Convenient and rapid DNA extracting method of peanut health tissues and diseased tissues

An extraction method and technology of healthy tissue, which is applied in the field of simple and rapid DNA extraction of peanut healthy tissue and diseased tissue, can solve the problems of time-consuming, etc., and achieve the effect of short time-consuming, low cost, fast and effective research process

Active Publication Date: 2010-08-18
SHANDONG PEANUT RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are few reports on the use of peanut seeds to extract DNA, which takes a long time

Method used

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  • Convenient and rapid DNA extracting method of peanut health tissues and diseased tissues
  • Convenient and rapid DNA extracting method of peanut health tissues and diseased tissues
  • Convenient and rapid DNA extracting method of peanut health tissues and diseased tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: DNA Extraction from Peanut Leaves

[0042] 1. Test materials and reagents

[0043] Materials: 3 genotypes were used in this experiment, including wild species (Arachis.duranensis), a hybrid offspring of a cultivated species and wild species A. glabrata, and a chemical mutant material.

[0044] Reagents: alkaline lysate, Tris-HCl buffer containing 5-8mg / ml PVP40.

[0045] 2. Test method

[0046] 1. Template Preparation

[0047] A. Select a small leaf on the peanut plant, and use a gel pen core to punch a hole on one side of the main vein to obtain a leaf disc with a diameter of about 1.12 mm. When the peanut material is relatively precious or the number of leaves in the early stage of peanut growth is small, in order to minimize the impact on its growth and development, it can be directly punched and sampled on the plant.

[0048] B. Place the leaf disk in a 1.5ml centrifuge tube (which can be frozen for future use) added with 60 μl of alkaline lysate, and u...

Embodiment 2

[0061] Example 2: Extracting DNA from Peanut Germ

[0062] 1. Test materials and reagents

[0063] Materials: 5 genotypic materials were used in this experiment, including the hybrid offspring of 1 cultivated species and wild species A. rigonii, 1 chemical mutant, and 3 peanut cultivars.

[0064] Reagents: alkaline lysate, Tris-HCl buffer containing 5-8mg / ml PVP40.

[0065] 2. Test method

[0066] 1. Template Preparation

[0067] Take a peanut seed, peel off two cotyledons, and use a knife to cut off the germ part, that is, several immature leaves. The following steps are the same as Scheme 1 B, C, D, E.

[0068] 2.PCR

[0069] PCR system: 12.5 μl 2×Taq PCR MasterMix, 0.5 μL each of 10 nM primers (TBPfex1 / TBPrex1), 2 μL DNA template, and 9.5 μL sterile deionized water.

[0070] PCR conditions: 35 cycles of pre-denaturation at 94°C for 3 min, 50 sec at 94°C, 1 min at 55°C, 1.5 min at 72°C, and 7 min at 72°C.

[0071] 3. Electrophoretic separation of PCR products

[0072...

Embodiment 3

[0075] Example 3: Cloning fungal 18S rDNA from scab disease tissue

[0076] 1. Test materials and reagents

[0077] Material: Peanut scab diseased tissue.

[0078] Reagents: alkaline lysate, Tris-HCl buffer containing 5-8mg / ml PVP40.

[0079] 2. Test method

[0080] 1. Template Preparation

[0081] Select a piece of scab diseased leaf on the peanut plant, punch a hole on the side of the main vein with a neutral pen core, and obtain a leaf disc with a diameter of about 1.12 mm; cut the diseased tissue of the stem into a thin slice with a diameter of about 4 mm with a razor blade. The following steps are the same as steps B, C, D, and E of "template preparation" in Example 1.

[0082] 2.PCR

[0083] PCR system: 25 μl 2×Tiangen Taq PCR MasterMix, 10 μM fungal-specific primers (NS26: 5’-CTGCCCTATCAACTTTCGA-3’ / R518: 5’-ATTACCGCGGCTGCTGG-3’) 2 μl each, 1 μL DNA template, 20 μl sterile deionized water.

[0084] PCR conditions: 95°C pre-denaturation for 6min, 94°C for 1min, 55°C...

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Abstract

The invention relates to a convenient and rapid DNA extracting method of peanut health tissues and diseased tissues. The method comprises two steps of: sample materials preparation: testing the leaf and stem tissues, shell tissues or peanut cotyledons, the weight of which is 3-10 mg, adding the sample materials into a 1.5ml centrifugal tube preset with an alkaline lysate or enzymolysis liquid, and grinding by a grinding rod till the visible particles do not exist; DNA extracting liquid preparation: treating the ground sample materials by an alkaline decoction method or a enzymolysis method to obtain the DNA extracting liquid. The method is also suitable for peanut health tissues and diseased tissues. Compared with the DNA extracting method, the invention has short required time, the required time of single sample by the alkaline decoction method does not exceed 20min, and the required time of single sample by the enzymolysis method does not exceed 0.5h; the dosage of the sample is little, and each sample only needs 3-10mg; the method has no need of liquid nitrogen to grind, therefore the cost is low, and the requirements of peanut molecule marker auxiliary breeding and transgene breeding are satisfied. The invention has no influence on the plant growth or seed germination, and has significant meaning on the practicality of peanut molecule breeding technology.

Description

technical field [0001] The invention relates to the category of DNA extraction from plant tissues, in particular to a simple and rapid DNA extraction method for peanut healthy tissues and diseased tissues. Background technique [0002] In the prior art, it is common to adopt etiolated seedling leaves, whole or half seeds as starting materials, and liquid nitrogen grinding to extract the DNA of peanut tissue, which includes at least 10 steps and takes more than 2 hours (Kochert et al.1991 ; Choi et al.1999; Burrow et al.2001; Wang Chuantang et al.2002; Chen Jing et al.2008). When processing a large number of samples, even using a ball mill is very inconvenient, and cannot meet the requirements of high-throughput research such as molecular marker-assisted breeding, map construction, and transformant screening. If it is not directly used for enzyme digestion and Southern hybridization, the DNA extraction step can be greatly simplified. [0003] PCR technology has become an in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07H21/04C07H1/08
Inventor 王传堂王秀贞唐月异张建成陈殿绪崔凤高禹山林于树涛
Owner SHANDONG PEANUT RES INST
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