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Screening method for active substances of plants

A screening method and technology for active substances, applied in the field of plant active substance screening, can solve the problems of difficulty in obtaining transgenic plants, low success rate, long transformation period, etc., and achieve the effects of short experimental period, simple operation, and reduced screening difficulty.

Inactive Publication Date: 2016-09-28
SOUTHWEST UNIVERSITY
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Problems solved by technology

This method requires the construction of a tubulin fluorescently labeled cell line, which requires genetic transformation, but it is difficult to obtain transgenic plants, involving steps such as callus generation, Agrobacterium-mediated genetic transformation, and plant regeneration. Not only the transformation cycle long and less successful

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  • Screening method for active substances of plants
  • Screening method for active substances of plants
  • Screening method for active substances of plants

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Embodiment 1

[0045] 1. Extraction of substances to be screened:

[0046] Dry the citrus fruit at 50°C and grind it into powder with a pulverizer; extract the above powder with 90% ethanol at 80°C for 2 hours; filter the extract through Whatman No. 1 filter paper, and then use a rotary evaporator at 40°C to gradually reduce The filtrate was concentrated by means of pressure, and finally freeze-dried to obtain the substance to be screened, which was stored at -20°C;

[0047] 2. Separation of tubulin:

[0048] Tubulin assembly / disassembly was achieved using two temperature-dependent cycles, and tubulin was purified from fresh porcine brain (Shelanski et al., 1973). The detailed steps are:

[0049]Fresh porcine brains were isolated within 2 hours after the pigs were slaughtered, and the porcine brains were placed on ice; the porcine brains were minced and soaked in a reassembly solution at 4°C. The reassembly solution included: 0.1M sulfo fatty acid formazan Esters sodium salt, 1 mM ethylene...

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Abstract

The invention provides a screening method for active substances of plants. The screening method comprises the following steps: (a) separating and purifying tubulins from pig brains; (b) performing fluorescence labeling on the tubulins to obtain fluorescence labeled tubulins; (c) mixing the tubulins, the fluorescence labeled tubulins and substances to be screened and performing a polymerization reaction; (d) performing microscopic observation on a polymerization product to obtain a microscopic picture; quantifying the quantity, length and roughness of the tubulins in the microscopic picture through applying computer software; carrying out conversion analysis on data and determining the activity of the substances to be screened according to a result. According to the screening method, the tubulins of the pig brains are subjected to the fluorescence labeling under an in-vitro condition, and the density, length frequency distribution and the like of the tubulins after the polymerization reaction are quantified by applying the computer software and are used as evaluation indexes for measuring factors influencing nucleation, elongation or reduction of the tubulins, so that the influences on the polymerization of the tubulins by the active substances of the plants are judged and a method model is provided for high-throughput screening researches for digging the active substances of the plants.

Description

technical field [0001] The invention belongs to the technical field of utilization of plant active ingredients, and in particular relates to a screening method of plant active substances. Background technique [0002] With the development and maturity of molecular biology, immunology, active ingredient extraction and separation and other technologies, the research on active substances has also developed rapidly, especially the screening of anti-tumor drugs. [0003] The prior art discloses two screening methods for active ingredients, one is to use molecular bioinformatics methods to construct cell line mutants (Vajihe Akbari et al., 2011), or to propose a theoretically assumed action site through sequence comparison (Dostal & Libusova, 2014), to discover the relevant response genes (such as: anti-cancer related genes, disease resistance related genes) stimulated by external signals (such as: secondary metabolites, pathogenic bacteria), the typical process is as follows fig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/84
CPCG01N21/84
Inventor 关心彼得·尼克谭飔王嘉钰胡楠尹秋月
Owner SOUTHWEST UNIVERSITY
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