CN108531477B

CN108531477B - Method for extracting chloroplast DNA of main vegetable crops of allium and establishment of quality evaluation system thereof

Info

Publication number: CN108531477B
Application number: CN201810421963.7A
Authority: CN
Inventors: 霍雨猛; 吴雄; 杨妍妍; 刘冰江; 高莉敏; 孔素萍; 杨宁; 孙雨晴
Original assignee: Vegetable Research Institute of Shandong Academy of Agricultural Sciences
Current assignee: Vegetable Research Institute of Shandong Academy of Agricultural Sciences
Priority date: 2018-05-04
Filing date: 2018-05-04
Publication date: 2020-07-14
Anticipated expiration: 2038-05-04
Also published as: CN108531477A

Abstract

The invention discloses a chloroplast DNA extraction method of main vegetable crops of allium and establishment of a quality evaluation system thereof. In addition, the effect of the high-salt low-pH method and the sucrose DNase method on extracting the chloroplast DNA content of the green Chinese onions is evaluated through a fluorescence quantitative ratio evaluation system of the chloroplast DNA content, and then the high-salt low-pH method is used for testing the onion, the garlic and the leek of the allium vegetable crops, so that the expected effect is obtained. The DNA extraction method of the invention has high DNA yield; the purity is high, and the pollution of impurities such as polysaccharide, protein and the like is avoided; the chloroplast DNA content is high, and the foundation is laid for further extracting high-purity chloroplast DNA without nuclear pollution and sequencing the whole genome of an organelle.

Description

Method for extracting chloroplast DNA of main vegetable crops of allium and establishment of quality evaluation system thereof

Technical Field

The invention relates to the technical field of molecular biology experiments, in particular to a method for extracting chloroplast DNA of main vegetable crops of allium and establishment of a quality evaluation system thereof.

Background

Chloroplasts are semi-autonomous organelles for photosynthesis of plants, participate in metabolism of various substances such as nucleotides, amino acids, pigment proteins and the like (Chenchumei and Chenliang, 2014), are originally found in autotrophic cyanobacteria, and exist in plants, algae and partial protists in the form of plastids after evolution (Neuhaus and Emes,2000), and Hans and Walter (1962) prove the existence of chloroplast genomes with the development of plant molecular biology. The chloroplast genome of higher plants is mostly 120-160 kb, the DNA of the chloroplast genome exists in a covalent, closed and circular double-stranded form, and the chloroplast DNA (chloroplastDNA, cpDNA) is relatively conservative, so the chloroplast DNA is widely used for various researches such as cytoplasmic inheritance, plant phylogeny, genetic diversity and genetic relationship (Palmer, 1985; Du et al, 2017).

With the development of High-throughput sequencing technology, 1411 kinds of green plants which have currently completed chloroplast whole genome sequencing are provided, and the obtainment of High-quality cpDNA is the basis for further research (luogong et al, 2015; evergweason et al, 2016). at present, cpDNA extraction methods mainly include a High-salt low-pH method (High-salt L ow-pH, HS L p), a sucrose DNase method and a density gradient centrifugation method (bookjankjans et al, 1984; Shi et al, 2012; Triboush et al, 1998; Vieira et al, 2014). existing research shows that the High-salt low-pH method has a wider application range, and the High-salt low-pH method is successfully applied to tea trees (old spring plum and old eucalyptus), sugarcane (dnesli et al, 2014), pine (Vieira, et al, chloroplast), wheat (chloroplast etc., chloroplast, li), urophysa et al (tenuifolia, etc.), eucalyptus, cauda et al, 2017), a High-density DNA extraction method (2017, 201 et al, a High-leaf centrifugation method for obtaining High-yield of High-purity DNA by centrifugation of plants after the simple and High-purity DNA extraction of a High-yield of a High-purity and High-yield of a plant DNA extraction method by centrifugation method of a High-yield of a High-salt low-yield plant such as a plant DNA extraction method after centrifugation method and a simple centrifugation method for obtaining High-yield of a High-purity plant DNA purification of a High-enriched sperm plant such as a low-enriched sperm plant.

Disclosure of Invention

In view of the above-described deficiencies of the prior art, the present invention provides an improved high salt, low pH process for extracting cpDNA from allium vegetable crops. Gerpc in Shuoshi paper of RAPD marker for cytoplasmic male sterility in Welsh onion scrapes off mucus in Welsh onion with a blunt knife, extracts cpDNA with high-salt buffer solution, and obtains high-purity cpDNA through the steps of differential centrifugation, lysis, protein extraction, purification and the like, but the DNA yield is about 20 mu g/100g of material, but the mucus scraping is required firstly, and the yield is low. The high-salt low-pH method is used for extracting the cpDNA of the allium vegetable crop, and the direct extraction is realized without scraping mucus, so that the operation process is simple and convenient; the DNA yield is up to 48.2 mu g/100g of material, which is 2.41 times higher than that of the Geroc method; the DNA has higher purity and no pollution of impurities such as polysaccharide, protein and the like; the cpDNA content is 5.05 times higher than that of the sucrose DNase method.

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention provides a method for extracting chloroplast DNA of main vegetable crops of allium by a high-salt low-pH method, which comprises the following steps:

(1) cleaning and crushing fresh leaves after dark treatment, putting the fresh leaves into a homogenizer, and adding Buffer I into the homogenizer for homogenizing;

(2) filtering, homogenizing and centrifuging;

(3) filtering the supernatant obtained by the centrifugation again, centrifuging, discarding the supernatant, and retaining the precipitate to obtain the crude chloroplast;

(4) adding Buffer II into the precipitate, suspending to constant volume, centrifuging, and retaining the precipitate.

(5) Adding Buffer III into the precipitate, suspending, centrifuging, and retaining the precipitate to obtain purified chloroplast.

The method for extracting the DNA is an extraction method and a reagent which are suitable for the cpDNA of allium vegetable crops and are screened based on a Vieira and other (2014) high-salt low-pH value method.

The formula of the extraction buffer solution is as follows:

Buffer I(pH 3.8)：0.5-2mol/L NaCl,0.25mol/L ascorbic acid(Vc),10mmol/Lsodium metabisulfite，0.0125mol/L Borax,50mmol/L Tris-HCl(pH 8.0),7mmol/LEDTA,1％PVP-40,0.1％BSA；

Buffer II(pH 8.0)：0.5-2mol/L NaCl,0.0125mol/L Borax,1％PVP-40,50mmol/L Tris-HCl(PH 8.0),25mmol/L EDTA,0.1％BSA；

Buffer III：0.5-2mol/L NaCl,100mmol/L Tris-HCl(PH 8.0),50mmol/L EDTA,10mmol/Lβ-Me；

preferably, the optimum concentration of NaCl in Buffer I, Buffer II and Buffer III is 1.5 mol/L.

Preferably, the amount of the fresh leaves in the step (1) is 45-55 g;

further, preferably, the fresh leaves in step (1) are 50 g;

preferably, the dark treatment time in the step (1) is 24-48 h;

preferably, the length of the cut pieces in the step (1) is 0.4-0.6 cm;

preferably, the homogenizer in the step (1) is homogenized, the homogenizer needs to be pre-cooled in advance, and the homogenizer is firstly homogenized for 20s at a low speed and then is homogenized for 15s at a high speed for two times;

preferably, the homogenate is filtered as described in step (2) by a method comprising: filtering with 2 layers of gauze, homogenizing, adding into a precooled beaker, filtering with 1 layer of Miracloth, and packaging the filtrate into a centrifuge bottle;

preferably, the centrifugation in the step (2) is performed at 4 ℃ and 200g for 8-12 min;

preferably, the filtration described in step (3) is a 2-layer Miracloth filtration;

preferably, the centrifugation in the step (3) is performed at 3220g at 4 ℃ for 8-12 min;

preferably, the centrifugation in the step (4) is performed at 3220g at 4 ℃ for 8-12 min;

preferably, the centrifugation in the step (5) is 2000g centrifugation at 4 ℃ for 8-12 min;

according to the characteristic that allium vegetable crops are rich in flavonoids and polysaccharides, the concentration of NaCl in buffers Buffer I, Buffer II and Buffer III of a conventional high-salt low-pH method is creatively increased from 1.25 to 1.5 mol/L, so that the yield of the extracted cpDNA is improved, the NaCl can influence the osmotic pressure of cells, the allium vegetable crops are rich in polysaccharides, the osmotic pressure in the cells is higher, and the osmotic pressure outside the cells is low under the condition of relatively low Buffer salt concentration, so that the cells and organelles absorb water in homogenate to be broken in advance to cause DNA loss.

The invention provides a group of primers for evaluating the copy number of organelles and nuclear genes of allium vegetable crops, wherein the organelle genes are chloroplast gene MatK and mitochondrial gene ATP6, and the nuclear gene L FY gene, the gene primer sequences are respectively SEQ ID NO.1 and SEQ ID NO.2 for MatK gene upstream and downstream primers, SEQ ID NO.3 and SEQ ID NO.4 for ATP6 gene upstream and downstream primers, and SEQ ID NO.5 and SEQ ID NO.6 for L FY gene upstream and downstream primers.

The application of the primer in the evaluation of the copy number of the organelle and nucleus genes of the allium vegetable crops is also within the protection scope of the invention.

The invention also provides a fluorescence quantitative ratio evaluation system of chloroplast DNA content, and the method comprises the following steps: and (3) taking the total DNA extracted by the kit as a control, calculating the extraction effect of the chloroplast DNA by the two methods according to the amplification efficiency of the organelle genes and the nuclear genes amplified by the primers, and determining the chloroplast DNA extraction method.

The evaluation system is applied to screening of a chloroplast DNA extraction method.

The invention has the beneficial effects that:

(1) the invention provides a method for extracting chloroplast DNA of main vegetable crops of allium by a high-salt low-pH method, which has high DNA yield of 0.482 mu g/g & FW (48.2 mu g/100g & FW), and the yield of the chloroplast DNA extracted by Gekkonio in the RAPD marker of cytoplasmic male sterility of green Chinese onion is 20 mu g/100g and is 2.41 times higher than that of the chloroplast DNA extracted by Gekkonio; the yield of the cpDNA extracted by the sucrose DNase method is 8.17 times higher than that of the cpDNA extracted by the sucrose DNase method, wherein the yield of the cpDNA is 0.059 mu g/g & FW.

(2) The invention provides a method for extracting chloroplast DNA of main vegetable crops of allium by a high-salt low-pH method, wherein the extracted DNA has high purity and is free from the pollution of impurities such as polysaccharide, protein and the like;

(3) the invention provides a fluorescence quantitative ratio evaluation system of chloroplast DNA content, which evaluates the effect of a high-salt low-pH method and a sucrose DNase method on extracting the chloroplast DNA of green Chinese onions, and the result shows that the high-salt low-pH method is superior to the sucrose DNase method, namely the evaluation of the chloroplast DNA content shows that the high-salt low-pH method is 5.05 times of the sucrose DNase method, and the mitochondrial DNA pollution of high-salt low-pH normal is less than that of the sucrose DNase method.

(4) The method is applied to other allium vegetable crops, and a good extraction effect is also obtained. The method is rapid, time-saving and low in cost.

(5) The invention provides a basis for the efficient, high-purity and nuclear pollution-free chloroplast DNA extraction research of allium crops in the future and provides a method basis for the whole genome sequencing of allium vegetable crop organelles.

Drawings

FIG. 1 shows the NaCl concentration gradient test, where M: D L15000 DNA Marker, 1: 2.0 mol/L, 2: 1.5 mol/L, 3: 1.0 mol/L, and 4: 0.5 mol/L.

FIG. 2 shows two electrophoretic images of cpDNA extracted by two methods, wherein M: D L15000 DNA Marker, 1: high-salt low-pH method, and 2: sucrose DNase method.

FIG. 3 is a melting curve assessment of chloroplast MatK, mitochondrial ATP6, and nuclear L FY specific gene detection primer amplification.

FIG. 4 is a standard curve of chloroplast MatK, mitochondrial ATP6, and nuclear L FY specific gene amplification and sample assays thereof.

FIG. 5 is a cPDNA electrophoresis test of different Allium vegetable crop leaves extracted by high salt low pH method, wherein M is 15000 bpMarker; 1: scallion; 2: garlic; 3: onion; 4: and (4) leek.

Detailed Description

The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions specified in the examples are not indicated, they are generally according to the conventional conditions, or according to the conditions recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.

1 Material

The chapter-dune-shaped green onions 2015B, 108M, garlic and leeks were collected from vegetable and flower institute of academy of agricultural sciences of Shandong province, and the tissue type was mature green leaves grown in the field.

2 preparation of reagents

2.1 sucrose DNase method buffer reagent

STE：0.4mol/L sucrose,50mmol/L Tris(pH 7.8),20mmol/L EDTA,0.2％BSA,0.2％β-Me

ST：0.4mol/L sucrose,50mmol/L Tris(pH 7.2),0.1％BSA

NET：50mmol/L Tris(pH 8.0),50mmol/L EDTA,1.25mol/L NaCl

2.2DNA extraction reagent

100 mmol/L Tris pH 8.0, 50 mmol/L EDTApH 8.0, 500 mmol/L NaCl, 20% SDS, phenol chloroform isoamyl alcohol (25:24:1), isopropanol, 75% ethanol, TE, DNase I, plant DNA extraction kit (Tiangen Biochemical technology Co., Ltd., Beijing, DP 321-03).

Example 1 high salt Low pH Process (ref. Vieira, et al, 2014)

The buffer reagent is:

Buffer I(pH 3.8):1.5mol/L NaCl,0.25mol/L ascorbic acid(Vc),10mmol/Lsodiummetabisulfite，0.0125mol/L Borax,50mmol/L Tris-HCl(pH 8.0),7mmol/L EDTA,1％PVP-40,0.1％BSA；

Buffer II(pH 8.0):1.5mol/L NaCl,0.0125mol/L Borax,1％PVP-40,50mmol/LTris-HCl(PH 8.0),25mmol/L EDTA,0.1％BSA；

Buffer III：1.5mol/L NaCl,100mmol/L Tris-HCl(PH 8.0),50mmol/L EDTA,10mmol/Lβ-Me。

the method comprises the following steps:

a. taking 50g of fresh leaves, carrying out dark treatment for 24-48h, washing the fresh leaves with sterile double-distilled water, absorbing residual water with absorbent paper, cutting the leaves into 0.5cm in length, putting the leaves into a precooled homogenizer, adding L Buffer I of 400m, homogenizing the pulp at a low speed for 20s once, and homogenizing the pulp at a high speed for 15s twice.

b. Filtering with 2 layers of gauze, homogenizing, adding 1 layer of Miracloth, filtering, packaging the filtrate into centrifuge bottles, and centrifuging at 4 deg.C for 10min at 200 g.

c. And filtering the supernatant through 2 layers of Miracloth into a new centrifugal tube, centrifuging for 15min at the temperature of 4 ℃ by 3220g, and removing the supernatant to obtain precipitate, namely the crude chloroplast.

d. 2m of L precooled Buffer II is added into the precipitate, after the precipitate is fully suspended, the volume is adjusted to 40m L, the mixture is centrifuged at 3220g at 4 ℃ for 10min, and the supernatant is discarded.

e. Adding 20m L Buffer III into the precipitate, fully suspending and precipitating, centrifuging at 4 ℃ for 10min at 2000g, and removing supernatant to obtain the purified chloroplast.

f. Purified chloroplasts were subjected to DNA extraction using SDS, reference (Shi, et al, 2012) 0.8% agarose gel electrophoresis in 0.5 × TBE electrophoresis buffer, EB staining for visualization and photography.

EXAMPLE 2 treatment with NaCl at different concentrations

This example differs from example 1 in that steps d and e are omitted and the NaCl concentrations in Buffer I are set to 2.0 mol/L, 1.5 mol/L, 1.0 mol/L and 0.5 mol/L, respectively, i.e., the effect of NaCl concentration on the amount of extraction is screened by directly performing the DNA extraction process after obtaining chloroplast crude material.

As can be seen from FIG. 1, in the NaCl concentration gradient of 0.5-2.0 mol/L, the yield of DNA is gradually increased with the increase of NaCl concentration, and there is a significant difference between 0.5 mol/L and 1.5 mol/L, and the difference between 1.5 mol/L and 2.0 mol/L is smaller, therefore, the optimal NaCl concentration is selected to be 1.5 mol/L.

Comparative example 1 sucrose DNase method (Triboush, et al.,1998)

a. Sample processing was performed by the high salt low pH method using STE, ST and NET buffers (prepared as described above for buffer reagent for the 2.1 sucrose DNase method).

b. The crude chloroplast pellet was suspended thoroughly in 2ml STE buffer, then the volume was adjusted to 40m L, centrifuged at 4 ℃ 3200g for 15min, and the supernatant was discarded.

c. Each 0.1g of precipitate was suspended in 200. mu.l of ST and 0.02 mol/L MgCl was added₂The DNA was digested at 37 ℃ for 20min with a final concentration of 25. mu.g/m L DNase I, and the nuclear DNA was eliminated.

d. Adding EDTA to final concentration of 0.2 mol/L, mixing, standing for 10-20min, and terminating digestion.

e. Adding NET into the reaction solution to the maximum volume of a centrifugal tube, centrifuging for 10min at the temperature of 4 ℃ at 3220g, removing DNase I, and obtaining the precipitate, namely the purified chloroplast.

As can be seen from FIG. 2, the molecular weights of the cpDNA extracted by the two methods are similar and are both larger than 15Kbp, and the cpDNA is not polluted by impurities such as polysaccharide, protein, RNA and the like. However, under the condition that the fresh weight, the dissolution volume and the loading amount of the plant sample are the same, the cpDNA electrophoretic band brightness extracted by the high-salt low-pH method is far greater than that of the sucrose DNase method, and is consistent with the yield detection result, and the beneficial effect of the high-salt low-pH method is further proved by the result of FIG. 4.

EXAMPLE 3 determination of the purity and yield of DNA extracted by two methods

OD measurement Using a Spectrophotometer₂₃₀、OD₂₆₀And OD₂₈₀And calculating the yield.

OD of onion cpDNA₂₆₀/OD₂₈₀、OD₂₆₀/OD₂₃₀And concentration values (Table 1) As can be seen, cpDNAod extracted by the two methods₂₆₀/OD₂₈₀All values are greater than 1.8, OD₂₆₀/OD₂₃₀The value is about 2, and the parameters show that the DNA extracted by the two methods has higher purity and is consistent with the agarose gel electrophoresis picture. Further yield analysis showed that the cpDNA yield extracted by the high-salt low-pH method was 0.482. mu.g/g.FW (Fresh Weight, FW), the sucrose DNase method was 0.059. mu.g/g.FW, and the high-salt low-pH method was 8.17 times that of the sucrose DNase method. Therefore, the yield of the onion cpDNA extracted by the high-salt low-pH method is obviously higher than that of the onion cpDNA extracted by the sucrose DNase method.

TABLE 1 purity and yield of cpDNA extracted by two methods

Example 4DNA copy number detection

The method refers to (Hao, et al, 2014), firstly, segments of chloroplast gene MatK, nuclear gene L FY and mitochondrial gene ATP6 are amplified by utilizing a PCR technology, a plurality of sets of organelle evaluation primers are designed, preferably, the sequences of the primers are respectively SEQ ID No.1 and SEQ ID No.2 for the upstream and downstream primers of the MatK gene, SEQ ID No.3 and SEQ ID No.4 for the upstream and downstream primers of ATP6 gene, and SEQ ID No.5 and SEQ ID No.6 for the upstream and downstream primers of L FY gene, the amplification effect of the primers is stable by carrying out PCR detection on a sample, the melting point temperature corresponding to the peak value of a melting curve is consistent (shown in figure 3), test results prove that the set of primers can be well applied to copy number detection of allium fistulosum organelles (shown in figure 4), then, a standard curve of three segments is prepared simultaneously by taking a PCR product as a standard, the amplification efficiency E value is obtained, the chloroplast extraction effect (the ratio of DNA to chloroplast DNA to nuclear DNA) is evaluated as formula (1), the total DNA (total DNA) is set as a sample, the result of the comparative DNA is obtained by adopting a comparative sample dilution method, the comparative method is changed into a comparative method for obtaining the comparative result of obtaining the comparative sample of the comparative gene analysis of the residual gene analysis of the chloroplast extraction efficiency E value of the chloroplast extraction efficiency (the chloroplast extraction efficiency is changed from the comparative example of the chloroplast extraction efficiency is shown in which is equal to the comparative example of the.

The reaction system comprises ddw 8 mu L, Mix 10 mu L, upstream and downstream primers 0.5 mu L respectively, template 1 mu L and 20 mu L in total, and the reaction program comprises 95 ℃ for 3min, denaturation at 95 ℃ for 10s, denaturation at 60 ℃ for 10s, and denaturation at 72 ℃ for 20s, and the reaction program comprises 39 cycles, and preservation at 72 ℃ for 10min and 4 ℃.

From the standard curves of chloroplast MatK, mitochondrial ATP6 and nuclear L FY specific gene amplification, it can be seen that their amplification efficiencies are 90.1%, 93.8% and 92.9%, respectively, and R is²Are all greater than 0.99 (fig. 4). And (3) taking the total DNA extracted by the kit as a control, calculating the extraction effect of the chloroplast DNA by the two methods according to the amplification efficiency of each organelle primer, and determining the chloroplast DNA extraction method. The ratio of the chloroplast DNA extraction effect of the high-salt low-pH method and the sucrose DNase method to the kit extraction effect is 78.38 and 15.51 respectively, and the high-salt low-pH method is 5.05 times of the sucrose DNase method. However, the samples extracted by both methods contained different levels of mitochondrial DNA contamination (Table 2), and from the contamination level analysis, the high-salt low-pH method was 0.87, the sucrose DNase method was 2.49, and the contamination level of mitochondrial DNA was much lower than that of the sucrose DNase method and the Kit method.

TABLE 2 comparison of the Effect of the two methods on cpDNA extraction

Note that Cq is the number of cycles, E is the Efficiency of amplification, HS L p is the High Salt L ow pH, the High Salt low pH method, the SucDNase is the sucrose DNase method, and Kit is the total DNA extraction Kit.

Comparative example 2

The other primer set designed by the invention comprises upstream and downstream primers of MatK gene of SEQ ID NO.7 and SEQ ID NO.8, upstream and downstream primers of ATP6 gene of SEQ ID NO.9 and SEQ ID NO.10, upstream and downstream primers of L FY gene of SEQ ID NO.11 and SEQ ID NO.12, PCR detection is carried out on fragment samples of chloroplast gene MatK, nuclear gene L FY and mitochondrial gene ATP6 in the embodiment 4, and the amplification condition is the same as that in the embodiment 4.

Amplification shows that the primer group is unstable in amplification, is easy to generate nonspecific amplification and dimer phenomena, and cannot be used for copy number detection tests.

Therefore, the primer group can be well applied to the evaluation of the organelle and nucleus gene copy number of the allium vegetable crops.

Example 5 evaluation of the application of the high-salt Low-pH method in chloroplast DNA extraction of different Allium vegetable crops

To verify the applicability of the high salt low pH method to chloroplast DNA samples of different allium vegetable crops, we subsequently tested onion, garlic, and leek samples. The sample extracted by the high-salt low-pH method has clear sample application holes and clear and bright DNA bands without diffusion phenomenon, and the method can be used for the extraction research of chloroplast DNA of a plurality of allium vegetable crops (figure 5).

Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive faculty, based on the technical solutions of the present invention.

SEQUENCE LISTING

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Claims

1. A method for extracting chloroplast DNA of allium vegetable crops is characterized by comprising the following steps:

(1) cleaning and cutting fresh leaves after dark treatment, putting the leaves into a homogenizer, and adding Buffer I into the leaves for homogenizing; the fresh leaves are 45-55 g; the dark treatment time is 24-48 h; pre-cooling the homogenizer in advance, homogenizing for 20s at a low speed during homogenizing, and then homogenizing for 15s at a high speed for two times;

(2) filtering, homogenizing and centrifuging; the filtration homogenate comprises the following specific steps: filtering with 2 layers of gauze, homogenizing, adding into a precooled beaker, filtering with 1 layer of Miracloth, and packaging the filtrate into a centrifuge bottle; the centrifugation is carried out at 4 ℃ for 8-12min under 200 g;

(3) filtering the supernatant through 2 layers of Miracloth again to a new centrifugal bottle, centrifuging for 15min at 3220g at 4 ℃, and removing the supernatant to obtain precipitate, namely crude chloroplast;

(4) adding Buffer II into the precipitate, suspending to a constant volume, centrifuging, and keeping the precipitate; the centrifugation is carried out for 8-12min at 3220g at 4 ℃;

(5) adding Buffer III into the precipitate, suspending, centrifuging, and retaining the precipitate to obtain purified chloroplast; the centrifugation is 2000g at 4 ℃ for 8-12 min;

1.5 mol/L NaCl, 0.25 mol/L ascorbyl acid, 10 mmol/L sodium metabisulfite, 0.0125 mol/L Borax, 50 mmol/L Tris-HCl with pH of 8.0, 7 mmol/L EDTA,1% PVP-40 and 0.1% BSA, wherein the pH value of the Buffer I is 3.8;

1.5 mol/L NaCl, 0.0125 mol/L Borax, 1% PVP-40, 50 mmol/L Tris-HCl with pH of 8.0, 25 mmol/L EDTA and 0.1% BSA, wherein the pH value of the Buffer II is 8.0;

the Buffer III is 1.5 mol/L NaCl, 100 mmol/L Tris-HCl with pH 8.0, 50 mmol/L EDTA, 10 mmol/L β -Me.

2. The method of claim 1, wherein the purified chloroplast is assessed for DNA extracted from the chloroplast using primers for assessing the copy number of organelle and nuclear genes from allium vegetable crops, the organelle gene being a chloroplast geneMatKGene and mitochondrial geneATP6Nuclear gene of cell nucleusLFYA gene; the gene primer sequences are respectively as follows:MatKthe upstream and downstream primers of the gene are SEQ ID number 1 and SEQ ID number 2;ATP6the upstream and downstream primers of the gene are SEQ ID number 3 and SEQ ID number 4;LFYthe upstream and downstream primers of the gene are SEQ ID number 5 and SEQ ID number 6.

3. The method according to claim 2, wherein the total DNA extracted from the kit is used as a control, and the effect of extracting chloroplast DNA is calculated based on the amplification efficiency of the organelle gene and the nuclear gene amplified by the primers.