刺五加GPS基因克隆及表达特征与皂苷含量相关性研究
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摘要
目的克隆刺五加香叶基焦磷酸合成酶(geranyl pyrophosphate synthase,GPS)基因的cDNA序列并分析基因序列特征、不同器官中基因表达水平及其与皂苷含量的相关性。方法提取刺五加的RNA,逆转录为cDNA。根据转录组测序结果中编码GPS的unigene(c37362.graph_c0),设计特异性引物,经过PCR扩增GPS基因的cDNA全长。利用实时荧光定量PCR(qRT-PCR)分析GPS基因在不同器官中的表达水平,并通过分光光度法检测刺五加总皂苷含量。结果克隆得到长1260bp、编码419个氨基酸的刺五加GPS基因cDNA。GPS蛋白定位于线粒体内且不存在跨膜区域。GPS基因在各个器官中均有表达,在叶片中的表达量最高,是根中表达量的5.26倍。GPS基因的相对表达量与皂苷量呈现出同升同降的变化趋势,表现为显著正相关关系(r=0.851,P<0.05)。结论首次克隆获得刺五加GPS基因的cDNA全长序列,明确了刺五加GPS基因的表达量与皂苷含量之间存在正相关关系。
Objective To clone cDNA sequence of geranyl pyrophosphate synthase(GPS) gene from Eleutherococcus senticosus and analyze genetic characteristics, gene expression level in different organs and the correlation between GPS gene expression and saponins content. Methods RNA was extracted from E. senticosus and reverse transcribed into cDNA. Gene specific primers were designed according to the unigene(c37362.graph_c0) of GPS from transcriptome sequencing data. The full length of the GPS gene cDNA was amplified by PCR. The expression level of GPS gene in different organs was analyzed by qRT-PCR. The content of E. senticosus saponins was detected by spectrophotometry method. Results GPS gene cDNA was cloned from E. senticosus and encodes 419 amino acids with full length of 1 260 bp. GPS protein located in mitochondria does not have transmembrane region. The GPS gene was expressed in each organ and had the highest expression in blade, which is 5.26 times in root. The relative expression of GPS gene and content of saponin showed the same trend and significant positive correlation(r = 0.851, P < 0.05). Conclusion The whole length of c DNA sequence of GPS gene is cloned for the first time, and there is a positive correlation between the expression level of GPS gene and the saponin content of E. senticosus.
Objective To clone cDNA sequence of geranyl pyrophosphate synthase(GPS) gene from Eleutherococcus senticosus and analyze genetic characteristics, gene expression level in different organs and the correlation between GPS gene expression and saponins content. Methods RNA was extracted from E. senticosus and reverse transcribed into cDNA. Gene specific primers were designed according to the unigene(c37362.graph_c0) of GPS from transcriptome sequencing data. The full length of the GPS gene cDNA was amplified by PCR. The expression level of GPS gene in different organs was analyzed by qRT-PCR. The content of E. senticosus saponins was detected by spectrophotometry method. Results GPS gene cDNA was cloned from E. senticosus and encodes 419 amino acids with full length of 1 260 bp. GPS protein located in mitochondria does not have transmembrane region. The GPS gene was expressed in each organ and had the highest expression in blade, which is 5.26 times in root. The relative expression of GPS gene and content of saponin showed the same trend and significant positive correlation(r = 0.851, P < 0.05). Conclusion The whole length of c DNA sequence of GPS gene is cloned for the first time, and there is a positive correlation between the expression level of GPS gene and the saponin content of E. senticosus.
引文
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[18]Zhou J,Wang C,Yang L,et al.Geranyl diphosphate synthase:An important regulation point in balancing a recombinant monoterpene pathway in Escherichia coli[J].Enzyme Microb Technol,2015,68:50-55.
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[3]Wang P,Cui J,Du X,et al.Panax notoginseng saponins(PNS)inhibits breast cancer metastasis[J].JEthnopharmacol,2014,154(3):663-671.
[4]陈永斌,董玉华.三七总皂苷治疗高脂血症的临床效果及药理作用分析[J].中国医药科学,2016,6(1):51-53.
[5]Zhang W,Wang X,Tang H,et al.Triterpenoid saponins from Clematis tangutica and their cardioprotective activities[J].Fitoterapia,2013,84(1):326-331.
[6]Choi D W,Jung J,Ha Y I,et al.Analysis of transcripts in methyl jasmonate-treated ginseng hairy roots to identify genes involved in the biosynthesis of ginsenosides and other secondary metabolites[J].Plant Cell Rep,2005,23(8):557-566.
[7]马颖瑞,龚一富,周静,等.绿色杜氏藻香叶基焦磷酸合成酶基因(GPS)生物信息学与诱导表达分析[J].核农学报,2017,31(2):248-254.
[8]Yin J L,Wong W S,Jang I C,et al.Co-expression of peppermint geranyl diphosphate synthasesmall subunit enhances monoterpene production in transgenic tobacco plants[J].New Phytol,2017,213(3):1133-1144.
[9]向蓓蓓,李晓雪,王勇,等.川西獐牙菜牻牛儿基焦磷酸合成酶基因的克隆及表达分析[J].中草药,2017,48(5):962-970.
[10]宋菊,国红玉,李志栋,等.刺五加转录组和差异性表达分析[J].中草药,2016,47(22):4049-4053.
[11]邢朝斌,龙月红,何闪,等.刺五加法尼基焦磷酸合酶基因的克隆、生物信息学及表达分析[J].中国中药杂志,2012,37(12):1725-1730.
[12]邢朝斌,龙月红,劳凤云,等.内生真菌对刺五加皂苷合成关键酶基因表达及皂苷含量的影响[J].中国中药杂志,2012,37(14):2041-2045.
[13]邢朝斌,劳凤云,龙月红,等.刺五加鲨烯合酶和鲨烯环氧酶基因单核苷酸多态性及其与总皂苷量的相关性研究[J].中草药,2012,43(10):2020-2024.
[14]Li G,Xi J,Ji X,et al.Non-plastidial expression of a synthetic insect geranyl pyrophosphate synthase effectively increases tobacco plant biomass[J].J Plant Physiol,2018,221:144-155.
[15]Xi J,Rossi L,Lin X,et al.Overexpression of a synthetic insect-plant geranyl pyrophosphate synthase gene in Camelina sativa alters plant growth and terpene biosynthesis[J].Planta,2016,244(1):215-230.
[16]Liu Z,Zhou J,Wu R,et al.Mechanism of assembling isoprenoid building blocks 1.elucidation of the structural motifs for substrate binding in geranyl pyrophosphate synthase[J].J Chem Theory Comput,2014,10(11):5057-5067.
[17]Schmidt A,W?chtler B,Temp U,et al.A bifunctional geranyl and geranylgeranyl diphosphate synthase is involved in terpene oleoresin formation in Picea abies[J].Plant Physiol,2010,152(2):639-655.
[18]Zhou J,Wang C,Yang L,et al.Geranyl diphosphate synthase:An important regulation point in balancing a recombinant monoterpene pathway in Escherichia coli[J].Enzyme Microb Technol,2015,68:50-55.