Cloning and Expression Analysis of the SmGGPPS3 Gene from Salvia miltiorrhiza
-
摘要: 香叶基香叶基焦磷酸合酶（Geranylgeranyl pyrophosphate synthase，GGPPS）是植物细胞二萜类物质合成的重要调节靶点。本研究从药用植物丹参中克隆了一条新的GGPPS基因（SmGGPPS3），基因全长2908 bp，包含一个931 bp的内含子和一个960 bp的编码序列。推测的氨基酸序列与蓖麻、橡胶、拟南芥等植物GGPPS一致性达到67%以上。实时定量PCR结果显示，SmGGPPS3基因在丹参不同发育时期不同器官中表达差异显著，同时受茉莉酸甲酯和病原菌的诱导。遗传互补实验也表明，SmGGPPS3编码蛋白具有GGPP合酶的活性。
-
关键词:
- 丹参 /
- 香叶基香叶基焦磷酸合酶(GGPPS) /
- 基因克隆 /
- 基因表达
Abstract: Geranylgeranyl pyrophosphate synthase(GGPPS) is an important target site for regulating diterpenoid biosynthesis in plant cells. Now,a novel geranylgeranyl pyrophosphate synthase gene (SmGGPPS3) was cloned from the medicinal plant Salvia miltiorrhiza. The full-length SmGGPPS3 consists of 2908 nucleotides,including a 931-bp intron and one 960-bp ORF encoding region. The deduced protein of SmGGPPS3 showed more than 67% similarity with GGPPSs from Ricinus communis,rubber and Arabidopsis.Quantitative RT-PCR analysis revealed that SmGGPPS3 was significantly expressed in different development stages and different tissues examined,and could be induced by methyl jasmonate(MeJA) and pathogens.Furthermore,the genetic complementation expression of SmGGPPS3 in Escherichia coli revealed that SmGGPPS3 encoded a functional GGPP synthase.-
Keywords:
- Salvia miltiorrhiza /
- GGPPS /
- Gene clone /
- Gene expression
-
-
[1] Kojima N,Sitthithaworn W,Viroonchatapan E,Suh DY,Iwanami N,Hayashi T,Sankaw U.Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus.cDNA cloning,functional expression,and conversion to a farnesyl diphosphate synthase[J].Chem Pharm Bull(Tokyo),2000,48(7):1101-1103.
[2] Laskaris G,De Jong CF,Jaziri M,Van Der Heijden R,Theodoridis G,Verpoorte R.Geranylgeranyl diphosphate synthase activity and taxane production in Taxus baccata cells[J].Phytochemistry,1999,50(6):939-946.
[3] Aharoni A,Giri AP,Deuerlein S,Griepink F,de Kogel WJ,Verstappen FW,Verhoeven HA,Jongsma MA,Schwab W,Bouwmeester HJ.Terpenoid metabolism in wild-type and transgenic Arabidopsis plants[J].The Plant Cell,2003,15(12):2866-2884.
[4] Lin TH,Hsieh CL.Pharmacological effects of Salvia miltiorrhiza(Danshen) on cerebral infarction[J].Chin Med,2010,5:22.
[5] Li YG,Song L,Liu M,Wang ZT.Advancement in analysis of Salviae miltiorrhizae Radix et Rhizoma(Danshen) [J].J Chromatogr A,2009,1216(11):1941-1953.
[6] Wang X,Cheung CM,Lee WY,Or PM,Yeung JH.Major tanshinones of Danshen(Salvia miltiorrhiza) exhibit different modes of inhibition on human CYP1A2,CYP2C9,CYP2E1 and CYP3A4 activities in vitro[J].Phytomedicine,2010,17(11):868-875.
[7] Yang W,Ju JH,Jeon MJ,Han X,Shin I.Danshen(Salvia miltiorrhiza) extract inhibits proliferation of breast cancer cells via modulation of Akt activity and p27 level[J].Phytother Res,2010,24(2):198-204.
[8] 张蕾.丹参牻牛儿基牻牛儿基焦磷酸合酶基因的克隆与功能研究[D].北京:中国人民解放军军事医学科学院,2009. [9] Kai G,Liao P,Zhang T,Zhou W,Wang J,Xu H,Liu YY,Zhang Lin. Characterization,expression profiling,and functional identification of a gene encoding geranylgeranyl diphosphate synthase from Salvia miltiorrhiza[J].Biotechnol Bioproc E,2010,15(2):236-245.
[10] Hua WP,Song J,Li CQ,Wang ZZ.Molecular cloning and characterization of the promoter of SmGGPPs and its expression pattern in Salvia miltiorrhiza[J].Molecular Biology Reports,2012,39(5):5775-5783.
[11] Hua WP,Zhang Y,Song J,Zhao LJ,Wang ZZ.De novo transcriptome sequencing in Salvia miltiorrhiza to identify genes involved in the biosynthesis of active ingredients[J].Genomics,2011,98(4):272-279.
[12] Stewart CN,Via LE.A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications[J].Biotechniques,1993,14(5):748-750.
[13] Song J,Wang ZZ.Molecular cloning,expression and characterization of a phenylalanine ammonia-lyase gene([STXFX]SmPAL1 ) from Salvia miltiorrhiza[J].Molecular Biology Reports,2009,36(5):939-952.
[14] Vandesompele J,De Preter K,Pattyn F,Poppe B,Van Roy N,De Paepe A,Speleman F.Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J].Genome Biology,2002,3(7):research0034.
[15] Emanuelsson O,Brunak S,von Heijne G, Niel-sen H.Locating proteins in the cell using TargetP,SignalP and related tools[J].Nat Protoc,2007,2(4):953-971.
[16] Bendtsen JD,Nielsen H,von Heijne G,Brunak S. Improved prediction of signal peptides:SignalP 3.0[J].J Mol Biol,2004,340(4):783-795.
[17] Claros MG,Vincens P.Computational method to predict mitochondrially imported proteins and their targeting sequences[J].Eur J Biochem,1996,241(3):779-786.
[18] Geourjon C,Deleage G.SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Comput Appl Biosci,1995,11(6):681-684.
[19] Misawa N,Nakagawa M,Kobayashi K,Yamano S,Izawa Y,Nakamura K,Harashima K.Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli[J].J Bacte-riol,1990,172(12):6704-6712.
[20] Kainou T,Kawamura K,Tanaka K,Matsuda H,Kawamukai M.Identification of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human[J].Biochim Biophys Acta,1999,1437(3):333-340.
[21] Ament K,Van Schie CC,Bouwmeester HJ,Haring MA,Schuurink RC.Induction of a leaf speci-fic geranylgeranyl pyrophosphate synthase and emission of(E, Tholl D,Lee S.Terpene specialized metabolism in Arabidopsis thaliana[J].The Arabidopsis Book,2011,9:e0143.
[26] Okada K,Saito T,Nakagawa T,van der Heijden R,Verpoorte R,Jaziri M.Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis[J].Plant Physiology,2000,122:1045-1056.
计量
- 文章访问数: 1313
- HTML全文浏览量: 0
- PDF下载量: 2248
下载:
