,高东尧,李琳,于卓,陈明,李连城,马有志,2008,植物基因启动子的克隆及其功能研究进展,植物遗传资源学报,9(3):385-391)
Sharma R.,Priya P.,and Jain M.,2013,Modified expression of an auxin-responsive rice CC-type glutaredoxin gene affects multiple abiotic stress responses,Planta,238(5):871-884
Su H.H.,Li T.,Li Z.X.,Wang Y.F.,and Li Y.F.,2015,Identification and gene expression analysis of GRX gene family in tomato,Henongxue Bao(Journal of Nuclear Agricultural Sciences),29(4):663-673(苏慧慧,李涛,黎振兴,李植良,王永飞,2015,番茄GRX基因家族的鉴定及表达分析,核农学报,29(4):663-673)
Sun Q.,Sun J.H.,Wang S.J.,Li H.L.,and Wang J.B.,2016,Cloning and transient expression assay of FKBP16-2 gene promoter from Litchi,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),37(4):736-741(孙琴,孙进华,王树军,李焕苓,王家保,2016,荔枝FKBP16-2基因启动子的克隆与瞬时表达分析,热带作物学报,37(4):736-741)
Wang H.,Wei M.,and Li C.H.,2016,Cloning and functional i dentification of promoter region of Pt AREB1 from Populus trichocarpa,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),44(4):18-24(王含,魏明,李成浩,2016,毛果杨Pt AREB1基因启动子的克隆及功能,东北林业大学学报,44(4):18-24)
Wang L.,Li Y.,Jacquot J.P.,Rouhier N.,and Xia B.,2014,Characterization of poplar Grx S14 in different structural forms,Protein Cell,5(5):329-333
Wang S.J.,Liu B.H.,Sun J.H.,Xiao Q.,Li H.L.,and Wang J.B.,2015,Cloning and function analysis of the promoter of PPOgene from litchi(Litchi chinensis Sonn.),Guoshu Xuebao(Journal of Fruit Science),32(2):427-433(王树军,刘保华,孙进华,肖茜,李焕苓,王家保,2015,荔枝多酚氧化酶基因启动子克隆与功能分析,果树学报,32(2):427-433)
Wang Z.,Xing S.,Birkenbihl R.P.,and Zachgo S.,2009,Conserved functions of Arabidopsis and rice CC-type glutaredoxins in flower development and pathogen response,Molecular Plant,2(2):323-335
Wu Q.,Lin J.,Liu J.Z.,Wang X.,Lim W.,Oh M.,Park J.,Rajashekar C.B.,Whitham S.A.,Cheng N.H.,Hirschi K.D.,and Park S.,2012,Ectopic expression of Arabidopsis glutaredoxin At GRXS17 enhances thermo tolerance in tomato,Plant Biotechnol.J.,10(8):945-955
Yang L.Y.,Wang M.L.,Zhao Y.,He L.X.,and Xia X.J.,2016,Cloning and expression analysis of rice gene promoter Ospz4,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(7):1648-1654(杨莉颖,王曼玲,赵洋,贺利雄,夏新界,2016,水稻基因启动子Ospz4的克隆与表达分析,分子植物育种,14(7):1648-1654)
Zhao Y.,Liu X.X.,Zhai Y.,Cao H.,and Wu Q.,2017,Expression pattern analysis of soybean lox2 gene and prediction of promoter,Xi'nan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),30(3):505-510(赵艳,刘晓鑫,翟莹,曹慧,吴琼,2017,大豆lox2基因表达方式分析及其启动子预测,西南农业学报,30(3):505-510)
Zhong T.Q.,Li H.L.,Tian W.M.,and Peng S.Q.,2008,Cloning and function analysis of 5'regulatory region of Lc VSP1 gene from Litchi chinensis,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),28(8):1507-1512(钟廷琼,李辉亮,田维敏,彭世清,2008,荔枝Lc VSP1基因5'调控序列的克隆及功能初步鉴定,西北植物学报,28(8):1507-1512)
Zhou L.Y.,Bu L.L.,Yang CH.L.,Gao T.,Tian T.,and Yang Z.A.,2016,Cloning and sequence analysis of AGPL1 promoters in watermelon fruit tissue,Yaredai Zhiwu Kexue(Subtropical Plant Science),45(2):122-126(周丽英,卜璐璐,杨春雷,高婷,田甜,杨正安,2016,西瓜果实AGPL1基因启动子的序列分析,亚热带植物科学,45(2):122-126)
Guo S.S.,Wen S.Y.,Qiao Y.Q.,Wu Y.M.,Duan J.,and Li H.Y.,2017,Bioinformatics analysis of BAS1 genes from foxtail millet(Setaria italic(L.)Beauv.)and its expression under drought stress,Shanxi Nongye Kexue(Journal of Shanxi A-gricultural Sciences),45(2):151-155(郭莎莎,温思钰,乔亚琪,武懿茂,段杰,李红英,2017,谷子BAS1基因生物信息学分析及其在干旱胁迫下的表达分析,山西农业科学,45(2):151-155)
Guo Y.S.,Yu J.,Pu W.,Zou J.,Fu Q.,Yu S.Z.,Lin S.F.,Zhang X.L.,Zhao J.H.,and Xia F.J.,2017,Subcellular localization of Nb GRX1 and its role in plant response to drought stress,Nanjing Nongye Daxue Xuebao(Journal of Nanjing Agricultural University),40(1):34-39(郭玉双,余婧,蒲伟,邹颉,付强,余世洲,林世锋,张孝廉,赵杰宏,夏范讲,2017,烟草谷氧还蛋白Nb GRX1的亚细胞定位及抗旱功能研究,南京农业大学学报,40(1):34-39)
He F.,Mei W.L.,Li H.L.,Guo D.,Peng S.Q.,and Dai H.F.,2014,Cloning and function analysis of the sesquiterpene guaiene synthase gene(As SGS)promoter in Aquilaria sinensis,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),35(4):712-717(何访,梅文莉,李辉亮,郭冬,彭世清,戴好富,2014,白木香愈创木烯合酶基因(As SGS)启动子的克隆及功能初步鉴定,热带作物学报,35(4):712-717)
Hu Y.,Wu Q.,Sprague S.A.,Park J.,Oh M.,Rajashekar C.B.,Koiwa H.,Nakata P.A.,Cheng N.,Hirschi K.D.,White F.F.,and Park S.,2015,Tomato expressing Arabidopsis glutaredoxin gene At GRXS17 confers tolerance to chilling stress via modulating cold responsive components,Horticulture Research,2:15051
Jin Q.F.,Chen Z.D.,Sun W.J.,Lin F.M.,Xue Z.H.,Huang Y.,and Tang X.H.,2016,Cloning and bioinformatical analysis of anthocyanin synthase gene and its promoter in Camellia sinensis,Chaye Kexue(Journal of Tea Science),36(2):219-228(金琦芳,陈志丹,孙威江,林馥茗,薛志慧,黄艳,唐秀华,2016,茶树Cs ANS基因及其启动子的克隆与生物信息学分析,茶叶科学,36(2):219-228)
Kang G.J.,Li Y.,Zeng R.Z.,and Nie Z.Y.,2013,Cloning and sequence analysis of Hb NTL1 promoter from Hevea brasiliensis,Shengwu Jishu Tongbao(Biotechnology Bulletin),(1):83-88(康桂娟,黎瑜,曾日中,聂智毅,2013,巴西橡胶树Hb NTL1基因启动子的克隆与序列分析,生物技术通报,(1):83-88)
Kong Y.B.,Li X.H.,and Zhang C.Y.,2017,Construction and activity analysis of the promoter of purple acid phosphatase gene Gm PAP4 in soybean,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),50(3):582-590(孔佑宾,李喜焕,张彩英,2017,大豆紫色酸性磷酸酶基因Gm PAP4启动子结构与活性分析,中国农业科学,50(3):582-590)
Lescot M.,Dehais P.,Thijs G.,Marchal K.,Moreau Y.,Peer Y.V.,Rouze P.,and Rombauts S.,2002,Plant CARE:a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences,Nucleic Acids Res.,30(1):325-327
Lian C.L.,Lu B.G.,Lai Z.X.,Feng X.,Lin Y.L.,Chen Y.K.,and Zhang X.H.,2015,Cloning and functional analysis of Lc Cu/SOD3 promoter from embryogenic callus of the ancient Litchi tree,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),35(1):16-22(练从龙,卢秉国,赖钟雄,冯新,林玉玲,陈裕坤,张梓浩,2015,荔枝古树胚性愈伤组织Lc Cu/Zn-SOD3基因启动子的克隆及功能验证,西北植物学报,35(1):16-22)
Minakuchi K.,Yabushita T.,Masumura T.,Ichihara K.,and Tanaka K.,1944,Cloning and sequence analys in of a c DNAencoding rice glutaredoxin,FEBS Lett.,337(2):157-160
Na S.H.,Wang Y.,and Liu G J.,2010,Cloning and analysis of Ps GRX gene from Polygonum sibiricum laxm,and its expression in response to Na HCO3,Zhiwu Shenglixue Tongxun(Plant Physiology Communications),46(4):341-346(那守海,王垠,刘关君,2010,西伯利亚蓼Ps GRX基因的克隆及其在Na HCO3胁迫下的表达,植物生理学通讯,46(4):341-346)
Nie L.N.,Xia L.Q.,Yao M.S.,Gao D.Y.,Li L.,Yu Z.,Chen M.,Li L.,CH.,and Ma Y.Z.,2008,Progress on cloning and functional study of plant gene promoters,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),9(3):385-391(聂丽娜,夏兰琴,徐兆师,高东尧,李琳,于卓,陈明,李连城,马有志,2008,植物基因启动子的克隆及其功能研究进展,植物遗传资源学报,9(3):385-391)
Sharma R.,Priya P.,and Jain M.,2013,Modified expression of an auxin-responsive rice CC-type glutaredoxin gene affects multiple abiotic stress responses,Planta,238(5):871-884
Su H.H.,Li T.,Li Z.X.,Wang Y.F.,and Li Y.F.,2015,Identification and gene expression analysis of GRX gene family in tomato,Henongxue Bao(Journal of Nuclear Agricultural Sciences),29(4):663-673(苏慧慧,李涛,黎振兴,李植良,王永飞,2015,番茄GRX基因家族的鉴定及表达分析,核农学报,29(4):663-673)
Sun Q.,Sun J.H.,Wang S.J.,Li H.L.,and Wang J.B.,2016,Cloning and transient expression assay of FKBP16-2 gene promoter from Litchi,Redai Zuowu Xuebao(Chinese Journal of Tropical Crops),37(4):736-741(孙琴,孙进华,王树军,李焕苓,王家保,2016,荔枝FKBP16-2基因启动子的克隆与瞬时表达分析,热带作物学报,37(4):736-741)
Wang H.,Wei M.,and Li C.H.,2016,Cloning and functional i dentification of promoter region of Pt AREB1 from Populus trichocarpa,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),44(4):18-24(王含,魏明,李成浩,2016,毛果杨Pt AREB1基因启动子的克隆及功能,东北林业大学学报,44(4):18-24)
Wang L.,Li Y.,Jacquot J.P.,Rouhier N.,and Xia B.,2014,Characterization of poplar Grx S14 in different structural forms,Protein Cell,5(5):329-333
Wang S.J.,Liu B.H.,Sun J.H.,Xiao Q.,Li H.L.,and Wang J.B.,2015,Cloning and function analysis of the promoter of PPOgene from litchi(Litchi chinensis Sonn.),Guoshu Xuebao(Journal of Fruit Science),32(2):427-433(王树军,刘保华,孙进华,肖茜,李焕苓,王家保,2015,荔枝多酚氧化酶基因启动子克隆与功能分析,果树学报,32(2):427-433)
Wang Z.,Xing S.,Birkenbihl R.P.,and Zachgo S.,2009,Conserved functions of Arabidopsis and rice CC-type glutaredoxins in flower development and pathogen response,Molecular Plant,2(2):323-335
Wu Q.,Lin J.,Liu J.Z.,Wang X.,Lim W.,Oh M.,Park J.,Rajashekar C.B.,Whitham S.A.,Cheng N.H.,Hirschi K.D.,and Park S.,2012,Ectopic expression of Arabidopsis glutaredoxin At GRXS17 enhances thermo tolerance in tomato,Plant Biotechnol.J.,10(8):945-955
Yang L.Y.,Wang M.L.,Zhao Y.,He L.X.,and Xia X.J.,2016,Cloning and expression analysis of rice gene promoter Ospz4,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(7):1648-1654(杨莉颖,王曼玲,赵洋,贺利雄,夏新界,2016,水稻基因启动子Ospz4的克隆与表达分析,分子植物育种,14(7):1648-1654)
Zhao Y.,Liu X.X.,Zhai Y.,Cao H.,and Wu Q.,2017,Expression pattern analysis of soybean lox2 gene and prediction of promoter,Xi'nan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),30(3):505-510(赵艳,刘晓鑫,翟莹,曹慧,吴琼,2017,大豆lox2基因表达方式分析及其启动子预测,西南农业学报,30(3):505-510)
Zhong T.Q.,Li H.L.,Tian W.M.,and Peng S.Q.,2008,Cloning and function analysis of 5'regulatory region of Lc VSP1 gene from Litchi chinensis,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),28(8):1507-1512(钟廷琼,李辉亮,田维敏,彭世清,2008,荔枝Lc VSP1基因5'调控序列的克隆及功能初步鉴定,西北植物学报,28(8):1507-1512)
Zhou L.Y.,Bu L.L.,Yang CH.L.,Gao T.,Tian T.,and Yang Z.A.,2016,Cloning and sequence analysis of AGPL1 promoters in watermelon fruit tissue,Yaredai Zhiwu Kexue(Subtropical Plant Science),45(2):122-126(周丽英,卜璐璐,杨春雷,高婷,田甜,杨正安,2016,西瓜果实AGPL1基因启动子的序列分析,亚热带植物科学,45(2):122-126)