刺五加鲨烯环氧酶基因DNA和启动子的克隆与分析
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摘要
本研究以刺五加鲨烯环氧酶(squalene epoxidase,SE)基因的c DNA序列为基础设计特异性引物,利用PCR和TAIL-PCR扩增SE DNA的全长序列,结合Plant CARE等软件,对启动子中的作用元件进行生物信息学分析。克隆得到长6 307 bp的刺五加SE DNA和启动子序列,包含8段外显子、7段内含子及两端的非翻译区,编码554个氨基酸;上游启动子序列长1 907 bp,含36个TATA-box,27个CAAT-box,此外还有茉莉酸甲酯调控元件、脱落酸调控元件以及光调控元件等多种顺势作用元件,表明SE基因表达受植物激素、光照以及温度等多种因素的调控。
In this paper,based on the c DNA sequence of squalene epoxidase(SE) gene in E.senticosus,we designed specific primers.PCR and TAIL-PCR were used to amplify DNA full-length sequence of SE.Then combined with Plant CARE and other softwares,bioinformatics analysis was performed on the functional elements in the promoter.DNA and promoter sequence of E.senticosus SE gene with 6 307 bp was cloned,containing 8exons,7 segment introns and untranslated regions at both ends and encoding 554 amino acids.The upstream promoter sequence was 1 907 bp long,containing 36 TATA-box and 27 CAAT-box.Besides,it also contained a variety of cis-acting elements,such as jasmonic acid regulatory element,abscisic acid regulatory element,and light regulatory elements,which indicated that the expression of SE gene was regulated by many factors,such as plant hormone,light and temperature.
In this paper,based on the c DNA sequence of squalene epoxidase(SE) gene in E.senticosus,we designed specific primers.PCR and TAIL-PCR were used to amplify DNA full-length sequence of SE.Then combined with Plant CARE and other softwares,bioinformatics analysis was performed on the functional elements in the promoter.DNA and promoter sequence of E.senticosus SE gene with 6 307 bp was cloned,containing 8exons,7 segment introns and untranslated regions at both ends and encoding 554 amino acids.The upstream promoter sequence was 1 907 bp long,containing 36 TATA-box and 27 CAAT-box.Besides,it also contained a variety of cis-acting elements,such as jasmonic acid regulatory element,abscisic acid regulatory element,and light regulatory elements,which indicated that the expression of SE gene was regulated by many factors,such as plant hormone,light and temperature.
引文
Ha n J.Y.,In J.G.,Kwon Y.S.,and Choi Y.E.,2011,Regulation of ginsenoside and phytosterol biosynthesis by RNA inter ferences of squalene epoxidase gene in Panax ginseng,Phy tochemistry,71(1):36-46
Lev-Maor G.,Sorek R.,Shomron N.,and Ast G.,2003,The birth of an alternatively spliced exon:3'splice-site selection in Alu exons,Science,300(5623):1288-1291
Liu Y.G.,and Chen Y.L.,2007,High-efficiency thermal asym metric interlaced PCR for amplification of unknown flank ing sequences,Bio.Techniques,43(5):649-650,652,654
Long Y.L.,Li F.F.,Yang G.,and Xing Z.B.,2015,Expression profiles analysis of two member of squalene epoxidase gen family from Eleutherococcus senticosus,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medi ca),40(1):59-62(龙月红,李非非,杨果,邢朝斌,2015,刺五加鲨烯环氧酶基因家族2成员表达特性分析,中国中药杂志,40(1):59-62)
Lu R.J.,Liu D.Q.,Ge F.,and Chen C.Y.,2014,Cloning and se quence analysis of promoter of SE in Panax notoginseng Zhiwu Shengli Xuebao(Plant Physiology Journal),50(10)1535-1540(卢荣江,刘迪秋,葛锋,陈朝银,2014,三七SE基因启动子的克隆及序列分析,植物生理学报,50(10)1535-1540)
Nasrollahi V.,Mirzaie-asi A.,Piri K.,Nazeri S.,and Mehrabi R.2015,The effect of drought stress on the expression of key genes involved in the biosynthesis of triterpenoid saponin in liquorice(Glycyrrhiza glabra),Phytochemistry,103(7)32-37
Nie L.N.,Xia L.Q.,Xu Z.S.,Gao D.X.,Li L.,Yu Z.,Chen M.,LL.C.,and Ma Y.Z.,2008,Progress on cloning and functiona study of plant gene promoters,Zhiwu Yichuan Ziyuan Xue bao(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)
Rance I.,Norre F.,Gruber V.,and Theisen M.,2002,Combina tion of viral promoter sequences to generate highly activ promoters for heterologous therapeutic protein over-expres sion in plants,Plant Science,162(5):833-842
Tu Z.W.,Zhou W.W.,Shan Q.,Xin N.,and Hou W.B.,2011Advances in studies on chemical constituents of Acan thopanax senticosus and their pharmacological effects Yaowu Pingjia Yanjiu(Drug Evaluation Research),34(3)213-216(涂正伟,周渭渭,单淇,辛宁,侯文彬,2011,刺五加的研究进展,药物评价研究,34(3):213-216)
Wang Y.B.,Lang Z.H.,and Huang D.F.,2008,Effects of Pre-m RNA introns on regulation of eukaryotic gene expression Shengwu Jishu Tongbao(Biotechnology Bulletin),4:1-4,8(王悦冰,郎志宏,黄大昉,2008,内含子对真核基因表达调控的影响,生物技术通报,4:1-4,8)
Xing Z.B.,Cao L.,Chen L.,He S.,Li B.C.,and Zhu J.L.2012,Cloning and sequence analysis on c DNA of squalene e poxidase gene in Eleutherococcus senticosus,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medi ca),37(2):172-175(邢朝斌,曹蕾,陈龙,何闪,李宝财,朱金丽,2012,刺五加鲨烯环氧酶基因c DNA的克隆及序列分析,中国中药杂志,37(2):172-175)
Zhang C.X.,Wang W.Q.,Jiang X.Y.,and Chen X.M.,2004,Re view on plant gene promoters,Yichuan Xuebao(Acta Ge netica Sinica),31(12):1455-1464(张春晓,王文棋,蒋湘宇,陈雪梅,2004,植物基因启动子研究进展,遗传学报31(12):1455-1464)
Zhao C.,Guo L.N.,Peng Y.S.,Liu C.,and Wang R.F.,2015,Re search progress in key enzymes involved in biosynthesis o Panax notoginseng saponins and their regulation,Zhong caoyao(Chinese Traditional and Herbal Drugs),46(19)2954-2965(赵灿,郭丽娜,彭玉帅,刘辰,王如峰,2015,三七总皂苷生物合成的关键酶及其调控研究进展,中草药46(19):2954-2965)
Lev-Maor G.,Sorek R.,Shomron N.,and Ast G.,2003,The birth of an alternatively spliced exon:3'splice-site selection in Alu exons,Science,300(5623):1288-1291
Liu Y.G.,and Chen Y.L.,2007,High-efficiency thermal asym metric interlaced PCR for amplification of unknown flank ing sequences,Bio.Techniques,43(5):649-650,652,654
Long Y.L.,Li F.F.,Yang G.,and Xing Z.B.,2015,Expression profiles analysis of two member of squalene epoxidase gen family from Eleutherococcus senticosus,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medi ca),40(1):59-62(龙月红,李非非,杨果,邢朝斌,2015,刺五加鲨烯环氧酶基因家族2成员表达特性分析,中国中药杂志,40(1):59-62)
Lu R.J.,Liu D.Q.,Ge F.,and Chen C.Y.,2014,Cloning and se quence analysis of promoter of SE in Panax notoginseng Zhiwu Shengli Xuebao(Plant Physiology Journal),50(10)1535-1540(卢荣江,刘迪秋,葛锋,陈朝银,2014,三七SE基因启动子的克隆及序列分析,植物生理学报,50(10)1535-1540)
Nasrollahi V.,Mirzaie-asi A.,Piri K.,Nazeri S.,and Mehrabi R.2015,The effect of drought stress on the expression of key genes involved in the biosynthesis of triterpenoid saponin in liquorice(Glycyrrhiza glabra),Phytochemistry,103(7)32-37
Nie L.N.,Xia L.Q.,Xu Z.S.,Gao D.X.,Li L.,Yu Z.,Chen M.,LL.C.,and Ma Y.Z.,2008,Progress on cloning and functiona study of plant gene promoters,Zhiwu Yichuan Ziyuan Xue bao(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)
Rance I.,Norre F.,Gruber V.,and Theisen M.,2002,Combina tion of viral promoter sequences to generate highly activ promoters for heterologous therapeutic protein over-expres sion in plants,Plant Science,162(5):833-842
Tu Z.W.,Zhou W.W.,Shan Q.,Xin N.,and Hou W.B.,2011Advances in studies on chemical constituents of Acan thopanax senticosus and their pharmacological effects Yaowu Pingjia Yanjiu(Drug Evaluation Research),34(3)213-216(涂正伟,周渭渭,单淇,辛宁,侯文彬,2011,刺五加的研究进展,药物评价研究,34(3):213-216)
Wang Y.B.,Lang Z.H.,and Huang D.F.,2008,Effects of Pre-m RNA introns on regulation of eukaryotic gene expression Shengwu Jishu Tongbao(Biotechnology Bulletin),4:1-4,8(王悦冰,郎志宏,黄大昉,2008,内含子对真核基因表达调控的影响,生物技术通报,4:1-4,8)
Xing Z.B.,Cao L.,Chen L.,He S.,Li B.C.,and Zhu J.L.2012,Cloning and sequence analysis on c DNA of squalene e poxidase gene in Eleutherococcus senticosus,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medi ca),37(2):172-175(邢朝斌,曹蕾,陈龙,何闪,李宝财,朱金丽,2012,刺五加鲨烯环氧酶基因c DNA的克隆及序列分析,中国中药杂志,37(2):172-175)
Zhang C.X.,Wang W.Q.,Jiang X.Y.,and Chen X.M.,2004,Re view on plant gene promoters,Yichuan Xuebao(Acta Ge netica Sinica),31(12):1455-1464(张春晓,王文棋,蒋湘宇,陈雪梅,2004,植物基因启动子研究进展,遗传学报31(12):1455-1464)
Zhao C.,Guo L.N.,Peng Y.S.,Liu C.,and Wang R.F.,2015,Re search progress in key enzymes involved in biosynthesis o Panax notoginseng saponins and their regulation,Zhong caoyao(Chinese Traditional and Herbal Drugs),46(19)2954-2965(赵灿,郭丽娜,彭玉帅,刘辰,王如峰,2015,三七总皂苷生物合成的关键酶及其调控研究进展,中草药46(19):2954-2965)