甘蓝型油菜BnGPAT9基因启动子克隆与表达载体构建
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摘要
甘油-3-磷酸酰基转移酶(glycerol-3-phosphate acyltransferase, GPAT)在植物三酰甘油脂(TAGs)等油脂合成中起着重要作用,而GPAT9基因是GPAT基因家族中参与油脂合成的重要基因。本研究采用电子克隆技术,以甘蓝型油菜湘油15为模板克隆了BnGPAT9基因的启动子序列pBnGPAT9,长度为900 bp。经过PlantCARE在线分析表明:该序列含有CAAT-box和TATA-box等区域特征元件,和多个参与光应答、激素诱导响应、逆境胁迫响应的元件,以及分生组织表达相关元件CAT-box与胚乳表达所需的顺式作用元件Skn-1_motif等,推测其可能在种子中表达较强烈,且在油菜生殖生长中有重要作用。通过双酶切,将pBnGPAT9启动子序列替换p BI121植物表达载体上的CaMV35S,使该启动子与GUS基因融合,构建了pBnGPAT9:GUS植物表达载体。
Glycerol-3-phosphate acyltransferase(GPAT) plays an important role in oil synthesis such as plant tri acylglycerols(TAGs), and GPAT9 gene is an important gene involved in lipid synthesis in the GPAT gene family. In this study, the promoter sequence p BnGPAT9 of BnGPAT9 gene was cloned from Brassica napus Xiangyou 15 using electron cloning technology and the length was 900 bp. PlantCARE online analysis showed that the sequence contained region-specific elements such as CAAT-box and TATA-box, and multiple elements involved in photoresponse, hormone-induced response, and stress response, and cis-acting elements Skn-1_motif required for the expression of CAT-box and endosperm in meristem. It was speculated that the sequence might be strongly expressed in seeds and might play an important role in the reproductive growth of rapeseed. By double enzyme digestion,pBnGPAT9 promoter sequence was substituted for CaMV35 S on pBI121 plant expression vector, and the promoter was fused with GUS gene to construct pBnGPAT9: GUS plant expression vector.
Glycerol-3-phosphate acyltransferase(GPAT) plays an important role in oil synthesis such as plant tri acylglycerols(TAGs), and GPAT9 gene is an important gene involved in lipid synthesis in the GPAT gene family. In this study, the promoter sequence p BnGPAT9 of BnGPAT9 gene was cloned from Brassica napus Xiangyou 15 using electron cloning technology and the length was 900 bp. PlantCARE online analysis showed that the sequence contained region-specific elements such as CAAT-box and TATA-box, and multiple elements involved in photoresponse, hormone-induced response, and stress response, and cis-acting elements Skn-1_motif required for the expression of CAT-box and endosperm in meristem. It was speculated that the sequence might be strongly expressed in seeds and might play an important role in the reproductive growth of rapeseed. By double enzyme digestion,pBnGPAT9 promoter sequence was substituted for CaMV35 S on pBI121 plant expression vector, and the promoter was fused with GUS gene to construct pBnGPAT9: GUS plant expression vector.
引文
Brown A.P.,Kroon J.T.,Swarbreck D.,Febrer M.,Larson T.R.,Grah am I.A.,Caccamo M.,and Slabas A.R.,2012,Tissue-specific whole transcriptome sequencing in castor,directed at understanding triacylglycerol lipid biosynthetic pathways,PLo S One,7(2):e30100
Chapman K.D.,and Ohlrogge J.B.,2012,Compartmentation of triacylglycerol ac cumulation in plants,J.Biol.Chem.,287(4):2288-2294
Chen X.,Snyder C.L.,Truksa M.,Shah S.,and Weselake R.J.,2011,Sn-glycer ol-3-phosphate acyltransferases in plants,Plant Signal.Behav.,6(11):1352-1359
Gidda S.K.,Shockey J.M.,Rothstein S.J.,Dyer J.M.,and Mullen R.T.,2009,Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals:functional divergence of the dilysine ER retrieval motif in plant cells,Plant Physiol.Biochem.,47(10):867-879
Jain R.K.,Coffey M.,Lai K.,Kumar A.,and Mac Kenzie S.L.,2000,Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes,Biochem.Soc.Trans.,28(6):958-961
Jako C.,Kumar A.,Wei Y.,Zou J.,Barton D.L.,Giblin E.M.,Covello P.S.,and Taylor D.C.,2001,Seed-specific over-expression of an Arabidopsis c DNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight,Plant Physiol.,126(2):861-874
Li N.,Xu C.,Li-Beisson Y.,and Philippar K.,2016,Fatty acid and lipid transport in plant cells,Trends Plant Sci.,21(2):145-158
Li X.C.,Zhu J.,Yang J.,Zhang G.R.,Xing W.F.,Zhang S.,and Yang Z.N.,2012,Glycerol-3-phosphate acyltransferase 6(GPAT6)is important for tapetum development in Arabidopsis and plays multiple roles in plant fertility,Mol.Plant,5(1):131-142
Peng P.F.,Hu Q.,Li Y.C.,Liu D.M.,Fu L.,Zhang H.,and Mei D.S.,2012,Cloning and expression analysis of Bn PPR636 and its promoter in rapeseed(brassica napus),Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),10(6):693-700(彭鹏飞,胡琼,李云昌,刘道敏,付丽,张洪,梅德圣,2012,甘蓝型油菜Bn PPR636及其启动子的克隆与表达分析,分子植物育种,10(6):693-700)
Radakovits R.,Jinkerson R.E.,Darzins A.,and Posewitz M.C.,2010,Genetic engineering of algae for enhanced biofuel production,Eukaryotic Cell,9(4):486-501
Shockey J.,Regmi A.,Cotton K.,Adhikari N.,Browse J.,and Bates P.D.,2016,Identification of arabidopsis GPAT9(At5g-60620)as an essential gene involved in triacylglycerol biosynthesis,Plant Physiol.,170(1):163-179
Singer S.D.,Chen G.,Mietkiewska E.,Tomasi P.,Jayawardhane K.,Dyer J.M.,and Weselake R.J.,2016,Arabidopsis GPAT9contributes to synthesis of intracellular glycerolipids but not surface lipids,J.Exp.Bot.,67(15):4627-4638
Sui N.,Li M.,Zhao S.J.,Li F.,Liang H.,and Meng Q.W.,2007,Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato,Planta,226(5):1097-1108
Xiao D.W.,Liu S.F.,Liu C.,Guan C.Y.,and Xiong X.H.,2016,Cloning and expression analysis of LPAT4 promoter from Brassica napus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(6):1455-1461(肖旦望,刘少锋,刘聪,官春云,熊兴华,2016,甘蓝型油菜Bn LPAT4启动子的克隆及表达分析,分子植物育种,14(6):1455-1461)
Xing M.,Liu S.F.,Wu X.M.,Guan C.Y.,and Xiong X.H.,2016,Cloning and bioinformatics analysis of GPAT9 gene in rapeseed,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(12):3282-3288(邢蔓,刘少锋,邬贤梦,官春云,熊兴华,2016,甘蓝型油菜GPAT9基因克隆与生物信息学分析,分子植物育种,14(12):3282-3288)
Xing M.,Zhou X.Q.,He T.,Zhang Z.R.,Yue N.Y.,Zeng X.G.,Wu X.M.,Guan C.Y.,and Xiong X.H.,2017,Expression pattern of Bn GPAT9 gene in Brassica napus and its expression under abiotic stresses,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),39(4):454-461(邢蔓,周雪晴,何婷,张泽荣,岳宁燕,曾祥拱,邬贤梦,官春云,熊兴华,2017,甘蓝型油菜Bn GPAT9基因表达模式及其苗期非生物胁迫表达分析,中国油料作物学报,39(4):454-461)
Yan K.,Chen N.,Qu Y.Y.,Dong X.C.,Meng Q.W.,and Zhao S.J.,2008,Overexpression of sweet pepper glycerol-3-phosphate acyltransferase gene enhanced thermotolerance of photosynthetic apparatus in transgenic tobacco,J.Integr.Plant Biol.,50(5):613-621
Yang W.,Simpson J.P.,Li-Beisson Y.,Beisson F.,Pollard M.,and Ohlrogge J.B.,2012,A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis:substrate specificity,sn-2 preference,and evolution,Plant Physiol.,160(2):638-652
Zhang F.,Cheng Y.,Gu T.C.,and Yin Y.,2011,Present status and countermeasure suggestions for rapeseed seed industry development of China,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and Technology),13(4):15-22(张芳,程勇,谷铁城,殷艳,2011,中国油菜种业发展现状及对策建议,中国农业科技导报,13(4):15-22)
Zhang N.,Xu R.H.,Liu X.Z.,and Liu A.Z.,2011,Cloning and sequence analysis of sn-glycerol-3-phosphate acyltransferase gene(Jc GPAT)from Jatropha curcas L.,Zhiwu Shengli Xuebao(Plant Physiology Journal),47(2):181-188(张楠,徐荣华,刘小烛,刘爱忠,2011,小桐子甘油-3-磷酸酰基转移酶(Jc GPAT)c DNA的克隆与序列分析,植物生理学报,47(2):181-188)
Zheng Z.F.,Xia Q.,Dauk M.,Shen W.Y.,Selvaraj G.,and Zou J.T.,2003,Arabidopsis At GPAT1,a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family,is essential for tapetum differentiation and male fertility,Plant Cell,15(8):1872-1887
Chapman K.D.,and Ohlrogge J.B.,2012,Compartmentation of triacylglycerol ac cumulation in plants,J.Biol.Chem.,287(4):2288-2294
Chen X.,Snyder C.L.,Truksa M.,Shah S.,and Weselake R.J.,2011,Sn-glycer ol-3-phosphate acyltransferases in plants,Plant Signal.Behav.,6(11):1352-1359
Gidda S.K.,Shockey J.M.,Rothstein S.J.,Dyer J.M.,and Mullen R.T.,2009,Arabidopsis thaliana GPAT8 and GPAT9 are localized to the ER and possess distinct ER retrieval signals:functional divergence of the dilysine ER retrieval motif in plant cells,Plant Physiol.Biochem.,47(10):867-879
Jain R.K.,Coffey M.,Lai K.,Kumar A.,and Mac Kenzie S.L.,2000,Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes,Biochem.Soc.Trans.,28(6):958-961
Jako C.,Kumar A.,Wei Y.,Zou J.,Barton D.L.,Giblin E.M.,Covello P.S.,and Taylor D.C.,2001,Seed-specific over-expression of an Arabidopsis c DNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight,Plant Physiol.,126(2):861-874
Li N.,Xu C.,Li-Beisson Y.,and Philippar K.,2016,Fatty acid and lipid transport in plant cells,Trends Plant Sci.,21(2):145-158
Li X.C.,Zhu J.,Yang J.,Zhang G.R.,Xing W.F.,Zhang S.,and Yang Z.N.,2012,Glycerol-3-phosphate acyltransferase 6(GPAT6)is important for tapetum development in Arabidopsis and plays multiple roles in plant fertility,Mol.Plant,5(1):131-142
Peng P.F.,Hu Q.,Li Y.C.,Liu D.M.,Fu L.,Zhang H.,and Mei D.S.,2012,Cloning and expression analysis of Bn PPR636 and its promoter in rapeseed(brassica napus),Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),10(6):693-700(彭鹏飞,胡琼,李云昌,刘道敏,付丽,张洪,梅德圣,2012,甘蓝型油菜Bn PPR636及其启动子的克隆与表达分析,分子植物育种,10(6):693-700)
Radakovits R.,Jinkerson R.E.,Darzins A.,and Posewitz M.C.,2010,Genetic engineering of algae for enhanced biofuel production,Eukaryotic Cell,9(4):486-501
Shockey J.,Regmi A.,Cotton K.,Adhikari N.,Browse J.,and Bates P.D.,2016,Identification of arabidopsis GPAT9(At5g-60620)as an essential gene involved in triacylglycerol biosynthesis,Plant Physiol.,170(1):163-179
Singer S.D.,Chen G.,Mietkiewska E.,Tomasi P.,Jayawardhane K.,Dyer J.M.,and Weselake R.J.,2016,Arabidopsis GPAT9contributes to synthesis of intracellular glycerolipids but not surface lipids,J.Exp.Bot.,67(15):4627-4638
Sui N.,Li M.,Zhao S.J.,Li F.,Liang H.,and Meng Q.W.,2007,Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato,Planta,226(5):1097-1108
Xiao D.W.,Liu S.F.,Liu C.,Guan C.Y.,and Xiong X.H.,2016,Cloning and expression analysis of LPAT4 promoter from Brassica napus,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(6):1455-1461(肖旦望,刘少锋,刘聪,官春云,熊兴华,2016,甘蓝型油菜Bn LPAT4启动子的克隆及表达分析,分子植物育种,14(6):1455-1461)
Xing M.,Liu S.F.,Wu X.M.,Guan C.Y.,and Xiong X.H.,2016,Cloning and bioinformatics analysis of GPAT9 gene in rapeseed,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(12):3282-3288(邢蔓,刘少锋,邬贤梦,官春云,熊兴华,2016,甘蓝型油菜GPAT9基因克隆与生物信息学分析,分子植物育种,14(12):3282-3288)
Xing M.,Zhou X.Q.,He T.,Zhang Z.R.,Yue N.Y.,Zeng X.G.,Wu X.M.,Guan C.Y.,and Xiong X.H.,2017,Expression pattern of Bn GPAT9 gene in Brassica napus and its expression under abiotic stresses,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),39(4):454-461(邢蔓,周雪晴,何婷,张泽荣,岳宁燕,曾祥拱,邬贤梦,官春云,熊兴华,2017,甘蓝型油菜Bn GPAT9基因表达模式及其苗期非生物胁迫表达分析,中国油料作物学报,39(4):454-461)
Yan K.,Chen N.,Qu Y.Y.,Dong X.C.,Meng Q.W.,and Zhao S.J.,2008,Overexpression of sweet pepper glycerol-3-phosphate acyltransferase gene enhanced thermotolerance of photosynthetic apparatus in transgenic tobacco,J.Integr.Plant Biol.,50(5):613-621
Yang W.,Simpson J.P.,Li-Beisson Y.,Beisson F.,Pollard M.,and Ohlrogge J.B.,2012,A land-plant-specific glycerol-3-phosphate acyltransferase family in Arabidopsis:substrate specificity,sn-2 preference,and evolution,Plant Physiol.,160(2):638-652
Zhang F.,Cheng Y.,Gu T.C.,and Yin Y.,2011,Present status and countermeasure suggestions for rapeseed seed industry development of China,Zhongguo Nongye Keji Daobao(Journal of Agricultural Science and Technology),13(4):15-22(张芳,程勇,谷铁城,殷艳,2011,中国油菜种业发展现状及对策建议,中国农业科技导报,13(4):15-22)
Zhang N.,Xu R.H.,Liu X.Z.,and Liu A.Z.,2011,Cloning and sequence analysis of sn-glycerol-3-phosphate acyltransferase gene(Jc GPAT)from Jatropha curcas L.,Zhiwu Shengli Xuebao(Plant Physiology Journal),47(2):181-188(张楠,徐荣华,刘小烛,刘爱忠,2011,小桐子甘油-3-磷酸酰基转移酶(Jc GPAT)c DNA的克隆与序列分析,植物生理学报,47(2):181-188)
Zheng Z.F.,Xia Q.,Dauk M.,Shen W.Y.,Selvaraj G.,and Zou J.T.,2003,Arabidopsis At GPAT1,a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family,is essential for tapetum differentiation and male fertility,Plant Cell,15(8):1872-1887
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