降解组测序技术对人参miRNA靶基因的分析鉴定
|
摘要
目的分析鉴定人参miRNA靶基因。方法采用降解组测序技术对石柱参(Shizhu ginseng)、园参(Yuan ginseng)的miRNA及靶基因进行检测;利用公共数据库KEGG/NR/GO数据库对降解组基因进行功能注释;通过荧光实时定量PCR(q RT-PCR)技术验证石柱参、园参的miRNA及靶基因表达量。结果共得到8个miRNA家族的13个靶基因;利用KEGG/NR/GO数据库分析发现该miRNA的靶基因类型主要是转录因子、应答因子及信号转导通路等。对5个差异表达mi RNAs:aqc-miR-159、bdi-miR162、cpa-miR319、pgi-miR4376、smo-mi R396及其靶基因进行的q RT-PCR验证结果与降解组测序结果一致。结论明确了部分人参miRNA的靶基因,为进一步研究人参miRNA的可能功能奠定基础。
Objective To analyze and identify ginseng miRNA. Methods The miRNA and target genes of Shizhu ginseng and Yuan ginseng were detected by the degradation sequencing technology; Functional annotation of Degradome genes was carried out using public databases of KEGG/NR/GO database; The expression of miRNA and target genes of Shizhu ginseng and Yuan ginseng was determinated by real time fluorescence quantitative PCR technique. Results A total of 13 target genes of eight miRNA families were obtained; The target gene type of miRNA was mainly transcriptional factor, response factor, and signal transduction pathway by means of KEGG/NR/GO database analysis. The results of real time fluorescence quantitative PCR verification of aqc-miR-159, bdi-miR162,cpa-mi R319, pgi-miR4376, smo-miR396 and its target genes were basically consistent with the expression of miRNA and target genes from the degradation group. Conclusion The target genes of partial Panax ginseng mi RNA is clarified, which lays the foundation for further study of the possible function of ginseng miRNA.
Objective To analyze and identify ginseng miRNA. Methods The miRNA and target genes of Shizhu ginseng and Yuan ginseng were detected by the degradation sequencing technology; Functional annotation of Degradome genes was carried out using public databases of KEGG/NR/GO database; The expression of miRNA and target genes of Shizhu ginseng and Yuan ginseng was determinated by real time fluorescence quantitative PCR technique. Results A total of 13 target genes of eight miRNA families were obtained; The target gene type of miRNA was mainly transcriptional factor, response factor, and signal transduction pathway by means of KEGG/NR/GO database analysis. The results of real time fluorescence quantitative PCR verification of aqc-miR-159, bdi-miR162,cpa-mi R319, pgi-miR4376, smo-miR396 and its target genes were basically consistent with the expression of miRNA and target genes from the degradation group. Conclusion The target genes of partial Panax ginseng mi RNA is clarified, which lays the foundation for further study of the possible function of ginseng miRNA.
引文
[1]Bartel D P.Micro RNAs:Genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[2]Fang Y N,Zheng B B,Wang L,et al.High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo(Citrus grandis)[J].BMC Genom,2016,17(1):591-605.
[3]Yang X,Wang L,Yuan D,et al.Small RNA and degradome sequencing reveal complex miRNA regulation during cotton somatic embryogenesis[J].J Exper Bot,2013,64(6):1521-1536.
[4]Yu X T,Wang S P.Clinical observation ontreatment for postoperative gastric cancer byginsenoside Rg3 combined with chemotherapy[J].Chin J Cancer Prev Treat,2010,17(10):779-781.
[5]Zhang G,Liu A,Zhou Y,et al.Panax ginseng ginsenoside-Rg2 protects memory impairment via antiapoptosis in a rat model with vascular dementia[J].JEthnopharmacol,2008,115(3):441-448.
[6]Sui D Y,Yu X F,Qu S C,et al.Effects of Panax quinquefolium 20S-2 protopanaxdiol saponins onexperimental ventricular remodeling in rat[J].Chin Pharm J,2007,42(2):108-112.
[7]William C S,Chung W S,Sally K W,et al.Ginsenoside Re of Panax ginseng possessessignificant antioxidant and antihyperlipidemic efficaciesin streptozotocin-induced diabetic rats[J].Eur J Pharm,2006,550(1/3):173-179.
[8]Kim S E,Lee Y H,Park H J,et al.Ginsenoside-Rs4,a new type of ginseng saponin concurrentlyinduces apoptosis and selectively elevates protein levels of p53and p21WAF1 in human hepatoma SK-HEP-1cells European[J].Eur J Cancer,1999,35(3):507-511.
[9]Wang W J,He Z H,Chen Y L,et al.Comparison of two miRNA methods for extraction of ginseng decoction[J].广东药科大学学报,2017,33(5):595-599.
[10]Addo-Quaye C,Miller W,Axtell M J.Cleave Land:Apipeline for using degradome data to find cleaved small RNA targets[J].Bioinformatics,2008,25(1):130-131.
[11]Elbashir S M,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs[J].Genes Dev,2001,15(2):188-200.
[12]Li B,Duan H,Li J,et al.Global identification of miRNAs and targets in Populus euphratica under salt stress[J].Plant Mol Biol,2013,81(6):525-539.
[13]Lu S,Sun Y H,Shi R,et al.Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis[J].Plant Cell,2005,17(8):2186-2203.
[14]Jiang J,Lv M,Liang Y,et al.Identification of novel and conserved miRNAs involved in pollen development in Brassica campestris ssp.chinensis by high-throughput sequencing and degradome analysis[J].BMC Genom,2014,15(1):146.
[15]Palatnik J F,Allen E,Wu X,et al.Control of leaf morphogenesis by micro RNAs[J].Nature,2003,425(6955):257-267.
[16]Zhang L,Hou D X,Chen X,et al.Exogenous plant MIR168a specifically targets mammalian LDLRAP1:Evidence of cross-kingdom regulation by microRNA[J].Cell Res,2012,22(22):107-126.
[17]Li J,Zhang Y J,Li D M,et al.Small non-coding RNAs transfer through mammalian placenta and directly regulate fetal gene expression[J].Protein Cell,2015,6(6):391-396.
[18]Zhou Z,Li X H,Liu J X,et al.Honeysuckle-encoded atypical microRNA2911 directly targets influenza Aviruses[J].Cell Res,2015,25(1):39-49.
[2]Fang Y N,Zheng B B,Wang L,et al.High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo(Citrus grandis)[J].BMC Genom,2016,17(1):591-605.
[3]Yang X,Wang L,Yuan D,et al.Small RNA and degradome sequencing reveal complex miRNA regulation during cotton somatic embryogenesis[J].J Exper Bot,2013,64(6):1521-1536.
[4]Yu X T,Wang S P.Clinical observation ontreatment for postoperative gastric cancer byginsenoside Rg3 combined with chemotherapy[J].Chin J Cancer Prev Treat,2010,17(10):779-781.
[5]Zhang G,Liu A,Zhou Y,et al.Panax ginseng ginsenoside-Rg2 protects memory impairment via antiapoptosis in a rat model with vascular dementia[J].JEthnopharmacol,2008,115(3):441-448.
[6]Sui D Y,Yu X F,Qu S C,et al.Effects of Panax quinquefolium 20S-2 protopanaxdiol saponins onexperimental ventricular remodeling in rat[J].Chin Pharm J,2007,42(2):108-112.
[7]William C S,Chung W S,Sally K W,et al.Ginsenoside Re of Panax ginseng possessessignificant antioxidant and antihyperlipidemic efficaciesin streptozotocin-induced diabetic rats[J].Eur J Pharm,2006,550(1/3):173-179.
[8]Kim S E,Lee Y H,Park H J,et al.Ginsenoside-Rs4,a new type of ginseng saponin concurrentlyinduces apoptosis and selectively elevates protein levels of p53and p21WAF1 in human hepatoma SK-HEP-1cells European[J].Eur J Cancer,1999,35(3):507-511.
[9]Wang W J,He Z H,Chen Y L,et al.Comparison of two miRNA methods for extraction of ginseng decoction[J].广东药科大学学报,2017,33(5):595-599.
[10]Addo-Quaye C,Miller W,Axtell M J.Cleave Land:Apipeline for using degradome data to find cleaved small RNA targets[J].Bioinformatics,2008,25(1):130-131.
[11]Elbashir S M,Lendeckel W,Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs[J].Genes Dev,2001,15(2):188-200.
[12]Li B,Duan H,Li J,et al.Global identification of miRNAs and targets in Populus euphratica under salt stress[J].Plant Mol Biol,2013,81(6):525-539.
[13]Lu S,Sun Y H,Shi R,et al.Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis[J].Plant Cell,2005,17(8):2186-2203.
[14]Jiang J,Lv M,Liang Y,et al.Identification of novel and conserved miRNAs involved in pollen development in Brassica campestris ssp.chinensis by high-throughput sequencing and degradome analysis[J].BMC Genom,2014,15(1):146.
[15]Palatnik J F,Allen E,Wu X,et al.Control of leaf morphogenesis by micro RNAs[J].Nature,2003,425(6955):257-267.
[16]Zhang L,Hou D X,Chen X,et al.Exogenous plant MIR168a specifically targets mammalian LDLRAP1:Evidence of cross-kingdom regulation by microRNA[J].Cell Res,2012,22(22):107-126.
[17]Li J,Zhang Y J,Li D M,et al.Small non-coding RNAs transfer through mammalian placenta and directly regulate fetal gene expression[J].Protein Cell,2015,6(6):391-396.
[18]Zhou Z,Li X H,Liu J X,et al.Honeysuckle-encoded atypical microRNA2911 directly targets influenza Aviruses[J].Cell Res,2015,25(1):39-49.