长白山人参microRNA鉴定及靶基因分析研究
发布时间：2019-05-23 09:20
【摘要】：目的:以15年生石柱参、6年生园参为研究对象,运用高通量测序技术构建人参small RNA、转录组及降解组数据库。鉴定两种人参中所含miRNA的种类,筛选差异表达miRNA,并进行靶基因预测、验证及分析。方法:1、选用Trizol法抽提园参、石柱参总RNA,经琼脂糖凝胶电泳、TGem微量分光光度计、Agilent 2100 Bioanalyzer检测总RNA的质量、纯度及完整性。2、利用Illumina HiSeqTM 2000测序仪进行园参、石柱参转录组测序,经短序列组装拼接获得两样本的Non-All-Unigene,将其与Nr、KOG、KEGG、GO等数据库比对分析,得到Unigene的蛋白功能注释及代谢通路信息。筛选样本间差异表达mRNA,进行GO、KEGG富集分析,利用Q-PCR对差异mRNA进行验证。3、构建园参、石柱参small RNA文库,以Unigene为参考基因组序列。将获得的Clean reads与Rfam、Gene、Repbase及miRBase 21.0数据库比对,得到已知miRNA并统计其表达量;未比对上的序列用于新miRNA的预测。筛选样本间差异miRNA,进行靶基因预测及KEGG、GO富集分析,采用Q-PCR对差异miRNA进行验证。4、构建两样本降解组数据库,获得原始序列与Rfam、Genbank、PolyN等数据库比对,得到cDNA-sense。cDNA-sense与Unigene、已知miRNA进行miRNA降解位点的鉴定与分析后,再与Nr、KEGG和GO数据库比对,获得降解基因的功能注释信息。结果:1、琼脂糖凝胶电泳显示,两样本总RNA的28S、18S条带清晰完整,亮度接近2倍;Agilent 2100 Bioanalyzer质检可见OD260nm/OD280nm比值在1.8~2.1范围,OD260nm/OD280nm比值在2.0~2.3,RIN7.0,RNA浓度900 ng/μL,28S/18S1.0,即总RNA质量满足高通量建库的标准。2、两样本转录组测序获得5千多万条reads,经De Novo拼接获得63875条去冗余Unigene。对Unigene进行功能注释,有19340条Unigene被归类到25个组蛋白直系KOG类别,20778条Unigene被归类为64个GO功能类别,7268条Unigene被归类到207个KEGG Pathway代谢通路。根据基因表达差异倍数FoldChange及P-value筛选出3216条差异mRNA,并选取差异显著的18条基因进行Q-PCR验证,验证结果与转录本表达量基本一致。3、通过small RNA高通量测序,在石柱参中鉴定235种已知miRNA,园参有246种已知miRNA,分属于71个miRNA家族;未被任何数据库比对上的序列进行新miRNA的预测,得到39个新miRNA。对已知和新miRNA进行靶基因预测,得到17个miRNA家族的169个靶基因,对这些靶基因进行富集分析,按其功能注释主要包括:蛋白转录因子、编码与植物生长发育有关的蛋白、生长素响应因子、泛素连接酶及非特征性蛋白等。根据miRNA表达差异倍数及P值,选取13条差异miRNA进行Q-PCR验证,验证结果与miRNA表达量基本一致。4、通过构建降解组文库及与数据库比对分析,在石柱参和园参中分别得到1千余万条cDNA-sense,用以降解位点的鉴定。根据cleaveland峰值,石柱参、园参中分别检测到812个、724个剪切位点。结合生物信息学miRNA靶基因预测方法,对cDNAsense的降解位点进行注释,共得到8个miRNA家族的13个靶基因,其中两个属于新miRNA的靶基因。结论:1、成功构建人参转录基因组、small RNA及降解组文库,鉴定石柱参、园参所含microRNA的种类,证实新鲜人参中富含microRNA。2、不同生长年限人参miRNA存在差异表达,显著差异miRNA主要调控DCL、E2-UBC、ACA8、AP2等靶基因的调节。3、差异miRNA靶基因分析表明所涉及的8个miRNA家族的13个靶基因功能注释为转录因子、F-Box生长素应答因子、DNA结合蛋白、Dicer酶、Ca 2+-ATP酶抑制剂、TIR-NBS-LRR抗病蛋白等,主要与人参自身能量代谢、生物胁迫及抗病免疫相关。
[Abstract]:Objective: To study the expression of ginseng small RNA, transcription and degradation group by high-throughput sequencing technique. The types of miRNAs contained in the two kinds of ginseng are identified, the differentially expressed miRNAs are screened, and the target gene prediction, the verification and the analysis are carried out. Methods:1. Trizol was used to extract the total RNA of the whole RNA. The quality, purity and integrity of total RNA were detected by agarose gel electrophoresis, TGem microspectrophotometer and Agilent 2100 Bioanalyzer. The Non-All-Uniene of two samples was obtained by short-sequence assembly and then compared with the database of Nr, KOG, KEGG, and GO to obtain the protein functional notes and the metabolic pathway information of Uniene. The differentially expressed mRNA between samples was selected for GO and KEGG enrichment analysis, and the differential mRNA was verified by Q-PCR. The obtained Clean threads are compared to the Rfam, Gene, Rebase, and the miRase 21.0 database to obtain known miRNAs and to count their amounts of expression; the sequence that is not above the pair is used for the prediction of the new miRNAs. In that invention, the difference miRNA between the sample is screened, the target gene prediction and the KEGG and GO enrichment analysis are carried out, and the differential miRNA is verified by using the Q-PCR.4, the database of the two degradation groups is constructed to obtain a database specific pair of the original sequence and the Rfam, Genbank, PolyN and the like to obtain the cDNA-sense. After the miRNA is known to perform the identification and analysis of the miRNA degradation site, the functional annotation information of the degradation gene is obtained by comparing with the Nr, the KEGG and the GO database. Results:1. The agarose gel electrophoresis showed that the 28S and 18S bands of the two total RNA were clear and intact, and the brightness was close to 2 times; the ratio of the OD260nm/ OD280nm to the Agilent 2100 Bioanalyzer was in the range of 1.8-2.1, the OD260nm/ OD280nm ratio was 2.0-2.3, the ratio of the OD260nm/ OD280nm was in the range of 2.0-2.3, RIN7.0, the RNA concentration of 900 ng/. mu.L, 28S/ 18S1.0, that is, the total RNA quality met the high-throughput building standard. Five thousand reads were obtained by sequencing two transcripts, and 63875 strips were obtained by De Novo. The functional notes to Uniene, with 19340 Uniene classified to 25 histone-line KOG categories, were classified as 64 GO functional categories, and 7268 Uniene was classified into 207 KEGG Pathways metabolic pathways. According to the gene expression difference multiple FoldChange and P-value,3216 difference mRNAs were selected, and 18 genes with significant difference were selected for Q-PCR verification, and the result of the verification was consistent with that of the transcript.3,235 known miRNAs were identified by small RNA high-throughput sequencing, and 246 known miRNAs were identified in the park. The fragment belongs to the 71 miRNA family, and the prediction of the new miRNA is not carried out by any database in comparison with the sequence, so that 39 new miRNAs are obtained. carrying out target gene prediction on the known and new miRNAs to obtain 169 target genes of 17 miRNA families, carrying out enrichment analysis on the target genes, Ubiquitin ligase and non-characteristic protein, etc. According to the difference of the expression of the miRNA and the P-value,13 different miRNAs were selected for Q-PCR, and the result of the validation was consistent with that of the miRNA. According to the clear and peak,812 and 724 shear sites were detected. According to the method for predicting the target gene of the bioinformatics miRNA, the degradation site of the cDNAsense is annotated to obtain 13 target genes of the 8 miRNA families, wherein the two target genes belonging to the new miRNA are obtained. Conclusion:1. The selection of microRNA contained in ginseng and garden ginseng is successfully constructed, and the microRNA in ginseng and garden is identified, and the microRNA is rich in fresh ginseng. The differential expression of ginseng miRNAs in different growth years is found, and the significant difference of the miRNA is mainly regulated by DCL, E2-UBC and ACA8. 3. The target gene analysis of the differentially expressed miRNA target showed that the 13 target gene functional notes of the 8 miRNA families involved were transcription factor, F-Box auxin response factor, DNA binding protein, Dicer enzyme, Ca 2 +-ATPase inhibitor, TIR-NBS-LRR disease-resistant protein, etc., mainly related to the energy metabolism of ginseng, And the biological stress and the disease-resistant immunity are related.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.51
本文编号：2483781
[Abstract]:Objective: To study the expression of ginseng small RNA, transcription and degradation group by high-throughput sequencing technique. The types of miRNAs contained in the two kinds of ginseng are identified, the differentially expressed miRNAs are screened, and the target gene prediction, the verification and the analysis are carried out. Methods:1. Trizol was used to extract the total RNA of the whole RNA. The quality, purity and integrity of total RNA were detected by agarose gel electrophoresis, TGem microspectrophotometer and Agilent 2100 Bioanalyzer. The Non-All-Uniene of two samples was obtained by short-sequence assembly and then compared with the database of Nr, KOG, KEGG, and GO to obtain the protein functional notes and the metabolic pathway information of Uniene. The differentially expressed mRNA between samples was selected for GO and KEGG enrichment analysis, and the differential mRNA was verified by Q-PCR. The obtained Clean threads are compared to the Rfam, Gene, Rebase, and the miRase 21.0 database to obtain known miRNAs and to count their amounts of expression; the sequence that is not above the pair is used for the prediction of the new miRNAs. In that invention, the difference miRNA between the sample is screened, the target gene prediction and the KEGG and GO enrichment analysis are carried out, and the differential miRNA is verified by using the Q-PCR.4, the database of the two degradation groups is constructed to obtain a database specific pair of the original sequence and the Rfam, Genbank, PolyN and the like to obtain the cDNA-sense. After the miRNA is known to perform the identification and analysis of the miRNA degradation site, the functional annotation information of the degradation gene is obtained by comparing with the Nr, the KEGG and the GO database. Results:1. The agarose gel electrophoresis showed that the 28S and 18S bands of the two total RNA were clear and intact, and the brightness was close to 2 times; the ratio of the OD260nm/ OD280nm to the Agilent 2100 Bioanalyzer was in the range of 1.8-2.1, the OD260nm/ OD280nm ratio was 2.0-2.3, the ratio of the OD260nm/ OD280nm was in the range of 2.0-2.3, RIN7.0, the RNA concentration of 900 ng/. mu.L, 28S/ 18S1.0, that is, the total RNA quality met the high-throughput building standard. Five thousand reads were obtained by sequencing two transcripts, and 63875 strips were obtained by De Novo. The functional notes to Uniene, with 19340 Uniene classified to 25 histone-line KOG categories, were classified as 64 GO functional categories, and 7268 Uniene was classified into 207 KEGG Pathways metabolic pathways. According to the gene expression difference multiple FoldChange and P-value,3216 difference mRNAs were selected, and 18 genes with significant difference were selected for Q-PCR verification, and the result of the verification was consistent with that of the transcript.3,235 known miRNAs were identified by small RNA high-throughput sequencing, and 246 known miRNAs were identified in the park. The fragment belongs to the 71 miRNA family, and the prediction of the new miRNA is not carried out by any database in comparison with the sequence, so that 39 new miRNAs are obtained. carrying out target gene prediction on the known and new miRNAs to obtain 169 target genes of 17 miRNA families, carrying out enrichment analysis on the target genes, Ubiquitin ligase and non-characteristic protein, etc. According to the difference of the expression of the miRNA and the P-value,13 different miRNAs were selected for Q-PCR, and the result of the validation was consistent with that of the miRNA. According to the clear and peak,812 and 724 shear sites were detected. According to the method for predicting the target gene of the bioinformatics miRNA, the degradation site of the cDNAsense is annotated to obtain 13 target genes of the 8 miRNA families, wherein the two target genes belonging to the new miRNA are obtained. Conclusion:1. The selection of microRNA contained in ginseng and garden ginseng is successfully constructed, and the microRNA in ginseng and garden is identified, and the microRNA is rich in fresh ginseng. The differential expression of ginseng miRNAs in different growth years is found, and the significant difference of the miRNA is mainly regulated by DCL, E2-UBC and ACA8. 3. The target gene analysis of the differentially expressed miRNA target showed that the 13 target gene functional notes of the 8 miRNA families involved were transcription factor, F-Box auxin response factor, DNA binding protein, Dicer enzyme, Ca 2 +-ATPase inhibitor, TIR-NBS-LRR disease-resistant protein, etc., mainly related to the energy metabolism of ginseng, And the biological stress and the disease-resistant immunity are related.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.51
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