独行菜法尼基焦磷酸合酶基因的克隆与原核表达
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摘要
从独行菜(Lepidium apetalum)中克隆强心苷合成途径的法尼基焦磷酸合酶(FPS)基因,并在大肠杆菌中表达,为进一步研究独行菜强心苷的合成途径提供参考。分析前期获得的独行菜幼苗转录组数据,选择其中注释为FPS且具有完整开放阅读框的序列,从独行菜叶片c DNA中通过PCR克隆该序列,并进行序列分析。结果表明,克隆得到独行菜FPS基因的cDNA序列,Gen Bank登录号KY366218,命名为La FPS。序列长度为1 332 bp,含有1 161 bp的开放阅读框,推测编码386个氨基酸。La FPS蛋白可能不含跨膜结构,定位于线粒体中,含有聚异戊二烯合成酶结构域。La FPS蛋白氨基酸序列与拟南芥(Arabidopsis thaliana)FPS1蛋白相似性高达92%,进化树分析结果表明,La FPS与同为十字花科的拟南芥FPS1、遏蓝菜(Noccaea caerulescens)FPS1、高山南芥(Arabis alpina)FPS亲缘关系最近。构建的载体p ET-32a-La FPS可在大肠杆菌中成功诱导表达。表明成功克隆了独行菜FPS基因,并建立了其原核表达体系。
This study obtained the farnesyl pyrophosphate synthase gene involved in the cardiac glycosides biosynthesis from Lepidium apetalum,analysed the sequence,and induced expression of the gene in E. coli,which provided new material for research of the biosynthetic pathway of cardiac glycoside in L. apetalum. Specific primers were designed for a gene fragment with complete ORF and annotated as FPS in the transcriptome data of L. apetalum,and the fragment was cloned from L. apetalum leaf c DNA template by PCR method. Then the sequence of cloned gene was analyzed. The cloned c DNA fragment of La FPS( Gen Bank accession No. KY366218) had an length of 1 332 bp,with an ORF of 1 161 bp encoding 386 amino acids. According to the sequence analysis result,La FPS had no transmembrane helices,located in mitochondria,and had an polyprenyl synthetase domain. Sequence of La FPS was 92% identical to that of Arabidopsis FPS1 protein. La FPS was closest to Arabidopsis FPS1,Noccaea caerulescens FPS1,Arabis alpine FPS in the phylogeny tree analysis,and all the proteins were from the Brassicaceae family. Expression ofLa FPS protein was successfully induced in E. coli strain BL21( DE3) with constructed expression vector p ET-32a-La FPS. La FPS gene was cloned from L. apetalum,and the prokaryotic expression system was established.
This study obtained the farnesyl pyrophosphate synthase gene involved in the cardiac glycosides biosynthesis from Lepidium apetalum,analysed the sequence,and induced expression of the gene in E. coli,which provided new material for research of the biosynthetic pathway of cardiac glycoside in L. apetalum. Specific primers were designed for a gene fragment with complete ORF and annotated as FPS in the transcriptome data of L. apetalum,and the fragment was cloned from L. apetalum leaf c DNA template by PCR method. Then the sequence of cloned gene was analyzed. The cloned c DNA fragment of La FPS( Gen Bank accession No. KY366218) had an length of 1 332 bp,with an ORF of 1 161 bp encoding 386 amino acids. According to the sequence analysis result,La FPS had no transmembrane helices,located in mitochondria,and had an polyprenyl synthetase domain. Sequence of La FPS was 92% identical to that of Arabidopsis FPS1 protein. La FPS was closest to Arabidopsis FPS1,Noccaea caerulescens FPS1,Arabis alpine FPS in the phylogeny tree analysis,and all the proteins were from the Brassicaceae family. Expression ofLa FPS protein was successfully induced in E. coli strain BL21( DE3) with constructed expression vector p ET-32a-La FPS. La FPS gene was cloned from L. apetalum,and the prokaryotic expression system was established.
引文
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[13]Xiang L,Zhao K G,Chen L Q.Molecular cloning and expression of Chimonanthus praecox farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral volatile sesquiterpenoids[J].Plant Physiology and Biochemistry,2010,48(10/11):845-850.
[14]Wang P,Liao Z,Guo L,et al.Cloning and functional analysis of a c DNA encoding Ginkgo biloba farnesyl diphosphate synthase[J].Molecules and Cells,2004,18(2):150-156.
[2]Kim S J,Kim H Y,Lee Y J,et al.Ethanol extract of Lepidium apetalum seed elicits contractile response and attenuates atrial natriuretic peptide secretion in beating rabbit atria[J].Evidence-Based Complementary and Alternative Medicine,2013.doi:10.1155/2013/404713.
[3]Kim B Y,Lee J,Kim N S.Helveticoside is a biologically active component of the seed extract of Descurainia sophia and induces reciprocal gene regulation in A549 human lung cancer cells[J].BMC Genomics,2015,16:713.
[4]Wu B,Li Y,Yan H,et al.Comprehensive transcriptome analysis reveals novel genes involved in cardiac glycoside biosynthesis and mlncRNAs associated with secondary metabolism and stress response in Digitalis purpurea[J].BMC Genomics,2012,13(1):1-22.
[5]VranováE,Coman D,Gruissem W.Network analysis of the MVA and MEP pathways for isoprenoid synthesis[J].Annual Review of Plant Biology,2013,64(1):665-700.
[6]Keim V,Manzano D,Fernández F J,et al.Characterization of Arabidopsis FPS isozymes and FPS gene expression analysis provide insight into the biosynthesis of isoprenoid precursors in seeds[J].PLo S One,2012,7(11):e49109.
[7]Closa M,VranováE,Bortolotti C,et al.The Arabidopsis thaliana FPP synthase isozymes have overlapping and specific functions in isoprenoid biosynthesis,and complete loss of FPP synthase activity causes early developmental arrest[J].The Plant Journal,2010,63(3):512-525.
[8]Chen Y,Shen Q,Wang Y,et al.The stacked over-expression of FPS,CYP71AV1 and CPR genes leads to the increase of artemisinin level in Artemisia annua L.[J].Plant Biotechnology Reports,2013,7(3):287-295.
[9]Wang Y,Jing F,Yu S,et al.Co-overexpression of the HMGR and FPS genes enhances artemisinin content in Artemisia annua L.[J].Journal of Medicinal Plants Research,2011,5(15):3396-3403.
[10]Banyai W,Kirdmanee C,Mii M,et al.Overexpression of farnesyl pyrophosphate synthase(FPS)gene affected artemisinin content and growth of Artemisia annua L.[J].Plant Cell Tissue and Organ Culture,2010,103(2):255-265.
[11]Zhao Y J,Chen X,Zhang M,et al.Molecular cloning and characterisation of farnesyl pyrophosphate synthase from Tripterygium wilfordii[J].PLo S One,2015,10(5):e0125415.
[12]Niu Y,Luo H,Sun C,et al.Expression profiling of the triterpene saponin biosynthesis genes FPS,SS,SE,and DS in the medicinal plant Panax notoginseng[J].Gene,2014,533(1):295-303.
[13]Xiang L,Zhao K G,Chen L Q.Molecular cloning and expression of Chimonanthus praecox farnesyl pyrophosphate synthase gene and its possible involvement in the biosynthesis of floral volatile sesquiterpenoids[J].Plant Physiology and Biochemistry,2010,48(10/11):845-850.
[14]Wang P,Liao Z,Guo L,et al.Cloning and functional analysis of a c DNA encoding Ginkgo biloba farnesyl diphosphate synthase[J].Molecules and Cells,2004,18(2):150-156.