CN113528505A - Rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption - Google Patents

Rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption Download PDF

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CN113528505A
CN113528505A CN202110691423.2A CN202110691423A CN113528505A CN 113528505 A CN113528505 A CN 113528505A CN 202110691423 A CN202110691423 A CN 202110691423A CN 113528505 A CN113528505 A CN 113528505A
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cucumber
filter membrane
gene dna
amplification
adsorption
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薛强
陈艳
邹明强
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China Inspection Laboratory Technologies Co ltd
Chinese Academy of Inspection and Quarantine CAIQ
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Chinese Academy of Inspection and Quarantine CAIQ
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Abstract

The invention discloses a rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption, which comprises the following steps: (1) cutting and cracking cucumber fruits; (2) preparing a filter membrane and adsorbing gene DNA; (3) cleaning the filter membrane sheet; (4) eluting the DNA adsorbed by the filter membrane in the PCR reaction solution; (5) PCR amplification and product electrophoresis. The method can stably extract the gene DNA from the epidermis, the surface layer pulp and the center pulp of the cucumber for amplification, has simple operation, low cost and quick extraction, can extract the cucumber gene DNA suitable for amplification within 10 minutes, and is suitable for quick identification and analysis of cucumber genes.

Description

Rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption
Technical Field
The invention relates to the technical field of cucumber gene DNA extraction and amplification, in particular to a rapid extraction and amplification method of cucumber gene DNA.
Background
Cucumber is an important vegetable throughout the world, the cucumber genome project is already completed, various genomics research methods are actively developed, particularly, the cucumber genome is transformed, and a lot of researches on the aspects of disease resistance, insect resistance, stress resistance, herbicide resistance, quality improvement and the like are carried out, so that varieties with excellent properties are obtained, but in order to better confirm the varieties, the identification and analysis of specific genes of cucumber are common experimental methods.
In the identification and analysis of cucumber specific genes, cucumber gene DNA is extracted first. The conventional extraction methods are a CTAB method and an SDS method, generally fresh leaves are used as raw materials, grinding and other steps are needed, time and labor are wasted, and the leaves of cucumbers sold in the market are difficult to find. The commercialized kit mostly adopts a silica adsorption centrifugation method and a magnetic bead adsorption method. In silica adsorption centrifugation, nucleic acids are bound to a solid silica support in the presence of a high concentration of a chaotropic salt (chaotropic salt), followed by a series of washing and centrifugation steps to remove impurities, and finally elution of nucleic acids from the silica in a low concentration salt solution. The magnetic bead adsorption method is to modify functional groups on the surface of magnetic beads to realize the combination with nucleic acids, then to adsorb the magnetic beads by using a magnet to separate and clean impurities, and then to elute the nucleic acids on the magnetic beads.
The filter membrane (cell membrane) adsorption method can also be used for extracting nucleic acid, the modified filter membrane can be used for extracting genome DNA from human blood, can also be used for realizing the rapid extraction of nucleic acid from various organisms, and can also be used for the long-term storage of DNA evidences in forensics. The membrane adsorption method is simple and convenient to operate, does not need auxiliary equipment such as a centrifugal machine, a magnet and the like, and has great application potential.
Disclosure of Invention
The invention aims to provide a rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption.
The principle of the rapid extraction and amplification method of cucumber gene DNA adopted by the invention is shown in figure 1, 10-100mg of the surface layer pulp or the central pulp of a cucumber fruit is taken and put into a small tube, the small tube is pinched by tweezers until no large fruit is obvious, 100 mu L of lysate is added, the mixture is fully mixed, and the incubation is carried out for 1 minute at room temperature. Putting the prepared filter membrane into the reaction kettle, and acting for 1 minute at room temperature; taking out the filter membrane sheet, putting the filter membrane sheet into a small tube filled with cleaning fluid for cleaning, and acting for 1 minute at room temperature; and taking out the filter membrane sheet, putting the filter membrane sheet into the prepared PCR reaction solution, incubating for 1 minute at room temperature, removing the filter membrane sheet, carrying out nucleic acid amplification, and carrying out agarose gel electrophoresis analysis on the amplified product.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption comprises the following steps:
(1) cutting and cracking cucumber fruits;
(2) preparing a filter membrane and adsorbing gene DNA;
(3) cleaning the filter membrane sheet;
(4) eluting the DNA adsorbed by the filter membrane in the PCR reaction solution;
(5) PCR amplification and product electrophoresis.
Wherein, the cutting and cracking of the cucumber fruit is to take 10-100mg of the skin, surface layer pulp or center pulp of the cucumber fruit, put the cucumber fruit into a small tube, knead the cucumber fruit with tweezers until no obvious large fruit blocks exist, add 100 mu L of lysate, fully mix the mixture, and incubate the mixture for 1 minute at room temperature.
The preparation method of the lysis solution used in the cucumber fruit lysis process comprises the following steps: for example, 1L of the lysate was prepared, 100mL of 8M guanidine hydrochloride (final concentration: 800 mM), 100mL of 500mM Tris [ pH 8] (final concentration: 50 mM), 0.5mL of Triton X100, and 1mL of Tween-20 were sequentially added, and the volume was adjusted to 1L with purified water.
Wherein, the preparation of the filter membrane sheet is as follows, and the filter membrane sheet which accords with the following characteristics is selected: the content of the cellulose is more than or equal to 98 percent, the aperture is approximately equal to 10 mu m, the thickness is approximately equal to 200 mu m, and the filtering speed is approximately equal to 150 s. Cutting the filter membrane into round pieces with the diameter of 2-3 mm. Cutting into 2-3mm diameter round pieces, and drying.
Wherein, the step of adsorbing the gene DNA by the filter membrane is as follows, adding 100 mu L of lysate into the kneaded cucumber tissue, fully mixing the lysate and the lysate, and incubating the mixture for 1 minute at room temperature.
The preparation method of the cleaning solution used in the cleaning process of the filter membrane sheet comprises the following steps: for example, 1L of the washing solution was prepared, 20mL of 500mM Tris [ pH 8] (final concentration: 10 mM), 10mL of Tween-20 were added, and the volume was adjusted to 1L with purified water.
The method comprises the following steps of: the filter membrane sheet having the gene DNA adsorbed thereon was put into a vial containing 500. mu.L of the washing solution and incubated at room temperature for 1 minute.
Wherein, the PCR reaction solution of the filter membrane adsorbing DNA in the elution of the PCR reaction solution comprises the following components: PCR amplification essential components such as Taq enzyme, dNTPs, MgCl2 and the like, and target gene specific primers.
Wherein, the step of adsorbing DNA by the filter membrane and eluting in the PCR reaction solution is as follows: and putting the cleaned filter membrane into the PCR mixed solution, incubating for 1 minute at room temperature, and directly eluting the cucumber gene DNA in the PCR mixed solution.
Wherein, the PCR amplification and product electrophoresis steps are as follows: setting PCR program according to the size of amplified target gene fragment and annealing temperature, after PCR amplification, making agarose gel electrophoresis of amplified product, using gel imaging instrument to observe the presence or absence of amplified product, in the PCR amplification process setting positive and negative control.
Compared with the prior art, the invention has the outstanding effects that:
at present, the common gene DNA extraction methods of vegetables such as cucumbers and the like are a CTAB method and an SDS method, generally fresh leaves are used as raw materials, and a plurality of steps such as grinding, centrifugation and the like are needed, so that time and labor are wasted. The commercial kit mostly adopts a silica adsorption centrifugation method or a magnetic bead adsorption method. The silica adsorption centrifugation method needs a series of steps of cleaning, centrifugation and the like to remove impurities, the magnetic bead adsorption method also needs a plurality of steps and an external magnetic field to extract nucleic acid, and the two methods are long in time consumption and high in cost and also need laboratory equipment such as a centrifuge and the like. The method provided by the invention utilizes the principle that a filter membrane adsorbs DNA, after the cucumber fruit is cracked, the filter membrane adsorbs free gene DNA, impurities which may influence PCR, such as endogenous nuclease, are rinsed, nucleic acid is eluted into PCR reaction liquid, and the cucumber gene is directly amplified. The method of the invention has the advantages of simple operation, rapidness, low cost, good specificity and sensitivity, and is a method which can be applied to rapid extraction and amplification of cucumber gene DNA.
The membrane adsorption-based rapid extraction and amplification method for cucumber gene DNA will be further described with reference to the accompanying drawings and specific examples.
Drawings
FIG. 1 is a schematic diagram of a membrane adsorption-based rapid extraction and amplification method of cucumber gene DNA;
FIG. 2 shows the result of the extraction of cucumber gene DNA and the amplification of Act1 gene using 5 different lysates;
FIG. 3 shows the DNA extraction of cucumber gene and the amplification of Act1 gene at different cleavage times;
FIG. 4 shows the result of extraction of cucumber gene DNA and amplification of Act1 gene when filter membrane sheets are adsorbed at different times;
FIG. 5 shows the result of extraction of cucumber gene DNA and amplification of Act1 gene when the filter membrane is eluted at different times;
FIG. 6 shows the result of gene DNA extraction and Act1 gene amplification in samples of 50mg and 100mg of 3 parts of cucumber fruit;
FIG. 7 shows the sensitivity of cucumber epidermal gene DNA extraction and Act1 gene amplification;
FIG. 8 shows the sensitivity of DNA extraction and Act1 gene amplification of cucumber surface flesh;
FIG. 9 shows the sensitivity of DNA extraction and Act1 gene amplification of cucumber center pulp gene.
Detailed Description
The materials and reagents used in this example are specifically as follows:
materials:
a filter membrane sheet: the content of the cellulose is more than or equal to 98 percent, the aperture is approximately equal to 10 mu m, the thickness is approximately equal to 200 mu m, and the filtering speed is approximately equal to 150 s.
Reagent:
guanidine hydrochloride (guanidine hydrochloride), Tris (hydroxymethyl) aminomethane (Tris), Triton X100, Tween-20, cucumber gene Act1 specific primer (base sequence shown in Table 1), PCR reaction mixture (containing Taq enzyme, dNTPs, MgCl, etc.)2Etc.), purified water.
TABLE 1 related DNA sequences
Name (R) Sequence of
Act1 upstream primer 5’-GTGGTGGTGAATGAGTAGCC-3’
Act1 downstream primer 5’-TTGGATTCTGGTGATGGTGTC-3’
Buffer solution:
lysis buffer: 800mM guanidine hydrochloride, 50mM Tris [ pH 8], 0.5% Triton X100, 1% Tween-20
Washing buffer solution: 10mM Tris [ pH8.0], 0.1% Tween-20
A rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption comprises the following steps:
(1) cutting and cracking of cucumber fruits
1L of cucumber fruit lysate was prepared, and 100mL of 8M guanidine hydrochloride (final concentration: 800 mM), 100mL of 500mM Tris [ pH 8] (final concentration: 50 mM), 0.5mL of Triton X100, and 1mL of Tween-20 were sequentially added thereto, and the volume was adjusted to 1L with purified water. Taking 10-100mg of cucumber fruit skin, surface layer pulp or central pulp, putting into a small tube, kneading with forceps until there is no big fruit, adding 100 μ L of lysate, mixing well, and incubating at room temperature for 1 min.
(2) Preparing a filter membrane and adsorbing gene DNA;
selecting filter membrane, making cellulose membrane into circular membrane with diameter of 3mm with puncher, adding into cucumber tissue lysate to ensure that the membrane is completely immersed in liquid, incubating at room temperature for 1min
(3) Cleaning the filter membrane sheet;
the membrane was removed with forceps and placed in 500. mu.L of washing buffer (10 mM Tris [ pH8.0], 0.1% Tween-20) and incubated for 1min at room temperature.
(4) Eluting the DNA adsorbed by the filter membrane in the PCR reaction solution;
PCR reaction was prepared at 200 μ L according to the PCR kit instructions, where 2 × PCR Mix: 100 μ L, forward primer 0.5 μ L (100 μ M), reverse primer 0.5 μ L (100 μ M), water: 99 μ L. The PCR reaction solution was dispensed in 25. mu.L/tube. Transferring the membrane in the washing buffer solution into a PCR mixed solution by using tweezers, incubating at room temperature for 1min, removing the membrane by using the tweezers, and carrying out PCR reaction.
(5) PCR amplification and product electrophoresis.
The cucumber genome extracted by the commercial kit was added to the positive control tube in an amount of 0.5. mu.L as a sample, and the washing solution was added to the negative control tube in an amount of 0.5. mu.L as a sample. The PCR reaction condition is 95 ℃ for 1 min; 95 ℃ for 15s, 60 ℃ for 15s, 40 cycles. After the reaction was completed, the PCR product was electrophoresed on 2% agarose gel, and the result was observed with a gel imager.
The key parameters in the quick detection method are determined as follows:
(1) formula of lysis solution
Preparing five kinds of lysate:
lysate 1: 800mM guanidine hydrochloride, 50mM Tris [ pH 8], 0.5% Triton X100, 1% Tween-20;
lysate 2: 50mM Tris [ pH8.0], 150mM NaCl, 2% PVP, 1% Tween-20;
lysate 3: 2% (m/V) CTAB (cetyltriethylammonium bromide), 100mM Tris Cl [ pH8.0], 20mM EDTA [ pH8.0], 1.4mM NaCl;
lysate 4: 10% (M/V) CTAB, 0.7M NaCl;
lysis solution 5: 1% (m/V) CTAB, 50mM Tris Cl [ pH8.0], 10mM EDTA [ pH8.0 ].
Taking 5 parts of cucumber fruit surface pulp, each 100mg, putting the cucumber fruit surface pulp into a small tube, kneading the cucumber fruit surface pulp with tweezers, adding lysis solution 1, lysis solution 2, lysis solution 3, lysis solution 4 and lysis solution 5 into each tube respectively, performing nucleic acid extraction and Act1 gene amplification according to the steps, performing agarose gel electrophoresis on an amplification product (shown in figure 2), wherein a lane 1 is a DNA Marker, lanes 2-6 are lysis solution 1, lysis solution 2, lysis solution 3, lysis solution 4, lysis solution 5, a lane 7 is a positive control, and a lane 8 is a negative control.
Only lysate 1 achieved efficient amplification.
(2) Cracking time of cucumber
Taking 4 parts of cucumber fruit surface flesh, each 100mg, putting into a small tube, kneading with forceps, adding 100 μ L of lysis solution into each tube, performing nucleic acid extraction and Act1 gene amplification according to the previous steps, performing agarose gel electrophoresis on amplification products (shown in figure 3), wherein lane 1 is a DNA Marker, lanes 2-5 are respectively lysis for 1min, 2min, 4min and 8min, lane 6 is a positive control, and lane 7 is a negative control.
The lysis times did not differ significantly from 1min to 8 min.
(3) Adsorption time of filter membrane
Taking 4 parts of cucumber fruit surface flesh, each 100mg, putting into a small tube, kneading with forceps, adding 100 μ L of lysis solution into each tube, acting at room temperature for 1min, adding a filter membrane, acting for 1min, 2min, 4min and 8min, respectively, then performing nucleic acid extraction and Act1 gene amplification according to the steps, performing agarose gel electrophoresis on an amplification product (shown in figure 4), wherein a lane 1 is a DNA Marker, lanes 2-5 are acting for 1min, 2min, 4min and 8min, a cracked lane 6 is a positive control, and a lane 7 is a negative control.
The adsorption time of the filter membrane pieces is not obviously different from 1min to 8 min.
(4) Elution time of filter membrane
Taking 3 parts of cucumber fruit surface layer pulp, each 100mg, putting the cucumber fruit surface layer pulp into a small tube, kneading the cucumber fruit surface layer pulp with tweezers, adding 100 mu L of lysis solution into each tube, acting at room temperature for 1min, adding a filter membrane, acting at room temperature for 1min, taking out the filter membrane, putting a cleaning solution into the tube for cleaning, then putting the tube into a PCR mixed solution for elution, wherein the elution time is 1min, 2min and 4min respectively, then performing Act1 gene amplification according to the steps, performing amplification product agarose gel electrophoresis (shown in figure 5), wherein a lane 1 is a DNA Marker, lanes 2-4 are respectively elution times of 1min, 2min and 4min, a lane 5 is a positive control, and a lane 6 is a negative control.
There was no significant difference in the elution time of the filter plates from 1min to 4 min.
(5) Extraction results of different parts of cucumber
The method comprises the steps of taking 50mg and 100mg of cucumber fruit epidermis, surface layer pulp and center pulp respectively, putting the cucumber fruit epidermis, surface layer pulp and center pulp into a small tube, kneading the cucumber fruit skin, surface layer pulp and center pulp into the small tube by using forceps, adding 100 mu L of lysate into each tube, carrying out nucleic acid extraction and Act1 gene amplification according to the steps, carrying out agarose gel electrophoresis on an amplification product (shown in figure 6), wherein a lane 1 is a DNA Marker, a lane 2 is 50mg of epidermis, a lane 3 is 100mg of epidermis, a lane 4 is 50mg of surface layer pulp, a lane 5 is 100mg of surface layer pulp, a lane 6 is 50mg of center pulp, a lane 7 is 100mg of center pulp, and a lane 8 is a negative control.
The amplification effects of 3 parts of the cucumber fruit have no obvious difference, and a 50mg sample and a 100mg sample have no obvious difference.
(6) Cucumber epidermal gene extraction sensitivity
50mg, 25mg, 13mg and 6mg of cucumber fruit epidermis are respectively placed in a small tube, the small tube is crushed by a pair of tweezers, 100 μ L of lysate is respectively added into each tube, nucleic acid extraction and Act1 gene amplification are carried out according to the steps, amplification products are subjected to agarose gel electrophoresis (shown in figure 7), lane 1 is a DNA Marker, lane 2 is 50mg of epidermis, lane 3 is 25mg of epidermis, lane 4 is 13mg of epidermis, lane 5 is 6mg of epidermis, lane 6 is a negative control, and lane 7 is a positive control.
Cucumber epidermis achieved amplification at a minimum of 13 mg.
(7) Cucumber surface layer pulp gene extraction sensitivity
50mg, 25mg, 13mg and 6mg of surface layer pulp of cucumber fruit are respectively put into a small tube, and are kneaded by tweezers, 100 μ L of lysis solution is respectively added into each tube, nucleic acid extraction and Act1 gene amplification are carried out according to the steps, and the amplification product is subjected to agarose gel electrophoresis (shown in figure 8), wherein lane 1 is a DNA Marker, lane 2 is 50mg of surface layer pulp, lane 3 is 25mg of surface layer pulp, lane 4 is 13mg of surface layer pulp, lane 5 is 6mg of surface layer pulp, lane 6 is a negative control, and lane 7 is a positive control.
The amplification of the surface layer pulp of the cucumber is realized at least 13 mg.
(8) Cucumber central pulp gene extraction sensitivity
50mg, 25mg, 13mg and 6mg of central pulp of cucumber fruit are respectively put into a small tube, and are crushed by tweezers, 100 μ L of lysis solution is respectively added into each tube, nucleic acid extraction and Act1 gene amplification are carried out according to the previous steps, and the amplification product is subjected to agarose gel electrophoresis (shown in figure 9), wherein lane 1 is a DNA Marker, lane 2 is 50mg of central pulp, lane 3 is 25mg of central pulp, lane 4 is 13mg of central pulp, lane 5 is 6mg of central pulp, lane 6 is a negative control, and lane 7 is a positive control.
Amplification was achieved with a minimum of 25mg of cucumber center pulp.
(9) Positive and negative control results
In the experimental process, the cucumber genome extracted by using the commercial kit is used as a positive sample and added into the PCR mixed solution, the sample is used as a template to realize the high-efficiency amplification of the Act1 gene, a strip of 150bp is presented in agarose gel electrophoresis, and the positive sample is used as a positive control in the experiment of cucumber genome extraction. In the experimental process, the cleaning solution is used as a sample and added into the PCR mixed solution, the sample is used as a template to realize the amplification of the Act1 gene, no visible band exists in agarose gel electrophoresis, and the sample is used as a negative control in the cucumber genome extraction experiment.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.

Claims (7)

1. A rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption is characterized by comprising the following steps:
(1) cutting and cracking cucumber fruits;
(2) preparing a filter membrane and adsorbing gene DNA;
(3) cleaning the filter membrane sheet;
(4) eluting the DNA adsorbed by the filter membrane in the PCR reaction solution;
(5) PCR amplification and product electrophoresis.
2. The rapid extraction method of cucumber gene DNA based on membrane adsorption as claimed in claim 1, which is characterized in that: the preparation method of the cucumber fruit lysate comprises the following steps: 800mM guanidine hydrochloride, 50mM Tris [ pH 8], 0.5% Triton X100, 1% Tween-20.
3. The rapid extraction method of cucumber gene DNA based on membrane adsorption as claimed in claim 3, characterized in that: the cracking method of the cucumber fruits comprises the steps of taking 10-100mg of cucumber fruit skin, surface layer pulp or center pulp, putting the cucumber fruit skin, surface layer pulp or center pulp into a small tube, kneading the cucumber fruit skin, surface layer pulp or center pulp into the small tube by using forceps until no large fruit blocks are obvious, adding 100 mu L of lysate, fully and uniformly mixing, and incubating for 1 minute at room temperature.
4. The rapid extraction method of cucumber gene DNA based on membrane adsorption as claimed in claim 2, characterized in that: the selection preparation and DNA adsorption of the filter membrane sheet have the following basic properties: the cellulose content is more than or equal to 98 percent, the aperture is approximately equal to 10 mu m, the thickness is approximately equal to 200 mu m, the filtering speed is approximately equal to 150s, the filter membrane sheet is cut into a wafer with the diameter of 2-3mm, the wafer is put into the cucumber tissue lysate, and the incubation is carried out for 1 minute at room temperature.
5. The rapid extraction method of cucumber gene DNA based on membrane adsorption as claimed in claim 4, characterized in that: cleaning the filter membrane sheet, wherein the formula of the cleaning solution is as follows: 10mM Tris [ pH 8], 1% Tween-20, the filter membrane sheet with the gene DNA adsorbed thereon was put into a vial containing a washing solution and incubated at room temperature for 1 minute.
6. The rapid extraction method of cucumber gene DNA based on membrane adsorption as claimed in claim 5, characterized in that: and adsorbing the elution of the DNA by the filter membrane, putting the washed filter membrane into a PCR mixed solution, incubating at room temperature for 1 minute, directly eluting the cucumber gene DNA into the PCR mixed solution, wherein the PCR mixed solution comprises necessary components for carrying out PCR reaction and primers for amplifying a target gene.
7. The rapid extraction method of cucumber gene DNA based on membrane adsorption as claimed in claim 6, characterized in that: and after the DNA adsorbed by the filter membrane is eluted in the PCR mixed solution, removing the filter membrane, selecting amplification parameters according to target genes, directly carrying out PCR reaction, and carrying out electrophoretic analysis on reaction products.
CN202110691423.2A 2021-06-22 2021-06-22 Rapid extraction and amplification method of cucumber gene DNA based on membrane adsorption Pending CN113528505A (en)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2018195594A1 (en) * 2017-04-24 2018-11-01 The University Of Queensland Simple nucleic acid extraction
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CN112430687A (en) * 2020-12-10 2021-03-02 广西大学 Method for rapidly detecting potato virus based on RT-PCR (reverse transcription-polymerase chain reaction) of filter paper strip capture virus nucleic acid

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018195594A1 (en) * 2017-04-24 2018-11-01 The University Of Queensland Simple nucleic acid extraction
CN109207475A (en) * 2018-10-22 2019-01-15 博迪泰（厦门）生物科技有限公司 A kind of Rapid nucleic acid extracting method
CN112430687A (en) * 2020-12-10 2021-03-02 广西大学 Method for rapidly detecting potato virus based on RT-PCR (reverse transcription-polymerase chain reaction) of filter paper strip capture virus nucleic acid

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Title
薛强 等: "基于纤维素膜吸附的黄瓜基因组DNA快速提取方法", 《中国瓜菜》 *

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Application publication date: 20211022