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    Home > Biochemistry News > Biotechnology News > Degradation group sequencing techniques were analyzed and identified for ginseng miRNA target genes.

    Degradation group sequencing techniques were analyzed and identified for ginseng miRNA target genes.

    • Last Update: 2020-08-05
    • Source: Internet
    • Author: User
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    Objective To analyze and identify ginseng miRNA target genes.
    method uses degradation group sequencing technology to detect the miRNA and target genes of Shizhuginseng, Yuanginseng, functional commentofon of degradation group genes using the kEGG/NR/GO database of public database, and verify the expression of the miRNA and target gene of the column ginseng, garden ginseng using fluorescent real-time quantitative PCR (qRT-PCR) technology.
    results results a total of 8 miRNA family of 13 target genes, using KEGG/NR/GO database analysis found that the target gene type of the miRNA is mainly transcription factors, response factors and signal transduction pathways.
    the qRT-PCR validation results of miRNAs for 5 differential syrotops: aqc-miR-159, bdi-miR162, cpa-miR319, pgi-miR4376, smo-miR396 and their target genes were consistent with the degradation group sequencing results.
    conclusions identify the target genes of human ginseng miRNA and lay the foundation for further study of the possible function of ginseng miRNA.
    microRNA (miRNA) is a class of endogenous, non-coding small RNAs with a length of 20 to 24 nucleotides.
    miRNA is widely present in plants and regulates gene expression by shearing target mRNA or inhibiting the translation of target mRNA.
    degradation group sequencing technology (degradodmeing) is capable of identifying the target gene of plant miRNA in high flux, and systematically commenting on the function of the target gene.
    ginseng is the application of a long history of valuable Chinese medicine, with the improvement of immunity, enhance memory, improve cardiovascular, delay aging, anti-tumor, and other pharmacological effects.
    the main active ingredients of ginseng are more studied, such as ginseng saponin and polysaccharides, and so on, it is not possible to fully reveal the clinical efficacy mechanism of ginseng.
    laboratory pre-study has detected a large amount of miRNAs in fresh ginseng.
    the use of degradation group sequencing methods, respectively, the fresh column ginseng, garden ginseng (ginseng) sequencing analysis, identify the differences related to the target gene and verify, to further study the possible role of ginseng miRNAs.
    1 material selection Changbaishan fresh 15-year-old raw stone column ginseng, 6-year-old raw garden ginseng are from Jilin Fusong Wanliang Changbaishan ginseng market, by the Guangdong University of Pharmacy, Associate Professor Ma Hongxuan identified as ginseng herb Panax ginseng C.A. Mey.
    the main root of 2 kinds of fresh ginseng is rinsed under tap water to remove the sticky soil, put 70% ethanol disinfection 1 min, and then rinsed by sterile water, placed on the disinfection filter paper to absorb dry water, chopped into pieces, after frozen liquid nitrogen, after storage and storage in the refrigerator of 80 degrees C to save.
    2 method 2.1 total RNA extraction and purification 2.1.1 total RNA extraction liquid nitrogen will have been cut spare (about 200 mg each) column ginseng and garden ginseng samples are ground in different abrasions to fine lysing, transferred to aseptic enzyme-free In the 1.5mL Eppendorf tube, the total RNA is extracted by Trizol method, the RNA is dissolved by deionized water treated by DEPC by 20 to 30 ?L, the spare is stored at 80 degrees C, or then DNase I is treated.
    2.1.2 Total RNA Purification Takes 20 to 50 sg RNA samples, 5?L10 x DNase Buffer I, 2?L (10 units) Recombinant DNase I., 1'L (20units) RNase Put, finally added deionized water to 50?L reaction system, at 37 degrees C reaction of 20 to 30 min, heat treatment inactivated Recombinant DNase I.
    add 10 sl 3 mol/L sodium acetate and 250?L cold ethanol, mixed, and placed at 20min at 80 oC.
    4 degrees C, 12 000r/min centrifugation 10 min, discard edified (do not bottom).
    add 70% cold ethanol washing of 1 mL, 4 degrees C, 12 000 r/min centrifugation 5 min, discarded.
    dry precipitation.
    dissolved RNA with the right amount of deionized water treated with DEPC, and used agar-sugar gel electrophoresis to detect total RNA integrity.
    2.2 Library Construction and Sequencing Commissioned illumina HiSeq TM2000 sequencing by Shanghai Ouyi Bio, which will detect qualified total RNA.
    2.3 data analysis high-throughput sequencing data, go to connectors, go to low-quality reads, decontamination, etc. to get clean purpose sequence (clean tags).
    selected The Rfam and Genbank databasetoretrieve the clean tags sequence information, as far as possible to remove the ncRNA on the comments: rRNA, scRNA, snoRNA, etc., using unnotated tags and reference genome (Unigenes) comparison, to obtain cDNAsense, the prediction and analysis of midegrade RNA site.
    use CleaveLand3 (10) software analysis (P 0.05) to identify miRNA target genes.
    2.4 Real-time fluorescence quantitative PCR (qRT-PCR) to verify differential expression miRNAs and its target gene selection of pillar ginseng, garden ginseng in 5 differential expression miRNA: aqc-miR-159, bdi-miR162, cpa-miR319, pgi-miR4376, smo-miR396, 5mi AsRN and its target sq.
    in accordance with QuantiFast® SYBR® Green PCR Kit (Qiagen, Germany).
    3 results and analysis of 3.1 ginseng RNA extraction and identification results as shown in Figure 1, the total RNA in the total RNA extracted by the column on the gel position, the total RNA 28 S and 18 S 2 characteristic strip slicing clear, brightness ratio close to 2:1, 5 S strip show fuzzy, indicating that the total RNA is more complete, no degradation, there is no obvious genomic DNA contamination.
    Nanodrop test results showed that the total RNA had a normal single peak at 260 nm and a total Of260/A280 was at 1.8 to 2.2.
    Agilent2100 test results show that 28 S/18 S are greater than 1.5, RNA integrity index (RNA integrity index, RIN) is greater than 8, the test results are A, indicating that the total RNA degradation of the sample is low, integrity is good, to meet the next step of the library and sequencing requirements.
    3.2 degradation gene function annotation stoices analyzed the two samples of common and especially specific genes noted in the degradation library (tables 2 and Figure 2, 3), and identified a total of 3 target genes of miR156, namely, SPL17_ORYSJ, SPL7_ORYSI, DNJH_CUCSA, the three target gene functions are SPL translvirus factor, SBP (squamosa promoter-bindingprotein) protein family and regulatory DNA.
    identified miR164 in the five target genes, there are 3 coding NAC functional domain transcription factors, and some coding non-characteristic proteins, the verification of these target genes and the existing reports of basic match (Table 3), explainthe the authenticity of this degradation library.
    3.3 Conservative miRNA target genes combined the miRNA sequence with the mating prediction of the mRNA sequence and the results of the degradation group density file (degradome densityfile) for accurate prediction of the target gene, a total of 8 miRNAs corresponding to 13 target genes (Table 4).
    3.4 qRT-PCR verification differential expression miRNAs and target gene selection aqc-miR159, bdi-miR162, cpa-miR319, pgi-miR4376, smo-miR396 5 differential expression miRNAs, using qRT-PCR technology to verify its participation in the expression of stone column in the garden.
    include GRF1_ARATH for smo-miR396 regulation, DCL1_ARATH for bdi-miR162 regulation, ACA8_ARATH for pgi-miR4376 regulation and GAM1_ORYSI for aqc-miR159/cpa-mi319 regulation.
    results show that the expression pattern of most target genes is negatively regulated with the corresponding miRNA, and the ACA8_ARATH results of pgi-miR4376 regulation show a non-negative regulatory relationship, indicating that there is a complex miRNA regulation in different varieties, and because there are multiple target genes of smo-miR396, selectone target gene verification, the results show that there is no negative regulatory relationship, the GRF1_ARATH is not a human parameter miR396 gene target (Figure 4).
    5 miRNAs verification results are basically consistent with high-throughput sequencing, indicating that the high-throughput sequencing results in this study are highly accurate.
    4 discussion along with the development of high-throughput sequencing technology, degradation group sequencing technology can successfully identify low expression abundance of miRNA target gene, in the prediction of plant miRNA target gene is widely used in the prediction of plant miRNA target genes.
    the results of the mating prediction of the miRNA sequence and the mRNA sequence and the results of the degradation group density file (degradome density file) were combined with the
    experiment, and a total of 13 target genes corresponding to 8 miRNAs were found.
    include six known miRNAs: aqc-miR-159, bdi-miR162, cpa-miR319, cme-miR166i, pgi-miR4376, smo-miR396, and 2 new miRNAs: comp65451, comp46638.
    using Nr, KEGG, GO and other databases to carry out functional annotation and metabolic pathway analysis of these target genes, found that the target gene types of these miRNAs are mainly transcription factors, response factors and signal transduction pathways, and miRNA mainly in the gene after the level of regulation is basically consistent.
    miR159 and miR319 jointly target the MYB domain protein family, miR396's five target genes are mainly GRF growth factor (growth-regulatingfactor), homologous with the target gene in the amoeba; and ginseng-specific non-conservative Pgi-miR4376 target base because of ACA8_ARATH, belongs to the coded calcium channel blocker type, the new miRNA comp65451 target gene and THE TIR NBS-LRR disease-resistant protein regulation, may be the ginseng species specific new miRNA, need further study.
    has been shown that exogenous miRNAs can be absorbed and detected from mammalian tissue.
    some miRNAs in chinese medicine, such as dansan and yellowing, can enter the human body through oral water frying solution, showing significant biological activity and tissue targeting.
    miR2911 in the water frying liquid of gold and silver flowers can enter the mice by feeding in the stomach and play a direct role in the influenza virus.
    miRNA in medicinal plants may be one of the mechanisms by which traditional Chinese medicine acts.
    , ginseng miRNA may enter the body to play a regulatory role, become one of the effective material bases of ginseng clinical efficacy.
    to verify this hypothesis, the study identified the target gene of fresh ginseng miRNA through degradation group sequencing analysis, and verified by qRT-PCR to produce consistent results.
    lays the foundation for further revealing the possible regulatory effects of ginseng miRNA and its target genes in the body.
    References (Slightly) Source: Wang Yingfang, Wang Wenxuan, Peng Yuxuan, Chen Yanlin, He Zhihua, Cao Jingjing, Dai Wangqiang, Lin Zhiyun, Yang Zemin, Yu Mengxuan, Yu Yongqin. Degradation group sequencing technology for the analysis and identification of ginseng miRNA target genes. J. Chinese herbal medicine, 2019, 50 (4): 945-950.Source:Wang Yingfang, Wang Wenxuan Chinese Herbal Medicine Magazine.
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