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Method for screening notoginsenoside synthesis key genes based on metabolites and gene expression

A key gene and gene expression technology, applied in the field of screening key genes of notoginsenoside synthesis based on metabolites and gene expression

Inactive Publication Date: 2016-06-15
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 notoginseng triterpene saponins based on the content of triterpene saponins and the expression of key genes in their synthesis pathways

Method used

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  • Method for screening notoginsenoside synthesis key genes based on metabolites and gene expression
  • Method for screening notoginsenoside synthesis key genes based on metabolites and gene expression
  • Method for screening notoginsenoside synthesis key genes based on metabolites and gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Metabolic profiling.

[0027] 1) Materials and methods

[0028] The plant materials used come from the cultivation of Panax notoginseng in our laboratory. The seeds of Panax notoginseng were purchased from Wenshan Prefecture, Yunnan Province. They were sown in a greenhouse in December 2013. The planting standards of Panax notoginseng were strictly followed to ensure a cool, dark and suitable environment for the growth of Panax notoginseng. temperature and humidity. The leaves, petioles and roots of the annual Panax notoginseng plant were collected in June of the following year. The samples were quick-frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0029] 2) Sample extraction

[0030] Each sample was ground into powder and extracted with methanol after 48 hours of freeze-drying. Accurately weigh 50 mg of the sample and dissolve it in 1.5 mL of methanol, vortex for 1 min, ultrasonically extract at room temperature for half an hour, leave the ...

Embodiment 2

[0037] Example 2 Gene expression analysis.

[0038] Quantitative detection of expression levels of key genes in saponin synthesis pathway

[0039] The total RNA of the sample was extracted according to the instructions of the RNApreppureplantkit from BioTeke Company. After extraction, the integrity of the extracted RNA was detected by 1% agarose gel electrophoresis, and the values ​​of OD260 / OD280 and OD260 / OD230 were measured to detect the purity and concentration of RNA. The first strand of cDNA was synthesized according to the instructions of PrimeScriptRTreagentkit (Takara). Subsequently, using the first strand of cDNA as a template, the 96 real-time fluorescent quantitative PCR instrument was used for relative quantitative analysis of the key genes FPS, SQS, DS, SE and CYP716A53 in the saponin synthesis pathway of annual Panax notoginseng. Quantitative PCR according to SYBRPremixExTaq TM (Takara) instructions to prepare the reaction system, wherein each gene-specific ...

Embodiment 3

[0040] Example 3 Construction of gene-metabolite relationship network.

[0041] Through the Pearson correlation coefficient in the canonical correlation analysis (canonical correlation analysis), the saponin content of annual Panax notoginseng and the expression of key genes in the saponin synthesis pathway in different parts were correlated, and the annual Panax notoginseng gene-metabolite relationship network was constructed to screen for the Genes with a large contribution to the synthesis of notoginseng saponins. The cut-off value of the variable correlation coefficient for key gene screening is 0.5.

[0042] results and analysis

[0043] Differences of Saponin Content in Different Parts of Annual Panax notoginseng

[0044] Such as figure 2 and image 3 As shown, there are significant differences in the saponin metabolism spectrum and saponin distribution in different parts of annual Panax notoginseng. In addition, as shown in Table 2, the content of each saponin and...

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Abstract

The invention discloses a method for screening key genes of notoginseng saponin synthesis based on metabolites and gene expression. Specifically, the high-performance liquid chromatography-mass spectrometry system was used to analyze the metabolite content of different parts of the annual Panax notoginseng, and the fluorescence quantitative PCR technology was used to quantitatively analyze the gene expression of key enzymes in the saponin synthesis pathway. Gene-metabolite correlation graph to screen key genes. The invention provides important information for further elucidating the synthesis mechanism of saponins in Panax notoginseng, and at the same time provides an important basis for increasing the content of Panax notoginseng saponins by means of gene regulation and the like.

Description

technical field [0001] The present invention relates to the screening of key genes in the synthesis pathway of triterpene saponins in Panax notoginseng, specifically performing canonical correlation analysis (canonical correlation analysis) in combination with the content of metabolites of annual Panax notoginseng and the expression of key genes in the synthesis pathway of triterpene saponins, and screening out Panax notoginseng The key genes of the triterpenoid saponin synthesis pathway that contribute greatly to the saponin content. Background technique [0002] Notoginseng (Panax notoginseng (Burk.) F.H.Chen) is a plant of the genus Panax notoginseng in the Araliaceae family. It is a traditional precious medicinal material in my country. The application of Panax notoginseng in ancient times was mainly used as an important medicine for gold wounds, and it was used for the treatment of various hemorrhages, bruises, stasis, swelling and pain inside and outside the body. Mod...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N30/02
CPCC12Q1/686G01N30/02G01N2030/027C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 齐炼文陆续王宁李萍
Owner CHINA PHARM UNIV
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