JPH06197762A - Method for extracting dna of onion - Google Patents

Abstract

PURPOSE:To enhance the extraction rate of the highly pure DNA of the onion by solving the subjects of conventional techniques. CONSTITUTION:The method for extracting the DNA of onion is characterized by adding the solution of a composition not decomposing the nuclei of the onion, collecting only the nuclei, finely grinding the collected nuclei and subsequently starting the extraction of the DNA with an extraction solution containing a surfactant, after lyophilizing the sliced scaly leave of the mother bulb of the onion and before adding the DNA extraction solution. The addition of the solution not decomposing the nuclei enables to collect the nuclei and remove organelle and polysaccharides in the cells, thereby permitting to provide the DNA having higher purity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an RFLP map for assisting onion breed improvement and a DNA extraction method for use in developing recombinant DNA technology.

[0002]

2. Description of the Related Art Upon successful extraction of onion DNA,
Using this DNA, a new gene can be cloned by a genetic engineering technique using a known gene as a probe. Then, a new variety can be bred by modifying this cloning gene or introducing a gene of another organism into the genome. Further, extraction of onion DNA makes it possible to create an RFLP map and shorten the breeding period. Like this onion DN
Successful extraction of A has immeasurable advantages in developing techniques for improving onion varieties. Moreover, more pure DN
When A is obtained, this DNA can be reacted with a smaller amount of restriction enzyme, ligase, etc., and in the breeding improvement research utilizing genetic engineering, the amount of DNA can be accurately estimated, the experimental accuracy is improved, and As a result, breeding costs using the accumulated technology can be reduced. But onion D
Until now, the method of extracting NA has not been known.

According to "Biochemistry Experiment Course 2, Nucleic Acid Chemistry I", page 76, edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin, May 1975, a method for separating nuclei for DNA extraction into liver was used. A method for separating nuclei using a sugar-CaCl ₂ aqueous solution is described. However, this method uses living animal cells as a material, and since it is necessary to destroy the cell wall and at the same time separate intact nuclei, it is difficult to directly apply it to plants. In addition, since high concentration of sucrose is used, it is difficult to operate.

Further, the present inventors previously lyophilized sliced scaly leaves of onion mother spheres, pulverized them into fine powder, and then started DNA extraction with an extract containing a surfactant. Invented a DNA extraction method (Japanese Patent Application No.
-129473). However, in this method, since DNA is directly extracted without separating nuclei from cells, polysaccharides are often mixed, which often hinders subsequent studies.

[0005]

If a purer DNA can be obtained, a smaller amount of this restriction enzyme, ligase, etc. and this DN can be obtained.
A can be reacted, the amount of DNA can be accurately estimated in the breeding improvement research using genetic engineering, the accuracy of the experiment can be improved, and the breeding cost using the accumulated technology can be reduced as a result. Therefore, an object of the present invention is to solve the problems of the above-mentioned conventional techniques and to establish a method for increasing the extraction rate of highly pure onion DNA.

[0006]

The above object of the present invention is to freeze-dry sliced scaly leaves of onion mother spheres, and then add a solution having a composition that does not destroy the nucleus before adding the DNA extract. , Collect the nuclei only, and then start DNA extraction with an extract containing detergent DN of onion
A method of extraction.

[0007]

In general, as a method for extracting plant DNA, a method in which plant tissue is crushed in the presence of liquid nitrogen to extract DNA is used. However, this method makes it difficult to crush the tissue when freezing it in liquid nitrogen such as scales because the water in the tissue freezes. The parts may melt or freeze again. Therefore, it causes significant damage to the chromosome. Therefore, in the case of extracting nuclear DNA from onion mother spheres, the scaly leaves of the mother spheres are sliced into a width of 1 to 3 mm, frozen with chilled acetone or the like, and then freeze-dried to remove the tissue in the tissue. The water of the above does not freeze, the subsequent fine pulverization of the tissue is facilitated, and the DNA extraction with the extract becomes possible.

The above findings are as disclosed in the present inventors' previous patent application (Japanese Patent Application No. 4-129473). However, in the present invention, the nucleus is further added before adding the DNA extract. It is characterized by adding a non-breaking solution. This solution that does not destroy the nuclei makes it possible to collect the nuclei and remove the organelles and polysaccharides in the cells, so that a highly purified DNA can be obtained.

[0009]

EXAMPLE An example of the present invention will be described. The procedure for extracting onion DNA is shown below. The epidermal root part of the onion (variety: Cloisonne sweet bulb) mother ball that had been stored refrigerated is excised, and 176 g of scaly tissue is sliced. The slice of the scaly tissue obtained in the above step is placed in a 500 ml eggplant-shaped flask and frozen in acetone that has been cooled in advance. After that, freeze dryer (Tokyo Rika Kikai Co., Ltd.)
FD-1) manufactured for 4 days. Crush the freeze-dried sample with a mortar and pestle. Put 1 g of sample in a 50 ml Falcon tube, add 20 ml of solution A below, and stir. After centrifuging this solution at 3000 rpm for 15 minutes, the supernatant liquid is removed, and 15 ml of the following solution B is added to suspend the precipitate.

Next, the solution was transferred to a centrifuge tube, and
Add 10 ml of 10% sodium dodecyl sulfate (SDS) and stir slowly. After stirring at 60 ° C. for 10 minutes, 5 ml and 5 mol of potassium acetate are added and stirred. Then, after 10 minutes of ice cooling, centrifugation was performed at 10,000 rpm for 20 minutes, the supernatant was transferred to a new tube, an equal amount of isopropanol was added, and 10,000 rp was added.
Centrifuge at m for 20 minutes to obtain a pellet. The pellet obtained was rinsed twice with 70% ethanol, then vacuum dried and 1 ml TE buffer (10 mM
Dissolve the pellet in Tris-HCl (pH 8.0).

Solution A Solution B 0.2 M sucrose 100 mM Tris-HCl (pH 8.0) 2.5 mM EDTA 50 mM EDTA 2.5 mM DTT 500 mM NaCl 10 mM NaCl 10 mM 2-mercaptoethanol 10 mM KCl

The yield of onion DNA of this example obtained by the above process was 103 μg / 10 g fresh weight (1 g dry weight). This DNA shows a peak near 262 nm according to the absorbance shown in FIG.
Can be said to have been obtained. The yield of the present inventors' previous invention (Japanese Patent Application No. 4-129473) is 37 μg / fresh weight 1
Since the amount was g, the yield of the method of this example is about 1/4, but a large amount of DNA can be extracted by using a large amount of onion as a raw material, and the yield is not a problem. Rather, it is more important that the method of the present example can significantly reduce the polysaccharide, as compared with the previous invention (Japanese Patent Application No. 4-129473).

Next, the onion DN related to the present invention described above.
The A extraction method will be described. Leeks generally contain mucilage, and constituent substances of the mucilage are cellulose, hemicellulose, protopectin, water-soluble pectin, etc., and their binding modes are α-1,4-glucoside bond and β-1. , 4- or β-1,3-glucosidic bond. By the way, "Experimental Biology Course 17, Plant Physiology [I
II] "278 pp, after the issuance Maruzen extracted DNA crude extraction liquid containing containing SDS, Bundou a method of adding various enzymes for the purpose of removal of today matters (Phaseolu
a method for extracting DNA of s aureus) is disclosed.
Although α-amylase is used in this method, this α-amylase decomposes α-1,4-glucoside bonds and cannot decompose cellulose and pectin in the mucilage of leek.

Therefore, in order to extract a large amount of DNA from a plant body containing a mucilage such as onion, an attempt was made to extract DNA by the following means. That is, it is a DNA extraction method characterized by adding a hydrolase to a crude DNA solution. The above-mentioned hydrolase is α-1,4-such as onion.
Glucoside bond, β-1,4-glucoside bond and β
An enzyme that randomly hydrolyzes the 1,3-glucoside bond is used. Specific examples thereof include α-amylase (α-1,4
-Cleavage of bond), cellulase (cleavage of β-1,4-bond), pectinase (cleavage of α-1,4-bond).

The concentration of the enzyme to be used is such that the DNase activity contained in the enzyme preparation shows the same level of activity as in the usual DNA extraction method, and preferably 100 μg / ml of α-amylase and 50 μg / cellulase. ml, pectinase less than 200 μg / ml. The pH of the buffer solution on which the hydrolase acts is 5.5 to 6.5, preferably 6.0. This adjustment of pH is carried out by adjusting the pH of the TE buffer solution having a pH of 7 to 8 in the steps of the above-mentioned example
Is replaced by pH 6 on the way.

Generally, an enzyme often used for extraction and purification of DNA is RNase (RNA degrading enzyme), but this enzyme has strong activity and low specificity to pH. Therefore, no special buffer is required, so RNase
Is used, the pH of the DNA extraction buffer may be 7 to 8 in the steps of the above-mentioned example, and the buffer replacement is not necessary. However, when the above-mentioned hydrolase is used, it can be allowed to act at the optimum pH of the enzyme, and a small amount of enzyme can remove the polysaccharide. Thus, by the DNA extraction method, which is characterized by adding a hydrolase to the above-mentioned crude DNA solution, the removal of polysaccharides, which is limited by the physical method, can be effectively performed by the chemical method, and the physical cutting force Since it is not added to DNA, it is possible to avoid lowering the molecular weight of DNA.

A DNA extraction method in which a hydrolase is added to the above-mentioned crude DNA solution will be described as a specific example. (1) The epidermal root of the onion (variety: Cloisonne sweet bud) mother ball that had been stored refrigerated was excised, and the scaly tissue was sliced to obtain 300
Place in a ml eggplant flask. (2) Add about 50 ml of pure water (dDW) and freeze-dry for 7 days. (3) After 7 days, crush in a mortar and 0.84m /
After sieving with a m (mesh No. 20) and passing through the mesh, it is transferred to a 50 ml tube at -20 ° C.
(If you are going to the next hoop immediately, this cryopreservation is not necessary), and 1 g of it is put in a 50 ml centrifuge tube. (4) Add 25 ml of the following buffer solution C, stir it, and let it stand for 10 minutes at 1500 rpm.
Centrifuge at 10 minutes and discard the supernatant. (5) After repeating the operation of (4), 15 ml of the buffer solution D described below is added and suspended, and 2150 μl of 10% SDS is added.

(6) This solution is slowly stirred to 60 ° C.
Allow to stand for 10 minutes, add 5.4 ml of 5 molar potassium acetate and stir slowly. (7) Next, leave it in ice for 10 minutes, and slowly stir on the way. (8) After 10 minutes, 10,000 rpm for 20 minutes, 4
Centrifuge at ° C. (9) The supernatant liquid is filtered with two pieces of Miracloth and transferred to a 50 ml tube. (10) Add the same volume of isopropanol, stir slowly, and let stand for 30 minutes. (11) Centrifuge at 2500 rpm for 20 minutes at 15 ° C. Wash the precipitate twice with cold 70% ethanol, then
Air dry for 40 minutes and vacuum dry for 5 minutes. (12) To the dried pellet, 5 ml of the following buffer solution F is added, and the mixture is left standing at 4 ° C. for one day and then transferred to a 15 ml tube. (13) 105 μl of 1 mg / ml RNase, 25 μ
1 20 mg / ml α-amylase and 25 μl of the following enzyme solution were added, and the mixture was kept at 37 ° C. for 90 minutes.

(14) Next, 150 μl of 10% SD
S, add 10 μl of 20 mg / ml proteinase K and place at 37 ° C. for 90 minutes. (15) Then, add the same volume of phenol-chloroform, stir slowly for 20 minutes, and then add 2800
Centrifuge at rpm for 15 minutes. (16) Transfer the obtained aqueous phase to a new tube, add the same volume of chloroform, and stir for 20 minutes. (17) Then, after centrifuging at 2800 rpm for 20 minutes, the aqueous phase is transferred to a new tube. (18) 1/10 volume of 3 mol sodium acetate was added, and the same volume of isopropanol was further added, and the mixture was carefully stirred and then allowed to stand still for 30 minutes to give 280
Centrifuge at 0 rpm for 20 minutes at 4 ° C. The obtained precipitate is washed twice with cold 70% ethanol, air-dried for 30 minutes, placed in a desiccator for 5 minutes, added with 1 ml of TE buffer solution, and stored at 4 ° C.

D buffer solution E buffer solution 0.2 M sucrose 100 mM Tris-HCl (pH 8) 1.0 mM EDTA 50 mM EDTA 2.5 mM DTT 500 mM NaCl 10 mM pH buffer MES 10 mM 2-mercaptoethanol 10 mM NaCl (pH 6) 10 mM KCl (pH 6)

F buffer solution Enzyme solution 1 mM EDTA 1% Cellulase Onozuka RS 10 mM MES (pH 6) 0.5% Macerozyme R-10 0.2% Pectolyase Y-23 10 mM MES (pH 6) 37 ° C., 1 hr. Incubation of Tris-HCl is an aqueous solution of tris (hydroxymethyl) aminomethane whose pH is adjusted with HCl.

In this way, when the above-mentioned hydrolase is used, the enzyme can be allowed to act at the optimum pH and the polysaccharide can be removed with a small amount of the enzyme. In addition, by the DNA extraction method characterized by adding a hydrolase to the crude DNA solution, the removal of polysaccharides, which is limited by the physical method, can be effectively performed by the chemical method, and the physical cutting force is Since it is not added to the DNA, it is possible to avoid lowering molecular weight of DNA.

[0023]

EFFECTS OF THE INVENTION Freezing with liquid nitrogen is unnecessary, and complicated operations are not required. In addition, organelles and polysaccharides in the cells can be removed by collecting the nuclei, so that a highly purified DNA can be obtained. Although nuclei can be collected from protoplasts, expensive enzymes are required to obtain a large amount of nuclei, but such a method is not necessary in this method.

[Brief description of drawings]

FIG. 1 is an absorbance distribution of onion DNA solutions obtained in Examples of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Haruki Otsuki 4818 Ami, Ami-cho, Inashiki-gun, Ibaraki Izumi Seki Agricultural Machinery Co., Ltd. Tsukuba Research Institute (72) Koichi Katsuyama 4818 Ami, Ami-cho, Inami-gun, Ibaraki Prefecture Machine Tsukuba Research Institute

Claims (1)

[Claims] 1. A slice of onion mother scaly leaves is freeze-dried, and then a solution having a composition that does not destroy the nuclei is added before the addition of the DNA extract, and only the nuclei are collected, and then the surfactant is added. A method for extracting onion DNA, which comprises initiating DNA extraction with an extract containing