Method for extracting plant polysaccharide polyphenol small RNA (Ribonucleic Acid)

Abstract

The invention discloses a plant polysaccharide polyphenol small RNA (Ribonucleic Acid) extraction method, belongs to the technical field of RNA extraction, and solves the problem that impurities interfere with small molecule RNA reverse transcription during separation due to the fact that polyphenol in cells is easy to oxidize when cell walls of plant tissues are broken during splitting of the plant tissues in the existing method. The method comprises the following steps: adding a cell lysis solution with the volume 3 times that of plant tissue powder into the plant tissue powder, and pre-preparing auxiliary reaction magnetic beads to obtain a supernatant A and a precipitate A; stirring and mixing a Licl solution with the supernatant A to obtain a precipitate B, carrying out precipitation washing on the precipitate B by using a washing solution, centrifuging, and discarding the supernatant to obtain a small molecule RNA precipitate; according to the method disclosed by the invention, high-efficiency recovery treatment of the small-molecule RNA in the plant tissue sample is realized through a technical means of synergistic cooperation of the cell lysis solution and the auxiliary reaction magnetic beads, a strong reducing environment is provided for recovery of the small-molecule RNA in a manner of reducing and inhibiting the cell lysis solution, and reverse transcription of the small-molecule RNA is ensured not to be inhibited.

Description

Technical Field

[0001] The invention belongs to the technical field of RNA extraction, and particularly relates to a method for extracting plant polysaccharide and polyphenol small RNA. Background Art

[0002] Plant small RNA is a type of non-coding RNA that plays an important regulatory role in plants. Small RNA mainly includes microRNAs, phased small interfering RNAs and heterochromatic siRNAs. They play a vital role in plant growth and development, stress response and epigenetic regulation. During the extraction of plant small RNA, the various types and structures of polyphenols and polysaccharides in plant tissues are easily oxidized to form quinones, which irreversibly bind to small RNA, resulting in a decrease in the extracted small RNA.

[0003] At present, the methods for extracting plant small molecule RNA include oxidant method and reductant method. The oxidant RNA extraction methods include Trizol method, guanidine isothiocyanate method, and perchlorate method, and the reductant extraction methods include SDS-acid phenol method and CTAB-LiCl method. However, when using the oxidant method to extract plant small molecule RNA, polyphenols and polysaccharides will not only interfere with plant small molecule RNA, but also combine with plant small molecule RNA to form precipitates, thus resulting in uncontrollable plant small molecule RNA extraction yield and purity.

[0004] Chinese patent CN114774406B discloses a method for extracting total RNA from rose plant tissues, the method comprising grinding the rose plant tissues with liquid nitrogen, and then performing three consecutive lysis and extraction steps, wherein the three lysis and extraction steps are performed using solution A, solution B and solution C respectively, and the components of solution A, solution B and solution C are disclosed; however, when the plant tissues are lysed by the existing method, the polyphenols in the cells are easily oxidized when the cell walls of the plant tissues are broken, so that impurities interfere with the reverse transcription of small molecule RNA during separation, affecting the extraction rate and integrity of the small molecule RNA. In view of the above problems, we propose a method for extracting plant polysaccharide polyphenol small RNA. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting small RNA from plant polysaccharides and polyphenols in view of the shortcomings of the prior art, thereby solving the problem that when the plant tissue cell walls are broken during the lysis of plant tissues by the prior method, the polyphenols in the cells are easily oxidized, so that impurities interfere with the reverse transcription of small molecule RNA during separation, affecting the extraction rate and integrity of the small molecule RNA.

[0006] The present invention is achieved by providing a method for extracting small RNA from plant polysaccharides and polyphenols, the method comprising:

[0007] Pre-treating the plant tissue sample, cutting the plant tissue sample with ophthalmic surgical scissors after high-temperature sterilization to obtain plant tissue fragments, freezing the plant tissue fragments in a -80°C liquid nitrogen environment for 2 hours, taking a mortar, filling the mortar with diatomaceous earth, and then grinding the diatomaceous earth with a grinding rod, removing the diatomaceous earth after grinding for 10 minutes, pre-treating the mortar, taking the pre-treated mortar, and grinding the plant tissue fragments with liquid nitrogen to obtain plant tissue powder;

[0008] Prepare cell lysis solution in advance, take plant tissue powder and transfer it into a sterilized reaction tube, add 3 times the volume of cell lysis solution to the plant tissue powder, and prepare auxiliary reaction magnetic beads in advance, react in a water bath at 35-40°C for 20 minutes, and oscillate 10-15 times during the reaction. After the reaction, separate the magnetic beads adsorbed with small molecule RNA from the cell lysis solution, and then centrifuge at 2-8°C for 20 minutes to obtain supernatant A and precipitate A;

[0009] Take the magnetic beads adsorbed with small molecule RNA, elute the magnetic beads three times with magnetic bead elution solution to obtain eluent A, and mix the eluent A with supernatant A;

[0010] A LiCl solution is stirred and mixed with supernatant A at a stirring speed of 10-15 r / min for 30 min. After the reaction is completed, the reaction solution is allowed to stand for 2 h to obtain supernatant B and precipitate B containing small molecule RNA. The supernatant B is mixed with precipitate A to obtain a polysaccharide-polyphenol mixture.

[0011] The precipitate B was precipitated and washed with 3 times the volume of washing solution, and the precipitate B was retained. After centrifugation for 10 minutes, the supernatant was discarded to obtain a small molecule RNA precipitate, DEPC solution was added to dissolve the precipitate, and the precipitate was stored at low temperature to obtain a small molecule RNA extract.

[0012] Take a polysaccharide-polyphenol mixture, use a 1:1:1 volume mixture of phenol, isoamyl alcohol and chloroform to extract the polysaccharide-polyphenol mixture, extract the sugar-phenol mixture after standing, recover the plant polysaccharides in the sugar-phenol mixture, and separate to obtain a plant polysaccharide extract.

[0013] Preferably, the method for pretreating the mortar specifically comprises:

[0014] Take a mortar and soak the mortar and grinding rod in DEPC solution for 24 hours;

[0015] Use 75% ethanol to disinfect the mortar and grinding rod 2-4 times, and then sterilize the mortar and grinding rod at a high temperature of 260°C;

[0016] The sterilized mortar and grinding rod were precooled with liquid nitrogen and set aside.

[0017] Preferably, the cell lysate comprises the following raw materials in percentage by weight:

[0018] 3mol / L guanidine thiocyanate: 10%;

[0019] Hexadecyl ammonium bromide: 20%;

[0020] 1 mol / L sodium chloride: 40%;

[0021] Polyvinylpyrrolidone: 5%;

[0022] Extraction buffer: 15%;

[0023] [AMIM][BPh4] ionic liquid: 5%;

[0024] 2-Ethylhexyl phosphate monoester: 3%;

[0025] Antioxidants: 2%.

[0026] Preferably, the method for pre-preparing a cell lysate specifically comprises:

[0027] Under nitrogen protection, 1-methylimidazole and allyl chloride are mixed in a reactor at a molar ratio of 3:1, and dichloromethane is used as a solvent. The mixed solution is heated to 35-45° C. and mixed for 10 minutes. Acetone and 3-chloropropylene are added to the reactor. After additional reaction for 4-5 hours, dichloromethane is removed by rotary evaporation, and the mixture is washed with toluene to obtain 1-allyl-3-methylimidazole chloride.

[0028] Add lithium tetraphenylborate and sodium tetraphenylborate to a reaction kettle containing 1-allyl-3-methylimidazolium chloride, evacuate the kettle, fill it with methane gas, adjust the temperature of the reaction kettle to 45-50° C., control the speed of the reaction kettle to 200-300 r / min, stir the reaction, and then vacuum-evaporate to obtain [AMIM][BPh4] ionic liquid;

[0029] Mix 2-ethylhexyl phosphate monoester, [AMIM][BPh4] ionic liquid and sodium chloride, stir for 1 hour at a temperature of 30-35°C, and stand for 4 hours to obtain a pyrolysis component 1;

[0030] Take guanidine isothiocyanate and hexadecyl ammonium bromide, add the guanidine isothiocyanate and hexadecyl ammonium bromide dropwise into a sodium chloride solution under stirring, then add polyvinyl pyrrolidone and an antioxidant into the mixed solution of guanidine isothiocyanate and hexadecyl ammonium bromide, and adjust the pH to 6-6.5 with an extraction buffer to obtain a cleavage component 2;

[0031] The lysis component 1 and the lysis component 2 were mixed, ultrasonicated for 20 minutes, allowed to stand for 30 minutes, and heated and stirred at 50° C. for 50 minutes to obtain a cell lysate.

[0032] Preferably, the method for pre-preparing auxiliary reaction magnetic beads specifically comprises:

[0033] Solid zinc acetate and ethylene glycol solution are mixed for reaction, and the mixture is stirred in a water bath at 75°C for 1.5 hours. Then, 0.5 mol / L sodium hydroxide solution is added to the reaction solution of zinc acetate and ethylene glycol, and the mixture is stirred in a water bath at 75°C for 4 hours. Then, the mixed solution is put into a reactor, and the mixture is reacted at 120°C for 48 hours, and then cooled to room temperature. Then, the reaction product is washed and ground with anhydrous ethanol to obtain zinc oxide.

[0034] Ferric chloride, polyacrylic acid, sodium acetate and propylene glycol were stirred and mixed for 1 hour to form an iron mixed solution, and then ultrasonically treated for 20 minutes under an ultrasonic environment to obtain an iron mixed emulsion, undispersed particles were separated by sieving, the iron mixed emulsion was added to a reactor, sodium diisooctyl succinate sulfonate was added to the iron mixed emulsion, and the mixture was kept warm at 200°C for 24 hours, and then the emulsion in the reactor was magnetically separated to obtain ferrosoferric oxide nanoparticles;

[0035] The prepared zinc oxide was dispersed in 8 times the volume of deionized water and stirred for 30 minutes, and then the zinc oxide dispersion and the ferroferric oxide nanoparticles were placed in a ball mill for ball milling for 1 hour, and then polyethylene glycol was added to the ball mill, and the ball milling liquid was centrifuged and washed with anhydrous ethanol, and dried at 110°C for 14 hours, and placed in a muffle furnace and heated to 350°C at a heating rate of 5°C / min for 4 hours, and cooled to room temperature to obtain modified ferroferric oxide nanoparticles;

[0036] The modified ferroferric oxide nanoparticles are placed in an atomic deposition chamber, and the atomic deposition chamber is evacuated to make the vacuum degree of the chamber 0.04-0.1 Pa, and then the rotation speed of the modified ferroferric oxide nanoparticles in the atomic deposition chamber is 12-20 r / min, and the modified ferroferric oxide nanoparticles are heated to 120-150° C., and then argon gas is introduced into the atomic deposition chamber for cleaning, and the flow rate of argon gas is 120 sccm;

[0037] Using monosilane and water vapor as silicon dioxide precursors, gaseous monosilane and water vapor are introduced into the atomic deposition chamber by alternating pulses, the bias voltage of the atomic deposition chamber is adjusted to 120-500V, the pulse frequency is 120-180kHz, and deposition is carried out for 2 hours to obtain auxiliary reaction magnetic beads containing silanol groups.

[0038] Preferably, the antioxidant is a mixture of β-mercaptoethanol and ascorbic acid in a volume ratio of 1:1, and the extraction buffer is a Tris base-malic acid buffer solution.

[0039] Preferably, the method for recovering plant polysaccharides from the para-glycoside phenol mixture and separating and obtaining plant polysaccharide extracts specifically comprises:

[0040] A polyphenol separation liquid is prepared, the sugar-phenol mixture is mixed with the polyphenol separation liquid, 0.02 times the volume of glycerol is added thereto, the mixture is placed on a heated magnetic stirrer at 80° C. and stirred for 30 minutes, cooled to room temperature, and centrifuged for 10 minutes, so that the polyphenol separation liquid adsorbs the polyphenols in the sugar-phenol mixture to obtain a polyphenol adsorbed liquid and a supernatant C containing polysaccharides;

[0041] Take the supernatant C, mix the supernatant C with 3 times the volume of petroleum ether, and put the mixed solution into the reactor, keep it warm in a 75°C water bath for 4 hours, stir the reactor with a glass rod every 10 minutes, then heat the reactor to 150°C, stir for 20 minutes, extract the plant polysaccharide with hot water at 50°C, and collect the polysaccharide supernatant;

[0042] The polysaccharide supernatant was suspended in pre-cooled anhydrous ethanol and allowed to stand at 25°C for 48 hours to obtain a plant polysaccharide precipitate.

[0043] Preferably, the polyphenol separation liquid comprises the following raw materials in parts by weight: 10-30 parts of polyvinyl pyrrolidone, 1-5 parts of dithioerythritol, 2-5 parts of isoamyl alcohol, 20-40 parts of sodium chloride, 10-15 parts of sodium dodecyl sulfate, and 10-30 parts of methanol.

[0044] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0045] The embodiment of the present invention realizes efficient recovery and processing of small molecule RNA in plant tissue samples by means of technical means of coordinated cooperation between cell lysis solution and auxiliary reaction magnetic beads, and adopts reduction inhibition cell lysis solution to provide a strong reducing environment for the recovery of small molecule RNA, thereby avoiding the interference of polyphenols and polysaccharides in plant tissues with the extraction of small molecule RNA during small molecule RNA extraction, and ensuring that the reverse transcription of small molecule RNA is not inhibited.

[0046] In an embodiment of the present invention, a polyphenol separation liquid is provided, in which dithioerythritol, isopentanol, sodium chloride, sodium dodecyl sulfate, and methanol cooperate to maintain a high affinity for polyphenols in a sugar-phenol mixture, thereby effectively removing polyphenols by adsorption, while also avoiding the oxidation of polyphenols, thereby facilitating the separation and recovery of polyphenols and polysaccharides.

[0047] In an embodiment of the present invention, a method for isolating and obtaining a plant polysaccharide extract is provided. By isolating and obtaining the plant polysaccharide extract, the plant polysaccharides in the plant tissue can be recycled and utilized while recovering small molecule RNA, and the purity of the plant polysaccharides can be ensured, which effectively solves the problem of difficulty in recovering plant polysaccharides and low recovery rate when isolating and recovering small molecule RNA from plant tissues. The high-purity plant polysaccharides provide a basis for enhancing immunity, anti-oxidation and other effects.

[0048] In an embodiment of the present invention, a cell lysis solution is provided. The cell lysis solution can act on plant tissue powder to quickly complex and precipitate with proteins, polyphenols, and polysaccharides in the plant tissue powder. The antioxidant can also prevent the oxidation of polyphenols and avoid excessive small molecule RNA impurities during separation. At the same time, the cell lysis solution contains [AMIM][BPh4] ionic liquid. The [AMIM][BPh4] ionic liquid can accelerate the cell wall of plant tissue, thereby allowing cellulose and small molecule RNA to be quickly released from the cell. The [AMIM][BPh4] ionic liquid affects the activity of cellulose degrading enzymes to avoid cellulose degradation, thereby ensuring the cleanliness of small molecule RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the implementation process of the method for extracting small RNA from plant polysaccharides and polyphenols provided by the present invention.

[0050] Figure 2 The present invention provides a schematic flow diagram of a method for recovering and treating plant polysaccharides in a glycophenol mixture and separating and obtaining a plant polysaccharide extract.

[0051] Figure 3 It is a test chart of the sugar yield and polysaccharide purity of plant polysaccharide separation in Examples 1-5 of the present invention and Comparative Examples 1-3.

[0052] Figure 4 It is a schematic diagram of the implementation process of the pretreatment mortar provided by the present invention.

[0053] Figure 5 It is a schematic diagram of the implementation process of the pre-formulated cell lysate provided by the present invention.

[0054] Figure 6 It is a schematic diagram of the implementation process of pre-preparing auxiliary reaction magnetic beads provided by the present invention.

[0055] Figure 7 It is a test chart of the concentration and purity of small molecule RNA extracted in Examples 1-5 and Comparative Examples 4-6 of the present invention.

[0056] Figure 8 It is a schematic diagram of the electrophoresis test results of Example 5 of the present invention. DETAILED DESCRIPTION

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of this application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0058] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0059] When the existing method is used to lyse plant tissue, the polyphenols in the cells are easily oxidized when the cell walls of the plant tissue are broken, so that impurities interfere with the reverse transcription of small molecule RNA during separation, affecting the extraction rate and integrity of small molecule RNA. In view of the above problems, we propose a method for extracting small molecule RNA from plant polysaccharide polyphenols. In short, when the method is used to extract small molecule RNA, the plant tissue sample is first pretreated to obtain plant tissue fragments, and the pretreated mortar is taken and the plant tissue fragments are ground with liquid nitrogen to obtain plant tissue powder. Then, cell lysis solution and auxiliary reaction magnetic beads are pre-prepared, and the magnetic beads adsorbed with small molecule RNA are separated from the cell lysis solution. Licl solution is used to stir and mix with supernatant A to obtain supernatant B and precipitate B containing small molecule RNA, and 3 times the volume of washing solution is used to precipitate and wash the precipitate B. After centrifugation for 10 minutes, the supernatant is discarded to obtain a small molecule RNA precipitate. The embodiment of the present invention realizes efficient recovery and processing of small molecule RNA in plant tissue samples by means of technical means of coordinated cooperation between cell lysis solution and auxiliary reaction magnetic beads, and adopts reduction inhibition cell lysis solution to provide a strong reducing environment for the recovery of small molecule RNA, thereby avoiding the interference of polyphenols and polysaccharides in plant tissues with the extraction of small molecule RNA during small molecule RNA extraction, and ensuring that the reverse transcription of small molecule RNA is not inhibited.

[0060] Example 1

[0061] The embodiment of the present invention provides a method for extracting small RNA from plant polysaccharides and polyphenols. Figure 1 The schematic diagram of the implementation process of the method for extracting small RNA from plant polysaccharides and polyphenols is shown. The method for extracting small RNA from plant polysaccharides and polyphenols specifically comprises:

[0062] Step S10, pre-treating the plant tissue sample, cutting the plant tissue sample with ophthalmic surgical scissors that have been sterilized at high temperature to obtain plant tissue fragments, freezing the plant tissue fragments in a -80°C liquid nitrogen environment for 2 hours, taking a mortar, filling the mortar with diatomaceous earth, and then grinding the diatomaceous earth with a grinding rod, removing the diatomaceous earth after grinding for 10 minutes, pre-treating the mortar, taking the pre-treated mortar, and grinding the plant tissue fragments with liquid nitrogen to obtain plant tissue powder;

[0063] It should be noted that the plant tissue can come from a variety of plants including but not limited to potatoes, cotton, apples, citrus, grapes, strawberries, etc., and the plant tissue can be plant seeds, fruits, epidermal tissues and rhizome tissues. The diatomaceous earth is a commercially available ultra-fine white diatomaceous earth product. The use of diatomaceous earth to treat the mortar can assist in inhibiting the activity of RNases and protect small molecule RNA from being degraded, thereby ensuring that the extracted small molecule RNA is clean and pollution-free.

[0064] Step S20, pre-preparing a cell lysis solution, taking a plant tissue powder and transferring it into a sterilized reaction tube, adding 3 times the volume of the cell lysis solution to the plant tissue powder, and pre-preparing auxiliary reaction magnetic beads, reacting in a water bath at 35-40°C for 20 minutes, and oscillating 10-15 times during the reaction. After the reaction is completed, the magnetic beads adsorbed with small molecule RNA are separated from the cell lysis solution, and then centrifuged at 2-8°C for 20 minutes to obtain supernatant A and precipitate A;

[0065] In this embodiment, when separating the magnetic beads adsorbed with small molecule RNA from the cell lysate, an external magnetic field can be applied to the magnetic beads in the cell lysate to attract the magnetic beads to the outer edge of the reaction tube and keep them stationary.

[0066] Step S30, taking the magnetic beads adsorbed with the small molecule RNA, eluting the magnetic beads three times with a magnetic bead elution solution to obtain an eluent A, and mixing the eluent A with a supernatant A;

[0067] It should be noted that the magnetic bead elution solution can be a 1:1 volume mixture of borate buffer and phosphate buffer, and the single elution time is controlled to be 10-15 minutes, which can achieve rapid elution and separation of small molecule RNA on the surface of the magnetic beads.

[0068] Step S40, stirring and mixing the LiCl solution with the supernatant A at a stirring speed of 10-15 r / min for 30 min. After the reaction is completed, the reaction solution is allowed to stand for 2 h to obtain a supernatant B and a precipitate B containing small molecule RNA. The supernatant B is mixed with the precipitate A to obtain a polysaccharide-polyphenol mixture.

[0069] In this embodiment, the mass fraction of the Licl solution can be 10-20%, the Licl manufacturer is Shanghai Binlian Industrial Co., Ltd., and the Licl solution and supernatant A can be stirred and mixed using an SJ-3 constant temperature magnetic stirrer produced by Changzhou Zhibo Instrument Manufacturing Co., Ltd., China.

[0070] Step S50, using 3 times the volume of washing solution to precipitate and wash the precipitate B, retaining the precipitate B, centrifuging for 10 minutes and discarding the supernatant to obtain a small molecule RNA precipitate, adding DEPC solution water to dissolve the precipitate, and storing it at low temperature to obtain a small molecule RNA extract.

[0071] It should be noted that when the precipitate B is washed, the washing liquid can be pre-cooled 70% ethanol, the temperature of 70% ethanol is 4°C, and the mixture of the washing liquid and the precipitate B is stirred during the precipitation washing, and the stirring speed is controlled to be 10-12Kr / min.

[0072] Step S60, taking the polysaccharide-polyphenol mixture, using a 1:1:1 volume mixture of phenol, isoamyl alcohol and chloroform to extract the polysaccharide-polyphenol mixture, extracting the sugar-phenol mixture after standing, recovering the plant polysaccharides in the sugar-phenol mixture, and separating to obtain a plant polysaccharide extract.

[0073] The embodiment of the present invention realizes efficient recovery and processing of small molecule RNA in plant tissue samples by means of technical means of coordinated cooperation between cell lysis solution and auxiliary reaction magnetic beads, and adopts reduction inhibition cell lysis solution to provide a strong reducing environment for the recovery of small molecule RNA, thereby avoiding the interference of polyphenols and polysaccharides in plant tissues with the extraction of small molecule RNA during small molecule RNA extraction, and ensuring that the reverse transcription of small molecule RNA is not inhibited.

[0074] In this embodiment, the method for recovering plant polysaccharides from the glycophenol mixture and separating and obtaining plant polysaccharide extracts specifically includes:

[0075] Step S101, preparing a polyphenol separation liquid, mixing the sugar-phenol mixture with the polyphenol separation liquid, adding 0.02 times the volume of glycerol thereto, placing the mixture on a heated magnetic stirrer at 80° C. and stirring for 30 minutes, cooling to room temperature, and centrifuging for 10 minutes, wherein the polyphenol separation liquid adsorbs the polyphenols in the sugar-phenol mixture to obtain a polyphenol adsorbed liquid and a supernatant C containing polysaccharides;

[0076] In an embodiment of the present invention, a polyphenol separation liquid is provided, in which dithioerythritol, isopentanol, sodium chloride, sodium dodecyl sulfate, and methanol cooperate to maintain a high affinity for polyphenols in a sugar-phenol mixture, thereby effectively removing polyphenols by adsorption, while also avoiding the oxidation of polyphenols, thereby facilitating the separation and recovery of polyphenols and polysaccharides.

[0077] Step S102, taking the supernatant C, mixing the supernatant C with 3 times the volume of petroleum ether, and loading the mixture into a reactor, keeping it warm in a 75°C water bath for 4 hours, stirring the reactor with a glass rod every 10 minutes, then heating the reactor to 150°C, stirring for 20 minutes, extracting plant polysaccharides with hot water at 50°C, and collecting the polysaccharide supernatant;

[0078] It should be noted that the supernatant C can be defatted and impurities removed by mixing petroleum ether with the supernatant C. The polar small molecules in the supernatant C can be removed during reflux degreasing by petroleum ether, thereby ensuring the purity of the recovered plant polysaccharides.

[0079] Step S103, suspending the polysaccharide supernatant in pre-cooled anhydrous ethanol, and standing at 25° C. for 48 hours to obtain a plant polysaccharide precipitate.

[0080] In this embodiment, the purpose of suspending the polysaccharide supernatant in pre-cooled anhydrous ethanol is to adsorb and recover the plant polysaccharide, and the anhydrous ethanol combines with the plant polysaccharide to produce a plant polysaccharide precipitate. After heating and recovering the anhydrous ethanol, the plant polysaccharide precipitate is washed and recovered with acetone or ether, and the plant polysaccharide precipitate is placed in a freeze dryer, the temperature of the freeze dryer is controlled to -50°C, the freezing time is 48h, and the plant polysaccharide dry powder with uniform particles is obtained by grinding with a mortar. The plant polysaccharide can be other plant polysaccharides such as cellulose, pectin, glucose, and arabinoxylan.

[0081] It should be noted that the polyphenol separation liquid includes the following raw materials in parts by weight: 10 parts of polyvinyl pyrrolidone, 1 part of dithioerythritol, 2 parts of isoamyl alcohol, 20 parts of sodium chloride, 10 parts of sodium dodecyl sulfate, and 10 parts of methanol.

[0082] In this embodiment, when preparing the polyphenol separation liquid, polyvinyl pyrrolidone, dithioerythritol, and isoamyl alcohol can be put into a reactor containing methanol in sequence, and then the reactor temperature is maintained at 35°C, the stirring speed is controlled to 250r / min, and stirring and mixing are carried out for 30 minutes. Then, sodium dodecyl sulfate and sodium chloride are put into the reactor, the reactor temperature is raised to 60°C, the stirring speed is controlled to 200r / min, and stirring and mixing is carried out for 30 minutes to obtain the polyphenol separation liquid.

[0083] In an embodiment of the present invention, a method for isolating and obtaining a plant polysaccharide extract is provided. By isolating and obtaining the plant polysaccharide extract, the plant polysaccharides in the plant tissue can be recycled and utilized while recovering small molecule RNA, and the purity of the plant polysaccharides can be ensured, which effectively solves the problem of difficulty in recovering plant polysaccharides and low recovery rate when isolating and recovering small molecule RNA from plant tissues. The high-purity plant polysaccharides provide a basis for enhancing immunity, anti-oxidation and other effects.

[0084] Example 2

[0085] The embodiment of the present invention provides a method for extracting small RNA from plant polysaccharides and polyphenols. Figure 1 The schematic diagram of the implementation process of the method for extracting small RNA from plant polysaccharides and polyphenols is shown. The method for extracting small RNA from plant polysaccharides and polyphenols specifically comprises:

[0086] Step S10, pre-treating the plant tissue sample, cutting the plant tissue sample with ophthalmic surgical scissors that have been sterilized at high temperature to obtain plant tissue fragments, freezing the plant tissue fragments in a -80°C liquid nitrogen environment for 2 hours, taking a mortar, filling the mortar with diatomaceous earth, and then grinding the diatomaceous earth with a grinding rod, removing the diatomaceous earth after grinding for 10 minutes, pre-treating the mortar, taking the pre-treated mortar, and grinding the plant tissue fragments with liquid nitrogen to obtain plant tissue powder;

[0087] Step S20, pre-preparing a cell lysis solution, taking a plant tissue powder and transferring it into a sterilized reaction tube, adding 3 times the volume of the cell lysis solution to the plant tissue powder, and pre-preparing auxiliary reaction magnetic beads, reacting in a water bath at 35-40°C for 20 minutes, and oscillating 10-15 times during the reaction. After the reaction is completed, the magnetic beads adsorbed with small molecule RNA are separated from the cell lysis solution, and then centrifuged at 2-8°C for 20 minutes to obtain supernatant A and precipitate A;

[0088] Step S30, taking the magnetic beads adsorbed with the small molecule RNA, eluting the magnetic beads three times with a magnetic bead elution solution to obtain an eluent A, and mixing the eluent A with a supernatant A;

[0089] Step S40, stirring and mixing the LiCl solution with the supernatant A at a stirring speed of 10-15 r / min for 30 min. After the reaction is completed, the reaction solution is allowed to stand for 2 h to obtain a supernatant B and a precipitate B containing small molecule RNA. The supernatant B is mixed with the precipitate A to obtain a polysaccharide-polyphenol mixture.

[0090] Step S50, using 3 times the volume of washing solution to precipitate and wash the precipitate B, retaining the precipitate B, centrifuging for 10 minutes and discarding the supernatant to obtain a small molecule RNA precipitate, adding DEPC solution water to dissolve the precipitate, and storing it at low temperature to obtain a small molecule RNA extract.

[0091] Step S60, taking the polysaccharide-polyphenol mixture, using a 1:1:1 volume mixture of phenol, isoamyl alcohol and chloroform to extract the polysaccharide-polyphenol mixture, extracting the sugar-phenol mixture after standing, recovering the plant polysaccharides in the sugar-phenol mixture, and separating to obtain a plant polysaccharide extract.

[0092] In this embodiment, the method for recovering plant polysaccharides from the glycophenol mixture and separating and obtaining plant polysaccharide extracts specifically includes:

[0093] Step S101, preparing a polyphenol separation liquid, mixing the sugar-phenol mixture with the polyphenol separation liquid, adding 0.02 times the volume of glycerol thereto, placing the mixture on a heated magnetic stirrer at 80° C. and stirring for 30 minutes, cooling to room temperature, and centrifuging for 10 minutes, wherein the polyphenol separation liquid adsorbs the polyphenols in the sugar-phenol mixture to obtain a polyphenol adsorbed liquid and a supernatant C containing polysaccharides;

[0094] Step S102, taking the supernatant C, mixing the supernatant C with 3 times the volume of petroleum ether, and loading the mixture into a reactor, keeping it warm in a 75°C water bath for 4 hours, stirring the reactor with a glass rod every 10 minutes, then heating the reactor to 150°C, stirring for 20 minutes, extracting plant polysaccharides with hot water at 50°C, and collecting the polysaccharide supernatant;

[0095] Step S103, suspending the polysaccharide supernatant in pre-cooled anhydrous ethanol, and standing at 25° C. for 48 hours to obtain a plant polysaccharide precipitate.

[0096] It should be noted that the polyphenol separation liquid includes the following raw materials in parts by weight: 30 parts of polyvinyl pyrrolidone, 5 parts of dithioerythritol, 5 parts of isoamyl alcohol, 40 parts of sodium chloride, 15 parts of sodium dodecyl sulfate, and 30 parts of methanol.

[0097] Example 3

[0098] The embodiment of the present invention provides a method for extracting small RNA from plant polysaccharides and polyphenols. Figure 1 The schematic diagram of the implementation process of the method for extracting small RNA from plant polysaccharides and polyphenols is shown. The method for extracting small RNA from plant polysaccharides and polyphenols specifically comprises:

[0099] Step S10, pre-treating the plant tissue sample, cutting the plant tissue sample with ophthalmic surgical scissors that have been sterilized at high temperature to obtain plant tissue fragments, freezing the plant tissue fragments in a -80°C liquid nitrogen environment for 2 hours, taking a mortar, filling the mortar with diatomaceous earth, and then grinding the diatomaceous earth with a grinding rod, removing the diatomaceous earth after grinding for 10 minutes, pre-treating the mortar, taking the pre-treated mortar, and grinding the plant tissue fragments with liquid nitrogen to obtain plant tissue powder;

[0100] Step S20, pre-preparing a cell lysis solution, taking a plant tissue powder and transferring it into a sterilized reaction tube, adding 3 times the volume of the cell lysis solution to the plant tissue powder, and pre-preparing auxiliary reaction magnetic beads, reacting in a water bath at 35-40°C for 20 minutes, and oscillating 10-15 times during the reaction. After the reaction is completed, the magnetic beads adsorbed with small molecule RNA are separated from the cell lysis solution, and then centrifuged at 2-8°C for 20 minutes to obtain supernatant A and precipitate A;

[0101] Step S30, taking the magnetic beads adsorbed with the small molecule RNA, eluting the magnetic beads three times with a magnetic bead elution solution to obtain an eluent A, and mixing the eluent A with a supernatant A;

[0102] Step S40, stirring and mixing the LiCl solution with the supernatant A at a stirring speed of 10-15 r / min for 30 min. After the reaction is completed, the reaction solution is allowed to stand for 2 h to obtain a supernatant B and a precipitate B containing small molecule RNA. The supernatant B is mixed with the precipitate A to obtain a polysaccharide-polyphenol mixture.

[0103] Step S50, using 3 times the volume of washing solution to precipitate and wash the precipitate B, retaining the precipitate B, centrifuging for 10 minutes and discarding the supernatant to obtain a small molecule RNA precipitate, adding DEPC solution water to dissolve the precipitate, and storing it at low temperature to obtain a small molecule RNA extract.

[0104] Step S60, taking the polysaccharide-polyphenol mixture, using a 1:1:1 volume mixture of phenol, isoamyl alcohol and chloroform to extract the polysaccharide-polyphenol mixture, extracting the sugar-phenol mixture after standing, recovering the plant polysaccharides in the sugar-phenol mixture, and separating to obtain a plant polysaccharide extract.

[0105] In this embodiment, a method for recovering plant polysaccharides from a sugar-phenol mixture and separating and obtaining a plant polysaccharide extract is also provided. Figure 2 The present invention shows a schematic flow diagram of a method for recovering plant polysaccharides from a glycophenol mixture and separating and obtaining a plant polysaccharide extract. The method for recovering plant polysaccharides from a glycophenol mixture and separating and obtaining a plant polysaccharide extract specifically comprises:

[0106] Step S101, preparing a polyphenol separation liquid, mixing the sugar-phenol mixture with the polyphenol separation liquid, adding 0.02 times the volume of glycerol thereto, placing the mixture on a heated magnetic stirrer at 80° C. and stirring for 30 minutes, cooling to room temperature, and centrifuging for 10 minutes, wherein the polyphenol separation liquid adsorbs the polyphenols in the sugar-phenol mixture to obtain a polyphenol adsorbed liquid and a supernatant C containing polysaccharides;

[0107] Step S102, taking the supernatant C, mixing the supernatant C with 3 times the volume of petroleum ether, and loading the mixture into a reactor, keeping it warm in a 75°C water bath for 4 hours, stirring the reactor with a glass rod every 10 minutes, then heating the reactor to 150°C, stirring for 20 minutes, extracting plant polysaccharides with hot water at 50°C, and collecting the polysaccharide supernatant;

[0108] Step S103, suspending the polysaccharide supernatant in pre-cooled anhydrous ethanol, and standing at 25° C. for 48 hours to obtain a plant polysaccharide precipitate.

[0109] It should be noted that the polyphenol separation liquid includes the following raw materials in parts by weight: 12 parts of polyvinyl pyrrolidone, 2 parts of dithioerythritol, 3 parts of isoamyl alcohol, 22 parts of sodium chloride, 11 parts of sodium dodecyl sulfate, and 12 parts of methanol.

[0110] Example 4

[0111] The embodiment of the present invention provides a method for extracting small RNA from plant polysaccharides and polyphenols. Figure 1 The schematic diagram of the implementation process of the method for extracting small RNA from plant polysaccharides and polyphenols is shown. The method for extracting small RNA from plant polysaccharides and polyphenols specifically comprises:

[0112] Step S10, pre-treating the plant tissue sample, cutting the plant tissue sample with ophthalmic surgical scissors that have been sterilized at high temperature to obtain plant tissue fragments, freezing the plant tissue fragments in a -80°C liquid nitrogen environment for 2 hours, taking a mortar, filling the mortar with diatomaceous earth, and then grinding the diatomaceous earth with a grinding rod, removing the diatomaceous earth after grinding for 10 minutes, pre-treating the mortar, taking the pre-treated mortar, and grinding the plant tissue fragments with liquid nitrogen to obtain plant tissue powder;

[0113] Step S20, pre-preparing a cell lysis solution, taking a plant tissue powder and transferring it into a sterilized reaction tube, adding 3 times the volume of the cell lysis solution to the plant tissue powder, and pre-preparing auxiliary reaction magnetic beads, reacting in a water bath at 35-40°C for 20 minutes, and oscillating 10-15 times during the reaction. After the reaction is completed, the magnetic beads adsorbed with small molecule RNA are separated from the cell lysis solution, and then centrifuged at 2-8°C for 20 minutes to obtain supernatant A and precipitate A;

[0114] Step S30, taking the magnetic beads adsorbed with the small molecule RNA, eluting the magnetic beads three times with a magnetic bead elution solution to obtain an eluent A, and mixing the eluent A with a supernatant A;

[0115] Step S40, stirring and mixing the LiCl solution with the supernatant A at a stirring speed of 10-15 r / min for 30 min. After the reaction is completed, the reaction solution is allowed to stand for 2 h to obtain a supernatant B and a precipitate B containing small molecule RNA. The supernatant B is mixed with the precipitate A to obtain a polysaccharide-polyphenol mixture.

[0116] Step S50, using 3 times the volume of washing solution to precipitate and wash the precipitate B, retaining the precipitate B, centrifuging for 10 minutes and discarding the supernatant to obtain a small molecule RNA precipitate, adding DEPC solution water to dissolve the precipitate, and storing it at low temperature to obtain a small molecule RNA extract.

[0117] Step S60, taking the polysaccharide-polyphenol mixture, using a 1:1:1 volume mixture of phenol, isoamyl alcohol and chloroform to extract the polysaccharide-polyphenol mixture, extracting the sugar-phenol mixture after standing, recovering the plant polysaccharides in the sugar-phenol mixture, and separating to obtain a plant polysaccharide extract.

[0118] In this embodiment, the method for recovering plant polysaccharides from the glycophenol mixture and separating and obtaining plant polysaccharide extracts specifically includes:

[0119] Step S101, preparing a polyphenol separation liquid, mixing the sugar-phenol mixture with the polyphenol separation liquid, adding 0.02 times the volume of glycerol thereto, placing the mixture on a heated magnetic stirrer at 80° C. and stirring for 30 minutes, cooling to room temperature, and centrifuging for 10 minutes, wherein the polyphenol separation liquid adsorbs the polyphenols in the sugar-phenol mixture to obtain a polyphenol adsorbed liquid and a supernatant C containing polysaccharides;

[0120] Step S102, taking the supernatant C, mixing the supernatant C with 3 times the volume of petroleum ether, and loading the mixture into a reactor, keeping it warm in a 75°C water bath for 4 hours, stirring the reactor with a glass rod every 10 minutes, then heating the reactor to 150°C, stirring for 20 minutes, extracting plant polysaccharides with hot water at 50°C, and collecting the polysaccharide supernatant;

[0121] Step S103, suspending the polysaccharide supernatant in pre-cooled anhydrous ethanol, and standing at 25° C. for 48 hours to obtain a plant polysaccharide precipitate.

[0122] It should be noted that the polyphenol separation liquid includes the following raw materials in parts by weight: 28 parts of polyvinyl pyrrolidone, 4 parts of dithioerythritol, 4 parts of isoamyl alcohol, 38 parts of sodium chloride, 14 parts of sodium dodecyl sulfate, and 27 parts of methanol.

[0123] Example 5

[0124] The embodiment of the present invention provides a method for extracting small RNA from plant polysaccharides and polyphenols. Figure 1The schematic diagram of the implementation process of the method for extracting small RNA from plant polysaccharides and polyphenols is shown. The method for extracting small RNA from plant polysaccharides and polyphenols specifically comprises:

[0125] Step S10, pre-treating the plant tissue sample, cutting the plant tissue sample with ophthalmic surgical scissors that have been sterilized at high temperature to obtain plant tissue fragments, freezing the plant tissue fragments in a -80°C liquid nitrogen environment for 2 hours, taking a mortar, filling the mortar with diatomaceous earth, and then grinding the diatomaceous earth with a grinding rod, removing the diatomaceous earth after grinding for 10 minutes, pre-treating the mortar, taking the pre-treated mortar, and grinding the plant tissue fragments with liquid nitrogen to obtain plant tissue powder;

[0126] Step S20, pre-preparing a cell lysis solution, taking a plant tissue powder and transferring it into a sterilized reaction tube, adding 3 times the volume of the cell lysis solution to the plant tissue powder, and pre-preparing auxiliary reaction magnetic beads, reacting in a water bath at 35-40°C for 20 minutes, and oscillating 10-15 times during the reaction. After the reaction is completed, the magnetic beads adsorbed with small molecule RNA are separated from the cell lysis solution, and then centrifuged at 2-8°C for 20 minutes to obtain supernatant A and precipitate A;

[0127] Step S30, taking the magnetic beads adsorbed with the small molecule RNA, eluting the magnetic beads three times with a magnetic bead elution solution to obtain an eluent A, and mixing the eluent A with a supernatant A;

[0128] Step S40, stirring and mixing the LiCl solution with the supernatant A at a stirring speed of 10-15 r / min for 30 min. After the reaction is completed, the reaction solution is allowed to stand for 2 h to obtain a supernatant B and a precipitate B containing small molecule RNA. The supernatant B is mixed with the precipitate A to obtain a polysaccharide-polyphenol mixture.

[0129] Step S50, using 3 times the volume of washing solution to precipitate and wash the precipitate B, retaining the precipitate B, centrifuging for 10 minutes and discarding the supernatant to obtain a small molecule RNA precipitate, adding DEPC solution water to dissolve the precipitate, and storing it at low temperature to obtain a small molecule RNA extract.

[0130] Step S60, taking the polysaccharide-polyphenol mixture, using a 1:1:1 volume mixture of phenol, isoamyl alcohol and chloroform to extract the polysaccharide-polyphenol mixture, extracting the sugar-phenol mixture after standing, recovering the plant polysaccharides in the sugar-phenol mixture, and separating to obtain a plant polysaccharide extract.

[0131] In this embodiment, the method for recovering plant polysaccharides from the glycophenol mixture and separating and obtaining plant polysaccharide extracts specifically includes:

[0132] Step S101, preparing a polyphenol separation liquid, mixing the sugar-phenol mixture with the polyphenol separation liquid, adding 0.02 times the volume of glycerol thereto, placing the mixture on a heated magnetic stirrer at 80° C. and stirring for 30 minutes, cooling to room temperature, and centrifuging for 10 minutes, wherein the polyphenol separation liquid adsorbs the polyphenols in the sugar-phenol mixture to obtain a polyphenol adsorbed liquid and a supernatant C containing polysaccharides;

[0133] Step S102, taking the supernatant C, mixing the supernatant C with 3 times the volume of petroleum ether, and loading the mixture into a reactor, keeping it warm in a 75°C water bath for 4 hours, stirring the reactor with a glass rod every 10 minutes, then heating the reactor to 150°C, stirring for 20 minutes, extracting plant polysaccharides with hot water at 50°C, and collecting the polysaccharide supernatant;

[0134] Step S103, suspending the polysaccharide supernatant in pre-cooled anhydrous ethanol, and standing at 25° C. for 48 hours to obtain a plant polysaccharide precipitate.

[0135] It should be noted that the polyphenol separation liquid includes the following raw materials in parts by weight: 20 parts of polyvinyl pyrrolidone, 3 parts of dithioerythritol, 3 parts of isoamyl alcohol, 30 parts of sodium chloride, 13 parts of sodium dodecyl sulfate, and 20 parts of methanol.

[0136] Comparative Example 1

[0137] The other steps of this comparative example are the same as those of Example 5, except that sodium lauryl sulfate and methanol are not included in the raw materials of the polyphenol separation liquid. The polyphenol separation liquid includes the following raw materials in parts by weight: 20 parts of polyvinyl pyrrolidone, 3 parts of dithioerythritol, 3 parts of isoamyl alcohol, and 30 parts of sodium chloride.

[0138] Comparative Example 2

[0139] The other steps of this comparative example are the same as those of Example 5, except that dithioerythritol and sodium lauryl sulfate are not included in the raw materials of the polyphenol separation liquid. The polyphenol separation liquid includes the following raw materials in parts by weight: 20 parts of polyvinyl pyrrolidone, 3 parts of isoamyl alcohol, 30 parts of sodium chloride, and 20 parts of methanol.

[0140] Comparative Example 3

[0141] The other steps of this comparative example are the same as those of Example 5, except that dithioerythritol, sodium lauryl sulfate and methanol are not included in the raw materials of the polyphenol separation liquid. The polyphenol separation liquid includes the following raw materials in parts by weight: 20 parts of polyvinyl pyrrolidone, 3 parts of isoamyl alcohol and 30 parts of sodium chloride.

[0142] In order to verify the sugar yield of plant polysaccharide separation in Examples 1-5 and Comparative Examples 1-3 of the present invention, the phenol-concentrated sulfuric acid method was used to determine the content of plant polysaccharides. During the test, the plant tissue was Phellinus igniarius, the plant polysaccharide was Phellinus igniarius polysaccharide, deionized water was used as a reference, and the absorbance of the embodiments and comparative examples was measured at a wavelength of 620 nm using a double-beam ultraviolet-visible spectrophotometer. The sugar yield and polysaccharide purity of plant polysaccharide separation in Examples 1-5 and Comparative Examples 1-3 of the present invention were calculated respectively. The test results are shown in Tables 1 and Figure 3 shown.

[0143] Table 1

[0144]

[0145]

[0146] It can be concluded from Table 1 that the sugar yield and purity of plant polysaccharides in Examples 1-5 of the present invention are higher than those in Comparative Examples 1-3. The synergistic combination of polyvinyl pyrrolidone, dithioerythritol, isopentanol, sodium chloride, sodium dodecyl sulfate and methanol in the embodiments of the present invention can avoid the oxidation of polyphenols and thus affect the purity and extraction rate of polysaccharides, while also avoiding the adsorption of plant polysaccharides by other impurities.

[0147] In this embodiment, if Figure 4 As shown, the method for pretreating the mortar specifically comprises:

[0148] Step S201, taking a mortar, and soaking the mortar and the grinding rod in a DEPC solution for 24 hours;

[0149] Step S202, using 75% ethanol to disinfect the mortar and the grinding rod for 2-4 times, and then sterilizing the mortar and the grinding rod at a high temperature of 260°C;

[0150] Step S203, using liquid nitrogen to pre-cool the sterilized mortar and grinding rod for standby use.

[0151] In the embodiment of the present invention, the DEPC solution is used to statically soak the mortar and the grinding rod to ensure that the mortar and the grinding rod effectively inhibit the activity of RNase when grinding plant tissues, thereby avoiding the problem of degradation of small molecule RNA during grinding.

[0152] It should be noted that the cell lysate includes the following raw materials in percentage by weight:

[0153] 3mol / L guanidine thiocyanate: 10%;

[0154] Hexadecyl ammonium bromide: 20%;

[0155] 1 mol / L sodium chloride: 40%;

[0156] Polyvinylpyrrolidone: 5%;

[0157] Extraction buffer: 15%;

[0158] [AMIM][BPh4] ionic liquid: 5%;

[0159] 2-Ethylhexyl phosphate monoester: 3%;

[0160] Antioxidants: 2%.

[0161] The antioxidant is a mixture of beta-mercaptoethanol and ascorbic acid in a volume ratio of 1:1, and the extraction buffer is a Tris base-malic acid buffer solution.

[0162] In an embodiment of the present invention, a cell lysis solution is provided. The cell lysis solution can act on plant tissue powder to quickly complex and precipitate with proteins, polyphenols, and polysaccharides in the plant tissue powder. The antioxidant can also prevent the oxidation of polyphenols and avoid excessive small molecule RNA impurities during separation. At the same time, the cell lysis solution contains [AMIM][BPh4] ionic liquid. The [AMIM][BPh4] ionic liquid can accelerate the cell wall of plant tissue, thereby allowing cellulose and small molecule RNA to be quickly released from the cell. The [AMIM][BPh4] ionic liquid affects the activity of cellulose degrading enzymes to avoid cellulose degradation, thereby ensuring the cleanliness of small molecule RNA.

[0163] In this embodiment, if Figure 5 As shown, the method for pre-preparing a cell lysate specifically comprises:

[0164] Step S301, 1-methylimidazole and allyl chloride are mixed in a reactor at a molar ratio of 3:1 under a nitrogen protection environment, dichloromethane is used as a solvent, and the mixed solution is heated to 35-45° C. After mixing for 10 minutes, acetone and 3-chloropropylene are added to the reactor, and after additional reaction for 4-5 hours, dichloromethane is removed by rotary evaporation, and washed with toluene to obtain 1-allyl-3-methylimidazole chloride;

[0165] Step S302, adding lithium tetraphenylborate and sodium tetraphenylborate to a reaction kettle containing 1-allyl-3-methylimidazolium chloride, evacuating the kettle, filling the kettle with methane gas, adjusting the temperature of the reaction kettle to 45-50° C., controlling the speed of the reaction kettle to 200-300 r / min, stirring the reaction, and then vacuum-evaporating the kettle to obtain [AMIM][BPh4] ionic liquid;

[0166] In this embodiment, when 1-allyl-3-methylimidazolium chloride is mixed with lithium tetraphenylborate and sodium tetraphenylborate for reaction, the allyl compound can effectively inhibit the action of cellulose degrading enzymes / groups on cellulose, thereby improving the recovery rate of plant polysaccharides.

[0167] Step S303, 2-ethylhexyl phosphate monoester, [AMIM][BPh4] ionic liquid and sodium chloride are mixed, stirred for 1 hour at a temperature of 30-35° C., and allowed to stand for 4 hours to obtain a pyrolysis component 1;

[0168] It should be noted that the manufacturer of 2-ethylhexyl monophosphate is Guangzhou Yuanda New Materials Co., Ltd. The mixture of 2-ethylhexyl monophosphate and [AMIM][BPh4] ionic liquid can promote the complexation of polysaccharides, polyphenols, and lipids, and accelerate the separation of small molecule RNA.

[0169] Step S304, taking guanidine isothiocyanate and hexadecyl ammonium bromide, adding the guanidine isothiocyanate and hexadecyl ammonium bromide dropwise into a sodium chloride solution under stirring, then adding polyvinyl pyrrolidone and an antioxidant into the mixed solution of guanidine isothiocyanate and hexadecyl ammonium bromide, and adjusting the pH to 6-6.5 with an extraction buffer to obtain a cleavage component 2;

[0170] Step S305, taking the lysis component 1 and the lysis component 2, mixing them, ultrasonicating them for 20 minutes, letting them stand for 30 minutes, heating and stirring them at 50° C. for 50 minutes, and obtaining a cell lysis solution.

[0171] The present invention also provides a method for pre-preparing auxiliary reaction magnetic beads. Figure 6 The schematic diagram of the implementation process of pre-preparing auxiliary reaction magnetic beads is shown, and the method of pre-preparing auxiliary reaction magnetic beads specifically includes:

[0172] Step S401, taking solid zinc acetate and ethylene glycol solution for mixed reaction, maintaining the reaction in a water bath at 75°C for 1.5 hours with stirring, then adding 0.5 mol / L sodium hydroxide solution to the reaction solution of zinc acetate and ethylene glycol, maintaining the reaction in a water bath at 75°C for 4 hours with stirring, then putting the mixed solution into a reactor, reacting at 120°C for 48 hours and then cooling to room temperature, then washing and grinding the reaction product with anhydrous ethanol to obtain zinc oxide;

[0173] Step S402, taking ferric chloride, polyacrylic acid, sodium acetate and propylene glycol and stirring them for 1 hour to form an iron mixed solution, then ultrasonically treating them for 20 minutes under an ultrasonic environment to obtain an iron mixed emulsion, sieving to separate undispersed particles, adding the iron mixed emulsion to a reactor, adding sodium diisooctyl succinate sulfonate to the iron mixed emulsion, keeping the temperature at 200° C. for 24 hours, and then magnetically separating the emulsion in the reactor to obtain ferrosoferric oxide nanoparticles;

[0174] It should be noted that sodium acetate is a sodium acetate solution of 3 mol / L and pH 6.5 dissolved in ultrapure water, while the manufacturer of ferric chloride, zinc acetate and ethylene glycol is Tianjin Fuchen Chemical Reagent Factory, China.

[0175] Step S403, dispersing the prepared zinc oxide in 8 times the volume of deionized water and stirring and mixing for 30 minutes, then placing the zinc oxide dispersion and the ferrosoferric oxide nanoparticles in a ball mill for ball milling for 1 hour, then adding polyethylene glycol to the ball mill, centrifuging and washing the ball milling liquid with anhydrous ethanol, drying at 110° C. for 14 hours, placing in a muffle furnace and heating to 350° C. at a heating rate of 5° C. / min, keeping warm for 4 hours, and cooling to room temperature to obtain modified ferrosoferric oxide nanoparticles.

[0176] In this embodiment, the modified ferroferric oxide nanoparticles are prepared by combining zinc oxide with nano-iron oxide to form nano-composite particles that not only retain the advantages of each of them, but also exhibit new excellent properties. This composite structure increases the specific surface area and provides more active sites, thereby enhancing the adsorption capacity for small molecule RNA. The addition of zinc oxide can enhance the mechanical strength of nano-iron oxide, so that the auxiliary reaction magnetic beads have better durability and reusability in practical applications. The combination of zinc oxide and nano-iron oxide makes the auxiliary reaction magnetic beads have good antibacterial properties, thereby avoiding contamination of small molecule RNA when extracting small molecule RNA, thereby improving the purity of small molecule RNA.

[0177] Step S404, placing the modified ferroferric oxide nanoparticles in an atomic deposition chamber, evacuating the atomic deposition chamber to a vacuum degree of 0.04-0.1 Pa, rotating the modified ferroferric oxide nanoparticles in the atomic deposition chamber at a speed of 12-20 r / min, heating the modified ferroferric oxide nanoparticles to 120-150° C., and then introducing argon gas into the atomic deposition chamber for cleaning, with a flow rate of 120 sccm;

[0178] Step S405, using monosilane and water vapor as silicon dioxide precursors, passing gaseous monosilane and water vapor into the atomic deposition chamber by alternating pulses, adjusting the bias voltage of the atomic deposition chamber to 120-500V, the pulse frequency to 120-180kHz, and depositing for 2 hours to obtain auxiliary reaction magnetic beads containing silanol groups.

[0179] Comparative Example 4

[0180] The method of this comparative example is the same as that of Example 5. The difference from Example 5 is that the cell lysis solution in this comparative example is replaced by Trizol reagent, and the other steps are the same as those of Example 5.

[0181] Comparative Example 5

[0182] The method of this comparative example is the same as that of Example 5. The difference from Example 5 is that the cell lysis solution in this comparative example is replaced by CTAB reagent, and the other steps are the same as those of Example 5.

[0183] Comparative Example 6

[0184] The method of this comparative example is the same as that of Example 5. The difference from Example 5 is that in this comparative example, the cell lysate is replaced by Trizol reagent, the auxiliary reaction magnetic beads are silicon-coated amino magnetic beads, and the other steps are the same as those of Example 5.

[0185] The concentration and purity of the small molecule RNA extracted from Examples 1-5 and Comparative Examples 4-6 of the present invention were measured by an ultra-micro spectrophotometer. The plant tissue extracted from Examples 1-5 and Comparative Examples 4-6 was cactus. The small molecule RNA concentration and OD260 / OD280 test results are shown in Table 2 and Table 3. Figure 7 shown.

[0186] Table 2

[0187]

[0188]

[0189] From Table 2 and Figure 7 It can be seen that when Examples 1-5 of the present invention are used to extract cactus tissue, the concentration of small molecule RNA is relatively moderate, indicating that Examples 1-5 of the present invention have less small molecule RNA loss and degradation than Comparative Examples 4-6, while when Comparative Example 6 extracts small molecule RNA, other impurities such as proteins are present in the small molecule RNA. The OD260 / OD280 ratio shows that the purity of small molecule RNA in Examples 1-5 of the present invention is very good compared to Comparative Examples 4-6.

[0190] 1% agarose gel electrophoresis was used to detect whether the small molecule RNA extracted in Example 5 of the present invention was intact, wherein the plant tissue was the stem and root of wheat. 5 μL of the small molecule RNA sample was taken, and 1 μL of 10× Loading Buffer was added to the small molecule RNA sample and mixed evenly. The voltage was 120V, and the electrophoresis was performed for 20 minutes. The electrophoresis test results were recorded using a gel imager. The results are as follows: Figure 8 As shown, from Figure 8 It can be seen that the small molecule RNA extracted in Example 5 of the present invention is relatively complete.

[0191] In summary, the present invention provides a method for extracting small RNA from plant polysaccharides and polyphenols. The embodiments of the present invention realize efficient recovery and processing of small molecule RNA in plant tissue samples by means of technical means of coordinated cooperation between cell lysate and auxiliary reaction magnetic beads. The reduction inhibition of cell lysate is used to provide a strong reducing environment for the recovery of small molecule RNA, thereby avoiding the interference of polyphenols and polysaccharides in plant tissues with the extraction of small molecule RNA during the extraction of small molecule RNA, and ensuring that the reverse transcription of small molecule RNA is not inhibited.

[0192] It should be noted that, for the above-mentioned embodiments, for the sake of simplicity, they are all described as a series of action combinations, but those skilled in the art should know that the present invention is not limited by the described order of actions, because according to the present invention, some steps may be performed in other orders or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present invention.

[0193] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on these embodiments, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in this field can still combine, add, delete or make other adjustments to the features in the various embodiments of the present invention according to the circumstances without conflict, without making creative work, so as to obtain different other technical solutions that do not deviate from the concept of the present invention in essence, and these technical solutions also belong to the scope of protection of the present invention.

Claims

1. A method for extracting small RNA from plant polysaccharides and polyphenols, characterized in that: The plant polysaccharide and polyphenol small RNA extraction method comprises: Pre-treating the plant tissue sample, cutting the plant tissue sample with ophthalmic surgical scissors after high-temperature sterilization to obtain plant tissue fragments, freezing the plant tissue fragments in a -80°C liquid nitrogen environment for 2 hours, taking a mortar, filling the mortar with diatomaceous earth, and then grinding the diatomaceous earth with a grinding rod, removing the diatomaceous earth after grinding for 10 minutes, pre-treating the mortar, taking the pre-treated mortar, and grinding the plant tissue fragments with liquid nitrogen to obtain plant tissue powder; Prepare cell lysis solution in advance, take plant tissue powder and transfer it into a sterilized reaction tube, add 3 times the volume of cell lysis solution to the plant tissue powder, and prepare auxiliary reaction magnetic beads in advance, react in a water bath at 35-40°C for 20 minutes, and oscillate 10-15 times during the reaction. After the reaction, separate the magnetic beads adsorbed with small molecule RNA from the cell lysis solution, and then centrifuge at 2-8°C for 20 minutes to obtain supernatant A and precipitate A; Take the magnetic beads adsorbed with small molecule RNA, elute the magnetic beads three times with magnetic bead elution solution to obtain eluent A, and mix the eluent A with supernatant A; A LiCl solution is stirred and mixed with supernatant A at a stirring speed of 10-15 r / min for 30 min. After the reaction is completed, the reaction solution is allowed to stand for 2 h to obtain supernatant B and precipitate B containing small molecule RNA. The supernatant B is mixed with precipitate A to obtain a polysaccharide-polyphenol mixture. The precipitate B was precipitated and washed with 3 times the volume of washing solution, and the precipitate B was retained. After centrifugation for 10 minutes, the supernatant was discarded to obtain a small molecule RNA precipitate, DEPC solution was added to dissolve the precipitate, and the precipitate was stored at low temperature to obtain a small molecule RNA extract.

2. The method for extracting small RNA from plant polysaccharides and polyphenols according to claim 1, characterized in that: The plant polysaccharide and polyphenol small RNA extraction method further comprises: Take a polysaccharide-polyphenol mixture, use a 1:1:1 volume mixture of phenol, isoamyl alcohol and chloroform to extract the polysaccharide-polyphenol mixture, extract the sugar-phenol mixture after standing, recover the plant polysaccharides in the sugar-phenol mixture, and separate to obtain a plant polysaccharide extract.

3. The method for extracting small RNA from plant polysaccharides and polyphenols as claimed in claim 1, characterized in that: The method for pretreating the mortar specifically comprises: Take a mortar and soak the mortar and grinding rod in DEPC solution for 24 hours; Use 75% ethanol to disinfect the mortar and grinding rod 2-4 times, and then sterilize the mortar and grinding rod at a high temperature of 260°C; The sterilized mortar and grinding rod were precooled with liquid nitrogen and set aside.

4. The method for extracting small RNA from plant polysaccharides and polyphenols as claimed in claim 3, characterized in that: The cell lysate comprises the following raw materials in percentage by weight: 3mol / L guanidine thiocyanate: 10%; Hexadecyl ammonium bromide: 20%; 1 mol / L sodium chloride: 40%; Polyvinylpyrrolidone: 5%; Extraction buffer: 15%; [AMIM][BPh4] ionic liquid: 5%; 2-Ethylhexyl phosphate monoester: 3%; Antioxidants: 2%.

5. The method for extracting small RNA from plant polysaccharides and polyphenols as claimed in claim 4, characterized in that: The method for pre-preparing a cell lysate specifically comprises: Under nitrogen protection, 1-methylimidazole and allyl chloride are mixed in a reactor at a molar ratio of 3:1, and dichloromethane is used as a solvent. The mixed solution is heated to 35-45° C. and mixed for 10 minutes. Acetone and 3-chloropropylene are added to the reactor. After additional reaction for 4-5 hours, dichloromethane is removed by rotary evaporation, and the mixture is washed with toluene to obtain 1-allyl-3-methylimidazole chloride. Add lithium tetraphenylborate and sodium tetraphenylborate to a reaction kettle containing 1-allyl-3-methylimidazolium chloride, evacuate the kettle, fill it with methane gas, adjust the temperature of the reaction kettle to 45-50° C., control the speed of the reaction kettle to 200-300 r / min, stir the reaction, and then vacuum-evaporate to obtain [AMIM][BPh4] ionic liquid; Mix 2-ethylhexyl phosphate monoester, [AMIM][BPh4] ionic liquid and sodium chloride, stir for 1 hour at a temperature of 30-35°C, and stand for 4 hours to obtain a pyrolysis component 1; Take guanidine isothiocyanate and hexadecyl ammonium bromide, add the guanidine isothiocyanate and hexadecyl ammonium bromide dropwise into a sodium chloride solution under stirring, then add polyvinyl pyrrolidone and an antioxidant into the mixed solution of guanidine isothiocyanate and hexadecyl ammonium bromide, and adjust the pH to 6-6.5 with an extraction buffer to obtain a cleavage component 2; The lysis component 1 and the lysis component 2 were mixed, ultrasonicated for 20 minutes, allowed to stand for 30 minutes, and heated and stirred at 50° C. for 50 minutes to obtain a cell lysate.

6. The method for extracting small RNA from plant polysaccharides and polyphenols according to claim 1, characterized in that: The method for pre-preparing auxiliary reaction magnetic beads specifically comprises: Solid zinc acetate and ethylene glycol solution are mixed for reaction, and the mixture is stirred in a water bath at 75°C for 1.5 hours. Then, 0.5 mol / L sodium hydroxide solution is added to the reaction solution of zinc acetate and ethylene glycol, and the mixture is stirred in a water bath at 75°C for 4 hours. Then, the mixed solution is put into a reactor, and the mixture is reacted at 120°C for 48 hours, and then cooled to room temperature. Then, the reaction product is washed and ground with anhydrous ethanol to obtain zinc oxide. Ferric chloride, polyacrylic acid, sodium acetate and propylene glycol were stirred and mixed for 1 hour to form an iron mixed solution, and then ultrasonically treated for 20 minutes under an ultrasonic environment to obtain an iron mixed emulsion, undispersed particles were separated by sieving, the iron mixed emulsion was added to a reactor, sodium diisooctyl succinate sulfonate was added to the iron mixed emulsion, and the mixture was kept warm at 200°C for 24 hours, and then the emulsion in the reactor was magnetically separated to obtain ferrosoferric oxide nanoparticles; The prepared zinc oxide was dispersed in 8 times the volume of deionized water and stirred for 30 minutes. The zinc oxide dispersion and ferrosoferric oxide nanoparticles were then placed in a ball mill for ball milling for 1 hour. Polyethylene glycol was then added to the ball mill. The ball milling liquid was centrifuged and washed with anhydrous ethanol, dried at 110°C for 14 hours, placed in a muffle furnace and heated to 350°C at a heating rate of 5°C / min for 4 hours, and cooled to room temperature to obtain modified ferrosoferric oxide nanoparticles.

7. The method for extracting small RNA from plant polysaccharides and polyphenols according to claim 6, characterized in that: The method for pre-preparing auxiliary reaction magnetic beads specifically also includes: The modified ferroferric oxide nanoparticles are placed in an atomic deposition chamber, and the atomic deposition chamber is evacuated to make the vacuum degree of the chamber 0.04-0.1 Pa, and then the rotation speed of the modified ferroferric oxide nanoparticles in the atomic deposition chamber is 12-20 r / min, and the modified ferroferric oxide nanoparticles are heated to 120-150° C., and then argon gas is introduced into the atomic deposition chamber for cleaning, and the flow rate of argon gas is 120 sccm; Using monosilane and water vapor as silicon dioxide precursors, gaseous monosilane and water vapor are introduced into the atomic deposition chamber by alternating pulses, the bias voltage of the atomic deposition chamber is adjusted to 120-500V, the pulse frequency is 120-180kHz, and deposition is carried out for 2 hours to obtain auxiliary reaction magnetic beads containing silanol groups.

8. The method for extracting small RNA from plant polysaccharides and polyphenols according to claim 4 or 5, characterized in that: The antioxidant is a mixture of beta-mercaptoethanol and ascorbic acid in a volume ratio of 1:1, and the extraction buffer is a Tris base-malic acid buffer solution.

9. The method for extracting small RNA from plant polysaccharides and polyphenols as claimed in claim 2, characterized in that: The method for recovering and treating the plant polysaccharide in the glycophenol mixture to separate and obtain the plant polysaccharide extract specifically comprises: A polyphenol separation liquid is prepared, the sugar-phenol mixture is mixed with the polyphenol separation liquid, 0.02 times the volume of glycerol is added thereto, the mixture is placed on a heated magnetic stirrer at 80° C. and stirred for 30 minutes, cooled to room temperature, and centrifuged for 10 minutes, so that the polyphenol separation liquid adsorbs the polyphenols in the sugar-phenol mixture to obtain a polyphenol adsorbed liquid and a supernatant C containing polysaccharides; Take the supernatant C, mix the supernatant C with 3 times the volume of petroleum ether, and put the mixed solution into the reactor, keep it warm in a 75°C water bath for 4 hours, stir the reactor with a glass rod every 10 minutes, then heat the reactor to 150°C, stir for 20 minutes, extract the plant polysaccharide with hot water at 50°C, and collect the polysaccharide supernatant; The polysaccharide supernatant was suspended in pre-cooled anhydrous ethanol and allowed to stand at 25°C for 48 hours to obtain a plant polysaccharide precipitate.

10. The method for extracting small RNA from plant polysaccharides and polyphenols according to claim 9, characterized in that: The polyphenol separation liquid comprises the following raw materials in parts by weight: 10-30 parts of polyvinyl pyrrolidone, 1-5 parts of dithioerythritol, 2-5 parts of isoamyl alcohol, 20-40 parts of sodium chloride, 10-15 parts of sodium dodecyl sulfate, and 10-30 parts of methanol.

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