水葫芦EcGS1b基因启动子的克隆及表达分析
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摘要
利用染色体步移技术从水葫芦基因组DNA中扩增得到谷氨酰胺合成酶EcGS1b基因5'端上游约1.7 kb的一段序列,经PlantCARE序列分析表明,该序列中除了包括TATA-box和CAAT-box等基本元件外,还含有TATC-box、HSE、LTR和多种光反应相关的元件,初步推测其为EcGS1b基因启动子,命名为pEcGS1b。进一步通过PCR扩增了4个5'缺失的pEcGS1b片段,命名为p1200、p900、p600、p400。以pBI121为基础,构建了4个以GUS为报告基因的植物表达载体,命名为p1200::GUS、p900::GUS、p600::GUS、p400::GUS。利用农杆菌介导的叶盘法将4个植物表达载体转化烟草叶片,进行瞬时表达。结果表明4个缺失片段均具有启动功能,但弱于组成型表达的花椰菜花叶病毒(CaMV) 35S启动子,其中p1200的启动活性最强。
A 5' flanking sequence of EcGS1 b gene which is about Glutamine Synthetase was amplified by genome walking from the genomic DNA of Water hyacinth(Eichhornia crassipes). The length of fragment is approximately1.7 kb. The results derived from PlantCARE analysis showed that the sequence contained a TATA-box core element, a CAAT-box, and other cis-acting elements, including an TATC-box, HSE, LTR and a variety of light-responsive elements. Therefore, it was speculated preliminarily to be the promoter of EcGS1 b gene. The 5'end different deletion fragment of pEcGS1 b was cloned by PCR, called p1700, p1200, p700 and p300 respectively.To investigate function of this promoter, four fragments were fused to GUS reporter gene of pBI121, generating four plant expression vector, named p1200::GUS, p900::GUS, p600::GUS, p400::GUS. With tobacco leaves for the receptor, the results of transient expression that Agrobacterium-mediated that showed all 5'deletion fragment can drive the expression of GUS reporter gene,but their activities were weaker than that of cauliflower mosaic virus(CaMV) 35 S promoter, and the activation activity of p1200 was the strongest.
A 5' flanking sequence of EcGS1 b gene which is about Glutamine Synthetase was amplified by genome walking from the genomic DNA of Water hyacinth(Eichhornia crassipes). The length of fragment is approximately1.7 kb. The results derived from PlantCARE analysis showed that the sequence contained a TATA-box core element, a CAAT-box, and other cis-acting elements, including an TATC-box, HSE, LTR and a variety of light-responsive elements. Therefore, it was speculated preliminarily to be the promoter of EcGS1 b gene. The 5'end different deletion fragment of pEcGS1 b was cloned by PCR, called p1700, p1200, p700 and p300 respectively.To investigate function of this promoter, four fragments were fused to GUS reporter gene of pBI121, generating four plant expression vector, named p1200::GUS, p900::GUS, p600::GUS, p400::GUS. With tobacco leaves for the receptor, the results of transient expression that Agrobacterium-mediated that showed all 5'deletion fragment can drive the expression of GUS reporter gene,but their activities were weaker than that of cauliflower mosaic virus(CaMV) 35 S promoter, and the activation activity of p1200 was the strongest.
引文
An G.,1987,Binary ti vectors for plant transformation and pro moter analysis,Methods in Enzymology,153(153C):292-305
Cheng X.Y.,Sardana R.,Kaplan H.,and Altosaar I.,1998,A-grobacterium-transformed rice plants expressing synthetic cryIA(b)and cryIA(c)genes are highly toxic to striped stem borer and yellow stem borer,Proceedings of the National A-cademy of Sciences of the United States of America,95(6):2767-2772
Chen X.W.,Zheng C.M.,Li X.D.,and Chen G.,2012,Effect of different concentrations of nitrogen nutrition on root activity in Eichhornia crassipes,Anhui Nongye Kexue(Journal of Agricultural Biotechnology),40(3):1657-1659(陈雄伟,郑春梅,李晓丹,陈刚,2012,不同氮营养水平对水葫芦根系活力的影响,安徽农业科学,40(3):1657-1659)
Fu M.H.,YAN G.H.,Li Y.M.,and Peng J.P.,2014,Cloning and expression analysis of three ammonium transporter gene fragments from Eichhornia crassipes,Shengwu Jishu(Biotechnology),24(3):32-35(傅明辉,严国花,李园枚,彭进平,2014,水葫芦三种铵转运蛋白基因片段的克隆及表达,生物技术,24(3):32-35)
Gao Y.T.,Ban S.Y.,and Wang A.M.,2015,Separation and functional analysis of the promoter of PpAGO7 gene from physcomitrella patens,Xibei Zhiwu Xuebao(Journal of Botany),35(3):465-471(高运通,潘沈元,王爱民,2015,小立碗藓PpAGO7基因启动子分离及其启动活性分析,西北植物学报,35(3):465-471)
Gao Y.,Yi N.,Wang Y.,Ma T.,Zhou Q.,Zhang Z.H.,and Yan S.H.,2014,Effect of Eichhornia crassipes on production of N2 by denitrification in eutrophic water,Ecological Engineering,68:14-24
Ghosh S.,2010,Role of light in the appearance of glutamine synthetase in leaves of Pennisetum glaucum,Journal of Plant Biochemistry and Biotechnology,19(2):239-242
Jefferson R.A.,Kavanagh T.A.,and Bevan M.W.,1987,GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants,Embo Journal,6(13):3901-3907
Jin Q.F.,Chen Z.D.,Sun W.H.,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,薛志慧,黄艳,唐秀华,茶树CsANS基因及其启动子的克隆与生物信息学分析,茶叶科学,2016,36(2):219-228)
Korobczak A.,Aksamit A.,覵ukaszewicz M.,Lorenc K.,Rorat T.,and Szopa J.,2005,The potato glucosyltransferase gene promoter is environmentally regulated,Plant Science,168(2):339-348
Li T.,Sun J.K.,and Liu J.T.,2015,Research advances on plant promoter,Shengwu Jishu Tongbao(Biotechnology Bulletin),31(2):18-25(李田,孙景宽,刘京涛,2015,植物启动子研究进展,生物技术通报,31(2):18-25)
Li C.X.,2016,Analysis of molecular characteristics and functional evaluation of efficiency of a glutamihe syhthetaseⅠtransgenic event for low-nitrogen tolerance in Maize,Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Xie C.X.,pp.1-6(李楚曦,2016,转Glutamine Synthetase基因耐低氮玉米分子特征分析与功能效率评价,中国农业科学院,导师:谢传晓,pp.1-6)
Lu X.,Dun H.,Lian C.L.,Zhang X.F.,Yin W.L.,and Xia X.L.,2017,The role of peu-miR164 and its target PeNAC genes in response to abiotic stress in Populus euphratica,Plant Physiology and Biochemistry,115:418-438
Ning H.,Tu W.G.,and Wang Q.Y.,2016,Research review on application of microorganism in resources utilization of water hyacinth,Anhui Nongye Kexue(Journal of Agricultural Biotechnology),44(18):7-10,20(宁华,涂卫国,王琼瑶,2016,微生物在水葫芦资源化利用中的应用研究综述,安徽农业科学,44(18):7-10,20)
Shahmuradov I.A.,Solovyev V.V.,and Gammerman A.J.,2005,Plant promoter prediction with confidence estimation,Nucleic Acids Research,33(3):1069-1076
Sun H.,Huang Q.M.,and Su J.,2005,Highly effective expression of glutamine synthetase genes GS1 and GS2 in transgenic rice plants increases nitrogen-deficiency tolerance,Journal of Plant Physiology and Molecular Biology,31(5):492-498
Wei R.,2014,Cloning and funtional analysis ofβVPE promoter from grape,Thesis for M.S.,Northwest A&F University,Supervisor:Zhang C.H.,pp.17-19(魏蓉,2014,葡萄βVPE基因启动子的克隆与功能分析,硕士学位论文,西北农林科技大学,导师:张朝红,pp.17-19)
Zhang Z.Y.,Yan S.H.,Xu C.F.,Liu H.Q.,and Zhang Y.Y.,2017,The functions of water hyacinth(Eichhornia crassipes(Mart)Solms)in remediation of polluted waters and challenges in engineering application,Shengtai Huanjing Xuebao(Ecology and Environmental Sciences),26(9):1612-1618(张志勇,严少华,徐寸发,刘海琴,张迎颖,2017,水葫芦修复污染水体的功能及其在工程应用中所面临的挑战,生态环境学报,26(9):1612-1618)
Zhang Y.L.,Zhao X.L.,Yi F.,Zhang W.D.,Xu L.,Liu J.,Chen Y.B.,and Liu S.Q.,2013,Study on anaerobic digestion of Eichhornia crassipes in a new way,Anhui Nongye Kexue(Journal of Agricultural Biotechnology),41(9):4005-4007,4025(张映兰,赵兴玲,尹芳,张无敌,许玲,柳静,陈玉保,刘士清,2013,水葫芦沼气利用新工艺研究,安徽农业科学,41(9):4005-4007,4025)
Zhang C.X.,Wang W.Q.,Jiang X.N.,and Chen X.M.,2004,Review on plant gene promoters,Yichuan Xuebao(Journal of Genetics and Genomics),31(12):1455-1464(张春晓,王文棋,蒋湘宁,陈雪梅,植物基因启动子研究进展,遗传学报,31(12):1455-1464)
Zhao K.Q.,Luo Y.Q.,Zu F.,Li J.F.,and Wang J.Q.,2012,Advances in application of glutamine synthetase in plant genet-ic engineering,Nanfang Nongye Xuebao(Journal of South-ern Agriculture),43(4):435-438(赵凯琴,罗延青,俎峰,李劲峰,王敬乔,2012,谷氨酰胺合成酶及其在植物基因工程中应用研究进展,南方农业学报,43(4):435-438)
Zhou Z.,2008,Effect of water quanlity on the growth of water hyacinth(Eichhornia crassipes(Mart.)Solms.),Thesis for M.S.,Fujian Agriculture and Forestry Univercity,Supervisor:You M.S.,pp.2-6(周喆,2008,水质条件对外来入侵生物水葫芦生长的影响,硕士学位论文,福建农林大学,导师:尤民生,pp.2-6)
Cheng X.Y.,Sardana R.,Kaplan H.,and Altosaar I.,1998,A-grobacterium-transformed rice plants expressing synthetic cryIA(b)and cryIA(c)genes are highly toxic to striped stem borer and yellow stem borer,Proceedings of the National A-cademy of Sciences of the United States of America,95(6):2767-2772
Chen X.W.,Zheng C.M.,Li X.D.,and Chen G.,2012,Effect of different concentrations of nitrogen nutrition on root activity in Eichhornia crassipes,Anhui Nongye Kexue(Journal of Agricultural Biotechnology),40(3):1657-1659(陈雄伟,郑春梅,李晓丹,陈刚,2012,不同氮营养水平对水葫芦根系活力的影响,安徽农业科学,40(3):1657-1659)
Fu M.H.,YAN G.H.,Li Y.M.,and Peng J.P.,2014,Cloning and expression analysis of three ammonium transporter gene fragments from Eichhornia crassipes,Shengwu Jishu(Biotechnology),24(3):32-35(傅明辉,严国花,李园枚,彭进平,2014,水葫芦三种铵转运蛋白基因片段的克隆及表达,生物技术,24(3):32-35)
Gao Y.T.,Ban S.Y.,and Wang A.M.,2015,Separation and functional analysis of the promoter of PpAGO7 gene from physcomitrella patens,Xibei Zhiwu Xuebao(Journal of Botany),35(3):465-471(高运通,潘沈元,王爱民,2015,小立碗藓PpAGO7基因启动子分离及其启动活性分析,西北植物学报,35(3):465-471)
Gao Y.,Yi N.,Wang Y.,Ma T.,Zhou Q.,Zhang Z.H.,and Yan S.H.,2014,Effect of Eichhornia crassipes on production of N2 by denitrification in eutrophic water,Ecological Engineering,68:14-24
Ghosh S.,2010,Role of light in the appearance of glutamine synthetase in leaves of Pennisetum glaucum,Journal of Plant Biochemistry and Biotechnology,19(2):239-242
Jefferson R.A.,Kavanagh T.A.,and Bevan M.W.,1987,GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants,Embo Journal,6(13):3901-3907
Jin Q.F.,Chen Z.D.,Sun W.H.,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,薛志慧,黄艳,唐秀华,茶树CsANS基因及其启动子的克隆与生物信息学分析,茶叶科学,2016,36(2):219-228)
Korobczak A.,Aksamit A.,覵ukaszewicz M.,Lorenc K.,Rorat T.,and Szopa J.,2005,The potato glucosyltransferase gene promoter is environmentally regulated,Plant Science,168(2):339-348
Li T.,Sun J.K.,and Liu J.T.,2015,Research advances on plant promoter,Shengwu Jishu Tongbao(Biotechnology Bulletin),31(2):18-25(李田,孙景宽,刘京涛,2015,植物启动子研究进展,生物技术通报,31(2):18-25)
Li C.X.,2016,Analysis of molecular characteristics and functional evaluation of efficiency of a glutamihe syhthetaseⅠtransgenic event for low-nitrogen tolerance in Maize,Thesis for M.S.,Chinese Academy of Agricultural Sciences,Supervisor:Xie C.X.,pp.1-6(李楚曦,2016,转Glutamine Synthetase基因耐低氮玉米分子特征分析与功能效率评价,中国农业科学院,导师:谢传晓,pp.1-6)
Lu X.,Dun H.,Lian C.L.,Zhang X.F.,Yin W.L.,and Xia X.L.,2017,The role of peu-miR164 and its target PeNAC genes in response to abiotic stress in Populus euphratica,Plant Physiology and Biochemistry,115:418-438
Ning H.,Tu W.G.,and Wang Q.Y.,2016,Research review on application of microorganism in resources utilization of water hyacinth,Anhui Nongye Kexue(Journal of Agricultural Biotechnology),44(18):7-10,20(宁华,涂卫国,王琼瑶,2016,微生物在水葫芦资源化利用中的应用研究综述,安徽农业科学,44(18):7-10,20)
Shahmuradov I.A.,Solovyev V.V.,and Gammerman A.J.,2005,Plant promoter prediction with confidence estimation,Nucleic Acids Research,33(3):1069-1076
Sun H.,Huang Q.M.,and Su J.,2005,Highly effective expression of glutamine synthetase genes GS1 and GS2 in transgenic rice plants increases nitrogen-deficiency tolerance,Journal of Plant Physiology and Molecular Biology,31(5):492-498
Wei R.,2014,Cloning and funtional analysis ofβVPE promoter from grape,Thesis for M.S.,Northwest A&F University,Supervisor:Zhang C.H.,pp.17-19(魏蓉,2014,葡萄βVPE基因启动子的克隆与功能分析,硕士学位论文,西北农林科技大学,导师:张朝红,pp.17-19)
Zhang Z.Y.,Yan S.H.,Xu C.F.,Liu H.Q.,and Zhang Y.Y.,2017,The functions of water hyacinth(Eichhornia crassipes(Mart)Solms)in remediation of polluted waters and challenges in engineering application,Shengtai Huanjing Xuebao(Ecology and Environmental Sciences),26(9):1612-1618(张志勇,严少华,徐寸发,刘海琴,张迎颖,2017,水葫芦修复污染水体的功能及其在工程应用中所面临的挑战,生态环境学报,26(9):1612-1618)
Zhang Y.L.,Zhao X.L.,Yi F.,Zhang W.D.,Xu L.,Liu J.,Chen Y.B.,and Liu S.Q.,2013,Study on anaerobic digestion of Eichhornia crassipes in a new way,Anhui Nongye Kexue(Journal of Agricultural Biotechnology),41(9):4005-4007,4025(张映兰,赵兴玲,尹芳,张无敌,许玲,柳静,陈玉保,刘士清,2013,水葫芦沼气利用新工艺研究,安徽农业科学,41(9):4005-4007,4025)
Zhang C.X.,Wang W.Q.,Jiang X.N.,and Chen X.M.,2004,Review on plant gene promoters,Yichuan Xuebao(Journal of Genetics and Genomics),31(12):1455-1464(张春晓,王文棋,蒋湘宁,陈雪梅,植物基因启动子研究进展,遗传学报,31(12):1455-1464)
Zhao K.Q.,Luo Y.Q.,Zu F.,Li J.F.,and Wang J.Q.,2012,Advances in application of glutamine synthetase in plant genet-ic engineering,Nanfang Nongye Xuebao(Journal of South-ern Agriculture),43(4):435-438(赵凯琴,罗延青,俎峰,李劲峰,王敬乔,2012,谷氨酰胺合成酶及其在植物基因工程中应用研究进展,南方农业学报,43(4):435-438)
Zhou Z.,2008,Effect of water quanlity on the growth of water hyacinth(Eichhornia crassipes(Mart.)Solms.),Thesis for M.S.,Fujian Agriculture and Forestry Univercity,Supervisor:You M.S.,pp.2-6(周喆,2008,水质条件对外来入侵生物水葫芦生长的影响,硕士学位论文,福建农林大学,导师:尤民生,pp.2-6)