CN110643602A

CN110643602A - Method for extracting root total RNA of magnolia obavata

Info

Publication number: CN110643602A
Application number: CN201911017197.9A
Authority: CN
Inventors: 杨安娜; 贺淑蕊; 孙志艳; 石亚冉
Original assignee: Anhui Normal University
Current assignee: Anhui Normal University
Priority date: 2019-10-24
Filing date: 2019-10-24
Publication date: 2020-01-03

Abstract

The invention provides a method for extracting total RNA of a root system of magnolia obavata, which comprises the steps of grinding root system tissues of magnolia obavata into powder in liquid nitrogen, mixing the powder with an extraction solvent, and incubating to obtain incubation mixed solution; performing extraction treatment to obtain an extraction supernatant; mixing the extracted supernatant with a lithium chloride solution, standing overnight, and centrifuging to obtain a total RNA precipitate; dissolving the total RNA precipitate in an SSTE buffer solution, and then performing extraction treatment in a chloroform/isoamylol mixed solution to obtain a centrifugal supernatant; mixing with anhydrous ethanol, standing at-70 deg.C, and centrifuging to obtain precipitate; washing the precipitate with ethanol solution to obtain clean precipitate; and (4) placing the clean precipitate in an open place, and adding ultrapure water treated by DEPC to obtain the total RNA of the root of the magnolia xanthioides. Compared with the prior art, the method can successfully extract the root total RNA of the magnolia obavata, and has excellent purity and high extraction rate.

Description

Method for extracting root total RNA of magnolia obavata

Technical Field

The invention relates to an extraction method of RNA, in particular to an extraction method of total RNA of a root system of magnolia obavata.

Background

Magnolia xanthioides (Magnolia cylindra) is a tree of Magnolia (Magnolia) belonging to Magnoliaceae (Magnolia) genus (Magnolia) and is named because the model specimen is collected from Huangshan mountain. The yellow magnolia denudata has excellent material and beautiful tree shape, and the flowers have more medicinal value, are important garden ornamental plants and excellent aromatic plants, and are valuable national third-level protection endangered species. Therefore, the method has very important significance in exploring the germplasm resources of the Majorana Huangshan from the molecular level.

The magnolia xanthifolia is a perennial woody plant and is rich in secondary metabolites such as protein, polysaccharide, polyphenol and the like. Polysaccharide and RNA are easy to coprecipitate, polyphenol is easy to be oxidized to form quinone substances which are irreversibly combined with RNA, and the secondary metabolites cause great interference on the extraction of RNA. High quality RNA extraction is a necessary prerequisite for molecular biology experiments, such as cDNA library construction, fluorescence quantitative PCR detection Northern hybridization and the like. Therefore, the exploration of the high-quality RNA extraction method of the magnolia wushanensis is of great significance.

The conventional common RNA extraction techniques comprise a CTAB method, a Trizol method and a plurality of RNA extraction kits sold in the market, but the methods have the problems of poor timeliness and the like. In addition, although many reports indicate that the above method can extract RNA from many plants. However, since there are many differences in the composition of matter between different plants or tissues of the same plant, the above method is not applicable to all materials, and for some plant materials, it is necessary to develop an ideal extraction method in the search.

For example, "a study of a method for extracting RNA from a medicinal plant purslane" disclosed by zhao dan et al has a significant effect in extracting RNA from a medicinal plant purslane by using an improved Trizol method (a study of a method for extracting RNA from a medicinal plant purslane [ J ]. shizhen chinese medicine, 2013,24(1): 83-85.). However, the method is not successful in extracting root RNA of magnolia obavata. The extracted RNA sample is milky white and sticky in appearance, and the RNA precipitate is diluted by DEPC water, so that the bottom of the EP tube can be obviously found to have impurities which are obviously insoluble in water. The method can not meet the requirements of the magnolia wushanensis related genetic engineering experiments.

Therefore, a method for extracting high-quality root RNA of magnolia obavata with stronger pertinence is needed.

Disclosure of Invention

The invention aims to provide a method for extracting root total RNA of magnolia obavata, which can be used for extracting the root total RNA of the magnolia obavata with high purity and has high extraction rate and low cost.

The specific technical scheme of the invention is as follows:

a method for extracting root total RNA of magnolia obavata includes the following steps:

1) grinding fresh lateral roots of the magnolia obavata in liquid nitrogen into powder, mixing the obtained magnolia obavata root system powder with an extraction solvent, and incubating to obtain an incubation mixed solution;

2) adding a chloroform-isoamyl alcohol mixed solution into the incubation mixed solution obtained in the step 1), and extracting and centrifuging to obtain an extracted supernatant;

3) mixing the extracted supernatant obtained in the step 2) with a lithium chloride solution, standing overnight, and centrifuging to obtain a total RNA precipitate;

4) adding an ethanol solution into the total RNA precipitate obtained in the step 3), rinsing and centrifuging to obtain a precipitate;

5) dissolving the precipitate obtained in the step 4) in an SSTE buffer solution, then mixing the SSTE buffer solution with a chloroform-isoamylol mixed solution, and extracting and centrifuging to obtain a centrifugal supernatant;

6) mixing the centrifugal supernatant obtained in the step 5) with absolute ethyl alcohol, standing, and centrifuging to obtain a precipitate;

7) mixing the precipitate obtained in the step 6) with an ethanol solution, rinsing and centrifuging to obtain a precipitate, adding absolute ethanol into the obtained precipitate, rinsing and centrifuging to obtain a precipitate; namely the total RNA of the root system of the magnolia obavata.

The magnolia xanthifolium in the step 1) is greenhouse-cultured magnolia xanthifolium which grows for two years;

further, in the step 1), the newly developed lateral roots of the magnolia wushanensis are selected, cut off by scissors, washed clean in running water, placed in liquid nitrogen, and stored at-80 ℃ for RNA extraction.

Grinding in the step 1) until any granular root system can not be seen by naked eyes.

The extraction solvent in the step 1) contains CTAB extraction liquid and beta-mercaptoethanol; the volume ratio of the CTAB extract to the beta-mercaptoethanol is 100: 2; and (3) autoclaving the CTAB extract for 20min at 121 ℃, and then adding beta-mercaptoethanol.

Preferably, the CTAB extract comprises 2% CTAB cetyltrimethylammonium bromide, 2% PVP polyvinylpyrrolidone, 0.1mol/L Tris-HCl buffer solution with pH8.0, 2mol/L NaCl, 25mmol/L EDTA-Na₂(pH8.0) and DEPC-treated ultrapure water.

The specific preparation method of the CTAB extract comprises the following steps:

2.4g Tris in 100ml per thousand DEPC water, then hydrochloric acid to pH8, 1.86g EDTA-Na₂·2H₂O was dissolved in 100ml per thousand of DEPC water, hydrochloric acid was added to pH8, and after mixing, 4g CTAB, 4g PVP and 23.4g nacl were added.

In the step 1), the temperature of the extraction solvent is 65 ℃; in the step 1), the mixing refers to vortex mixing, and the vortex mixing time is not less than 30 s;

in step 1), the incubation conditions are as follows: incubating at 65 deg.C for 3-5 min; vortex 3-5 times during incubation, 10s per vortex.

Preferably, the ratio of the volume of the extraction solvent to the mass of the root powder of the magnolia amurensis in the step 1) is as follows: 6 mu L of 1.5-2 mg. Preferably, the extraction solvent is mixed with 150-200mg of the root powder of the yellow magnolia per 600 muL of the extraction solvent. In the present invention, the amount of plant sample material is not excessive in order to ensure that the sample tissue is uniformly and thoroughly dispersed in the extract, which would otherwise result in clumping of the sample tissue. The combination of factors verified that 150-200mg of sample material was most suitably mixed with 600. mu.L of CTAB extract.

And step 1), pre-cooling by using liquid nitrogen before using a mortar and a medicine spoon used in the grinding process. Liquid nitrogen should be added at any time during the milling process to ensure that the powder does not melt before the extraction solution is added, otherwise RNA degradation may result.

The CTAB extract contains PVP polyvinylpyrrolidone, the PVP with high quality concentration can effectively remove polysaccharide and polyphenol in plant sample tissues and can also remove other pigment substances and lipids, because the magnolia wufengensis plant tissues contain oil cells, polysaccharide, polyphenol and other substancesTherefore, the PVP with high mass concentration is used in the CTAB extract liquid of the invention to remove the lipid, polysaccharide and polyphenol and other substances in the sample tissue. In addition, most cells can only move within a narrow pH range, so that the cells are not easily inactivated when a normal pH environment is maintained, and the invention provides a buffer environment by using 0.1mol/L Tris-HCl buffer solution (pH8.0) to prevent nucleic acid from being damaged, thereby being beneficial to the stability of RNA and not easily degrading the RNA. In the present invention, CTAB acts as a detergent, dissolves cell membranes, forms a complex with nucleic acids, and is soluble in high salt solutions. Therefore, when the salt concentration of the solution is lowered to a certain degree (e.g., 2mol/L NaCl), the CTAB-nucleic acid complex is precipitated from the solution and separated from the protein or polysaccharide by centrifugation. The pH value of RNase is 8.0, and the invention is 25mmol L EDTA-Na₂(pH8.0) provides an optimum environment for inhibiting the activity of RNase.

The chloroform-isoamyl alcohol mixed solution in the step 2) has the volume ratio of chloroform to isoamyl alcohol of 24: 1;

preferably, the mixing volume ratio of the incubation mixed solution in the step 2) to the chloroform-isoamylol mixed solution is 1: 1;

more preferably, 600. mu.L of chloroform-isoamyl alcohol mixed solution is used per 600. mu.L of incubation mixture; the addition of 600. mu.L of the extract is the most suitable by combining various factors.

The step 2) is specifically as follows: adding chloroform-isoamyl alcohol mixed solution into the incubation mixed solution obtained in the step 1), carrying out vortex mixing, carrying out centrifugal treatment, centrifuging for 15min at room temperature of 10000r/min, and taking supernatant to obtain extracted supernatant.

Further, the vortex mixing in the step 2) is specifically as follows: vortex for 1min at room temperature.

Preferably, the extraction supernatant obtained in step 2) is treated again by the same extraction centrifugation method as in step 2).

The invention carries out vortex mixing, ensures that the tissues of the plant sample are thoroughly and uniformly suspended in the solution, ensures that the tissues of the sample are uniformly dispersed, prevents the material from being in a bulk shape, and improves the extraction rate and the purity. In addition, when the temperature of CTAB is lower than 15 ℃, CTAB is separated out, nucleoprotein can not be depolymerized, and nucleic acid is released, so that the CTAB extract is fully mixed with the tissue of the plant sample at room temperature for 1min, thereby separating RNA.

In the step 3), the volume ratio of the extraction supernatant to the lithium chloride solution is 3: 1; the concentration of the lithium chloride solution is 8 mol/L.

Preferably, in step 3), the standing overnight conditions: standing at 3-5 deg.C for 8-16 h;

in step 3), the centrifugal treatment is as follows: centrifuging at 4 deg.C at 10000r/min for 20 min.

In the step 4), the ethanol solution is 70% ethanol solution in volume concentration, and the RNase-free DEPC-treated water ultrapure water is used for preparation.

In the step 4), the rinsing centrifugation means centrifugation at 10000r/min for 5min at 4 ℃.

In the step 5), the dosage of the SSTE buffer solution is 500 mu L relative to the sediment prepared by every 600 mu L of incubation mixed solution;

preferably, the SSTE buffer consists of: 0.5% sodium dodecyl sulfate SDS, 1.0mol/L NaCl, 10mmol/L Tris-HCl (pH8.0), and 1mmol/L EDTA-Na₂(pH＝8.0)。

The specific preparation method of the SSTE buffer solution comprises the following steps: 0.12g Tris in 50ml of DEPC water, then hydrochloric acid was added to pH8, 0.037g EDTA-Na₂·2H₂Dissolving O in 50ml of DEPC water, adding hydrochloric acid until the pH value is 8, mixing the two solutions, adding 0.5g of SDS and 5.84g of NaCl, and autoclaving at 121 ℃ for 20 min.

The high-concentration SDS in the SSTE buffer solution can destroy cell membranes, so that RNase in cells can be quickly inhibited, the integrity of RNA is ensured, the combination of protein and nucleic acid can be destroyed by Tris-Cl, the nucleic acid is released, EDTA can effectively inhibit the activity of RNase, the RNA degradation is prevented, the salt concentration is increased by the action of NaCL, and the RNA degradation is prevented.

In the step 5), the volume ratio of the SSTE buffer solution to the chloroform-isoamylol mixed solution is 1: 1;

the step 5) is specifically as follows:

dissolving the precipitate obtained in the step 4) in an SSTE buffer solution, then vortexing the SSTE buffer solution with chloroform-isoamylol mixed solution of the same volume at room temperature for 1min, then centrifuging the mixture for 15min at 10000r/min at 4 ℃, and taking supernatant;

in the step 6), the volume ratio of the centrifugal supernatant to the absolute ethyl alcohol is 1: 2.

In the step 6), standing is carried out under the following conditions: standing at-70 deg.C for 30min, or at-20 deg.C for 2 h.

The centrifugation in the step 6) refers to: centrifuging at 4 deg.C at 10000r/min for 20 min.

Compared with DNA, RNA is easy to degrade, and the RNA content extracted from a sample with the same quality is lower, so that the standing and precipitating time needs to be prolonged to ensure that the RNA is completely precipitated. And RNA has low solubility in cold absolute ethanol and high protein solubility. RNA is more easily precipitated under the ultralow temperature environment.

In the step 7), the amount of the ethanol solution is 400 mu L relative to the precipitate obtained after the incubation mixed solution is processed in the steps 2) to 6);

the step 7) is specifically as follows: centrifuging the precipitate obtained in the step 6) for 5min at 4 ℃ at 10000r/min by using an ethanol solution to obtain a precipitate, adding absolute ethanol into the precipitate, centrifuging at 4 ℃ at 10000r/min for 5min to obtain the precipitate.

Placing the precipitate obtained in the step 7) in an open place, adding DEPC treated ultrapure water, and storing.

The open placement condition is as follows: standing at room temperature for 2-3 min;

the mass ratio of the precipitate to the DEPC-treated ultrapure water is 3.7-9.6: 10-20.

All the steps are carried out in a clean bench.

The invention successfully extracts the total RNA from the root material of the magnolia amurensis through the synergistic effect of the steps; moreover, the extraction method is simple and easy to implement, and further meets the requirements of subsequent molecular biology experiments and related researches. Meanwhile, the extraction method has high extraction rate.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is an agarose gel electrophoresis image of RNA extracted by the method of the present invention;

FIG. 2 is an agarose gel electrophoresis of RNA extracted by Trizol;

FIG. 3 shows the detection of the amplification products by agarose gel electrophoresis.

Detailed Description

The following detailed description of specific embodiments of the present invention is provided to illustrate and explain the present invention and to be understood not to limit the present invention.

The reagents, equipment and operation considerations used in the invention are as follows:

1) CTAB extract is prepared from CTAB, PVP, Tris, NaCl, EDTA-Na₂·2H₂Preparation of ultrapure water for O and DEPC treatment, wherein reagents required by the preparation are purchased from Biotechnology engineering (Shanghai) GmbH; before the CTAB extract is used, whether precipitates exist or not is checked, if the precipitates exist, the CTAB extract is dissolved at 65 ℃ and then is used; the lithium chloride solution was prepared with DEPC-treated ultrapure water, lithium chloride was purchased from bio-engineering (shanghai) gmbh; chloroform and isoamyl alcohol are utilized for preparing a chloroform-isoamyl alcohol mixed solution (24:1, v/v); chloroform was obtained from Suzhou Korea chemical Co., Ltd, isoamyl alcohol was obtained from Kanto chemical reagent Co., Ltd, Tianjin; 1.5ml centrifuge tube of RNase-free (RNase-free) was purchased from corning Corning, and 10-1000. mu.L tip of RNase-free used in the present invention was purchased from biosharp product of Woodful Hongtak Biotechnology, Inc.; the vortex instrument used by the vortex of the invention is a QL-861 vortex mixer; the ethanol solution was prepared using ultrapure water obtained by treating water with RNase-free DEPC.

2) The preparation method of the ultrapure water treated by DEPC comprises the following steps: adding DEPC diethyl pyrocarbonate into ultrapure water, standing overnight for 12h, and autoclaving at 121 deg.C for 20min to remove DEPC, to obtain the ultrapure water of RNase-free DEPC treated water. Wherein the volume ratio of the ultrapure water to the DEPC is 1000: 1. Unsterilized DEPC treated water and DEPC reagents were banned from use in the experiments, and sterilized DEPC treated water was not reused for treatment of pipettes and the like to avoid contamination.

3) The reagent, the suction head and other devices used in the invention are RNase-free, and the specific treatment steps are as follows: soaking the suction head, the centrifugal tube and the like in ultrapure water added with DEPC treatment overnight, taking out and autoclaving to remove DEPC, thus obtaining RNase-free which can be used for RNA experiments.

4) Before use, a mortar, a spoon and the like used in the experiment are soaked in ultrapure water added with DEPC overnight, taken out and autoclaved, and dried to remove RNase. Liquid nitrogen is used for precooling before the mortar and the medicine spoon are used. Liquid nitrogen should be added at any time during the milling process to ensure that the powder does not melt before the extraction solution is added, otherwise RNA degradation may result.

5) Before the operation process, a superclean workbench is wiped by using 75% ethanol and absorbent cotton, required objects such as a pipettor, a suction head, a mortar, a medicine spoon and the like are placed on a table top, an alcohol lamp is turned on, the superclean workbench is turned on, and the superclean workbench is used after being subjected to ultraviolet disinfection for 30 min; the alcohol lamp is kept not to be turned off during working.

6) In the implementation process of the invention, the gloves are required to be replaced frequently, and the gloves are required to be replaced when the gloves contact the surfaces of door handles, refrigerator doors, laboratory tables, tables and chairs and the like; keep the experimental clothes clean and tidy, avoid the personnel to walk around when working, close the door and window, reduce talk and so on to reduce the RNase (RNA enzyme) pollution probability.

Example 1

1) dissolving 2.4g of Tris in 100ml of DEPC water, and adding hydrochloric acid to adjust the pH value to 8 to obtain a Tris-HCl buffer solution; mixing 1.86g of EDTA-Na₂·2H₂Dissolving O in 100ml of DEPC water, adding hydrochloric acid to adjust the pH to 8 to obtain EDTA-Na₂Mixing the two solutions, adding 4g CTAB, 4g PVP and 23.4g NaCl, and mixing to obtain CTAB extractive solution; sterilizing the CTAB extractive solution at 121 deg.C under high pressure for 20min, adding 2 vol% beta-mercaptoethanol of CTAB extractive solution, mixing with CTAB extractive solution, and standing at 65 deg.C as extraction solventPreheating in a water bath kettle; 0.5g of fresh lateral root material of two-year-old Majorana huangshanensis cultured in a greenhouse is put into a mortar precooled by liquid nitrogen, and is fully ground by the liquid nitrogen until any granular root system can not be seen by naked eyes, and is powdered. The mortar was allowed to stand at room temperature for 2min (to prevent the mortar from cooling too much to crystallize CTAB); adding 1.5mL of the extraction solvent preheated to 65 ℃ into a mortar at room temperature, and grinding to uniformly mix the extraction solvent and the powder to obtain a mixture of the extraction solvent and the powder; and (3) putting 600 mu L of the mixture into a 1.5mL centrifuge tube, immediately shaking and mixing the mixture by using a vortex mixer for 30s, finally placing the mixture into a 65 ℃ water bath pot for incubation for 5min, taking the mixture out every other minute during incubation for 1 time, placing the mixture at room temperature for vortex, and vortexing the mixture for 10s every time to obtain an incubation mixed solution.

2) Taking 600 mu L of the incubation mixed solution prepared in the step 1) to a centrifugal tube, shaking up, cooling to room temperature, adding 600 mu L of chloroform-isoamyl alcohol mixed solution with the volume ratio of chloroform to isoamyl alcohol being 24:1 into the centrifugal tube, mixing by vortex at room temperature for 1min, and then centrifuging at room temperature for 15min at 10000 r/min; then sucking the supernatant; putting the obtained supernatant into a centrifugal tube, adding a chloroform-isoamylol mixed solution with the volume ratio of chloroform to isoamylol being 24:1 in the same volume into the centrifugal tube at room temperature, mixing by vortex at room temperature for 1min, and then centrifuging at room temperature for 15min at 10000 r/min; then sucking the supernatant, namely extracting the supernatant; when the supernatant is sucked, in order to reduce impurity pollution, the lower layer solute is prevented from being sucked, and the liquid at the bottom of the uppermost layer, which is contacted with the liquid surface of the second layer, is not sucked as much as possible.

3) Adding the extracted supernatant obtained in the step 2) into another new centrifugal tube of 1.5ml, adding 1/3 LiCl solution of 8mol/L in volume of the extracted supernatant, and turning upside down to mix the solution evenly; then placing the mixture at 4 ℃ overnight for 14h for precipitation; and finally, centrifuging the centrifuge tube at the rotation speed of 10000rpm at 4 ℃ for 20min, and taking the precipitate to obtain crude total RNA precipitate. Wherein, in order to prevent the RNA from being incompletely precipitated, the centrifuged supernatant is transferred to a new centrifugal tube without RNase, and the supernatant is discarded after the RNA is completely precipitated after centrifugation.

4) Rinsing the crude total RNA precipitate obtained in the step 3) with 50 mu L of 70% ethanol solution with volume concentration, centrifuging for 5min at 4 ℃ at 10000r/min, removing supernatant, and air-drying the precipitate at room temperature for 30 s;

5) placing the dried precipitate obtained in the step 4) into 500 mu L of SSTE buffer solution, uniformly mixing, adding chloroform and isoamylol with the same volume ratio of 24:1, vortex the chloroform-isoamyl alcohol mixed solution at room temperature for 1min, then centrifuge for 15min at 10000r/min under the condition of 4 ℃, and take supernatant;

6) Transferring the supernatant obtained in the step 5) into a new 1.5ml centrifuge tube, adding 2 times of absolute ethyl alcohol in volume of the supernatant, uniformly mixing, and standing and precipitating at-70 ℃ for 30 min; then, the mixture was centrifuged at 10000rpm at 4 ℃ for 20min, and the supernatant was discarded to obtain a precipitate.

7) Dissolving the precipitate prepared in the step 6) in 400 μ L of 70% ethanol solution, centrifuging at 10000rpm for 5min at 4 ℃, carefully pouring out the supernatant to obtain the precipitate, adding 400 μ L of anhydrous ethanol solution into the precipitate, centrifuging at 10000rpm at 4 ℃ for 5min, and sucking out the supernatant by a pipette gun to obtain the precipitate.

8) Opening a cover of the centrifugal tube obtained in the step 7), and placing and airing the centrifugal tube in a super clean bench at room temperature for 2 min; and completely volatilizing the ethanol to obtain the total RNA of the root system of the magnolia obavata.

9) Dissolving the root total RNA precipitate of the magnolia xanthioides prepared in the step 8) by using 20 mu L of ultrapure water treated by DEPC to obtain a root total RNA sample 1 of the magnolia xanthioides, and storing at-80 ℃.

Example 2

A method for extracting root system total RNA of magnolia obavata, in the extraction process of this embodiment, after adding LiCl solution in step 3), place overnight for 16h at 4 ℃; otherwise, as in example 1, the total RNA in roots of Magnolia liliiflora sample 2 was finally prepared.

Example 3

A method for extracting root system total RNA of magnolia obavata, in the extraction process of the embodiment, after LiCl solution is added in the step 3), the extract is placed at 4 ℃ and is kept overnight for 12 hours; otherwise, as in example 1, the total RNA in roots of Magnolia liliiflora will be finally obtained as sample 3.

Comparative example 1

A method for extracting root total RNA of magnolia obavata (Trizol method) comprises the following steps:

1) taking 0.3g of fresh lateral roots of 2-year-old Majorana huangshanensis cultured at room temperature, adding 1% polyvinylpyrrolidone (PVP) in the volume of a sample, grinding the mixture into powder in liquid nitrogen, taking 200mg of the powder, placing the powder into a 1.5mL centrifuge tube precooled in the liquid nitrogen, adding 1mL of a trizol reagent and 10 mu L of beta-mercaptoethanol with the mass volume ratio of 1%, standing the mixture for 10min at room temperature to fully crack the mixture, then, centrifuging the mixture for 5min at 4 ℃ and 12000r/min, and transferring supernatant into a new centrifuge tube;

2) adding 200 mu L of 5mol/L NaCl solution into the supernatant obtained in the step 1), shaking up, adding 200 mu L of chloroform, shaking up, mixing uniformly, standing at room temperature for 15min, centrifuging at 12000r/min at 4 ℃ for 15min, and absorbing the supernatant phase into a new centrifuge tube;

3) adding equal volume of chloroform into the clear liquid obtained in the step 2), extracting, carrying out 12000r/min, centrifuging for 15min at 4 ℃, and absorbing the upper-layer water phase into a new centrifugal tube;

4) adding the mixed solution of phenol, chloroform and isoamylol with the same volume into the clear liquid obtained in the step 3), uniformly mixing, standing for 5min at room temperature, carrying out 12000r/min at the temperature of 4 ℃, centrifuging for 15min, and taking the supernatant to another new centrifugal tube; wherein the volume ratio of the phenol to the chloroform to the isoamyl alcohol is 25:24: 1.

5) Adding 0.5mL of isopropanol into the supernatant obtained in the step 4), uniformly mixing, standing at room temperature for 5-10min, centrifuging at 12000r/min at 4 ℃ for 10min, and removing the supernatant to obtain an RNA precipitate;

6) adding 1mL of ethanol solution with volume concentration of 75% into the RNA precipitate in the step 5), centrifuging at 8000r/min at 4 ℃ for 5min, removing supernatant, and drying at room temperature;

7) adding 20 mu L of ultrapure water treated by DEPC into the dried precipitate in the step 6) to dissolve the clean precipitate to obtain a total RNA sample 4 of the root of the magnolia xanthioides; storing at-80 deg.C.

Comparative example 2

A method for extracting root total RNA of magnolia obavata (Trizol method) comprises the steps of adding an equal volume of phenol-chloroform-isoamyl alcohol mixed solution into a step 4) in the extraction process of the comparative example for extraction for 2 times, and finally preparing a magnolia obavata root total RNA sample 5 by the same steps as the comparative example 1.

And (3) detection:

detecting the total RNA samples 1-5 of the roots of the Majorana Huangshan by using a TECAN microplate reader, taking ultrapure water treated by 2 mu of LDEPC as a blank, absorbing 2 mu of LRNA samples for detection, wherein the specific detection sample indexes are shown in Table 1.

TABLE 1

RNA index extracted from detection sample 1-5 of TECAN microplate reader

Material	Sample 1	Sample 2	Sample 3	Sample No. 4	Sample No. 5
						RNA concentration (μ g/. mu.L)	303.02	476.65	187.04	-0.08	-0.56
OD₂₆₀/OD₂₈₀	2.23	2.25	2.22	0.33	1.27
						OD₂₆₀/OD₂₃₀	2.47	2.48	2.40	0.50	0.54

Detecting 1-5 total RNA samples of roots of the magnolia xanthifolia by agarose gel electrophoresis under the following detection conditions: 1% agarose gel, voltage 108V, current 100mA, time 30 min; the detection results are shown in FIG. 1 and FIG. 2(marker is molecular weight marker, and the sizes of the bands are from bottom to top: 100, 250, 500, 750, 1000, 1500 and 2000bp in sequence), FIG. 1 is the present invention, and FIG. 2 is Trizol method. As can be seen from FIG. 1, the total RNA electrophoresis band of the root of the magnolia xanthiifolia extracted by the method is clear and has good integrity, and the requirement of a downstream test can be met. The 28s and 18s bands are clearly visible in fig. 1, whereas fig. 2 is free of any bands and has a miscellaneous band tail.

Reverse transcription PCR experiment:

1. reverse transcription: reverse transcription was performed using the Tiangen "FastQuant cDNA first Strand Synthesis kit". A20. mu.L reaction was set up with 50 ng-2. mu.g total RNA:

(1) thawing template RNA on ice, 5 XgDNA Buffer, FQ-RT Primer Mix, 10 Xfast RT Buffer, RNase-Free ddH₂O was thawed at room temperature and placed on ice quickly after thawing. Before use, each solution is vortexed, oscillated and uniformly mixed, and centrifuged to collect liquid remained on the tube wall;

the following operation steps are carried out on ice, in order to ensure the accuracy of the preparation of the reaction solution, Mix is prepared firstly and then is subpackaged into each reaction tube when carrying out each reaction;

(2) a mixed solution was prepared according to the genomic DNA removal system of Table 2, mixed, centrifuged, incubated at 42 ℃ for 3min, and then placed on ice.

TABLE 2 gDNA removal reaction System

Composition of matter	Amount of the composition used
		5×g DNA Buffer	2μL
Total RNA	5μL
		RNase-Free ddH2O	Make up to 10. mu.L

(3) Preparing a mixture according to the reverse transcription reaction system of Table 3

TABLE 3 reverse transcription reaction System

Composition of matter	Amount of the composition used
		10×Fast RT Buffer	2μL
RT Enzyme Mix	1μL
		FQ-RT Primer Mix	2μL
RNase-Free ddH₂O	Make up to 10. mu.L

(4) Mix in the reverse transcription reaction was added to the reaction solution in the gDNA removal step and mixed well.

(5) Incubate at 42 ℃ for 15 min.

(6) After incubation for 3min at 95 ℃, the cDNA obtained can be used for subsequent experiments or stored at low temperature.

Reverse transcription PCR:

designing actin primer by using Magnolia wufengensis transcriptome gene,

the upstream primer F is 5'-CGAGTTGTACGTGGTCTC-3', and the upstream primer F is 5'-CGAGTTGTACGTGGTCTC-3',

5'-CATCCTGTCCTCCTTACTG-3' as the downstream R of the primer;

length of the amplified sequence: 543bp, 10-fold dilution of cDNA, and 10 μ M primer. And (3) PCR system: mu.L of cDNA obtained by reverse transcription of the RNA extracted in example 1; 1.25 mu L of upstream primer; 1.25 mu L of downstream primer; 2 XFastTaq Premix 12.5. mu.L (purchased from Toutou harbor Biotech Co., Ltd.) was supplemented with DNase-free ultrapure water to 25. mu.l. reaction conditions: denaturation at 94 deg.C for 3 min; melting at 94 ℃ for 30s, annealing at 52 ℃ for 1min, and extending at 72 ℃ for 1min for 30s reactions for 35 cycles; extension at 72 ℃ for 10 min.

The amplification product 1-amplification product 5 obtained from the cDNA reverse transcribed from the RNA extracted in example 1 was detected by agarose gel electrophoresis under the following conditions: 1% agarose gel, voltage 108V, current 100mA, time 30min, the detection results are shown in figure 3.

SEQUENCE LISTING

<110> university of teacher's university in Anhui

<120> extraction method of root system total RNA of magnolia xanthioides

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 18

<212> DNA

<213> upstream primer

<400> 1

5'-cgagttgtac gtggtctc-3' 18

<210> 2

<211> 19

<212> DNA

<213> downstream primer R

<400> 2

5'-catcctgtcc tccttactg-3' 19

Claims

1. The extraction method of the total RNA of the root system of the magnolia amurensis is characterized by comprising the following steps:

2. The extraction method according to claim 1, wherein the mixing in step 1) is vortex mixing, and the vortex mixing time is not less than 30 s.

3. The extraction method according to claim 1 or 2, wherein in step 1), the incubation conditions are: incubating at 65 deg.C for 3-5 min; vortex 3-5 times during incubation, 10s per vortex.

4. The extraction method according to claim 1, wherein the step 2) is specifically: adding chloroform-isoamyl alcohol mixed solution into the incubation mixed solution obtained in the step 1), carrying out vortex mixing, carrying out centrifugal treatment, centrifuging for 15min at room temperature of 10000r/min, and taking supernatant to obtain extracted supernatant.

5. The extraction method according to claim 1, wherein the ethanol solution is 70% ethanol solution by volume, and the water is treated with DEPC-based RNase-free ultrapure water.

6. The extraction process according to claim 1, characterized in that, in step 5), the SSTE buffer consists of: 0.5% sodium dodecyl sulfate SDS, 1.0mol/L NaCl, 10mmol/L Tris-HCl (pH8.0), and 1mmol/L LEDTA-Na₂(pH＝8.0)。

7. The extraction method according to claim 1 or 6, wherein the volume ratio of the SSTE buffer to the chloroform-isoamyl alcohol mixed solution in the step 5) is 1: 1.

8. The extraction method according to claim 1 or 6, wherein in step 6), the volume ratio of the centrifugal supernatant to the absolute ethyl alcohol is 1: 2.

9. The extraction method according to claim 1 or 6, characterized in that, in step 6), the standing is carried out under the conditions: standing at-70 deg.C for 30min, or at-20 deg.C for 2 h.

10. The extraction method according to claim 1 or 6, wherein step 7) is specifically: centrifuging the precipitate obtained in the step 6) for 5min at 4 ℃ at 10000r/min by using an ethanol solution to obtain a precipitate, adding absolute ethanol into the precipitate, centrifuging at 4 ℃ at 10000r/min for 5min to obtain the precipitate.