CN116850155B - A kind of transgenic tomato microvesicle nanoparticles and its preparation method and application - Google Patents

Abstract

The invention discloses microvesicle nano-particles of transgenic tomatoes, a preparation method and application thereof, and relates to the technical field of biology. The preparation method comprises the steps of (1) connecting lncENAF DNA molecules into a gene expression vector, then transforming into competent cells to obtain recombinant microorganism strains, (2) infecting tomato explants by using the recombinant microorganism strains, then carrying out tissue culture, identification and screening to obtain transgenic tomato plants, (3) culturing and setting the transgenic tomato plants to obtain transgenic tomato fruits, and (4) carrying out microvesicle extraction on the transgenic tomato fruits to obtain microvesicle nano-particles containing lncENAF. The invention successfully realizes genetic transformation of lncENAF of animal sources in plants, and the transgenic tomato fruits prepared by the method can extract the microvesicle nano-particles containing lncENAF and can be applied to preparation of medicines for relieving inflammation.

Description

Microvesicle nano-particle of transgenic tomato and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to microvesicle nano-particles of transgenic tomatoes, a preparation method and application thereof.

Background

Long non-coding RNA (lncRNA) is a Long non-coding RNA molecule which does not code protein per se and has a transcript length of more than 200nt, and can perform gene regulation from different layers such as epigenetic regulation, transcriptional regulation and posttranscriptional regulation. In recent years, the regulatory role of long non-coding RNAs is being increasingly focused and studied.

Long-chain non-coding RNA LNCENAF has been shown to bind to nuclear heterogeneous ribonucleoprotein F (hnRNPF). lncENAF and its binding protein hnRNPF can inhibit lipopolysaccharide-induced production of cytokines such as macrophage IL-6.

At present, no report of transfecting animal-derived non-coding RNA into tomatoes exists in the prior study, and the invention tries to obtain a transgenic tomato by using agrobacterium infection through a genetic engineering method by using mouse-derived non-coding RNA LNCENAF, so as to obtain microvesicles (containing lncENAF) of tomato fruits, so as to develop a novel method for relieving or treating inflammation-related diseases.

Disclosure of Invention

The invention aims to provide a micro-vesicle nano-particle of a transgenic tomato and a preparation method and application thereof, so as to solve the problems in the prior art, successfully realize genetic transformation of lncENAF of animal origin in plants, and the micro-vesicle nano-particle of the transgenic tomato prepared by the method can be applied to preparation of medicines for relieving inflammation.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a preparation method of microvesicle nano-particles of transgenic tomatoes, which comprises the following steps:

(1) Connecting lncENAF DNA molecules into a gene expression vector, and then converting into competent cells to obtain a recombinant microorganism strain;

(2) Infecting tomato explants by utilizing the recombinant microorganism strain, and obtaining transgenic tomato plants through tissue culture, identification and screening;

(3) Culturing the transgenic tomato plants, and setting fruits to obtain transgenic tomato fruits;

(4) Carrying out microvesicle extraction on the transgenic tomato fruits to obtain microvesicle nano-particles containing lncENAF;

In the step (1), the nucleotide sequence of the lncENAF DNA molecule is shown as SEQ ID NO. 1.

Further, in step (1), the gene expression vector is pCAMBIA1301.

Further, in step (1), the competent cells are E.coli competent cells.

Further, in step (2), the tomato explant is a leaf.

Further, in step (4), the extraction is performed by a density gradient centrifugation method.

The invention also provides the microvesicle nano-particles containing lncENAF prepared by the preparation method.

The invention also provides application of the microvesicle nano-particles containing lncENAF in preparation of medicines for relieving inflammation.

Further, the alleviation of inflammation refers to inhibition of the production of inflammatory cytokines IL-6, IL-1. Beta. And/or TNF-alpha.

The invention also provides a medicine for relieving inflammation, which comprises the microvesicle nano-particle containing lncENAF.

Further, the medicament also comprises pharmaceutically acceptable auxiliary materials.

The invention discloses the following technical effects:

According to the invention, lncENAF (SEQ ID NO. 1) of a mouse source is infected by agrobacterium to obtain lncENAF transgenic tomato seedlings, tomato seedlings are further cultivated to obtain tomato fruits, then microvesicle nano-particles of tomatoes are extracted from the tomato fruits, and after the microvesicle nano-particles are incubated with HEK-293T cells, HEK-293T cells which do not express lncENAF originally can be expressed lncEANF, and the expression of inflammatory cytokines caused by LPS stimulation can be relieved. The micro-vesicle nano-particles (containing lncENAF) of the transgenic tomatoes prepared by the invention can be applied to preparing medicines for relieving inflammation, so that the micro-vesicle nano-particles of the transgenic tomatoes can provide a new strategy for developing anti-inflammatory medicines.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram of an electrophoretic assay synthesized by lncENAF;

FIG. 2 is a diagram of a pCAMBIA1301 plasmid vector;

FIG. 3 is an electrophoresis chart of PCR detection after lncENAF is inserted into pCAMBIA1301 plasmid, wherein 1-6 are respectively negative control, blank control, positive sample 1, positive sample 2, positive sample 3 and negative sample;

FIG. 4 is a flow chart of the construction of transgenic tomato;

FIG. 5 is an identification chart of lncENAF in transgenic tomato leaf (A) and fruit (B), wherein 1-7 are positive sample 1, negative sample, positive sample 3, positive sample 4, positive sample 5, positive sample 6, and positive sample 7, respectively;

FIG. 6 is a flow chart of transgenic tomato fruit microvesicle nanoparticle extraction;

FIG. 7 is an identification of lncENAF in tomato fruit microvesicle nanoparticles;

FIG. 8 is a TEM (A) and particle size plot (B) of tomato fruit microvesicle nanoparticles, scale in A being 200nm;

fig. 9 is a graph showing the inhibition of inflammation by tomato fruit microvesicle nanoparticles.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

HEK-293T cells and Raw264.7 cells in the following examples were purchased from Shanghai cell Bank of China academy of sciences.

Example 1

Construction of lncenaf transgenic tomato

1.1 Gene Synthesis

(1) The sequence of lncENAF gene synthesized by the division of the biological engineering (Shanghai) of the principal engineering, lncENAF is shown as SEQ ID NO. 1;

SEQ ID NO.1:

ATTGTACACCATGCAGACAAAGCGCTCAAACACTTGCAATGCTGGGCTTTCTCCCAATATCCTCTGCCATCTTTCCTACCCTTATAAAAGTCCAGAAAGAAAATAATCATTCTATCTGGAGGTGGGGGCCACTTCTTTAATCCTGGCACTTGGGAAGCAGAGGTAGGTGTATTGCTTTGAGTTCAAGACCAGACTGGTCTACAAAGTGAGTTCCGGGACAGTCAGGACTGTTGAACTTGGAAGCCTTGTCCTCAAATTTCTGGCAATTTTACTAGCACCAGTCTTCCCGCCTCAGCCTCCAGTGTCTTCCTGAGATGATCTGACTGCATGAAATGCCCTCGCCTCATTTTAGTTGGCTGGCCCTAAGGTCAAGGTAAATCCGCGCCCAAGCTGCCCGGTGGAGGTGGTCTCAGAGGGTGCTGCGGGATCGAGGTAGTGAGGAGACTAGATCGCAAGACGTGATCCTCACA TTTATTTGCCTGGAGTTCTCATGCCAGAGAACCTGGCAGATTTTACTATTTCCCAATTGTTTACTCGCCAAGCTTTCAGGTCCACGCGCCTCAGGGCTGCGCCTCTCACTCTGAAACTTCATTCAAAGGCCAGGCAGGGAGGCCCAAGAGGTGGCGAATGGGCTTGAGTATGACCTCAAGGCC.

(2) And (3) PCR reaction:

PCR amplification was performed using the gene synthesized lncENAF (SEQ ID NO. 1) as a template and the ORF amplification primers CZ-lncENAF-Kpn I-F and CZ-lncENAF-BamH I-R to obtain an amplified product.

The upstream primer CZ-lncENAF-KpnI-F: AGGAGCTCCCGCGGGTCGACATTGTACACCATGCAGAC (SEQ ID NO. 2) and the downstream primer CZ-lncENAF-BamHI-R: AGCCTGCAGCCATGGGGCCTTGAGGTCATACTC (SEQ ID NO. 3), wherein the underlined portion is the homology arm primer attached to the vector.

The reaction system (total volume 50. Mu.L) is shown in Table 1:

TABLE 1

Component (A)	Volume of
		ddH2O	17μL
2×Phanta Max Buffer	25μL
		dNTP Mix(10mM each)	1μL
Template DNA	2μL
		Upstream primer (10. Mu.M)	2μL
Downstream primer (10. Mu.M)	2μL
		Phanta Max Super-Fidelity DNA Polymease(1U/μL)	1μL

The PCR reaction procedure was 95℃for 30s, 95℃for 15s, annealing temperature for 55℃for 15s, extension at 72℃for 1min,35 cycles, and finally extension at 72℃for 5min.

(3) Agarose gel electrophoresis

1% Agarose gel was prepared and the formulation is shown in Table 2.

TABLE 2

The above 1×TAE and agar powder were poured into a 250mL conical flask, and the mixture was heated and boiled in a microwave oven to dissolve the agarose sufficiently. After cooling to 60 ℃, one ten thousandth of GoldView I type nucleic acid dye is added dropwise. Shaking until the gel is fully and uniformly mixed, pouring the liquid into a clean electrophoresis tank, inserting a comb, and standing at room temperature for 15min until the gel is completely solidified;

Removing comb, placing agarose gel in 1 xTAE buffer solution, adding corresponding amount of 10 x Loading buffer into PCR reaction system, mixing uniformly, taking 5 μl with sample gun, and adding into small hole of gel;

electrophoresis, setting an electrophoresis program of 110V for 30min;

exposure to light after electrophoresis, agarose gel was placed in a gel imager for imaging, and the result is shown in fig. 1.

1.2 Vector construction

(1) Linearization with a KpnI and BamHI double cut vector pCAMBIA1301 (pCAMBIA 1301 vector map see FIG. 2);

the cleavage reaction system is shown in Table 3:

TABLE 3 Table 3

(2) After the enzyme digestion product is purified, carrying out recombination connection reaction with the PCR product obtained in 1.1;

The recombination ligation reaction system (total volume 10. Mu.L) is shown in Table 4:

TABLE 4 Table 4

Component (A)	Volume of
		Linearization carrier	4μL
Insertion fragment	1μL
		5×CE II Buffer	2μL
Exnase II	1μL
		ddH2O	Make up 10 mu L

The reaction solution is gently sucked and beaten by a pipette, and is collected to the bottom of a tube by centrifugation. The reaction was allowed to stand at 37℃for 30min, immediately followed by cooling on ice.

(3) Transforming the recombinant product into escherichia coli DH5 alpha cells;

a) Adding 10 mu L of the ligation product into 100 mu L of escherichia coli competent cells, and carrying out ice bath for 30min;

b) Heat-shocking at 42 ℃ for 60s, and ice-bathing for 2min;

c) Adding 800 μLLB liquid culture medium, and shake culturing at 37deg.C for 30min;

d) Centrifuging at 6000rpm for 3min, removing supernatant, coating Kana (50 mg/L) resistant culture medium plate, and culturing at 37deg.C for 16 hr in an inverted manner to obtain resistant colony;

e) In 96-well plates, 100 μΜ LLB (Kana-containing) liquid medium was added to each well;

f) 4 colonies were obtained from each plate, and subjected to amplification culture at 37℃for 2 hours at 180 rpm;

g) 1. Mu.L of bacterial liquid is taken for PCR positive detection:

Picking up PCR positive transformant, culturing and extracting plasmid by shaking, and sequencing amplified product. The amplification and sequencing primers are vector sequences inserted at two sides of the target gene, and are M13-F5'-GTTGTAAAACGACGGCCAG-3' (SEQ ID NO. 4) and 2301-R5'-GCTTCCGGCTCGTATGTTG-3' (SEQ ID NO. 5) respectively;

h) And (3) detecting electrophoresis, wherein the electrophoresis result is shown in figure 3.

1.3 Tomato transformation

(1) Taking a tomato aseptic seedling in a laboratory, cutting leaves to serve as explants, co-culturing with agrobacterium for transforming a vector (without light) for 2 days after pre-culturing, transferring into a hygromycin resistance screening culture medium plate, and carrying out 16:8 illumination at room temperature of 25 ℃;

The hygromycin resistance screening culture medium comprises 5g/L beef extract, 1g/L yeast extract, 5g/L peptone, 5g/L sucrose, 4g/L MgSO ₄·7H₂ O,15g/L agar powder and 5mg/L hygromycin b.

(2) After infection of agrobacterium, the agrobacterium is subjected to 4 times of screening/subculture (14 days each time interval) to induce resistant buds, and then the resistant buds are transferred into a growth medium (the growth medium is a 1/2MS modified plant medium purchased from Biyun biotechnology Co., ltd., product number: B5010);

(3) The normally elongated seedlings are transferred into a rooting medium (MS medium is used as the rooting medium and purchased from Qingdao Gaokou Haibo biotechnology Co., ltd., product number: HB 8469) again after root cutting until strong root hairs grow out.

The specific process is with reference to fig. 4.

1.4 Tomato seedling leaf DNA extraction and PCR identification

(1) Plant leaf DNA extraction was performed using plant DNA extraction kit (B518411) from Shanghai Co., ltd.

A) Plant leaves with a diameter of 0.5 were taken and placed in a 1.5mL centrifuge tube, and ground with a grinding rod. Adding 100 mu LQlysis-P Reagent, shaking and mixing well, and carrying out water bath at 85 ℃ for 20min;

b) Adding 100 mu LBuffer NST into the centrifuge tube after water bath, and shaking and uniformly mixing;

c) Centrifuging at room temperature for 5min at 12,000 rpm;

d) The supernatant was used as a template for PCR detection directly.

(2) PCR and electrophoresis

The PCR detection strategy is that the upstream vector primer, the downstream target gene inner primer and the product length is about 750bp. The PCR detection uses 35S-F GACGCACAATCCCACTATCC (SEQ ID NO. 6) and lncENAF-R GGCCTTGAGGTCATACTC (SEQ ID NO. 7);

The PCR reaction (total volume 25. Mu.L) is shown in Table 5:

TABLE 5

The PCR reaction procedure was 95℃for 30s, (95℃for 15s, annealing temperature for 52℃for 15s, extension for 72℃for 1 min). Times.35 cycles, and finally extension for 72℃for 5min.

Electrophoresis was performed using 1% agarose gel, and gel imaging was performed after 100v 30min, and the results are shown in fig. 5 a.

And (5) culturing the correctly identified tomato transformed seedlings after transplanting until the tomato transformed seedlings are firm, and obtaining tomato fruits.

2. Extraction and PCR identification of RNA of tomato fruit

2.1 Extraction of tomato fruit Total RNA

RNA was extracted from tomatoes obtained in 1.4 using a rapid extraction kit (B518631) for total RNA of plants from the company Shanghai, inc. of bioengineering (Shanghai).

(1) 600 Mu LBuffer Rlysis-P was added to a 1.5mL RNase-free centrifuge tube for use;

(2) Grinding 50mg of tomato into powder by liquid nitrogen, adding into the 1.5mL centrifuge tube, immediately shaking and uniformly mixing;

(3) Completely lysing the sample in a water bath at 65 ℃ for 5 min;

(4) 60 mu LBuffer PCA was added to the lysed sample and mixed well. Standing at-20deg.C for 3min;

(5) Centrifuging at room temperature of 12,000rpm at 4 ℃ for 5min, and collecting supernatant;

(6) Adding equal volume of phenol and chloroform (volume ratio of 25:24, pH 4.5) into the supernatant, and mixing. Centrifuging at 12,000rpm and 4 ℃ for 5min, and taking supernatant;

(7) Adding equal volume of chloroform into the supernatant, and mixing. Centrifuging at 12,000rpm and 4 ℃ for 5min, and taking supernatant;

(8) Adding 1/3 volume of absolute ethyl alcohol, mixing, standing at room temperature for 3min, centrifuging at 12,000rpm for 5min at 4 ℃, carefully pouring out the supernatant (after adding absolute ethyl alcohol, mixing, standing at-20 ℃ for 10min to improve the yield of RNA);

(9) The pellet was washed with 700 μl of 75% ethanol (please be formulated with DEPC water), centrifuged at 12,000rpm for 3min at 4 ℃, and the supernatant carefully decanted;

(10) Inversion is carried out for 10min at room temperature, so that the ethanol remained in the centrifuge tube is volatilized thoroughly as much as possible. The precipitate was dissolved by adding 50. Mu.L of DEPC water to obtain RNA.

2.2RNA concentration and purity determination

(1) The concentration and purity of the extracted RNA are detected by using a Nanodrop 2000, and the detection probe is washed 3 times by using 2 mu L of DEPC water;

(2) Zeroing 1 mu L of DEPC water, and after the concentration of RNA is regulated to +/-0.2 ng/. Mu.L, taking 1 mu L of RNA sample to detect the concentration and purity of the RNA sample, wherein the concentration and purity of RNA A _260/280 is generally required to be between 1.8 and 2.0, and A _260/230 is more than or equal to 1.5;

(3) The test probe was washed 5 times with 2. Mu.L of double distilled water.

2.3 Reverse transcription

1000Ng of total RNA was taken according to VazymeII Q RT SuperMix for qPCR (+ GDNA WIPER) the reverse transcription kit (R323-01) was operated with a total system of 20. Mu.L, the specific procedure being as follows:

(1) RNA samples were adjusted to the same concentration, e.g., 500 ng/. Mu.L, with DEPC water and 200. Mu.L of the De-enzyme EP tube was used to formulate a de-genome reaction system as shown in Table 6:

TABLE 6

(2) Gently stirring and mixing with a pipetting gun, and reacting for 2min at 42 ℃ with a PCR instrument

(3) Preparation of reverse transcription reaction System (Table 7)

TABLE 7

(4) The mixture was gently stirred with a pipette, and the mixture was put into a PCR instrument, and a reverse transcription reaction program was set in accordance with Table 8.

TABLE 8

After the reaction is finished, the subsequent experiment is continued or the reaction product is kept at-20 ℃ for standby.

2.4PCR amplification assay

The PCR detection uses lncENAF-F GGAAGCAGAGGTAGGTGTAT (SEQ ID NO. 8), lncENAF-R GGCTTCCAAGTTCAACAGTC (SEQ ID NO. 9) and the size of the target product is 112bp.

The PCR reaction system (total volume 25. Mu.L) is shown in Table 9:

TABLE 9

The PCR reaction procedure was 95℃for 30s, 95℃for 15s, annealing temperature for 55℃for 15s, extension for 72℃for 30s,35 cycles, and finally extension for 72℃for 5min.

Electrophoresis was performed using 1% agarose gel, and gel imaging was performed after 100v 30min, and the results are shown in fig. 5B.

3. Extraction of tomato microvesicles

(1) Washing, and weighing 50g of tomato fruit (obtained in part 1.4).

(2) The tomatoes and a proper amount of PBS are smashed and ground according to the mass ratio of 1:5, and the grinding is carried out for 5 times at the maximum rotating speed for 1min each time.

(3) The grinding fluid is poured into a 50mL centrifuge tube and centrifuged at 1000g for 10 min.

(4) After centrifugation, the supernatant was removed and the pellet was discarded. The supernatant was centrifuged at 3000g for 20 min.

(5) After centrifugation, the supernatant was removed and the pellet was discarded. The supernatant was centrifuged at 10000g for 40 min.

(6) The supernatant was removed after differential centrifugation and subjected to ultracentrifugation at 4℃at 150,000g for 90 min. Sucrose solutions with concentration gradients of 60wt%, 45wt%, 30wt% and 8wt% from bottom to top were prepared in advance, with 8mL of each gradient.

(7) The supernatant was discarded after the completion of the ultracentrifugation, the pellet was resuspended in an appropriate amount of PBS and ultracentrifuged at 150,000g, 4℃for 2h in the uppermost layer of sucrose solution.

(8) 2 Bands (between 8wt% and 30wt%,30wt% and 45 wt%) were present and sucrose was removed by centrifugation at 2,150000g with aspiration of band 1 and band, respectively.

(9) The pellet was resuspended with 1mL of PBS, filtered through a 0.22 μm filter, and stored at-80 ℃.

4. Extraction and identification of RNA in tomato microvesicles

4.1 Extraction of RNA from tomato microvesicles

(1) Respectively taking tomato microvesicles (strip 1 and strip 2), adding 200 mu L of Trizol, fully shaking and uniformly mixing, adding 40 mu L of chloroform, fully mixing by vortex, and standing on ice for 5min;

(2) Centrifuging at 14,000rpm for 15min at 4 ℃;

(3) Transferring the supernatant to a new 1.5mL of the enzyme-depleted EP tube, adding an equal volume of pre-chilled isopropanol;

(4) After being gently inverted and evenly mixed, the mixture is placed on ice for 10min;

(5) Centrifuging at 4 deg.C and 14,000rpm for 10min, discarding supernatant, and collecting white precipitate;

(6) 1mL of 75% absolute ethanol prepared with DEPC water was added, the pellet was gently blown up but not blown off, centrifuged at 14,000rpm for 5min at 4℃and repeated once;

(7) Removing the supernatant, centrifuging the liquid on the pipe wall to the bottom of the pipe, sucking the liquid by a gun head, opening an EP pipe cover, heating by a 42 ℃ metal bath to change the sediment from white to colorless, adding a proper amount of DPEC water according to the size of the sediment to dissolve the sediment, and detecting the concentration and the purity later.

4.2 Determination of RNA concentration and reverse transcription references sections 2.2 and 2.3.

4.3PCR identification of RNA expression in microvesicles

Identification of PCR reference part 2.4, the PCR products were electrophoresed using 1% agarose, and the electrophoresis results are shown in FIG. 7, wherein each of lanes 1 and 2 contains lncENAF, and lane 2 contains more lncENAF, so that lane 2 was used for the later functional verification.

5. Characterization of tomato microvesicles

(1) The sample of strip 2 was subjected to negative staining with phosphomolybdic acid and observed under an electron microscope (see a in fig. 8), which indicated that the extract of 4.1 was a nano-scale vesicle-like structure.

(2) The extracted band 2 was subjected to about 200. Mu.L of nanovesicles, and its particle size was checked on-machine using a nanoparticle size analyzer (see B in FIG. 8), and the result showed that the average particle size was 112.5.+ -. 59.9nm.

6. Influence of tomato microvesicles on cells

6.1 Effect of tomato microvesicles on HEK-293T cells

HEK-293T cells were seeded into 24 well plates (8 ten thousand per well), after 12h of cell wall culture, then 20 μg of extracted tomato microvesicles (strip 2) were incubated with the cells for 24h, the cells were collected, cellular RNAs were extracted by Trizol method, reverse transcribed (specific steps 2.1, 2.2 and 4.3 parts), and fluorescence quantitative analysis lncENAF levels were performed (results see table 10), which indicated that microvesicles of transgenic tomatoes entered HEK-293T cells, and lncENAF were expressed in the cells.

Table 10 CT values of lncENAF in cells after co-incubation of transgenic tomato microvesicles with HEK-293T cells

6.2 Inhibition of inflammatory cytokines by tomato microvesicles

(1) Raw264.7 cells were plated into 24 well plates (5 ten thousand per well), and after cell attachment, the cells were divided into 4 groups, a control group (no treatment), an LPS-stimulated group (10 μg/mL lipopolysaccharide stimulation for 6 h), an lps+normal tomato microvesicle group, and an lps+ lncENAF transgenic tomato microvesicle group.

Wherein, the control group does not carry out any treatment;

LPS-stimulated group, stimulated with 10. Mu.g/mL LPS (lipopolysaccharide) for 6h;

LPS+common tomato microvesicle group, namely adding 20 mug of common tomato microvesicles to incubate with cells for 24 hours, and then stimulating the cells for 6 hours by using 10 mug/mL LPS;

LPS+ lncENAF transgenic tomato microvesicle group after 24h incubation with cells with 20. Mu.g of lncENAF transgenic tomato microvesicles (carrying lncENAF), 6h stimulation with 10. Mu.g/mL LPS was performed.

(2) After LPS stimulation of each experimental group is finished, cells are collected, RNA extraction and reverse transcription are carried out (specific steps 2.1, 2.2 and 4.3), and real-time fluorescence quantitative PCR (RT-qPCR) is carried out to detect the expression quantity of inflammatory cytokines IL-6, IL-1 beta and TNF-alpha, and detection primers are shown in Table 13.

The real-time fluorescence quantitative PCR detection method comprises the following steps:

(1) Adding diluted cDNA (20 mu L cDNA is diluted by adding 20 mu L DEPC water) into 20 mu L reverse transcription reaction system, and mixing thoroughly with a pipette;

(2) Preparing N+1 qPCR reaction systems according to the requirement, fully and uniformly mixing, adding 18 mu L of the mixture into a pre-marked 96-well PCR reaction plate, and adding 2 mu L of a reverse transcription product, wherein the specific system is shown in Table 11:

TABLE 11

(3) After spotting the 96-well PCR reaction plate, the plate was placed in a fluorescence quantitative analyzer of Bio-Rad, and the program settings were as shown in Table 12:

Table 12

(4) After the program operation, the relative expression amount of mRNA was calculated by the ΔΔCt method.

The calculation steps are as follows:

delta Ct = Ct average of the gene of interest-Ct average of the reference gene;

ΔΔct= - Δct experimental group- Δct control group;

Wherein the reference gene is GAPDH.

TABLE 13

The results of real-time fluorescent quantitative PCR (RT-qPCR) detection of the expression levels of inflammatory cytokines IL-6, IL-1 beta and TNF-alpha are shown in figure 9, and according to figure 9, it can be seen that the production of inflammatory cytokines (IL-6, IL-1 beta and TNF-alpha) caused by LPS stimulation can be inhibited after the lncENAF transgenic tomato microvesicles are incubated with Raw264.7 cells, which indicates that the fruits of the transgenic tomato prepared by the invention can carry lncENAF, and the transgenic tomato fruit microvesicles carry lncENAF, thereby playing a role in inhibiting inflammation.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (10)

1. A method for preparing microvesicle nano-particles of transgenic tomatoes, which is characterized by comprising the following steps:

(1) Connecting lncENAF DNA molecules into a gene expression vector, and then converting into competent cells to obtain a recombinant microorganism strain;

(2) Infecting tomato explants by utilizing the recombinant microorganism strain, and obtaining transgenic tomato plants through tissue culture, identification and screening;

(3) Culturing the transgenic tomato plants, and setting fruits to obtain transgenic tomato fruits;

(4) Carrying out microvesicle extraction on the transgenic tomato fruits to obtain microvesicle nano-particles containing lncENAF;

In the step (1), the nucleotide sequence of the lncENAF DNA molecule is shown as SEQ ID NO. 1.

2. The method according to claim 1, wherein in the step (1), the gene expression vector is pCAMBIA1301.

3. The method of claim 1, wherein in step (1), the competent cells are e.

4. The method of claim 1, wherein in step (2), the tomato explant is a leaf.

5. The method of claim 1, wherein in step (4), the extraction is performed by density gradient centrifugation.

6. A microvesicle nanoparticle containing lncENAF prepared according to the method of any one of claims 1 to 5.

7. Use of microvesicle nanoparticles according to claim 6 for the preparation of a medicament for reducing inflammation.

8. The use according to claim 7, wherein said reducing inflammation is inhibiting the production of inflammatory cytokines IL-6, IL-1 β and/or TNF- α.

9. A medicament for reducing inflammation, comprising the microvesicle nanoparticle containing lncENAF according to claim 6.

10. The medicament of claim 9, further comprising a pharmaceutically acceptable excipient.