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Metabonomics and transcriptomics correlation analysis based method for screening key gene for synthesis of fritillaria alkaloid

A technology for fritillary alkaloids and gene synthesis, which is used in biochemical equipment and methods, determination/inspection of microorganisms, analytical materials, etc.

Inactive Publication Date: 2018-06-01
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before the present invention, there has not been any public report related to the screening of key genes for the synthesis of Fritillaria alkaloids based on the content of Fritillaria alkaloids and the expression of genes related to their synthesis pathways

Method used

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  • Metabonomics and transcriptomics correlation analysis based method for screening key gene for synthesis of fritillaria alkaloid
  • Metabonomics and transcriptomics correlation analysis based method for screening key gene for synthesis of fritillaria alkaloid
  • Metabonomics and transcriptomics correlation analysis based method for screening key gene for synthesis of fritillaria alkaloid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Metabolomics Analysis

[0023] 1) Material

[0024] The plant materials used were the fresh bulbs of Fritillaria fritillaria collected in Jinhua, Zhejiang, Fritillaria flatus in Panshi, Jilin, and Fritillaria dark purple in Aba Town, Sichuan. The collection time was May and June 2017. Part of the collected samples were quick-frozen in liquid nitrogen and stored in a -80°C refrigerator, while the other part was dried and stored for analysis.

[0025] 2) Sample extraction

[0026] Each sample was ground into powder, and 500 mg of dry powder was taken, basified with 3 mL of ammonia water (25%) for 1 hour, and ultrasonically extracted with 50 mL of chloroform:methanol (4:1, v / v) mixed solution for 1 hour. The extract was filtered with filter paper, and 10 mL of the filtrate was taken in an evaporating dish, and evaporated to dryness in a water bath. After evaporating to dryness, it was dissolved in 10 mL of 0.1M hydrochloric acid and passed through a 0.22 μm o...

Embodiment 2

[0038] Example 2 Transcriptomics analysis

[0039] The RNAprep Pure Plant Kit kit was used to extract the total RNA from the sample, and the concentration, integrity and purity of the obtained total RNA were tested. After the test was qualified, the eukaryotic mRNA was enriched with magnetic beads containing Oligo (dT), and then Add fragmentation buffer to fragment mRNA into short fragments, use mRNA as a template, use six base random primers (randomhexamers) to synthesize one-strand cDNA, then add buffer, NTPs, DNA polymerase I and RNase H to synthesize second-strand cDNA, and then use AMPure P beads for purification of double-stranded cDNA. The purified double-stranded cDNA was firstly repaired, A-tailed and sequenced adapters were ligated, and then fragment size selection was performed with AMPure XP beads. Finally, PCR amplification was performed, and the PCR product was purified with AMPureXP beads to obtain the final library.

[0040] After the library construction is ...

Embodiment 3

[0047] Example 3 Construction of metabolite-gene relationship network

[0048] The Pearson correlation coefficient in the correlation analysis (correlation analysis) was used to perform correlation analysis on the expression levels of genes related to different Fritillaria alkaloid content and alkaloid synthesis pathways, and to screen the genes that contribute more to Fritillaria alkaloid synthesis. The cut-off value of the variable correlation coefficient for key gene screening is 0.5.

[0049] results and analysis

[0050] Example 1 Metabolomics Analysis

[0051] Such as figure 2 A shows the total ion chromatogram (TIC) obtained from the full scan of UPLC-QTOF / MS. A total of 59 compounds were detected. After the compounds were classified, the MRM mode of UPLC-QQQ / MS was used for quantification, as shown in figure 2 B shows the quantitative MRM chromatograms of 64 compounds and internal standards. From the PCA analysis chart, it can be seen that Fritillaria, Fritillari...

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Abstract

The invention discloses a metabonomics and transcriptomics correlation analysis based method for screening a key gene for the synthesis of fritillaria alkaloid. Specifically, the differential alkaloidand the content information of the differential alkaloid are obtained through the metabonomics analysis of three types of fritillaria, and meanwhile, the expression condition of the related gene in the synthetic route of the alkaloid is obtained through the functional annotation of the transcriptomics. The differential metabolin-gene dependence relation network is constructed through the correlation analysis, and the key gene for the synthetic route of the alkaloid, having a greater contribution to the content of the alkaloid in the fritillaria, is screened. The method provides important information for the further clarification of the synthesis mechanism of the alkaloid substance in the fritillaria, and provides an important evidence for increasing the content of fritillaria alkaloid bymeans of gene control and the like.

Description

technical field [0001] The present invention relates to the screening of key genes of the alkaloid synthesis pathway in Fritillaria, specifically, the association analysis is carried out by combining the metabolomics analysis of three Fritillaria and the expression of genes related to the alkaloid synthesis pathway to screen out the alkaloids in Fritillaria. The key genes of alkaloid synthesis pathway with large content contribution. Background technique [0002] Fritillaria is a perennial herb of Liliaceae, whose bulbs are used for medicinal purposes, and are widely distributed in the Mediterranean, North America and Central Asia. Different varieties of Fritillaria are commonly used as traditional herbal medicines in Turkey, Pakistan and Southeast Asia. There are nearly 50 species and 19 varieties of Fritillaria plant resources in my country, which are mainly distributed in Zhejiang, Sichuan, Xinjiang, Gansu, Hubei and Anhui provinces. The dried bulbs of Fritillaria plants...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869G01N30/02
CPCC12Q1/6869G01N30/02C12Q2535/122
Inventor 李会军陆续张琳宁李萍辛贵忠
Owner CHINA PHARM UNIV
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