| Purpose: The root, stem and leaf of the five-year ginseng are researched in this thesis. Withhigh-throughput sequencing technology, the transcriptome dataset is built and thedifferentially expressed genes between the root and the stem and leaf are screened. Theresearch in this paper will supply theory basis to find out functional genes, clarify ginsengefficacy material and breed good varieties.Methods: Total RNA of the ginseng root, stem and leaf are distilled with improved Trizolmethod and tested with the ethidium bromide-stained agarose gel and the AgilentTechnologies2100Bioanalyzer. Transcriptome sequence is obtained with Illumina HiSeq2000system and assembled from the beginning, with the Trinity software. The assembledsequence redundancy is eliminated with Tgicl and then jointed together. By eliminating theassembled sequence redundancy with Tgicl method, further jointing the sequence togetherand clustering the homologous transcription, the final Unigenes are obtained. The sequenceinformation from different samples is operated by further splicing, redundancy eliminationand the homologous transcription, and the non-redundant All-Unigenes as long as possibileare obtained.BLASTX with the database of Nr, Swiss-Prot, KEGG and COG (E value<10-5), the sequencealignment is determined with the protein of the best BLASTX result. The gene annotationinformation, function category and metabolic pathway etc are obtained. The direction of theUnigene out of the above database is determined with ESTScan software. The high expressedgenes of root, stem and leaf are screened according to the expression level (FPKM value) inthe dataset. The specific genes high expressed and the non-difference expressed genes of theroot, in contrast with the stem and leaf, are screened according to the multiple ratio of geneexpression level. The accuracy of the transcriptome database is verified with q-PCR methodin this thesis.Results:1. The total RNA of root, stem and leaf was distilled with improved Trizol method and testedwith the ethidium bromide-stained agarose gel electrophoresis. The28s,18s band is clear and brightness ratio is closer to2. The result of Agilent Technologies2100Bioanalyzer was this:the O.D260/280value is between1.8and2.2, O.D260/230greater than1.8, RIN greater than6.5, total RNA greater than20μg. The RNA meets the standards of dataset building.2. On the HiSeq2000sequencing system platform, after the operation of sequence jointing andredundancy elimination with paired-end sequencing method, more than40,000,000highquality short sequences are obtained.53,870Unigenes of root,69,591ones of stem and66,045ones of leaf were obtained, with average length553nt,686nt and644nt accordingly.73,434Unigenes were obtained from All-Unigenes, with the average length877nt.3. BLASTX with NCBI protein database, there are39,475Unigenes of root,48,751ones ofstem and46,769ones of leaf returned to the Nr protein database, and accordingly, there are30,519ones,37,539ones,36,078ones categorized into61GO function categories;11,755ones,15,646ones,14,803ones categorized into25protein orthologous cluster (COG)function categories;21,532ones,27,098ones,25,767ones categorized into128KEGGclassic metabolic pathways. There are51,407Unigenes,37,640ones,17,735ones,29,197ones commented respectively to Nr, GO, COG function classification and KEGG classicmetabolic pathways.4.61,48and46high expressed genes (FPKM>1000) are obtained accordingly from the root,stem and leaf transcriptome database. In the meanwhile, the specific genes high expressed ofthe ginseng root, such as plant growth storage protein etc, and the specific genes highexpressed of the stem and leaf, such as phloem protein etc, as well as the non-differenceexpressed genes of the root, stem and leaf, such as ubiquitin-conjugating enzyme etc, arescreened.5. Some differentially expressed genes are chosen to do the q-PCR test, and the result accordswith the transcriptome database, proving the truth and reliability of the transcriptomedatabase.Conclusion:1. The ranscriptome of the ginseng root, stem and leaf is not obviously different in GOfunction annotation, COG gene function description and KEGG metabolic pathwaysannotation.2. The function of the ginseng root high expressed genes mainly relates to the energy metabolism itself, environment stress. The function of the ginseng stem and leaf highexpressed genes mainly relates to the chlorophyll metabolism.3. The ginseng root, stem and leaf transcriptome database is constructed with Illumina HiSeq2000sequencing platform successfully. The result of this thesis will provide theory basis forthe further research of gene structure and function of the ginseng medicinal part andnon-medicinal part. |
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