Identification Of Micro Rna And Analysis Of Target Genes In Changbai Mountain Ginseng

Objective: The Shizhu ginseng of fifteen-years and Yuan ginseng of six-years are researched in this thesis to built small RNA-Seq,RNA-Seq and Degradome database with highthroughput sequencing technology.Identify the kinds of mi RNAs contained in two ginsengs,screening the differential expression mi RNAs,as well as target gene prediction,validation and analysis.Methods: 1.Trizol method was used to extract total RNA from Yuan ginseng and Shizhu ginseng roots.The quality,purity and integrity of RNA were detected by agarose gel electrophoresis,TGem micro spectrophotometer and Agilent 2100 Bioanalyzer.2.Transcriptome sequence is obtained with Illumina Hi SeqTM 2000 system.Two samples of Non-All-Unigene were obtained by short sequence assembly and splicing,and compared with Nr,KOG,KEGG and GO databases to obtain the genes function annotation and metabolic pathway information.Differential expression of m RNA between samples was analyzed by GO and KEGG enrichment,and then verified by Q-PCR.3.Two samples of small RNA library were constructed,and unigene was used as the reference gene for sequence alignment.By comparing with Rfam,Gene,Repbase and mi RBase 21.0 databases,the known mi RNA sequences and their total expression were obatained.Those not on the sequence are predicted as the new mi RNA.Screening the differential mi RNA between samples,target gene prediction with KEGG,GO enrichment analysis,and using Q-PCR to verify differential expression of mi RNA.4.Degradome database of two samples was constructed,and the raw tags were compared with Rfam,Genbank,Poly N and other databases to get c DNA-sense.Compared c DNA-sense with unigene and known mi RNAs to identified and analyzed the degradation site,then compared with Nr,KEGG and GO databases to obtaine the functional annotation of degradation genes.Results: 1.It is display that the 28 S and 18 S bands of total RNA of two samples are clear and complete by Agarose gel electrophoresis,the brightness of them are nearly 2 times.The ratio of OD260nm/OD280 nm was 1.8~2.1 in the range of visible quality inspection by Agilent 2100 Bioanalyzer.OD260nm/OD280 nm ratio is range from 2.0 to 2.3,RIN>7.0,RNA concentration is more than 900 ng/μL,28S/18 S >1.0,so RNA quality can meets the high throughput sequencing standard.2.Two samples of the transcriptome each received 5 million reads,with De Novo to get 63875 redundant unigene splicing.Functional annotation of unigene shows that 19340 unigenes were classified into 25 groups of protein family with KOG vertical categories,20778 unigenes were classified into 64 GO functional categories,7268 unigenes were classified into 207 KEGG Pathway metabolic pathway,3216 differentially expressed genes were screened according to Fold Change and P-value.18 genes with significant difference were selected for Q-PCR,and the results were consistent with the transcript expression.3.Through small RNA sequencing,235 kinds of known mi RNAs were detected in Shizhu ginseng,and 246 kinds in Yuan ginseng,and they belong to 71 mi RNA families.Using the sequence that has not been matched by any database to be predicted,39 novel mi RNAs were obtained.The target genes of known and new mi RNA were predicted,and 169 target genes of the 17 known mi RNA families were obtained.The predicted target genes were mainly expressed in terms of protein transcription factors,proteins encoded by plant growth and development,auxin response factors,unknown functional products,or non-specific proteins.13 differentially expressed mi RNA were selected for Q-PCR verification,and the results were consistent with the expression of mi RNA.4.Through the construction of degradome library and database annotation,each of Shizhu ginseng and Yuan ginseng were obtained 1 million c DNA-sense for further identification of degradation sites.According to the peak value of Cleaveland,812 and 724 shear sites were found in Shizhu and Yuan ginseng.Through the identification and annotation of c DNA-sense degradation sites,13 target genes of the 8 mi RNA families were obtained,of which two belong to the target gene of the new mi RNA.Conclusion: 1.By using the Illumina Hi SeqTM2000 sequencing platform,we successfully constructed the RNA-Seq,small RNA-Seq and degradome library,identified the mi RNA species of ginseng,which showed that fresh ginseng was rich in micro RNA.2.There were differential expression in different growth years of ginseng micro RNA,and significant difference of mi RNA mainly regulated the expression of DCL,E2-UBC,ACA8,AP2 and other target genes.3.The analysis shows that 13 target genes functional annotation of 8 mi RNA families involved in the transcription factor,F-Box auxin response factor,DNA binding protein,Dicer enzyme and Ca2+-ATP enzyme inhibitor,TIR-NBS-LRR resistance protein and so on,which mainly related to energy metabolism,biological stress and disease resistance in ginseng.