Specific gene PgWOX5 used for detecting ginseng root tip stem cells and detection method thereof and application thereof

A technology of stem cells and ginseng roots, which is applied to the detection of the specific gene PgWOX5 of ginseng root tip stem cells and its detection field, can solve the problems of detection of ginseng stem cells that have not been seen yet, and achieve good research potential and application prospects

Pending Publication Date: 2019-02-15
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there has been no report on the detection of ginseng stem cells by in situ hybridization of stem cell marker genes and EdU staining.

Method used

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  • Specific gene PgWOX5 used for detecting ginseng root tip stem cells and detection method thereof and application thereof
  • Specific gene PgWOX5 used for detecting ginseng root tip stem cells and detection method thereof and application thereof
  • Specific gene PgWOX5 used for detecting ginseng root tip stem cells and detection method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1, the acquisition of PgWOX5 gene and its encoded protein

[0073] 1. Extraction of RNA

[0074] Ginseng fibrous root and adventitious root samples were ground and crushed with liquid nitrogen, total RNA was extracted using TRIzol Plant RNA Kit according to the instructions of TRIzol Plant RNA Kit, and the integrity of RNA was determined by 1% agarose gel electrophoresis, and the RNA content was determined by a nucleic acid / protein quantifier.

[0075] 2. Synthesis of cDNA

[0076] cDNA was synthesized using M-MLV RTase cDNA Synthesis Kit (Takara) according to the instructions of M-MLV RTase cDNA Synthesis Kit (Takara).

[0077] 3. PCR amplification

[0078] Using the cDNA obtained in step 2 as a template, WOX5-F and WOX5-R were used for PCR amplification to obtain PCR products. The primer sequences are as follows:

[0079] WOX5-F: 5′-ATGGATGAAGGAATGTCAGGC-3′:

[0080] WOX5-R: 5'-TTAAGATTGGAAGCTTAAACGC-3'.

[0081] 4. Cloning and sequencing

[0082] The P...

Embodiment 2

[0083] Example 2. Detection of ginseng adventitious root apical stem cells by whole-body in situ hybridization

[0084] 1. Preparation of PgWOX5 probe

[0085] 1. Preparation of probe template

[0086] Using ginseng cDNA as a template to carry out PCR amplification on the open reading frame of PgWOX5 gene, the obtained PCR product (herein the PCR product is the mixture of gene fragments shown in sequence 1 and sequence 3, the gene fragment shown in sequence 1 and sequence 3 The ratio is about 1:1) is the template DNA for probe synthesis. The primers for PCR amplification are as follows:

[0087] Forward primer: 5′-ATGGATGA AGGAATGTCAGGC-3′;

[0088] Reverse primer: 5'-TTAAGATTGGAAGCTTAAACGC-3'.

[0089] 2. Synthesis of single-stranded probes

[0090] Take 1 μg of the template DNA obtained in step 1, add water to make up to 11 μL, denature at 65°C for 5 minutes, and quickly place it on ice. Prepare the system for the synthesis of single-stranded probes: 4 μL of 5×trans bu...

Embodiment 3

[0103] Example 3, EdU staining method to detect the expansion of ginseng adventitious root apical stem cells

[0104] The EdU staining method was used to detect the expansion of ginseng adventitious root stem cells, and the specific steps were as follows:

[0105] 1. EdU marking

[0106] Take the root tip of ginseng adventitious root with a length of about 2cm, put it into 5mL MS liquid medium and shake it for 48 hours, and then use Click- EdU Imaging Kit (ThermoFisher) was used for EdU labeling. EdU staining method according to Click- The procedure described in EdU Imaging Kit (ThermoFisher) was carried out.

[0107] 2. The expansion of ginseng adventitious root stem cells

[0108] A two-photon laser confocal microscope (LSM880) was used to detect the expansion of ginseng adventitious root-tip stem cells. The excitation wavelength of EdU fluorescence was 590nm, and the emission wavelength was 615nm. The expansion of ginseng adventitious root-tip stem cells was observed ...

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Abstract

The invention discloses a specific gene PgWOX5 used for detecting ginseng root tip stem cells and a detection method thereof and application thereof. The specific gene PgWOX5 used for detecting the ginseng root tip stem cells can be used for marking root tip stem cells of ginseng adventitious roots and fibrous roots. The invention further discloses a method for detecting root tip stem cell parts or positions of the ginseng adventitious roots and the fibrous roots. The specific gene PgWOX5 used for detecting the ginseng root tip stem cells, disclosed by the invention, has the advantages that the blank in the non-mode plant stem cell detection field is filled, one set of feasible detection scheme is provided for positioning of non-mode plant stem cells, and very good research potential and application prospect are realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific gene PgWOX5 for detecting ginseng root tip stem cells, a detection method and application thereof. Background technique [0002] Ginseng (Panax ginseng C.A.Mey.) is a perennial herbaceous plant of Araliaceae. It has been used for more than 5,000 years in my country. It is listed as "Top Grade" in "Shen Nong's Materia Medica", and enjoys the reputation of "King of Herbs". Under natural conditions, wild ginseng has a strong repair ability and can resist the erosion of pathogenic bacteria and herbivores. The phenomenon of "mutation" of ginseng indicates that ginseng stem cells have strong recovery ability and regeneration potential. Root tip stem cells play an important role in maintaining plant growth and repairing damage, but how to detect ginseng root tip stem cells has not been reported yet. [0003] Observing the expression of plant stem cell marker genes by in situ hy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12Q1/6895C12N15/11
CPCC12Q1/6895C07K14/415
Inventor 黄璐琦刘娟蒋超袁媛陈同
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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