CN113121636B - Complex for enriching n-tridecanol conjugate and preparation method and application thereof - Google Patents

Complex for enriching n-tridecanol conjugate and preparation method and application thereof Download PDF

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CN113121636B
CN113121636B CN202110379735.XA CN202110379735A CN113121636B CN 113121636 B CN113121636 B CN 113121636B CN 202110379735 A CN202110379735 A CN 202110379735A CN 113121636 B CN113121636 B CN 113121636B
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tridecanol
complex
protein
plant
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CN113121636A (en
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周扬颜
许鹏昊
莫翱玮
陈文秀
孙卫健
郭鹏
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Shandong Dafengyuan Agricultural Co ltd
Yunnan Fengwo Agriculture Co., Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
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Abstract

The invention discloses a compound for enriching n-tridecanol conjugate, which comprises the following components: a n-tridecanol moiety and a carboxylic magnetic bead, the carboxylic magnetic bead moiety being capable of being driven by a magnetic field to separate a complex or a complex containing the complex from a mixture system. The invention also discloses a preparation method of the compound, which comprises the following steps: the n-tridecanol and the carboxyl magnetic beads are subjected to esterification reaction to generate the magnetic beads with the n-tridecanol distributed on the surface. The invention also discloses a method for enriching the n-tridecanol interaction protein from the total plant protein by the compound. The method can enrich the n-triacontanol interaction protein, so as to further research the biological function and related genetic pathways of the n-triacontanol interaction protein and promote the improvement of the plant production performance.

Description

Complex for enriching n-tridecanol conjugate and preparation method and application thereof
Technical Field
The invention belongs to the field of biochemistry, relates to a compound for enriching a n-tridecanol conjugate and a preparation method thereof, and particularly relates to a compound for enriching a n-tridecanol interaction protein based on magnetic separation and a preparation method and application thereof.
Background
The effects of the n-triacontanol (also called triacontanol, 1-triacontanol) are mainly embodied in promoting the plant to grow roots, promoting the plant to absorb mineral elements, promoting the photosynthesis of leaves, promoting the germination of flower buds and the like. In agricultural production, the application of n-tridecanol can increase the number of small ears and grains per ear of crops.
There is no report on the n-tridecanol interaction protein, and the main reason is that there is no effective method for finding the protein interacting with n-tridecanol.
It is possible that n-tridecanol functions together with its interacting protein, but the detailed mechanism thereof has not been reported at present.
If the components such as protein and the like interacting with the n-tridecanol in the plant tissues can be found, the further research on the composition and the properties of the components is facilitated, and further, the synthesis and decomposition routes of the n-tridecanol or the potential signal path initiated or participated by the n-tridecanol are intervened manually, so that the production performance of crops is improved.
Disclosure of Invention
In order to solve the problems of the prior art, the first aspect of the present invention provides a complex for enriching a n-tridecanol conjugate, the complex comprising a n-tridecanol moiety and a ligand moiety, the ligand moiety being capable of being driven to separate the complex or a complex containing the complex from a mixture system.
In some embodiments, the n-tridecanol conjugate is an n-tridecanol interacting protein.
In some embodiments, the n-tridecanol moiety and the ligand moiety are linked by a covalent bond.
In some embodiments, the n-tridecanol moiety and the ligand moiety are directly linked.
In some embodiments, the ligand moiety is capable of specifically binding to a ligand binder such that the complex or a complex containing the complex is separated from a mixture system by the ligand binder.
In some embodiments, the material of the ligand moiety is a modified biotin molecule capable of binding to n-tridecanol and the ligand conjugate is immobilized streptavidin.
In some embodiments, the material of the ligand moiety is biotin with a terminal carboxyl linker arm attached.
In some embodiments, the material of the ligand moiety is biocytin.
In some embodiments, the ligand binder is streptavidin sepharose.
In some embodiments, the n-tridecanol moiety and the ligand moiety are indirectly linked.
In some embodiments, the ligand moiety is a magnetic material, which can be actuated by a magnetic field to separate the complex or a complex containing the complex from a mixture system.
In some embodiments, the ligand moiety is a magnetic bead, the surface of which is modified with an active arm having a terminal group capable of reacting with an alcoholic hydroxyl group.
In some embodiments, the magnetic bead is a paramagnetic bead.
In some embodiments, the magnetic beads have a particle size of 30 to 100 microns (e.g., 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns).
In some embodiments, the magnetic beads are carboxyl magnetic beads.
In some embodiments, the active arm terminates with a carboxyl group.
In some embodiments, the active arm is C10-15The hydrocarbon chain carboxylic acid of (1).
In a second aspect, the present invention provides a method for preparing the complex of the first aspect, the method comprising: chemically reacting the n-tridecanol and the ligand moiety starting materials to form the complex comprising the n-tridecanol moiety and the ligand moiety.
In some embodiments, the chemical reaction is an esterification reaction.
In some embodiments, the esterification reaction is an esterification reaction between n-tridecanol and biotin having a terminal carboxyl linker arm attached thereto.
In some embodiments, the esterification reaction is an esterification reaction between n-tridecanol and biocytin.
In some embodiments, the esterification reaction is an esterification reaction between n-tridecanol and a carboxylic magnetic bead.
In some embodiments, the esterification reaction is: and mixing the n-tridecanol solution with the carboxyl magnetic bead solution, and carrying out esterification reaction to obtain the crude compound.
In some embodiments, the n-tridecanol solution is an ethanolic n-tridecanol solution.
In some embodiments, the amount of n-tridecanol solution to ethanol is in a mass ratio of 2 to 6:1 (e.g., 3:1, 4:1, 5: 1).
In some embodiments, the n-tridecanol solution includes a surfactant.
In some embodiments, the surfactant is Tween-20.
In some embodiments, the n-tridecanol solution has a volume ratio of the surfactant to the ethanol of 0.5 to 2: 1.
In some embodiments, the solvent of the carboxylic magnetic bead solution is MES solution.
In some embodiments, the MES solution is 2- (N-morpholine) ethanesulfonic acid in an aqueous solution at a concentration of 10 to 30 g/L.
In some embodiments, the concentration of the solution of carboxylic magnetic beads is 30-70 mg/ml (e.g., 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 55 mg/ml, 60 mg/ml, 65 mg/ml).
In some embodiments, the carboxylic magnetic beads are used after washing.
In some embodiments, the carboxyl magnetic beads are washed with MES solution and used.
In some embodiments, the solvent of the carboxylic magnetic bead solution is MES-Mix solution.
In some embodiments, the MES-Mix solution is an aqueous solution containing 10 to 30 mg/ml 2- (N-morpholino) ethanesulfonic acid and 3 to 7 mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
In some embodiments, the concentration of the solution of carboxylic magnetic beads is 15-35 mg/ml (e.g., 18 mg/ml, 20 mg/ml, 22 mg/ml, 24 mg/ml, 26 mg/ml, 28 mg/ml, 30 mg/ml, 32 mg/ml).
In some embodiments, in the esterification reaction, the amount by mass ratio of the n-tridecanol to the carboxyl magnetic beads is: 1:20-40 (e.g., 1:22, 1:24, 1:26, 1:28, 1:30, 1:32, 1:34, 1:36, 1: 38).
In some embodiments, the esterification reaction temperature 20-30 ℃ (e.g., 22 ℃, 24 ℃, 26 ℃, 28 ℃).
In some embodiments, the esterification reaction time is 60-180min (e.g., 80min, 100min, 120min, 140min, 160 min).
In some embodiments, the blending occurs once every 3-10min during the esterification reaction.
In some embodiments, during the esterification reaction, the Mix solution is added to the reaction system once every 10 to 30 minutes, and the volume ratio of each addition amount to the reaction system is: 1:10-25.
In some embodiments, the Mix solution is an aqueous solution containing 10 to 30 mg/ml 2- (N-morpholino) ethanesulfonic acid and 30 to 90 mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
In some embodiments, the reaction system containing the crude complex is magnetically adsorbed, and the liquid components of the reaction system are discarded to obtain a purified complex.
In some embodiments, the purified complex is washed with a wash solution to obtain a refined complex.
In some embodiments, the wash solution is a PBST solution.
In some embodiments, the method of cleaning is: mixing the washing solution with the purified compound, magnetically adsorbing a reaction system containing the crude compound, and discarding liquid components in the reaction system to obtain a refined compound.
In some embodiments, the refined complex is dissolved in a TE-TW solution to provide a solution of the complex for use.
In some embodiments, the concentration of the complex in the complex solution is 4.0-8.0 mg/ml (e.g., 4.5 mg/ml, 5.0 mg/ml, 5.5 mg/ml, 6.0 mg/ml, 6.5 mg/ml, 7.0 mg/ml, 7.5 mg/ml).
In some embodiments, the TE-TW solution is an aqueous solution containing 0.5 to 1.5 v/v% Tris, 0.2 to 0.5g/L disodium EDTA, and 0.005 to 0.015 v/v% Tween-20.
In some embodiments, the complex solution is stored at a temperature of 1-6 deg.C (e.g., 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C).
In a third aspect, the present invention provides a method for enriching a n-tridecanol conjugate, which comprises: using the complex according to the first aspect of the invention, n-tridecanol conjugates are enriched in plant tissue.
In some embodiments, the plant is a poplar.
In some embodiments, the tissue is a root.
In some embodiments, the enrichment method comprises the steps of:
s1, mixing the plant total protein solution with the compound solution, and incubating to obtain a first mixture;
s2, driving the ligand moiety in the first mixture, and separating the complex or a complex containing the complex from the first mixture to obtain a separation product;
s3, eluting the separation product to obtain the n-tridecanol conjugate.
In some embodiments, the solvent is a protein binding solution, and the concentration of the total plant protein in the solution is 5-15 μ g/mL (e.g., 6 μ g/mL, 7 μ g/mL, 8 μ g/mL, 9 μ g/mL, 10 μ g/mL, 11 μ g/mL, 12 μ g/mL, 13 μ g/mL, 14 μ g/mL).
In some embodiments, the protein binding solution comprises 0.1-0.3M Tris,0.4-0.6M NaCl, 15-25mM MgCl20.8-1.2 v/v% Tween-20 in water, pH 7.5.
In some embodiments, in the solution of total plant protein, the total plant protein is total protein in the plant tissue as a dry powder.
In some embodiments, the concentration of the complex in the complex solution is 4.0-8.0 mg/ml (e.g., 4.5 mg/ml, 5.0 mg/ml, 5.5 mg/ml, 6.0 mg/ml, 6.5 mg/ml, 7.0 mg/ml, 7.5 mg/ml).
In some embodiments, the complex solution is a TE-TW solution.
In some embodiments, the TE-TW solution is an aqueous solution containing 0.5 to 1.5 v/v% Tris, 0.2 to 0.5g/L disodium EDTA, and 0.005 to 0.015 v/v% Tween-20.
In some embodiments, the amount by mass ratio of the total plant protein to the complex is: 1:30-100 (e.g., 1:40, 1:50, 1:60, 1:70, 1:80, 1: 90).
In some embodiments, in step S2, the incubation time is 2-10min (e.g., 3min, 4min, 5min, 6min, 7min, 8min, 9 min).
In some embodiments, in step S2, the incubation condition is on ice.
In some embodiments, in step S2, the complex or a complex containing the complex is separated from the first mixture by magnetically attracting the ligand moiety to obtain the separation product.
In some embodiments, in step S3, the ligand moiety is magnetically attracted and the separation product is eluted with a protein eluent comprising the n-tridecanol conjugate.
In some embodiments, in step S3, the elution time is 2-10min (e.g., 3min, 4min, 5min, 6min, 7min, 8min, 9 min).
In some embodiments, in step S3, the elution condition is on ice.
In some embodiments, the eluent composition is 10-30mM Tris,5-15mM NaCl,0.08-0.12 v/v% Tween-20 in water, pH 7.5.
In a fourth aspect, the present invention provides a n-tridecanol conjugate obtained by using the enrichment method according to the third aspect of the present invention.
The fifth aspect of the invention provides the use of a complex according to the first aspect of the invention, a method of preparing a complex according to the second aspect of the invention, an enrichment method according to the third aspect of the invention or a combination of n-tridecanol according to the fourth aspect of the invention in the preparation of a formulation for any one of promoting rooting of a plant, promoting mineral uptake by a plant, promoting photosynthesis in leaves, promoting germination of flower buds, increasing the number of ears of a crop and increasing the number of grains per ear of a crop.
Compared with the prior art, the invention has the advantages that: the method is more convenient to find the interacting protein of a certain substance, and is simpler and more practical and less in cost compared with the prior yeast single hybridization, isothermal titration calorimetry, nuclear magnetic resonance and other technologies. Through an immunization method, a method for researching triacontanol interaction protein is found, and a new thought is provided for researching interaction protein of other substances.
Drawings
FIG. 1 shows the electrophoresis of protein, wherein lane 1 represents protein marker, the molecular weight from top to bottom is: 250kDa, 150kDa, 100kDa, 70kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa, 15kDa, lane 2 represents total plant protein and lane 3 represents n-tridecanol-interacting protein.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The invention provides a complex for enriching n-tridecanol and an enrichment method thereof, and the main technical concept is as follows:
binding of n-tridecanol to magnetic beads to form magnetic bead-n-tridecanol complex, incubating the complex with total plant protein to bind protein to the n-tridecanol moiety in the complex to form magnetic bead-n-tridecanol-protein complex. The magnetic beads are magnetically adsorbed by a magnetic frame so as to fix the magnetic bead-n-tridecanol-protein complex. And then incubating the complex and protein eluent together, and eluting the protein by changing the binding environment to obtain the protein interacted with the n-tridecanol.
The principle of the combination of magnetic beads and n-tridecanol is as follows: the carboxyl group of the magnetic beads and the hydroxyl group of the n-tridecanol are subjected to esterification reaction, and the n-tridecanol is tightly bonded to the magnetic beads through an ester bond.
Definition of
(1) N-tridecanol conjugate:
a biomolecule or biomolecule complex capable of specific binding to n-tridecanol through a non-covalent bond pattern, including but not limited to: proteins, glycoproteins, protein complexes, glycoprotein complexes, protein-glycoprotein complexes, and complexes of the foregoing with RNA.
When the n-tridecanol conjugate is a protein capable of specifically binding to n-tridecanol by a non-covalent bond pattern, the n-tridecanol conjugate can be referred to as a n-tridecanol interacting protein.
(2) N-tridecanol moiety:
also referred to as the n-tridecanol residue, refers to the moiety from n-tridecanol in the complex.
(3) Ligand moiety:
a ligand moiety refers to the moiety from a ligand in a complex, and when the ligand is a small molecule, the ligand moiety may also be referred to as a ligand residue.
The aforementioned n-tridecanol moiety is stably associated with the aforementioned ligand moiety, and in some cases, the two are associated by covalent bonding to form a complex for enriching the aforementioned n-tridecanol conjugate.
(4) Complex-containing complex:
in some cases, refers to a complex formed by combining a complex with an n-tridecanol conjugate.
(5) Is driven to separate the complex or complexes containing the complex from the mixture system:
including, but not limited to, 1) driving a ligand moiety (e.g., a magnetic bead, sometimes with a linker arm residue on its surface for binding to triacontanol) and driving the complex or complexes containing the complex to separate from the mixture system (which may contain the complex, potential triacontanol conjugates and other components) by a non-contact means (e.g., a magnetic field); 2) the complex or complex-containing complex is separated from the mixture system (which may contain a complex, a mixture of potential n-tridecanol conjugates and other components) by affinity driving of the ligand moiety using a solid phase (e.g., a support capable of immobilizing streptavidin) (e.g., an avidin biotin moiety), e.g., by binding the solid phase support to the ligand moiety of the complex or complex-containing complex, thereby removing the other components of the mixture system.
Example 1: preparation of the composite
Materials (I) and (II)
Carboxyl magnetic beads: commercially available from beaver heads, with a particle size of 30-100 microns, paramagnetic, and the surface of the magnetic beads is modified with a group of twelve carbon carboxylic acids.
Secondly, a solution preparation method comprises the following steps:
(1) MES solution preparation method:
19.5g of 2- (N-morpholine) ethanesulfonic acid was weighed into a 1L flask and made up to 1L with ultrapure water to form a MES solution.
(2) The Mix solution preparation method:
1 ml of MES solution was added to a 2 ml centrifuge tube, 60 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide was added thereto, and the mixture was sufficiently inverted to dissolve 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to form a Mix solution.
(3) The preparation method of the MES-Mix solution comprises the following steps:
add 58.4. mu.l of MES solution to 5.1. mu.l of Mix solution to form a MES-Mix solution.
(4) The preparation method of the n-tridecanol solution comprises the following steps:
weighing 2 mg of n-tridecanol, adding the n-tridecanol into a 2 ml centrifuge tube, adding 500 microliters of 60 ℃ absolute ethyl alcohol, fully inverting to dissolve the n-tridecanol, and adding 500 microliters of Tween-20 to form a n-tridecanol solution.
(5) The PBST solution formulation method:
8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate were dissolved in 800ml of deionized water, the pH was adjusted to 7.4 with hydrochloric acid, and 200. mu.l of Tween-20 was added to obtain a PBST solution.
(6) The preparation method of the TE-TW solution comprises the following steps:
adding 10 ml of tris aqueous solution (pH 8.0,1 mol/L) into a 1L conical flask, adding 0.336 g of disodium ethylene diamine tetraacetate, fixing the volume to 1L, and adding 100 microliters of Tween-20 to obtain a TE-TW solution. Storing at 4 ℃.
Thirdly, a complex synthesis process:
1. adding 30 microliters of carboxyl magnetic beads (50 mg/ml, wherein the solvent is MES solution) into a 1.5 ml centrifuge tube, adding 200 microliters of MES solution, reversing and mixing uniformly, adsorbing the carboxyl magnetic beads by using a magnetic frame, and adsorbing the MES solution; and adding 200 microliters of MES solution again, reversing and mixing uniformly, adsorbing the carboxyl magnetic beads by using a magnetic frame, and adsorbing the MES solution. The MES solution has the function of stabilizing the pH value and preventing the pH value of the solution from changing violently.
2. Add 60. mu.l MES-Mix solution to a 1.5 ml centrifuge tube and invert to resuspend the beads. The MES-Mix solution activates the magnetic beads.
3. 12.4. mu.l of magnetic beads were added to 200. mu.l centrifuge tubes, and 5. mu.l of n-tridecanol solution was added to each of the centrifuge tubes, for a total of five centrifuge tubes.
4. After blowing and mixing, the centrifuge tube was placed in a metal bath at 25 ℃ for 20 minutes and inverted every 5 minutes.
5. Then 1 microliter of Mix solution was added to each centrifuge tube. The Mix solution acts to activate the magnetic beads.
6. After vortexing, the tubes were placed in a 25 ℃ metal bath for 20 minutes, and inverted every 5 minutes.
7. An additional 1 microliter of Mix solution was added to each centrifuge tube.
8. After vortexing, the tubes were placed in a 25 ℃ metal bath for 80 minutes, and inverted every 5 minutes.
9. Adsorbing the carboxyl magnetic beads by using a magnetic frame, and adsorbing the solution in the centrifugal tube;
10. adding 200 microliters of PBST solution into the centrifugal tube, reversing and uniformly mixing, adsorbing carboxyl magnetic beads by using a magnetic frame, and adsorbing the PBST solution; adding 200 microliter PBST solution again, reversing and mixing evenly, adsorbing carboxyl magnetic beads by using a magnetic frame, and absorbing the PBST solution.
11. Adding 50 microliters of TE-TW solution into the centrifuge tube, reversing and uniformly mixing the solution and the resuspended magnetic beads to form a magnetic bead-n-tridecanol complex suspension, and storing at 4 ℃.
Example two: application of magnetic bead-n-tridecanol complex
1. Obtaining of Total plant protein
The 84K poplar root total protein dry powder was extracted according to the procedures described in the protocol of the plant protein extraction kit (C500053) of the Producer company.
2. Incubation
10 micrograms of dry plant total protein powder was dissolved in 1 ml of protein binding solution (0.2M Tris (pH 7.5),0.5M NaCl,20mM MgCl21 v/v% Tween-20 Detergent), 100. mu.l of a suspension of magnetic bead-n-tridecanol complex was added. On ice, bind for 5 minutes. Forming a magnetic bead-n-tridecanol-protein complex.
3. Magnetic treatment
The magnetic beads are magnetically adsorbed by a magnetic frame so as to fix the magnetic bead-n-tridecanol-protein complex.
The liquid component is removed.
4. Elution is carried out
Add 500. mu.l protein eluent (20mM Tris (pH 7.5),10mM NaCl,0.1 v/v% Tween-20 Detergent).
Place on ice for 5 minutes. The protein is eluted by changing the solution in such a way that the binding of n-tridecanol to the protein is disrupted.
The magnetic beads are magnetically adsorbed by a magnetic frame.
And sucking the liquid to obtain the protein interacted with the n-tridecanol.
5. Electrophoresis
Using conventional SDS-PAGE, stained with Coomassie Brilliant blue, protein Marker was added in lane 1 at molecular weights of 250, 150, 100, 70 (red), 50, 40, 30, 25 (red), 20, 15kDa, respectively, in sequence from top to bottom, the total plant protein sample obtained in step 1 above was added in lane 2, and the n-tridecanol interacting protein sample in lane 3. Electropherograms are shown in FIG. 1.
As can be seen from FIG. 1, the electrophoresis result in lane 3 shows two proteins, protein 1, with a molecular weight of about 70kDa and protein 2, with a molecular weight of about 20 kDa.
It can be seen that, in plants, n-tridecanol can be combined with protein 1 and protein 2, and protein 1 and protein 2 may act as plant hormones of n-tridecanol by forming a complex with n-tridecanol and other possible components. Up-regulating the expression of protein 1 and protein 2 in plants (e.g., by transgenically transferring protein 1 and protein 2 genes to stronger promoters) may increase the sensitivity of plants to n-tridecanol and promote plant growth.
More triacontanol interacting proteins may also be found when working with plant total proteins of different species, different tissues, and different developmental stages.
When whole cell homogenate is used instead of total plant protein, the magnetic bead-triacontanol complex may also separate other components involved in the protein, such as small RNA.
Therefore, the magnetic bead-n-tridecanol complex of the present invention has many potential uses.
EXAMPLE III use of biocytin-n-tridecanol Complex
The carboxyl of lysine in biocytin and the hydroxyl of n-tridecanol are subjected to esterification reaction to form a biocytin-n-tridecanol compound.
The compound is combined with the n-tridecanol interacting protein after being incubated with the total plant protein to form a biocytin-n-tridecanol interacting protein compound.
And (3) binding streptavidin agarose gel with biotin part of biocytin to capture biocytin-n-tridecanol interaction protein complex, further eluting and releasing n-tridecanol interaction protein.
It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.

Claims (18)

1. The compound for enriching the n-tridecanol conjugate consists of n-tridecanol and a ligand, wherein the n-tridecanol and the ligand are connected through an ester bond, the ligand is a magnetic bead, the magnetic bead is a carboxyl magnetic bead, the surface of the magnetic bead is modified with an active arm, the tail end of the active arm is a carboxyl group, the n-tridecanol conjugate is a n-tridecanol interaction protein, and the n-tridecanol interaction protein is a protein capable of being specifically combined with the n-tridecanol through a non-covalent bond mode.
2. The complex of claim 1, wherein the magnetic bead is a paramagnetic bead.
3. The composite of claim 1, wherein the magnetic beads have a particle size of 30 to 100 microns.
4. The complex of claim 1, wherein the active arm is C10-15The hydrocarbon chain carboxylic acid of (1).
5. A method of preparing a complex according to any one of claims 1 to 4, said method comprising: and carrying out esterification reaction on the n-tridecanol and the carboxyl magnetic beads to form the complex comprising the n-tridecanol and the ligand.
6. The method of claim 5, wherein the esterification reaction is: mixing a n-tridecanol solution and a carboxyl magnetic bead solution, and carrying out esterification reaction to obtain a crude compound;
the n-tridecanol solution is an ethanol solution of n-tridecanol;
in the n-tridecanol solution, the mass ratio of the n-tridecanol to the ethanol is 2-6: 1;
the n-tridecanol solution contains a surfactant;
the surfactant is Tween-20;
in the n-tridecanol solution, the volume ratio of the dosage of the surfactant to the dosage of the ethanol is 0.5-2: 1;
the solvent of the carboxyl magnetic bead solution is MES-Mix solution;
the MES-Mix solution is an aqueous solution containing 10-30 mg/ml of 2- (N-morpholine) ethanesulfonic acid and 3-7 mg/ml of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide;
the concentration of the carboxyl magnetic bead solution is 15-35 mg/ml;
in the esterification reaction, the mass ratio of the n-tridecanol to the carboxyl magnetic beads is as follows: 1:20-40.
7. The method according to claim 6, wherein the carboxyl magnetic beads are washed with MES solution and used;
the MES solution is a 2- (N-morpholine) ethanesulfonic acid aqueous solution with the concentration of 10-30 g/L.
8. The method of claim 6, wherein the esterification reaction is carried out at a temperature of 20 to 30 ℃;
the esterification reaction time is 60-180 min;
uniformly mixing once every 3-10min in the esterification reaction process;
in the esterification reaction process, adding a Mix solution into a reaction system once at intervals of 10-30 minutes, wherein the volume ratio of each addition amount to the reaction system is as follows: 1: 10-25;
the Mix solution is an aqueous solution containing 10-30 mg/ml 2- (N-morpholine) ethanesulfonic acid and 30-90 mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
9. The method according to claim 6, wherein the reaction system containing the crude complex is magnetically adsorbed, and a liquid component in the reaction system is discarded to obtain a purified complex.
10. The method according to claim 9, wherein the purified complex is washed with a washing solution to obtain a purified complex;
the washing solution is PBST solution;
the cleaning method comprises the following steps: mixing the washing solution with the purified compound, magnetically adsorbing a reaction system containing the crude compound, and discarding liquid components in the reaction system to obtain a refined compound.
11. The method according to claim 10, wherein the purified complex is dissolved in a TE-TW solution to obtain the complex solution for use;
in the complex solution, the concentration of the complex is 4.0-8.0 mg/ml;
the TE-TW solution is an aqueous solution containing 0.5-1.5 v/v% of tris (hydroxymethyl) aminomethane, 0.2-0.5g/L of disodium ethylenediaminetetraacetate and 0.005-0.015 v/v% of Tween-20.
12. The method according to claim 11, wherein the complex solution is stored at a temperature of 1 to 6 ℃.
13. An enrichment method of n-tridecanol conjugate, the enrichment method comprising the following steps: enriching n-tridecanol conjugates in plant tissue using the complex of any one of claims 1-4;
the plant is a poplar;
the tissue is a root;
the enrichment method comprises the following steps:
s1, mixing the plant total protein solution with the compound solution, and incubating to obtain a first mixture;
in the plant total protein solution, a solvent is a protein binding solution, and the concentration of the plant total protein is 5-15 mu g/mL;
the protein binding solution contains 0.1-0.3MTris,0.4-0.6M NaCl，15-25mM MgCl20.8-1.2 v/v% Tween-20 in water, pH 7.5;
in the complex solution, the concentration of the complex is 4.0-8.0 mg/ml;
in the complex solution, a solvent is TE-TW solution;
the TE-TW solution is an aqueous solution containing 0.5-1.5 v/v% of tris (hydroxymethyl) aminomethane, 0.2-0.5g/L of disodium ethylene diamine tetraacetate and 0.005-0.015 v/v% of Tween-20;
the dosage mass ratio of the total plant protein to the compound is as follows: 1: 30-100;
s2, attracting the ligand by magnetic force, and separating the complex or the complex containing the complex from the first mixture to obtain the separation product;
s3, attracting the ligand by magnetic force, eluting the separation product by protein eluent, wherein the eluent contains the n-tridecanol conjugate; the eluent comprises 10-30mM Tris,5-15mM NaCl,0.08-0.12 v/v% Tween-20 in water solution, and has pH of 7.5.
14. The enrichment method according to claim 13, wherein the total plant protein in the total plant protein solution is the dry powder of the total protein in the plant tissue.
15. The enrichment method of claim 13, wherein the incubation time is 2-10min in step S1.
16. The enrichment method of claim 13, wherein in step S1, the incubation condition is on ice.
17. The enrichment method of claim 13, wherein the elution time is 2-10min in step S3.
18. The enrichment method of claim 13, wherein the elution condition is on ice in step S3.
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