摘要
丹参酮是中药丹参的主要有效成分,其生物合成途径表明细胞色素CYP450酶在丹参酮生物合成途径结构后修饰过程中发挥着重要作用。长链非编码RNA(lncRNA)是一类长度大于200 nt的核苷酸,对药用植物的生长发育、次生代谢等具有重要调控作用。该研究通过对丹参毛状根进行诱导,利用高通量测序构建其长链非编码RNA文库,共获得8 942个差异lncRNA,其中6 755个基因间lncRNA,并鉴定出1 115 814个lncRNA-靶基因对,包括122个lncRNA-靶基因顺式对。对差异lncRNA与CYP450进行相关性分析,共鉴定出16 249个lncRNA-CYP450靶基因对。将其与丹参酮生物合成途径CYP76AH1,CYP76AH3,CYP76AK1基因进行靶向相关性分析,得到216个靶基因,这些候选基因为丹参酮生物合成途径下游调控机制研究奠定基础。
Tanshinones are abietane-type norditerpenoid quinones that make up the main bioactive ingredients of traditional Chinese medicine Salvia miltiorrhiza. Cytochrome CYP450 plays an important role in the post-structural modification of tanshinone biosynthesis pathway. Long non-coding RNA( lncRNA) have been defined as transcripts longer than 200 nucleotides,which have been functionally characterized in regulating the growth and development,secondary metabolism and stress of medicinal plants. In this study,we perform a comprehensive identification of lncRNAs in response to tanshinone metabolism induced by yeast extract( YE) and Ag~+ S. miltiorrhiza hairy roots. Deep RNA sequencing was used to identify a set of different 8 942 lncRNAs,of which 6 755 were intergenic lncRNAs. We predicted a total of 1 115 814 lncRNA-coding gene pairs,including 122 lncRNA-coding gene as cis pairs. The correlation analysis between lncRNA and CYP450 related to tanshinone biosynthesis was carried out and a total of 16 249 lncRNA-CYP450 target gene pairs were identified. Further analysis with functional known CYP76 AH1,CYP76 AH3 and CYP76 AK1 involved in tanshinone biosynthesis,we also identified a set of 216 target genes. These candidate genes will be the important target in the downstream regulation mechanism analysis of the tanshinone biosynthesis pathway.
引文
[1]陈芬燕,郭韧,张毕奎.丹参酮ⅡA的心血管药理作用研究进展[J].中国中药杂志,2015,40(9):1649.
[2]高伟,胡添源,郭娟,等.丹参酮合成生物学研究进展[J].中国中药杂志,2015,40(13):2486.
[3]马莹,郭娟,毛亚平,等.药用植物有效成分生物合成途径解析及其应用[J].中华中医药杂志,2017(5):2079.
[4] Gao W,Hillwig M L,Huang L,et al. A functional genomics approach to tanshinone biosynthesis provides stereochemical insights[J]. Org Lett,2009,11(22):5170.
[5] Guo J,Zhou Y J,Hillwig M L,et al.CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts[J]. Proc Natl Acad Sci USA,2013,110(29):12108.
[6] Guo J,Ma X,Cai Y,et al.Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones[J]. New Phytol,2016,210(2):525.
[7] Lee J T. Epigenetic regulation by long noncoding RNAs[J]. Science,2013,21(6/7):685.
[8] Rinn J L,Chang H Y. Genome regulation by long noncoding RNAs[J]. Annu Rev Biochem,2012,81(1):145.
[9] Tang T H,Polacek N,Zywicki M,et al. Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus[J].Mol Microbiol,2010,55(2):469.
[10] Huancamamani W,Ariascarrasco R,Cárdenasninasivincha S,et al. Long non-coding rnas responsive to salt and boron stress in the hyper-arid Lluteo Maize from Atacama Desert[J]. Genes,2018,9(3):170.
[11] Kwenda S,Birch P R J,Moleleki L N.Genome-wide identification of potato long intergenic noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection[J]. BMC Genomics,2016,17(1):614.
[12] Kosik K S. MicroRNAs and cellular phenotypy[J]. Cell,2010,143(1):21.
[13] Singh D,Kashyap A,Pandey R V,et al. Novel advances in cytochrome P450 research[J]. Drug Discov Today,2011,16(17):793.
[14] Gao W,Sun H X,Xiao H,et al. Combining metabolomics and transcriptomics to characterize tanshinone biosynthesis in Salvia miltiorrhiza[J]. BMC Genomics,2014,15(1):1.
[15] Miettinen K,Pollier J,Buyst D,et al. The ancient CYP716 family is a major contributor to the diversification of eudicot triterpenoid biosynthesis[J]. Nat Commun,2017,8:14153.
[16] Seki H,Tamura K,Muranaka T. P450s and UGTs:key players in the structural diversity of triterpenoid saponins[J]. Plant Cell Physiol,2015,56(8):1463.
[17] Imai T,Tanabe K,Kato T,et al. Localization of ferruginol,a diterpene phenol,in Cryptomeria japonica heartwood by time-of-flight secondary ion mass spectrometry[J]. Planta,2005,221(4):549.
[18] Sharp H,Latif Z,Bartholomew B,et al.Totarol,totaradiol and ferruginol:three diterpenes from Thujaplicata(Cupressaceae)[J].Biochem Syst Ecol,2001,29(2):215.
[19] Yatagai M,Takahashi T. Diterpenes of the ferruginol type from Chamaecyparis pisifera[J]. Phytochemistry,1979,18(1):176.
[20] Wu S J,Shi M,Wu J Y.Cloning and characterization of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza(Chinese sage)hairy roots[J]. Biotechnol Appl Biochem,2011,52(1):89.
[21] Yan X M,Zhang L,Wang J,et al.Molecular characterization and expression of 1-deoxy-D-xylulose 5-phosphate reductoisomerase(DXR)gene from Salvia miltiorrhiza[J]. Acta Physiol Plant,2009,31(5):1015.
[22] Song J,Wang Z. Molecular cloning,expression and characterization of a phenylalanine ammonia-lyase gene(SmPAL1)from Salvia miltiorrhiza[J]. Mol Biol Rep,2009,36(5):939.
[23] Huang B,Duan Y,Yi B,et al. Characterization and expression profiling of cinnamate 4-hydroxylase gene from Salvia miltiorrhiza in rosmarinic acid biosynthesis pathway[J]. Russ J Plant Physiol,2008,55(3):390.
[24]刘玉忠,申业,荣齐仙,等.丹参转录因子SmWRKY1蛋白的表达和纯化条件优化研究[J].中国中药杂志,2014,39(7):1214.
[25]吴文燕,蒋喜红,刘春生,等.丹参ERF转录因子SmERF1基因的表达分析和亚细胞定位[J].中国中药杂志,2013,38(7):957.
[26]汪琬宜,蒋喜红,张利华,等.丹参转录因子SmbhHLH1基因的克隆和表达分析[J].中国中药杂志,2011,36(24):3416.
[27] Hao D C,Yang L,Xiao P G,et al. Identification of Taxus microRNAs and their targets with high-throughput sequencing and degradome analysis[J]. Physiol Plantarum,2012,146(4):388.
[28] Masaharu M,Fumihiko S. Unusual P450 reactions in plant secondary metabolism[J]. Arch Biochem Biophys,2011,507(1):194.