阅读说明：本技术 一种田菁线粒体及其dna提取的有效方法 (Sesbania mitochondria and effective method for extracting DNA thereof ) 是由 贺亭亭 邢锦城 洪立洲 于 2019-12-23 设计创作，主要内容包括：本发明公开了一种田菁线粒体及其DNA提取的有效方法,属于植物生物技术领域。以田菁黄化苗为材料,优化操作后获得的田菁组织提取液通过调整后的转速离心去除杂质,分离并富集粗制的线粒体,经过蔗糖梯度离心法对线粒体进行纯化,再次通过离心富集纯化后的线粒体。利用改良后的SDS法提取的线粒体DNA其A260/A280约为1.80～1.88,限制性内切酶EcoR I酶切后条带清晰,说明本方法提取的线粒体DNA纯度高。利用该发明所公开的方法能够简单快捷的提取到纯净的田菁线粒体DNA,为田菁线粒体基因组及其内部基因的分子机理研究奠定了基础。(The invention discloses sesbania mitochondria and an effective method for extracting DNA thereof, belonging to the technical field of plant biology. And (3) taking the sesbania etiolated seedlings as materials, centrifuging the sesbania tissue extract obtained after optimized operation at an adjusted rotating speed to remove impurities, separating and enriching crude mitochondria, purifying the mitochondria by a sucrose gradient centrifugation method, and centrifuging again to enrich the purified mitochondria. The A260/A280 of the mitochondrial DNA extracted by the improved SDS method is about 1.80-1.88, and the band of the mitochondrial DNA extracted by the method is clear after the restriction enzyme EcoR I is cut, which shows that the purity of the mitochondrial DNA extracted by the method is high. The method disclosed by the invention can be used for simply and quickly extracting pure sesbania mitochondrial DNA, and lays a foundation for the molecular mechanism research of sesbania mitochondrial genomes and internal genes thereof.)

1. An effective method for extracting sesbania mitochondria and DNA thereof is characterized by comprising the following steps:

1) dark culture is carried out on sesbania seeds, a green light lamp is used when illumination is needed, grinding buffer solution is utilized to crush quantitative sesbania etiolated seedlings, and tissue homogenate is obtained by filtering through gauze and a nylon net in sequence;

2) removing tissue fragments and cell nucleuses from the homogenate by differential centrifugation, and removing DNA in a mitochondrial solution by using DNase I to obtain a crude mitochondria;

3) after being resuspended by buffer solution, the crude mitochondria are transferred to discontinuous density gradient liquid paved by cane sugar, and then purified mitochondria are obtained through ultra-high speed centrifugation;

4) mitochondrial DNA is extracted by an improved SDS method to obtain high-purity DNA without protein and RNA pollution;

5) and (3) carrying out concentration detection, electrophoresis band detection and purity detection on the extracted sesbania mitochondrial DNA.

2. The effective method for extracting sesbania mitochondria and DNA thereof as claimed in claim 1, wherein in step 1), sesbania cultured for 2 weeks is used as a mitochondrial extraction material including upper and lower hypocotyls and leaves due to the limitation of the growth rate in the seedling stage, cysteine and β -mercaptoethanol are added to the grinding buffer to reduce the oxidative damage of mitochondria, the homogenate is filtered by 2 layers of sterile absorbent gauze and then by 2 layers of 100 mesh nylon net, and the filtrate is collected on ice.

3. The method of claim 1, wherein the method comprises the steps of: in the step 2), the homogenate liquid is centrifuged for 15min in a high-speed centrifuge at the rotating speed of 2200 g; centrifuging the supernatant at high speed for 15min again at a rotation speed of 17000 g; adding a buffer solution and DNase I into the precipitate until the concentration of the DNase I is 50 mu g/mL, and adding EDTA to stop the reaction in ice bath for 1 h; centrifuging at high speed for 20min, and rotating at 18000g to obtain crude mitochondria.

4. The method of claim 1, wherein the method comprises the steps of: in step 3), the mitochondrial pellet was resuspended in buffer and transferred to a discontinuous density gradient solution spread with 5mL of 1.45M and 5mL of 1.2M sucrose; centrifuging for 90min by using an ultra-high speed centrifuge at the rotating speed of 20600 rpm; collecting mitochondria between sucrose gradient solutions; centrifuging for 20min by a high-speed centrifuge at the rotating speed of 20000g to obtain the precipitate as the purified mitochondria.

5. The method of claim 1, wherein the method comprises the steps of: and 4) adding a lysis buffer solution into the purified mitochondria, adding protease K, SDS and RNase into the purified mitochondria according to the system, then carrying out water bath at 37 ℃ for 3h, cooling to room temperature, adding NH4AC solution, then extracting proteins cleanly according to a conventional DNA extraction method, and dissolving and storing in sterile water to obtain the high-purity mitochondrial DNA.

6. The method of claim 1, wherein the method comprises the steps of: and 5) detecting the concentration and purity of sesbania mitochondrial DNA by using a microplate reader, detecting the integrity of the DNA by agarose (1%) gel electrophoresis, and carrying out enzyme digestion by using a restriction enzyme EcoR I to detect the purity of the mitochondrial DNA.

One, the technical field

The invention relates to the technical field of plant biology, in particular to an effective extraction method of sesbania mitochondria and DNA thereof, which is suitable for separation and purification of the sesbania mitochondria and the DNA thereof.

Second, background Art

Mitochondria are semi-autonomous organelles that, in eukaryotes, supply ATP to cells through oxidative phosphorylation processes. The mitochondrial genome has the characteristics of slow evolution, frequent sequence insertion and deletion, rapid recombination, complex and various structure, unique gene expression pattern and the like (Schuster and Brennicke 1994; Sanchez Puerta et al 2017; Kovar et al 2018; Sanchez Puerta et al 2019; Zhang et al 2019). Plant mitochondrial genomes vary widely in size, ranging from 187kb to 2400kb (Bentolila and Stefanov, 2012). The sequence exchange between mitochondria and the nucleus, plastids and the content of their self-repetitive sequences have a great influence on the size of the mitochondrial genome (Sternand Lonsdale 1982; Vaughn et al 1995; Goremykin et al 2012; Christensen 2014; Wynn and Christensen 2019). During aerobic respiration in plants, glucose releases energy in the mitochondria through glycolysis, the tricarboxylic acid cycle and oxidative phosphorylation. However, in an anaerobic environment, the cell will perform anaerobic respiration in the cytoplasmic matrix and the pyruvate produced by glycolysis will no longer enter the mitochondria for the tricarboxylic acid cycle. In addition to the major functions of ATP synthesis to supply energy to cells, mitochondria can also control apoptosis by regulating membrane potential.

Sesbania, a leguminous plant, has developed root system, high yield of fresh grass and rich nutrients, and has become an excellent summer green manure and animal feed (Xukailai et al, 2014; huangshiwu and Liaohong 1992). In addition, sesbania stalk fibers can be used for paper making and seeds can be used for the production of sesbania gum (yeanb et al 1986). Recent studies have shown that a novel cysteine enzyme in sesbania leaves may play a role in hemostasis and wound healing (Shivamadhu et al 2017). Therefore, the sesbania has higher ecological and economic utilization value. According to research, sesbania roots can form spongy ventilating tissues in a waterlogging stress environment, biomass is increased, and nodulation capacity is improved. In the process, whether the respiration of sesbania root mitochondria is affected or not and whether species-specific genes respond to waterlogging stress or not are unknown. The extraction of sesbania mitochondria and high-purity DNA thereof can lay a foundation for later research.

Third, the invention

Technical problem

The invention aims to disclose an effective method for extracting sesbania mitochondria and DNA thereof, which is suitable for separating and purifying the sesbania mitochondria and the DNA thereof. In order to obtain purified mitochondria, dark culture is carried out on sesbania seeds to reduce the production of chloroplasts, plant tissue impurities and cell nucleuses are removed by a differential centrifugation method to obtain crude mitochondria, plastids with the sedimentation coefficient similar to that of the mitochondria are removed by adopting a mode of combining sucrose density gradient and ultracentrifugation, and the purified mitochondria are separated. To obtain mitochondrial DNA in high purity, it is necessary to remove DNA from the nucleus and plastids as well as RNA from the tissues. DNA free and bound to the mitochondrial membrane was removed by a combination of differential centrifugation and DNase digestion. In the process of extracting DNA by breaking the walls of the purified mitochondria, the interference of RNA can be well removed by adding RNase and water bath for 3 h. The invention combines the extraction methods of different plant mitochondrial DNAs and continuous test and exploration, adjusts the tissue crushing mode, the components of the homogenate buffer solution and the DNA extraction buffer solution and the differential centrifugation combination, and combines the sucrose density gradient centrifugation method to obtain high-purity mitochondria and DNA thereof. The method has the characteristics of simple and convenient operation, low cost and high product purity, and lays a foundation for the research of sesbania mitochondrial genomes.

Technical scheme

The invention combines the improved tissue homogenate buffer solution component and differential centrifugation based on test exploration, obtains purified mitochondria by combining sucrose density gradient and ultracentrifugation, and obtains high-purity mitochondrial DNA through experiments by using the improved DNA extraction buffer solution. The specific experimental contents are as follows:

1. obtaining sesbania tissue homogenate:

1) adding 0.13g of cysteine and 1.3mL of β -mercaptoethanol into 260mL of precooled buffer solution A, pouring the mixture and 50g of sesbania yellow seedlings into a precooled juice extractor, and stirring for 90 s;

2) the homogenate was filtered through 2 layers of sterile absorbent gauze and then through 2 layers of 100 mesh nylon mesh, the filtrate was collected on ice and aliquoted into 50mL sterile centrifuge tubes.

2. Separation and purification of mitochondria:

1) centrifuging the homogenate in a high-speed centrifuge for 15min at the rotating speed of 2200g and the temperature of 4 ℃;

2) quickly transferring the supernatant into a 50mL sterile centrifuge tube, centrifuging for 15min at a rotation speed of 17000g and a temperature of 4 ℃ by using a high-speed centrifuge, and obtaining precipitate, namely the crude mitochondria;

3) adding a buffer solution B into the precipitate, slightly suspending the precipitate by using a brush pen, adding DNase I until the concentration of DNase I is 50 mu g/mL, carrying out ice bath for 1h, and adding EDTA to stop the reaction;

4) centrifuging for 20min by a high-speed refrigerated centrifuge at the rotating speed of 18000g and the temperature of 4 ℃;

5) gently resuspend the pellet in buffer C with a goat brush and transfer to a discontinuous density gradient of 5ml1.45m and 5ml1.2m sucrose;

6) lightly hanging the paved centrifugal tube on a horizontal rotor of an ultra-high speed centrifuge, centrifuging for 90min at the rotation speed of 20600rpm and at the temperature of 4 ℃;

7) collecting mitochondria between 1.45M and 1.2M sucrose density gradient solutions, and adding an equal volume of buffer solution C;

8) centrifuging for 20min with a high-speed refrigerated centrifuge at 20000g and 4 deg.C, and precipitating to obtain purified mitochondria.

3. Extraction of DNA from purified mitochondria:

1) adding lysis buffer solution into purified mitochondria, sucking and beating by using a pipette gun, uniformly mixing, adding protease K, SDS and RNase according to a system, carrying out water bath at 37 ℃ for 3h, and gently shaking every 30 min;

2) cooling to room temperature and adding NH₄Mixing AC solution and equal volume of chloroform-isoamyl alcohol (24: 1) by inversion for 10min, centrifuging at 12000g for 10min, and keeping the temperature at 4 ℃;

3) transferring the supernatant to a new centrifuge tube, adding equal volume of chloroform-isoamylol (24: 1) again, reversing and mixing uniformly for 10min, centrifuging at 12000g for 10min, and keeping the temperature at 4 ℃;

4) transferring the supernatant into a new centrifugal tube, and repeating the operation of the step 3) until the protein is completely extracted;

5) sucking the supernatant, transferring the supernatant into a new centrifuge tube, adding isopropanol with the volume of 0.7 times of that of the centrifuge tube, and storing the mixture at the temperature of minus 20 ℃ for a night;

6) centrifuging at 12000g for 15min, washing with 70% ethanol at 4 deg.C for 2 times, air drying to half dry at room temperature, and dissolving in TE or sterile water.

4. The obtained mitochondrial DNA was quantitatively and qualitatively detected:

1) mitochondrial DNA concentration detection: 2. mu.L of mitochondrial DNA solution was aspirated, and the concentration was detected by a microplate reader.

2) Mitochondrial DNA electrophoresis band detection: 2. mu.L of the mitochondrial DNA solution was subjected to agarose (1%) gel electrophoresis for detection, and the band pattern was observed.

3) Detecting the purity of mitochondrial DNA: a2. mu.L mitochondrial DNA solution was digested with restriction enzyme EcoR I, subjected to 1% agarose gel electrophoresis, and then the band pattern was observed.

Advantageous effects

The sesbania mitochondria and the effective method for extracting the DNA thereof have the following innovation points and beneficial effects:

1) in order to avoid interference of chloroplasts, sesbania seeds are cultured in a shading mode, the optimal culture time is 14 days, and quantitative tissues are placed on ice for precooling, so that the integrity of mitochondria and DNA thereof is guaranteed.

2) BSA is added into a cracking buffer solution for mitochondrial DNA extraction to remove free fatty acid, PVP is used for removing phenolic compounds, cysteine and β -mercaptoethanol are used as reducing agents to reduce oxidative damage, and sucrose can effectively adjust the osmotic pressure of mitochondria and control the cost.

3) Repeated experiments determine a differential centrifugal force combination suitable for sesbania mitochondria extraction to remove broken tissues and cell nuclei, DNase can sufficiently eliminate the interference of other DNA except mitochondria, and sucrose density gradient high-speed centrifugation further removes plastids from mitochondrial buffer.

4) In the mitochondrial DNA lysate, proteinase K degrades membrane proteins bound to DNA, and the DNA is sufficiently dissociated. RNase can degrade RNA and eliminate RNA contamination.

The method can effectively solve the technical problem of extraction of sesbania mitochondria, obtain high-quality and pollution-free mitochondrial DNA, lay a foundation for research of sesbania mitochondrial genomes, and effectively control the experiment cost. Through calculation, 0.3-0.35 mu g of mitochondrial DNA can be obtained from 1g of sesbania tissue, A260/A280 is 1.80-1.88, agarose (1%) has clear gel electrophoresis detection bands without degradation, and the enzyme digestion bands are clear without nuclear and plastid DNA pollution.

Description of the drawings

FIG. 1: sesbania mitochondrial DNA sucrose density gradient centrifugation results

In the figure, 1 is 1.2M sucrose; 2 is a plastid; 3 is 1.42M sucrose; 4 is a mitochondrion

FIG. 2: sesbania mitochondrion DNA and agarose gel electrophoresis detection result after enzyme digestion

In the figure, lanes 5 and 6 are sesbania mitochondrial DNA; lane 7 is DNA Marker; lane 8 is the result of agarose gel electrophoresis after restriction enzyme EcoRI digestion; lane 9 is DNA Marker

Fifth, detailed description of the invention

The sesbania seeds used in the research are provided by a saline soil agricultural research laboratory of agricultural science research institute in coastal areas of Jiangsu. Healthy and plump sesbania seeds are selected and sowed in a sterilized substrate (nutrient soil: vermiculite is 1: 3), the temperature is 27 ℃, the humidity is 65 percent, and dark culture is carried out. When illumination is needed, a green light lamp is used, the generation of chloroplasts is reduced as much as possible, and etiolated seedlings are obtained after 14 days of culture. All reagents of this study were pre-chilled in advance and all manipulations were performed on ice or below 4 ℃ without specification, and the manipulations were skilled and quick. Of the required reagents, sucrose for sucrose density gradient centrifugation was purchased from Sigma-Aldrich, USA, and the rest from Nanjing shoddy Biotech, Inc. The specific formulations of the buffer and the reagent are as follows:

and (3) buffer solution A: 171g of sucrose dissolved in ddH₂To this, 50mL of 1M Tris-HCl (pH8.0), 10mL of 0.5M EDTA (pH8.0), 1g of BSA, and 15g of PVP were added. After mixing uniformly, the pH value is adjusted to 7.5, and the volume is constant to 1L (BSA and PVP are added after being sterilized and cooled).

And (3) buffer solution B: 85.5g sucrose in ddH₂To this, 25mL of 1M Tris-HCl (pH8.0) and 3.05g of MgCl 2.6H2O were added. After mixing uniformly, the pH value is adjusted to 7.5, and the volume is adjusted to 500 mL.

And (3) buffer C: 102.5g sucrose in ddH₂To this, 5mL of 1M Tris-HCl (pH8.0) and 20mL of 0.5M EDTA (pH8.0) were added. After mixing uniformly, the pH value is adjusted to 7.5, and the volume is adjusted to 500 mL.

Lysis buffer: 5mL of 1M Tris-HCl (pH8.0) and 2mL of 0.5M EDTA (pH8.0) were combined to 100 mL.

1.2M sucrose solution: 20.53g of sucrose was dissolved in water, and 0.5mL of 1M Tris-HCl (pH8.0) and 2mL of 0.5M EDTA (pH8.0) were added thereto. After mixing uniformly, the pH value is adjusted to 7.5, and the volume is adjusted to 50 mL.

1.45M sucrose solution: 24.81g of sucrose was dissolved in water, and 0.5mL of 1M Tris-HCl (pH8.0) and 2mL of 0.5M EDTA (pH8.0) were added thereto. After mixing uniformly, the pH value is adjusted to 7.5, and the volume is adjusted to 50 mL.

10mg/mL of DNase I: 10mg of DNase I was dissolved in 1mL of sterile water, gently mixed and stored at-20 ℃.

10mg/mL proteinase K: 100mg of proteinase K solution was dissolved in 10mL of sterile water, dispensed and stored at-20 ℃.

The experimental procedure was as follows:

in order to increase sesbania mitochondrial yield and reduce the influence of cell debris and cell nuclei, the present study adjusted the combination of filtration mode, homogenization buffer composition and differential centrifugal force through multiple experiments. In order to extract high quality, contaminant-free mitochondrial DNA, the composition of the lysis buffer was adjusted. A series of measures ensures high quality access to sesbania mitochondria and their DNA.

1. Obtaining sesbania tissue homogenate:

1) adding 0.13g of cysteine and 1.3mL of β -mercaptoethanol into 260mL of precooled buffer solution A, pouring the mixture and 50g of sesbania yellow seedlings into a precooled juice extractor, and stirring for 40-80 s;

2) the homogenate was filtered through 2 layers of sterile absorbent gauze and then through 2 layers of 100 mesh nylon mesh, the filtrate was collected on ice and aliquoted into 50mL sterile centrifuge tubes.

2. Separation and purification of mitochondria:

1) centrifuging the homogenate at a speed of 2200g in an Avanti J-26S XP (Beckman, USA) precooled high-speed centrifuge at 4 ℃ for 15 min;

2) quickly transferring the supernatant into a 50mL sterile centrifuge tube, centrifuging for 15min at a rotation speed of 17000g and a temperature of 4 ℃ by using a high-speed centrifuge, and obtaining precipitate, namely the crude mitochondria;

3) adding a buffer solution B into the precipitate of each centrifuge tube, gently suspending the precipitate by using a writing brush pen, adding DNase I until the concentration of DNase I is 50 mu g/mL, carrying out ice bath for 1h, and adding EDTA until the concentration of EDTA is 50mmol/L to terminate the reaction;

4) transferring the sample to a 4 ℃ precooled high-speed refrigerated centrifuge for centrifugation for 20min, wherein the rotation speed is 18000 g;

5) gently resuspending the pellet in 1.5mL buffer C with a goat brush pen and transferring to a discontinuous density gradient solution of 5mL1.45M and 5mL1.2M sucrose;

6) lightly hanging the paved centrifuge tube on a horizontal rotor of a 4 ℃ precooled ultra-high speed centrifuge (XPN-100, Beckman company, USA), and centrifuging for 90min at a rotation speed of 20600 rpm;

7) collecting the mitochondria layer between 1.45M and 1.2M sucrose density gradient solutions by using a pipette, and adding an equal volume of buffer solution C;

8) transferring the sample to a 4 ℃ precooled high-speed refrigerated centrifuge for centrifugation for 20min at the rotation speed of 20000g, and obtaining the precipitate as the purified mitochondria.

3. Extraction of DNA from purified mitochondria:

1) adding lysis buffer solution into purified mitochondria, sucking and pumping the mitochondria by using a pipette gun, uniformly mixing, adding protease K according to a system to a final concentration of 200 mu g/mL, SDS to a final concentration of 1 percent and 7 mu LRNase, then carrying out water bath at 37 ℃ for 3h, and gently shaking every 30 min;

2) cooling to room temperature, adding NH4AC solution to final concentration of 0.5M and equal volume of chloroform-isoamyl alcohol (24: 1), mixing by inversion for 10min, and centrifuging at 12000g for 10min in a 4 deg.C precooled desktop centrifuge (CT15E/CT15RE, Hitachi, Japan);

3) carefully sucking the supernatant, transferring to a new centrifuge tube, adding equal volume of chloroform-isoamyl alcohol (24: 1) again, reversing and mixing uniformly for 10min, centrifuging at 12000g for 10min, and keeping the temperature at 4 ℃;

4) carefully sucking the supernatant liquid and transferring the supernatant liquid into a new centrifugal tube, and repeating the operation of the step 3) until the protein is completely extracted;

5) sucking the supernatant, transferring the supernatant into a new centrifuge tube, adding isopropanol with the volume of 0.7 times of that of the centrifuge tube, and storing the mixture at the temperature of minus 20 ℃ for a night;

6) centrifuging the sample in a 4 ℃ precooled centrifuge at a rotating speed of 12000g for 15min, washing the sample for 2 times by 70% alcohol, airing the sample to be semi-dry at room temperature, and adding 50 mu L of sterile water for dissolving.

4. The obtained mitochondrial DNA was quantitatively and qualitatively detected:

1) mitochondrial DNA concentration detection: sucking 2 mu L of mitochondrial DNA solution, and detecting the concentration of the mitochondrial DNA solution by using an enzyme-labeling instrument, wherein A260/A280 is about 1.80-1.88.

2) Mitochondrial DNA electrophoresis band detection: 2 mu L of mitochondrial DNA solution is taken to carry out agarose (1%) gel electrophoresis detection, the banding pattern is observed, the tailing phenomenon of the electrophoresis is avoided, the mitochondrial DNA is proved to be complete without degradation, the RNA band is avoided, and the mitochondrial DNA is proved to be free from RNA pollution.

3) Detecting the purity of mitochondrial DNA: to a sterilized PCR tube were added 5. mu.L of 10 XHbuffer, 2. mu.L of mitochondrial DNA solution, 1. mu.L of EcoR I and 42. mu.L of ddH in this order₂O constituted a 50. mu.L reaction system. And (3) uniformly mixing the reaction system, centrifuging, carrying out water bath at 37 ℃ for 6-8h, carrying out water bath at 70 ℃ for 15min to inactivate the restriction endonuclease EcoR I, detecting the enzyme digestion result by using 1% agarose gel electrophoresis, observing the band type, and uniformly distributing DNA bands after enzyme digestion without flooding to prove that the extracted mitochondrial DNA has high purity and no pollution of cell nucleus and plastid DNA.