金线莲转录组测序及其黄酮类合成相关基因分析
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摘要
首次采用RNA-seq高通量测序技术对不同种植时期的金线莲进行转录组测序,并通过Q-PCR和HPLC对其结果进行验证和分析。转录组测序共获得金线莲51 370条基因,并注释到Nr、GO、Swiss-Prot、KEGG和KOG数据库,根据同源序列比对,与油棕和海枣的同源性最高。通过比较不同生长时间的金线莲转录组,分析其差异基因主要集中在黄酮类生物合成相关基因,运用Q-PCR对6个黄酮类生物合成相关基因(反式肉桂酸4-单加氧酶、咖啡酰辅酶A O-甲基转移酶、查尔酮合成酶、黄酮醇合成酶、莽草酸O-羟基肉桂酰转移酶、黄酮类3',5'-羟化酶)表达量进行验证;并结合HPLC对相应的金线莲样品中6种主要黄酮类成分(芦丁、异槲皮苷、水仙苷、槲皮素、山柰酚和异鼠李素)进行含量测定。结果表明6种主要黄酮类成分含量随着金线莲黄酮类合成基因的表达量的升高而增加,并结合转录组数据,绘制出金线莲中黄酮类成分的生物合成途径。此研究为金线莲黄酮合成关键基因及黄酮类成分生物合成提供重要的遗传信息,同时为其药用价值开发提供依据。
Transcriptome sequencing was performed for the first time on Anoectochilus roxburghii( AR) in different harvesting periods using RNA-seq high-throughput sequencing technique,and the results were verified and analyzed by Q-PCR and HPLC. A total of 51,370 genes were obtained by transcriptome sequencing and annotated to the database of Nr,GO,Swiss-Prot,KEGG and KOG. The species that were sequenced according to the homology sequence were the same as AR monocotyledon plants. Through comparison of AR transcriptome in different periods,it was found that the differences were mainly in flavonoid biosynthesis-related genes. The expression levels of flavonoid biosynthesis-related genes( trans-cinnamate 4-monooxygenase,caffeoyl-CoA O-methyltransferase,chalcone synthase,flavonol synthase,shikimate O-hydroxycinnamoyltransferase and flavonoid 3',5'-hydroxylase) were verified by Q-PCR,and the results were consistent with those of transcriptome sequencing. The contents of 6 flavonoids( rutin,isoquercitrin,narcissin,quercetin,kaempferol and isorhamnrtin) were determined by HPLC. The results showed that the expression of flavonoid synthetic gene in AR increased with the growth time,and the variation trend of flavonoid compound content and gene expression were basically consistent. Combined with transcriptome data,the biosynthetic pathway of flavonoid content in AR was plotted. This study provides important genetic resources for the key genes of flavonoid synthesis in AR and the biosynthesis of flavonoids,as well as the basis for the development of its medicinal value.
Transcriptome sequencing was performed for the first time on Anoectochilus roxburghii( AR) in different harvesting periods using RNA-seq high-throughput sequencing technique,and the results were verified and analyzed by Q-PCR and HPLC. A total of 51,370 genes were obtained by transcriptome sequencing and annotated to the database of Nr,GO,Swiss-Prot,KEGG and KOG. The species that were sequenced according to the homology sequence were the same as AR monocotyledon plants. Through comparison of AR transcriptome in different periods,it was found that the differences were mainly in flavonoid biosynthesis-related genes. The expression levels of flavonoid biosynthesis-related genes( trans-cinnamate 4-monooxygenase,caffeoyl-CoA O-methyltransferase,chalcone synthase,flavonol synthase,shikimate O-hydroxycinnamoyltransferase and flavonoid 3',5'-hydroxylase) were verified by Q-PCR,and the results were consistent with those of transcriptome sequencing. The contents of 6 flavonoids( rutin,isoquercitrin,narcissin,quercetin,kaempferol and isorhamnrtin) were determined by HPLC. The results showed that the expression of flavonoid synthetic gene in AR increased with the growth time,and the variation trend of flavonoid compound content and gene expression were basically consistent. Combined with transcriptome data,the biosynthetic pathway of flavonoid content in AR was plotted. This study provides important genetic resources for the key genes of flavonoid synthesis in AR and the biosynthesis of flavonoids,as well as the basis for the development of its medicinal value.
引文
[1] Fujian food and drug administration. Standards of Chinese herbalmedicines in Fujian(福建省中药材标准)[M]. Fuzhou:Haifeng press(海风出版社),2006:154.
[2] Chinese academy of science flora of China Editorial Board. Floraof China,Vol. 17(中国植物志)[M]. Beijing:Science Press(科学出版社),1998:220.
[3] Ye S,Shao Q,Zhang A. Anoectochilus roxburghii:A review of itsphytochemistry,pharmacology,and clinical applications[J]. JEthnopharmacol,2017,39(209):184-202.
[4] Shen YM,Huang CQ,Liu ZY,et al. Study on quality standard ofimitation ecological planting Anoectochilus roxburghii under forestcovering[J]. China Tradit Herb Drugs(中草药),2018,49(02):450-454.
[5] Zhang ZM,Lu JG,Xie QS,et al. Establishment of fingerprint anddetermination of kinsenoside of herba anoectochili from differentsources by UHPLC-TOF-MS[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志),2018,24(01):31-37.
[6] Liu Z,Zhang J,Liu Q,et al. The vascular protective effects ofAnoectochilus roxburghii polysaccharose under high glucose condi-tions[J]. J Ethnopharmacol,2017,202:192-199.
[7] Li L,Li Y,Liu Z,et al. The renal protective effects of Anoectochi-lus roxburghii polysaccharose on diabetic mice induced by high-fat diet and streptozotocin[J]. J Ethnopharmacol,2016,178:58-65.
[8] Zeng B,Su M,Chen Q,et al. Protective effect of a polysaccharidefrom Anoectochilus roxburghii against carbon tetrachloride-induced acute liver injury in mice[J]. J Ethnopharmacol,2017,200:124-135.
[9] Jia Y,Zhang FS,Xiao SX,et al. Component Analyses of Tussilagofarfara in Different Development Stages by Metabonomic andComparative Transcriptomic Approaches[J]. Chin J BiochemMol Biol(中国生物化学与分子生物学报),2017,33(6):615-623.
[10] Grabherr MG,Haas BJ,Yassour M,et al. Full-length transcrip-tome assembly from RNA-Seq data without a reference genome[J]. Nat Biotechnol,2011,29(7):644.
[11] Iseli C,Jongeneel CV,Bucher P. ESTScan:a program for detec-ting,evaluating,and reconstructing potential coding regions inEST sequences.[J]. Proc Int Conf Intell Syst Mol Biol,1998,99(2):138-148.
[12] Conesa A,Gtz S,Garcíagómez JM,et al. Blast2GO:a universaltool for annotation,visualization and analysis in functional genom-ics research[J]. Bioinformatics,2005,21(18):3674-3676.
[13] Ye J,Fang L,Zheng H,et al. WEGO:a web tool for plotting GOannotations[J]. Nucleic Acids Res,2006,34(SI):W293-W297.
[14] Benjamini Y,Hochberg Y. Controlling the false discovery rateA practical and powerful approach to multiple testing[J]. J RoyStat Soc,1995,57(1):289-300.
[15] Chen Y,Huang JF,Lin N,et al. Simultaneous determination ofsix constituents in Anoectochilus roxburghii by HPLC[J]. ChinTradit Pat Med(中成药),2018,40(10):2222-2227.
[16] Li R,Fan W,Tian G,et al. The sequence and de novo assemblyof the giant panda genome[J]. Nature,2010,463(7279):311-317.
[17] Jiang FX,Wei PW,Kou YP,et al. Study on the Characterizationof the transcriptome of Anoectochilus formosanus Taiwan[J]. MolPlant Breeding(分子植物育种),2015,13(12):2743-2753.
[18] Zhang G,Zhao M,Song C,et al. Characterization of referencegenes for quantitative real-time PCR analysis in various tissues ofAnoectochilus roxburghii[J]. Mol Biol Rep,2012,39(5):5905-5912.
[19] Zhang JE,Deng HF. Analysis of transcriptome sequencing andqRT-PCR analysis of related genes of flavonoid biosynthesis fromEucommia ulmoides calli[J]. Chin Tradit Herbal Drugs(中草药),2018,49(16):3912-3917.
[20] Sun ML. Gene expression of catechins biosynthesis in tea plant[Camellia sinensis(L.)O. Kuntze]with real time fluorescencequantitative PCR(茶儿茶素生物合成相关基因表达的实时荧光定量PCR分析)[D]. Anhui:Anhui Agricultural University(安徽农业大学),2010.
[2] Chinese academy of science flora of China Editorial Board. Floraof China,Vol. 17(中国植物志)[M]. Beijing:Science Press(科学出版社),1998:220.
[3] Ye S,Shao Q,Zhang A. Anoectochilus roxburghii:A review of itsphytochemistry,pharmacology,and clinical applications[J]. JEthnopharmacol,2017,39(209):184-202.
[4] Shen YM,Huang CQ,Liu ZY,et al. Study on quality standard ofimitation ecological planting Anoectochilus roxburghii under forestcovering[J]. China Tradit Herb Drugs(中草药),2018,49(02):450-454.
[5] Zhang ZM,Lu JG,Xie QS,et al. Establishment of fingerprint anddetermination of kinsenoside of herba anoectochili from differentsources by UHPLC-TOF-MS[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志),2018,24(01):31-37.
[6] Liu Z,Zhang J,Liu Q,et al. The vascular protective effects ofAnoectochilus roxburghii polysaccharose under high glucose condi-tions[J]. J Ethnopharmacol,2017,202:192-199.
[7] Li L,Li Y,Liu Z,et al. The renal protective effects of Anoectochi-lus roxburghii polysaccharose on diabetic mice induced by high-fat diet and streptozotocin[J]. J Ethnopharmacol,2016,178:58-65.
[8] Zeng B,Su M,Chen Q,et al. Protective effect of a polysaccharidefrom Anoectochilus roxburghii against carbon tetrachloride-induced acute liver injury in mice[J]. J Ethnopharmacol,2017,200:124-135.
[9] Jia Y,Zhang FS,Xiao SX,et al. Component Analyses of Tussilagofarfara in Different Development Stages by Metabonomic andComparative Transcriptomic Approaches[J]. Chin J BiochemMol Biol(中国生物化学与分子生物学报),2017,33(6):615-623.
[10] Grabherr MG,Haas BJ,Yassour M,et al. Full-length transcrip-tome assembly from RNA-Seq data without a reference genome[J]. Nat Biotechnol,2011,29(7):644.
[11] Iseli C,Jongeneel CV,Bucher P. ESTScan:a program for detec-ting,evaluating,and reconstructing potential coding regions inEST sequences.[J]. Proc Int Conf Intell Syst Mol Biol,1998,99(2):138-148.
[12] Conesa A,Gtz S,Garcíagómez JM,et al. Blast2GO:a universaltool for annotation,visualization and analysis in functional genom-ics research[J]. Bioinformatics,2005,21(18):3674-3676.
[13] Ye J,Fang L,Zheng H,et al. WEGO:a web tool for plotting GOannotations[J]. Nucleic Acids Res,2006,34(SI):W293-W297.
[14] Benjamini Y,Hochberg Y. Controlling the false discovery rateA practical and powerful approach to multiple testing[J]. J RoyStat Soc,1995,57(1):289-300.
[15] Chen Y,Huang JF,Lin N,et al. Simultaneous determination ofsix constituents in Anoectochilus roxburghii by HPLC[J]. ChinTradit Pat Med(中成药),2018,40(10):2222-2227.
[16] Li R,Fan W,Tian G,et al. The sequence and de novo assemblyof the giant panda genome[J]. Nature,2010,463(7279):311-317.
[17] Jiang FX,Wei PW,Kou YP,et al. Study on the Characterizationof the transcriptome of Anoectochilus formosanus Taiwan[J]. MolPlant Breeding(分子植物育种),2015,13(12):2743-2753.
[18] Zhang G,Zhao M,Song C,et al. Characterization of referencegenes for quantitative real-time PCR analysis in various tissues ofAnoectochilus roxburghii[J]. Mol Biol Rep,2012,39(5):5905-5912.
[19] Zhang JE,Deng HF. Analysis of transcriptome sequencing andqRT-PCR analysis of related genes of flavonoid biosynthesis fromEucommia ulmoides calli[J]. Chin Tradit Herbal Drugs(中草药),2018,49(16):3912-3917.
[20] Sun ML. Gene expression of catechins biosynthesis in tea plant[Camellia sinensis(L.)O. Kuntze]with real time fluorescencequantitative PCR(茶儿茶素生物合成相关基因表达的实时荧光定量PCR分析)[D]. Anhui:Anhui Agricultural University(安徽农业大学),2010.