長白山人參microRNA鑒定及靶基因分析研究
發(fā)布時間：2019-05-23 09:20
【摘要】：目的:以15年生石柱參、6年生園參為研究對象,運用高通量測序技術構建人參small RNA、轉錄組及降解組數(shù)據(jù)庫。鑒定兩種人參中所含miRNA的種類,篩選差異表達miRNA,并進行靶基因預測、驗證及分析。方法:1、選用Trizol法抽提園參、石柱參總RNA,經(jīng)瓊脂糖凝膠電泳、TGem微量分光光度計、Agilent 2100 Bioanalyzer檢測總RNA的質量、純度及完整性。2、利用Illumina HiSeqTM 2000測序儀進行園參、石柱參轉錄組測序,經(jīng)短序列組裝拼接獲得兩樣本的Non-All-Unigene,將其與Nr、KOG、KEGG、GO等數(shù)據(jù)庫比對分析,得到Unigene的蛋白功能注釋及代謝通路信息。篩選樣本間差異表達mRNA,進行GO、KEGG富集分析,利用Q-PCR對差異mRNA進行驗證。3、構建園參、石柱參small RNA文庫,以Unigene為參考基因組序列。將獲得的Clean reads與Rfam、Gene、Repbase及miRBase 21.0數(shù)據(jù)庫比對,得到已知miRNA并統(tǒng)計其表達量;未比對上的序列用于新miRNA的預測。篩選樣本間差異miRNA,進行靶基因預測及KEGG、GO富集分析,采用Q-PCR對差異miRNA進行驗證。4、構建兩樣本降解組數(shù)據(jù)庫,獲得原始序列與Rfam、Genbank、PolyN等數(shù)據(jù)庫比對,得到cDNA-sense。cDNA-sense與Unigene、已知miRNA進行miRNA降解位點的鑒定與分析后,再與Nr、KEGG和GO數(shù)據(jù)庫比對,獲得降解基因的功能注釋信息。結果:1、瓊脂糖凝膠電泳顯示,兩樣本總RNA的28S、18S條帶清晰完整,亮度接近2倍;Agilent 2100 Bioanalyzer質檢可見OD260nm/OD280nm比值在1.8~2.1范圍,OD260nm/OD280nm比值在2.0~2.3,RIN7.0,RNA濃度900 ng/μL,28S/18S1.0,即總RNA質量滿足高通量建庫的標準。2、兩樣本轉錄組測序獲得5千多萬條reads,經(jīng)De Novo拼接獲得63875條去冗余Unigene。對Unigene進行功能注釋,有19340條Unigene被歸類到25個組蛋白直系KOG類別,20778條Unigene被歸類為64個GO功能類別,7268條Unigene被歸類到207個KEGG Pathway代謝通路。根據(jù)基因表達差異倍數(shù)FoldChange及P-value篩選出3216條差異mRNA,并選取差異顯著的18條基因進行Q-PCR驗證,驗證結果與轉錄本表達量基本一致。3、通過small RNA高通量測序,在石柱參中鑒定235種已知miRNA,園參有246種已知miRNA,分屬于71個miRNA家族;未被任何數(shù)據(jù)庫比對上的序列進行新miRNA的預測,得到39個新miRNA。對已知和新miRNA進行靶基因預測,得到17個miRNA家族的169個靶基因,對這些靶基因進行富集分析,按其功能注釋主要包括:蛋白轉錄因子、編碼與植物生長發(fā)育有關的蛋白、生長素響應因子、泛素連接酶及非特征性蛋白等。根據(jù)miRNA表達差異倍數(shù)及P值,選取13條差異miRNA進行Q-PCR驗證,驗證結果與miRNA表達量基本一致。4、通過構建降解組文庫及與數(shù)據(jù)庫比對分析,在石柱參和園參中分別得到1千余萬條cDNA-sense,用以降解位點的鑒定。根據(jù)cleaveland峰值,石柱參、園參中分別檢測到812個、724個剪切位點。結合生物信息學miRNA靶基因預測方法,對cDNAsense的降解位點進行注釋,共得到8個miRNA家族的13個靶基因,其中兩個屬于新miRNA的靶基因。結論:1、成功構建人參轉錄基因組、small RNA及降解組文庫,鑒定石柱參、園參所含microRNA的種類,證實新鮮人參中富含microRNA。2、不同生長年限人參miRNA存在差異表達,顯著差異miRNA主要調控DCL、E2-UBC、ACA8、AP2等靶基因的調節(jié)。3、差異miRNA靶基因分析表明所涉及的8個miRNA家族的13個靶基因功能注釋為轉錄因子、F-Box生長素應答因子、DNA結合蛋白、Dicer酶、Ca 2+-ATP酶抑制劑、TIR-NBS-LRR抗病蛋白等,主要與人參自身能量代謝、生物脅迫及抗病免疫相關。
[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.
【學位授予單位】：廣東藥科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S567.51
本文編號：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.
【學位授予單位】：廣東藥科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S567.51
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