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Method for using sts primer to identify ginseng species

A technology of ginseng and varieties, applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as poor differentiation and consistency, prevent variety mixing, and improve genetic stability Effect

Active Publication Date: 2010-02-24
韩国农村振兴厅
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  • Claims
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AI Technical Summary

Problems solved by technology

[0010] The purpose of the present invention is to solve the problem of poor differentiation and consistency of the RAPD and ISSR techniques utilizing arbitrary primers and the ITS techniques for analyzing the conserved rDNA regions of base sequences. For the object, use gene cloning and transformation methods to construct a genomic DNA library, select specific clones; and use these clones as objects for base sequence analysis, and then prepare STS primers based on these genetic information; use samples of different varieties of ginseng as templates, Use the primers to carry out PCR, combined with the genetic pattern shown by agarose gel electrophoresis to distinguish five main ginseng varieties including Tianfeng, thus providing a variety identification system with high stability and consistency

Method used

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  • Method for using sts primer to identify ginseng species
  • Method for using sts primer to identify ginseng species
  • Method for using sts primer to identify ginseng species

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Effect test

Embodiment 1

[0052]DNA (1 μg) extracted from young leaves of a standard variety Lianfeng grown in a ginseng field of the Ginseng and Herbal Research Institute of the Academy of Crop Science of the Rural Development Administration was cleaved with a methylation-insensitive restriction enzyme SpeI. The cleavage product was subjected to agarose gel electrophoresis, and after the DNA was recovered using a PCR product recovery kit (Qiagen), it was ligated with the prepared pGEM_7zf (Promega). The ligated mixture was transformed into Escherichia coli (Stratagene) with wild-type Mcr A and BC genes by electroporation, and the so-called "active genome region-enriched semi-genomic DNA library" (active genome region-enriched semi-genomic DNA library) was constructed. ”, thus obtaining a total of 2,039 clones.

Embodiment 2

[0054] Delegate Solgent( ) Co., Ltd. performed base sequence analysis on the obtained clones. STS primers were synthesized based on the analyzed base sequence. The size of the clones obtained above was 0.6kb-1.3kb, and a total of 343 clones were prepared, thereby preparing a total of 262 pairs of STS primers.

Embodiment 3

[0056] The composition of the PCR reaction mixture (50 μl) for observing the polymorphism among ginseng varieties is as follows: 20 ng of genomic DNA of ginseng, 0.4 μM of each primer, 250 μM of dNTPs, 10 × PCR reaction buffer solution (75 mM Tris-HCl (pH 9.0 ), 2.5mM MgCl 2 , 15mM (NH 4 ) 2 SO 4 , BSA 100 μg / ml), 0.5 units of DNA polymerase, and the rest as distilled water. In order to effectively utilize the synthetic STS primer pair and suppress the excessive appearance of non-specific amplification products, reasonable primer Tm values ​​were determined after PCR was performed at an annealing temperature range of 55-65°C. The results determined that the PCR reaction conditions were as follows: the template DNA was pre-denatured at 95°C for 5 minutes; then denatured at 95°C for 30 seconds, annealed at 65°C for 30 seconds, and extended at 72°C for 1 minute, repeated for a total of 40 cycles; finally , extended at 72°C for 5 minutes to obtain specific amplification produc...

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Abstract

The invention relates to an STS (sequence tagged site) primer pair for ginseng species identification, a kit including the primer pair for ginseng species identification, and a method using the primerpair to identify ginseng species. The STS primer pair for ginseng species identification includes more than one primer pair selected from four primer pairs shown by SEQ ID NO: 1 to 8. According to the invention, five ginseng species can be accurately distinguished by taking the four STS nucleic acid marker factors as the marker objects, so that the nucleic acid marker factors can be taken as themarker objects to be applied in varieties affirmation taking intellectual property protection of varieties as the objective. using four kinds of DNA markers STS factor can be the five varieties ofginseng, so described nucleic acid can be used as markers for marking factor applied to varieties of species of intellectual property protection for the purpose of confirmation.

Description

technical field [0001] The present invention relates to a method for identifying ginseng species using sequence tag site (Sequence Tagged Site, STS) primers. More specifically, the present invention relates to identifying 5 individuals including Tianfeng, Lianfeng, Gaofeng, Jinfeng and Xianfeng. The development of STS nucleic acid markers for ginseng varieties also involves preparing STS primers based on ginseng base sequence information, and then using these primers to analyze dominant inheritance among varieties to clearly distinguish each ginseng variety. Background technique [0002] There are more than 12 kinds of plants of the genus Panax reported in the world, among which Panax ginseng C.A. Meyer (Oriental ginseng), American ginseng (P.quinquefolium L. (American ginseng)) and Panax notoginseng (Burkill) F.H. Chen (Sanchi)) are mainly used as Chinese herbal medicines. Ginseng is grown in South Korea, China, Japan, Russia, etc. American ginseng is grown in the United S...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/13
Inventor 方景焕金永昌金玉泰丁智雄车善佑李济完
Owner 韩国农村振兴厅
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