CN108546289A - A kind of method and application preparing lunasin polypeptides using rice eukaryotic expression system - Google Patents

Abstract

The invention discloses a method and application for preparing lunasin polypeptide using a rice eukaryotic expression system. The method comprises the following steps: (1) cloning soybean lunasin gene and constructing a plant expression vector, cloning soybean lunasin gene, and constructing pCAMBIA2301 35s-lunasin plant An expression vector; (2) using the rice genetic transformation technology mediated by Agrobacterium to transfer the lunasin gene fragment into the rice genome, and performing molecular detection; and (3) extracting the transgenic rice protein, and isolating and purifying the lunasin polypeptide. In the application, for the first time, lunasin was transferred into the genome of rice. By detecting the content and biological activity of lunasin in rice, it was verified that exogenous lunasin can be successfully expressed in rice and has biological activities, including antioxidant and anti-inflammatory activities. In order to develop it into a functional food.

Description

A method and application of preparing lunasin polypeptide using rice eukaryotic expression system

technical field

The invention relates to the technical field of plant genetic engineering, in particular to a method and application for preparing lunasin polypeptide using a rice eukaryotic expression system.

Background technique

Lunasin, a new type of natural polypeptide containing 43 amino acids, was first discovered in soybeans, and later found to also exist in plants such as barley, wheat and black nightshade. Studies have shown that lunasin has a variety of physiological activities, including anti-oxidation, anti-hypertension, anti-inflammation, anti-cancer, lowering cholesterol, anti-obesity and immune regulation. Due to its huge application value, more and more scientists have begun to pay attention to how to use more efficient methods to produce lunasin.

At present, genetic engineering is developing rapidly. Using genetic engineering and plant genetic transformation methods to transfer lunasin gene fragments into plant genomes and using plant eukaryotic expression systems to produce lunasin has become an effective means. As a major food crop in my country, rice has a very mature genetic transformation system.

At present, there is no domestic report on how to use genetic engineering methods to clone and transfer lunasin gene fragments into rice to produce lunasin through the eukaryotic expression system of rice.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method and application for preparing lunasin polypeptide using the rice eukaryotic expression system, using genetic engineering methods to clone the gene fragments into the rice genome, and using the rice eukaryotic expression system to make It can be expressed and has high physiological activity.

The technical problem to be solved by the present invention is achieved through the following technical solutions:

A method for preparing a lunasin polypeptide using a rice eukaryotic expression system, comprising the following steps:

(1) Soybean lunasin gene cloning and plant expression vector construction, soybean lunasin gene was cloned, and pCAMBIA2301 35s-lunasin plant expression vector was constructed;

(2) Using Agrobacterium-mediated rice genetic transformation technology to transfer the lunasin gene fragment into the rice genome and perform molecular detection; and

(3) extract transgenic rice protein, separate and purify lunasin polypeptide.

Preferably, in the above technical solution, the step (1) includes:

(11) Use plant tissue RNA extraction kit to extract soybean fresh tissue RNA, reverse transcribe it into cDNA (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit), carry out PCR amplification, clone lunasin gene fragment; PCR product is agarose Gel electrophoresis, cut out the target fragment of expected size, purify, recover, and connect to T vector;

(12) Digest pCAMBIA2301 and pEASY-T1-lunasin with SacI and KnpI respectively; recover the large fragment of pCAMBIA2301 and the small fragment of pEASY-T1-lunasin respectively; connect the recovered two fragments, and complete the ligation reaction in a PCR tube at 16°C Water bath, ligated overnight; the ligated product was transformed into Escherichia coli competent cells; the recombinant was sequenced after enzyme digestion verification.

Preferably, in the above-mentioned technical scheme, the PCR amplification in the step (11) uses cDNA as a template, and Iun-F:CGAGCTCATGTCCAAATGGCAGCACCAGC and Iun-R:GGGGTACCTCAGTCGTCGTCATCATCATCATC with SacI and KnpI adapters as upstream and downstream primers.

Preferably, in the above technical scheme, the cloning reaction system in the step (11) is: 4 μl (1Kb20ng) of PCR recovered product, 1 μl of PEASY-T1 cloning vector, mixed gently, and reacted at 25°C for 5 minutes; after the reaction, Place the centrifuge tube on ice; add the ligation product to 50 μl Trans-T1 competent cells, transform Escherichia coli competent cells, perform colony PCR and enzyme digestion verification, and then sequence.

Preferably, in the above technical solution, the step (2) is to transfer the lunasin gene fragment into the rice genome by using Agrobacterium-mediated rice genetic transformation technology, extract the rice genome, and perform PCR amplification.

Preferably, in the above technical scheme, PCR amplification uses Iun-F:CGAGCTCATGTCCAAATGGCAGCACCAGC and Iun-R:GGGGTACCTCAGTCGTCGTCATCATC as upstream and downstream primers.

Preferably, in the above-mentioned technical scheme, the step (2) includes utilizing a kit to extract the rice genome, specifically comprising the following steps:

21). Take 100 mg of fresh rice leaves, add liquid nitrogen to the mortar and grind to a fine powder, add 400 μL buffer LP1 and 4 μL RNase A (10 mg/ml), vortex for 1 min, and place at room temperature for 10 min;

22). Add 130 μL buffer LP2, mix up and down, vortex for 1 minute, centrifuge at 12,000 rpm for 6 minutes, and absorb the supernatant;

23). Add 1.5 times the volume of LP3 in the supernatant, and mix by pipetting;

24). Put the mixture obtained in step 23) into the adsorption column CB3, put the adsorption column into the collection tube and centrifuge at 12,000 rpm for 60 seconds, discard the waste liquid in the collection tube, and put the adsorption column CB3 back into the collection tube;

25). Add 600 μL of washing solution WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid;

26). Repeat step 5;

27). Put the adsorption column CB3 back into the empty collection tube, centrifuge at 13,000rpm for 2 minutes, pour off the waste liquid; place the adsorption column at room temperature for 3 minutes, and remove the rinse solution;

28). Take out the adsorption column CB3, put it into a clean centrifuge tube, add 50-200μL ddH2O to the middle part of the adsorption membrane, let it stand at room temperature for 4 minutes, centrifuge at 13,000rpm for 2 minutes, collect the solution into the centrifuge tube, and place in- Store in a refrigerator at 20°C.

Preferably, in the above technical solution, the step (3) includes the following steps:

(31) The grains of the transgenic rice were ground in a pre-cooled mortar and pestle, then dissolved in PBS buffer, and extracted by shaking at 4°C for 48 hours.

(32) The homogenate was centrifuged at 12000g at 4°C for 15 minutes, and the supernatant was collected.

(33) Purify by ultrafiltration at 4°C, pass through columns with filter membrane pore sizes of 10KD and 1KD respectively, and lunasin polypeptide with a molecular weight cut-off of 5.5KD.

(34) The concentrated solution is subjected to vacuum freeze-drying to obtain highly pure lunasin polypeptide.

The application of the high-purity lunasin polypeptide prepared by a method for preparing lunasin polypeptide using a rice eukaryotic expression system in the production of functional food.

The technical scheme of the present invention has the following beneficial effects:

In this study, we transferred the lunasin gene fragment into the rice genome, and used the rice's own expression system to produce lunasin without complicated and cumbersome separation and purification steps. It can be used not only as a daily diet, but also as a functional food.

In addition, the application also has the following effects:

1. The transgenic rice with stable inheritance of lunasin has been obtained, and western blot blot hybridization analysis was performed on the transgenic rice, and the study showed that the lunasin polypeptide was successfully expressed in rice.

2. Separating and purifying lunasin, the content of lunasin in rice was identified by UPLC-MS/MS as 1.01 mg·kg-1 dry rice flour.

3. The antioxidant test showed that the antioxidant activity of polypeptide extracts from lunasin-transformed rice was significantly higher than that of wild-type rice.

4. In the inflammatory response induced by LPS, the anti-inflammatory activity of the polypeptide extract from lunasin-transformed rice was significantly higher than that of wild-type rice.

Description of drawings

Fig. 1 is the vector frame of the pCAMBIA2301 35s-lunasin plant expression vector of the present invention.

Fig. 2 is the PCR verification of pCAMBIA2301 35s-lunasin of the present invention.

Fig. 3 is the sequencing identification of pCAMBIA2301 35s-lunasin of the present invention.

Fig. 4 is the result of PCR verification among different transgenic lines of the transgenic rice of the present invention.

Fig. 5 is the western blot result of the present invention.

Figure 6 is the MRM chromatogram of lunasin standard.

Fig. 7 is the ESI mass spectrum of lunasin standard and transgenic rice lunasin polypeptide.

Fig. 8 is the MRM chromatogram of the lunasin polypeptide in the transgenic rice of the present invention.

Fig. 9 is a graph showing the measurement and analysis of DPPH free radicals of the lunasin polypeptide in the transgenic rice of the present invention.

Fig. 10 is an analysis diagram of ABTS+ free radical measurement and analysis of lunasin polypeptide in transgenic rice of the present invention.

Fig. 11 is an ORAC analysis chart of the lunasin polypeptide in the transgenic rice of the present invention.

Fig. 12 shows the inhibitory rate of NO by PEW of lunasin polypeptide in the transgenic rice of the present invention.

Fig. 13 shows the inhibition rate of IL-6 by LET of lunasin polypeptide in the transgenic rice of the present invention.

Fig. 14 shows the inhibition rate of TNF-α by LET of lunasin polypeptide in the transgenic rice of the present invention.

Fig. 15 shows the inhibition rate of MCP1 by PEW of lunasin polypeptide in the transgenic rice of the present invention.

Detailed ways

Specific embodiments of the present invention are described in detail below to facilitate a further understanding of the present invention.

All the experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained through commercial channels unless otherwise specified.

Embodiment 1 soybean lunasin gene cloning and plant expression vector construction

Soybean lunasin gene cloning and plant expression vector construction, soybean lunasin gene was cloned, and pCAMBIA2301 35s-lunasin plant expression vector was constructed.

(1) RNA was extracted from fresh soybean tissue using a plant tissue RNA extraction kit, reverse-transcribed into cDNA (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit), using cDNA as a template, and using Iun- F: CGAGCTCATGTCCAAATGGCAGCACCAGC and Iun-R: GGGGTACCTCAGTCGTCGTCATCATCATCATC are upstream and downstream primers for PCR amplification to clone the lunasin gene fragment. The PCR product was subjected to agarose gel electrophoresis, and the target fragment of the expected size was cut out, purified, recovered, and connected to the T vector.

The cloning reaction system is as follows: 4 μl of PCR recovered product (1Kb 20ng), 1 μl of PEASY-T1 cloning vector, mixed gently, and reacted at 25°C for 5 minutes. After the reaction, place the centrifuge tube on ice. Add the ligation product to 50 μl Trans-T1 competent cells, transform Escherichia coli competent cells, perform colony PCR and enzyme digestion verification, and then sequence.

(2) Digest pCAMBIA2301 and pEASY-T1-lunasin with SacI and KnpI respectively; recover the large fragment of pCAMBIA2301 and the small fragment of pEASY-T1-lunasin respectively; connect the recovered two fragments, and complete the ligation reaction in a PCR tube at 16°C Water bath, ligated overnight; the ligated product was transformed into Escherichia coli competent cells; the recombinant was sequenced after enzyme digestion verification.

Embodiment 2 The lunasin gene fragment is transferred to the rice genome

Using Agrobacterium-mediated rice genetic transformation technology, the lunasin gene fragment was transferred into the rice genome, and molecular detection was carried out.

Genetically modified material acquisition and identification:

Using Agrobacterium-mediated rice genetic transformation technology to transfer the lunasin gene fragment into the rice genome, extract the rice genome, use Iun-F:CGAGCTCATGTCCAAATGGCAGCACCAGC and Iun-R:GGGGTACCTCAGTCGTCGTCATCATC as upstream and downstream primers for PCR identification, PCR reaction system: 20uL

The PCR amplification program was: 95°C for 5min; 95°C for 30S, 58°C for 30S, 72°C for 30S; 72°C for 10min; 30 cycles. The expected target band is 135bp.

(1) Kit to extract rice genome (Plant Tissue Genomic DNA Extraction Kit-Tiangen Biochemical Technology)

1). Take 100mg of fresh rice leaves and add liquid nitrogen to a mortar and grind them to a fine powder. Add 400 μL buffer LP1 and 4 μL RNase A (10 mg/ml), vortex for 1 min, and place at room temperature for 10 min.

2). Add 130 μL of buffer solution LP2, mix up and down, vortex for 1 min, centrifuge at 12,000 rpm for 6 minutes, carefully pipette the supernatant into a new 1.5 ml centrifuge tube, and be careful not to suck the sediment.

3). Estimate the amount of supernatant, add 1.5 times the volume of LP3 (add absolute ethanol in advance), and immediately mix by pipetting.

4). Put the mixture obtained in the previous step into an adsorption column CB3, put the adsorption column into a collection tube and centrifuge at 12,000 rpm for 60 seconds, discard the waste liquid in the collection tube, and put the adsorption column CB3 back into the collection tube.

5). Add 600 μL of washing solution WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid.

6). Repeat step 5.

7). Put the adsorption column CB3 back into the empty collection tube, centrifuge at 13,000rpm for 2 minutes, and discard the waste liquid. Place the adsorption column at room temperature for 3 minutes, remove the rinse solution, and prevent the residual ethanol in the rinse solution from inhibiting downstream reactions.

8). Take out the adsorption column CB3, put it into a clean centrifuge tube, add 50-200μL ddH2O to the middle part of the adsorption membrane (ddH2O was preheated in a 65°C water bath), place it at room temperature for 4 minutes, and centrifuge at 13,000rpm for 2 minutes. Collect the solution into a centrifuge tube. Store in a -20°C refrigerator.

Embodiment 3 extraction and purification

Extract transgenic rice protein, separate and purify lunasin polypeptide.

(1) The grains of the transgenic material were ground in a pre-cooled mortar and pestle, then dissolved in PBS buffer, and extracted by shaking at 4°C for 48 hours.

(2) The homogenate was centrifuged at 12000g at 4°C for 15 minutes, and the supernatant was collected.

(3) Purify by ultrafiltration at 4°C, pass through columns with filter membrane pore sizes of 10KD and 1KD respectively, and lunasin polypeptide with a molecular weight cut-off of 5.5KD.

(4) The concentrated solution is subjected to vacuum freeze-drying to obtain highly pure lunasin polypeptide.

Dissolve the separated and purified lunasin extract LET (lunasin-enriched fraction from trans-lunasin rice) in ultrapure water, take an appropriate amount for SDS-PAGE protein electrophoresis, after electrophoresis, transfer to membrane, hybridize, and detect whether exogenous lunasin is expressed .

Similarly, the wild type rice grains were ground, ultrafiltered, and the peptide PEW (peptide-enriched fraction from wild type rice) with the same molecular weight was isolated and purified.

Example 4 Detection of lunasin content in transgenic rice

The content of lunasin in transgenic rice was detected by UPLC-MS/MS.

(1) Lunasin standard substance and lunasin polypeptide LET isolated and purified from rice were dissolved in ultrapure water according to a series of concentration gradients (0-1000pg·mL-1).

(2) UPLC was connected to XEVO TQ-S mass spectrometer (Waters; Etten-Leur, The Netherlands), which performed multiple reaction monitoring (MRM) in ion electrospray ionization mode.

The separation column is CSH C18 (1.7 μm, 2.1 mm×150 mm), the flow rate is 0.1 ml/min, phase A is 0.1% trifluoroacetic acid aqueous solution, and phase B is 0.1% trifluoroacetic acid acetonitrile. The flow conditions were: 80% A for 10 minutes, 40% A for 2 minutes, 20% A for 3.5 minutes, 20% A for 5.5 minutes, and 80% A for 7 minutes. The content of lunasin in transgenic rice was detected according to the peak area.

The detection of embodiment 5 antioxidant

The antioxidant activity of lunasin polypeptide in transgenic rice was evaluated by detecting the DPPH free radical scavenging ability, ABTS+ free radical scavenging ability and oxygen free radical scavenging ability of LET and PEW.

(1) Scavenging DPPH organic free radicals is a fast, simple and sensitive method to evaluate the antioxidant capacity. Configure LET and PEW to a series of gradient concentrations of 2, 4, 6, 8, 10, 12g L-1, take 2mL of DHHP solution, add 2mL of lunasin extract dissolved in the same solvent, and mix thoroughly. The absorbance was measured at 517nm after 30min. The scavenging rate of active ingredients in the extract to DPPH can be calculated by the following formula:

K=[1-(Ai-Aj)/Ac]×100% where Ai=2mL DPPH solution+2mL absorbance value of solution to be tested, Aj=absorbance value of 2mL solution to be tested+2mL solvent, Ac=2mLDPPH solution+ The absorbance value of 2mL of solvent.

Prepare the antioxidants to be tested into a series of solutions, measure the antioxidant quality and DPPH free radical scavenging rate, and draw a curve, and read the required antioxidant quality when the DPPH free radical scavenging rate is 50% from the curve. The ability of antioxidants to scavenge free radicals adopts the IC50 of scavenging DPPH.

DHHP solution: Take 8mg of DPPH, dissolve it with a small amount of methanol, and then dilute to volume (2×10-4mol/L) with 100mL of methanol.

(2) ABTS+ free radical scavenging experiment

Configure LET and PEW to 0.5, 1, 1.5, 2, 3, 4, 5g·L-1 series gradient concentration, prepare an appropriate amount of ABTS+ stock solution, mix ABTS+ stock solution and K2S2O8 stock solution by volume 1:1, Prepare ABTS+ working mother liquor. Store the prepared ABTS+ working mother solution at room temperature in the dark for 12–16 hours, and dilute it with 80% ethanol before use. After subtracting the 80% ethanol blank control from the absorbance of the ABTS+ working solution, the A734 is 0.7±0.05. Accurately measure 10 μL of the sample solution of each concentration and 200 μL of ABTS+ working solution in a 96-well plate, mix gently, and measure the absorbance value at 734 nm after reacting for 2–6 minutes. In the experiment, Trolox was used as the positive control, and reagent blank and sample blank were set at the same time, and the experiment was repeated 3 times. Calculate the clearance rate of ABTS according to the following formula: ABTS+ clearance rate (%)=[1-(At-B)/A0]×100% In the formula: A0 is no sample added; ABTS+ absorbance value; At is the sample The absorbance value after reacting with ABTS+·; B is the absorbance value of the sample blank.

ABTS stock solution (7.4mmol/L, 0.4mL): Take 3mg of ABTS, add 0.735mL of distilled water.

K2S2O8 stock solution (2.6mmol/L, 1.43mL): take K2S2O8 1mg, add distilled water 1.43mL.

Trolox standard solution (0.3mmol/L, 9.6mL, 0.1042mg/mL): take Trolox 1mg, add distilled water 9.6mL.

(3) Oxygen free radical scavenging ability

ORAC is the abbreviation of oxygen radical absorbance capacity (oxygen radical absorbance capacity), also known as the antioxidant capacity index, the free radicals in the ORAC analysis method mainly come from the azo compound 2,2'-azo-bis-(2 -Azobis (2-amidinopropane) dihydrochloride dihydrochloride [2,2'-azobis (2-amidinopropane) dihydrochloride, AAPH] thermal decomposition of hydrogen peroxide free radicals can also be produced in the process of Fenton (Fenton) reaction hydroxyl free radicals base, using sodium fluorescein (FL) as a fluorescent probe, observing the decline process of the fluorescence intensity of the probe after the interaction between free radicals and the fluorescent probe, using a water-soluble vitamin E analog (6-hydro-2,5 , 7,8-tetramethylchroman-2-carboxylic acid, Trolox) was used as an antioxidant standard substance to detect the ability of various antioxidants in the system to delay the decline of the fluorescence intensity of the probe, so as to evaluate the antioxidant capacity of the antioxidant. Configure LET and PEW to 0.5, 1, 1.5, 2, 3, 4g·L-1 series gradient concentration, and prepare appropriate amount of fluorescent sodium reagent and AAPH solution. Precisely draw 100uL of fluorescent sodium dilution solution into a 96-well fluorescent plate, then add 50uL of sample solutions of different concentrations and shake for 5min, incubate at 37°C for 10min, then quickly add 50uL of AAPH solution to start the reaction. The fluorescence value was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm, and the fluorescence value was measured every 1.5 min during the reaction (denoted as Fn). The fluorescence decay curves of different concentrations of lunasin were plotted with the measurement time as the abscissa and the fluorescence value as the ordinate. The ORAC value is obtained by comparing the protected area of the fluorescence decay curve with that of standard antioxidant substances. ORAC value = [(AUCSample–AUCBlank)/(AUCTrolox–AUCBlank)]×(molarity of Trolox/molarity of sample).

Preparation of AAPH solution: Accurately weigh 207 mg of AAPH, dissolve it in pH 7.4 phosphate buffer (75 mM) and set the volume to a 5 mL measuring bottle to obtain an AAPH solution with a concentration of 153 mmol/L.

Preparation of sodium fluorescein solution: Accurately weigh 40 mg of sodium fluorescein, dissolve it in phosphate buffer to prepare a sodium fluorescein solution of 4125 mmol/L, and record it as sodium fluorescein mother solution a. Precisely draw 50uL of sodium fluorescein mother solution a into a 50mL volumetric bottle, dilute to the mark with the above buffer solution, and record it as sodium fluorescein mother solution b; both a and b of sodium fluorescein mother solution are refrigerated at 4°C. During the experiment, accurately draw 500uL of the mother solution of fluorescein sodium and place it in a 25mL measuring bottle, and dilute to the mark with the above buffer solution to obtain a dilution of 8*10-5mmol/L.

Example 6 Anti-inflammatory Detection

Low immunity of the body can induce various diseases, and it is very important to enhance the immunity of the body. Nitric oxide is a multifunctional soluble gas signal molecule, an endogenous bioinformation molecule with important physiological and pathological functions, and has a wide range of biological functions, such as dilating blood vessels, inhibiting platelet aggregation, killing tumors and microorganisms, etc., NO The synthesis of is an important sign that macrophages are activated. Macrophages rapidly express TNF-α, IL-6 and MCP1 and other cytokines within a few hours after being activated to mediate host cell immunity. A certain concentration of NO, TNF-α, IL-6 and MCP1 expression can affect the body Immunity is positive. However, studies have shown that excessive production of NO, TNF-α, IL-6, and MCP1 in the body can trigger inflammatory reactions, cause pathological damage to tissue cells, change hemodynamics, cause microcirculatory disturbance, and can lead to shock and multiple organ failure in severe cases. In this study, the anti-inflammatory activity of lunasin polypeptide in transgenic rice was evaluated by analyzing the inhibitory ability of LET and PEW on LPS-induced overexpression of NO, TNF-α, IL-6 and MCP1.

Mouse macrophage Raw264.7 was cultured in a constant temperature incubator (37°C, 5% CO2, saturated humidity) with RP1640 medium, and was passaged every 2-3 days to maintain logarithmic growth. When the cultured cells cover 70-80% of the bottom area of the entire culture flask (in the logarithmic growth phase), wash twice with PBS of pH 7.4, add complete RP1640 culture medium, and collect the Raw264 in the logarithmic growth phase with a cell scraper. .7 cells, transferred to a centrifuge tube, 1000rpm, centrifuged for 5 minutes, adjusted the density to 2.5×106/mL with complete RPMI-1640 medium, according to 100μL/well, inoculated in a 96-well cell culture plate, and then set three For group experiments, 100 μL of different concentrations of samples (sample group), LPS (positive control group), and medium (blank control group) were added respectively. After the addition was completed, the 96-well plate was put into the incubator. After 2 hours, except for the blank control group, the other two groups were added with 100 μL of 2ug/mL LPS, and then placed in the incubator. After 24 hours, the OD value of the supernatant in the above treatments was measured by Griess reaction, and the normal growth The cell wells were used as the control group, and the LPS treatment group was used as the model group. The amount of NO produced in the culture medium was calculated according to the standard curve prepared by NaNO2. The determination of TNF-α, IL-6 and MCP1 expression was carried out with reference to BD Pharmingen ELISA Kit (Mouse tumor cell necrosis factor-α (TNF-α) SimpleStep Elisa Kit, Interleukin-6 (IL-6) Elisa Kit and Mouse monocyte chemoattractant protein chemokine (C-C motif) ligand 2 (MCP1/CCL2) SimpleStep Elisa Kit).

Result analysis:

1. Using the lunasin cDNA sequence as a template, simultaneously design lunasin sequence-specific primers, carry out PCR amplification, obtain the target sequence, and construct the pCAMBIA2301 35s-lunasin plant expression vector. Figure 1, Figure 2, and Figure 3 respectively show the vector framework, PCR verification and sequencing identification of the pCAMBIA2301 35s-lunasin plant expression vector. As shown in the figure, the target gene has been accurately inserted into the plant expression vector pCAMBIA2301.

2. Using Agrobacterium-mediated rice genetic transformation technology to transfer the lunasin gene fragment into the rice genome, extract the rice genomic DNA, and use this as a template to carry out PCR verification with lunF/lunR as upstream and downstream primers, and the PCR amplification result is 135bp The fragments were in line with expectations, indicating that a stable genetic transgenic rice with lunasin had been obtained. Figure 4 shows the results of PCR verification among different transgenic lines of transgenic rice.

3. Extract transgenic rice protein, separate and purify lunasin polypeptide. Three transgenic lines L07, L09 and L15 were selected for protein level verification. Western blot hybridization results showed that lunasin polypeptide was successfully expressed in rice. Figure 5 is a graph of western blot results.

4. Using UPLC-MS/MS method to detect lunasin content in transgenic rice. The MRM chromatogram (M+4H)4+ of the lunasin standard product is 1257.39m/z, and the peak appears at the retention time of 1.91min (Figure 6). The ESI mass spectrum associated with this peak revealed a multiple charge profile of the lunasin standard, which was consistent with previously reported studies (Fig. 7). The MRM chromatogram signal of transgenic rice lunasin extract is relatively complex, which may be due to the complex composition of substances in the extract. The machine recognizes lunasin and reaches a peak at the retention time of 1.78min (Figure 8). The content of lunasin was obtained according to the area of the peak diagram, and every kilogram of dry rice flour contained 1.01×10-3g lunasin.

5. By detecting the DPPH free radical scavenging ability of LET, PEW, ABTS+ free radical scavenging ability, oxygen free radical scavenging ability to evaluate the antioxidant activity of lunasin polypeptide in transgenic rice.

In the determination of DPPH free radicals, the DPPH free radical scavenging activity of LET was significantly higher, and its IC50 value was 8g·L-1; the DPPH free radical scavenging activity of PEW showed an IC50 value of 11g·L-1 (Figure 9). In the determination of ABTS+ free radicals, the IC50 value of LET was 1.18 g·L-1; while the ABTS+ free radical scavenging activity of PEW was significantly lower, with an IC50 value of 2 g·L-1 (Figure 10). In addition, in the ORAC analysis, the ORAC value of LET was significantly higher than that of PEW, and the antioxidant effect of 4g L-1 LET was equivalent to that of 170 μmol L-1 Trolox (Figure 11). The results show that the polypeptide extract (including lunasin polypeptide) obtained from the transgenic rice has stronger antioxidant activity than the polypeptide extract from wild-type rice, and the difference is significant.

6. To evaluate the anti-inflammatory activity of lunasin polypeptide in transgenic rice by analyzing the inhibitory ability of LET and PEW on LPS-induced intracellular NO, TNF-α, IL-6 and MCP1 overexpression through cell experiments.

Raw264.7 mouse macrophages were stimulated by LPS, the expression of NO, TNF-α, IL-6 and MCP1 in the cells increased, but this up-regulation can be significantly inhibited by LET and PEW, and the inhibitory effect of LET is obviously strong on PEW. The research results showed that when the concentrations of LET and PEW were both 6g·L-1, the inhibition rate of LET on NO could reach 34.3%, while the inhibition rate of PEW on NO was only 7.25% (Figure 12). When the concentrations of LET and PEW were both 4g·L-1, the inhibition rate of LET on IL-6 was 66.8%, and the inhibition rate on TNF-α was 76.2%; while the inhibition rate of PEW on IL-6 was 50.6%. The inhibition rate of TNF-α was 68.8% (Fig. 13, Fig. 14). When the concentrations of LET and PEW were both 8g·L-1, the inhibition rate of LET on MCP1 could reach 69.2%, while the inhibition rate of PEW on MCP1 was only 2.8% (Figure 15). The above results indicated that in the inflammatory response induced by LPS, the anti-inflammatory activity of polypeptide extracts from lunasin-transformed rice was significantly higher than that of wild-type rice.

Although the present invention has been disclosed as above with the embodiments, it is not intended to limit the present invention, and any person skilled in the art can make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection is defined by the claims and their equivalents.

Claims (9)

1. A method utilizing rice eukaryotic expression system to prepare lunasin polypeptide, is characterized in that, comprises the following steps: (1) Soybean lunasin gene cloning and plant expression vector construction, soybean lunasin gene was cloned, and pCAMBIA2301 35s-lunasin plant expression vector was constructed; (2) Using Agrobacterium-mediated rice genetic transformation technology to transfer the lunasin gene fragment into the rice genome and perform molecular detection; and (3) extract transgenic rice protein, separate and purify lunasin polypeptide. 2. the method utilizing rice eukaryotic expression system to prepare lunasin polypeptide according to claim 1, is characterized in that, described step (1) comprises: (11) Use plant tissue RNA extraction kit to extract soybean fresh tissue RNA, reverse transcribe into cDNA (TransScriptOne-Step gDNA Removal and cDNA Synthesis SuperMix kit), perform PCR amplification, clone lunasin gene fragment; Gel electrophoresis, cut out the target fragment of expected size, purify, recover, and connect to T vector; (12) Digest pCAMBIA2301 and pEASY-T1-lunasin with SacI and KnpI respectively; recover the large fragment of pCAMBIA2301 and the small fragment of pEASY-T1-lunasin respectively; connect the recovered two fragments, and complete the ligation reaction in a PCR tube at 16°C Water bath, ligated overnight; the ligated product was transformed into Escherichia coli competent cells; the recombinant was sequenced after enzyme digestion verification. 3. the method utilizing rice eukaryotic expression system to prepare lunasin polypeptide according to claim 2, is characterized in that, the PCR amplification in the described step (11) is template with cDNA, with the Iun with SacI and KnpI linker -F: CGAGCTCATGTCCAAATGGCAGCACCAGC and Iun-R: GGGGT ACCTCAG TCGTCGTCATCATCATC are upstream and downstream primers. 4. the method for utilizing rice eukaryotic expression system to prepare lunasin polypeptide according to claim 2, is characterized in that, in described step (11), cloning reaction system is: PCR recovery product 4 μ l (1Kb 20ng), PEASY-T1 clone 1 μl of the carrier, mix gently, and react at 25°C for 5 minutes; after the reaction, place the centrifuge tube on ice; add the ligation product to 50 μl Trans-T1 competent cells, transform E. coli competent cells, and perform colony PCR and sequenced after digestion. 5. the method utilizing rice eukaryotic expression system to prepare lunasin polypeptide according to claim 1, is characterized in that, described step (2) utilizes the rice genetic transformation technique mediated by Agrobacterium to transfer lunasin gene fragment into rice genome In , the rice genome was extracted and PCR amplified. 6. the method utilizing rice eukaryotic expression system to prepare lunasin polypeptide according to claim 5, is characterized in that, PCR amplification is with Iun-F:CGAGCTCATGTCCAAATGGCAGCACCAGC and Iun-R:GGGGTACCTCAGTCGTCGTCATCATCATCATC as upstream and downstream primers. 7. the method utilizing rice eukaryotic expression system to prepare lunasin polypeptide according to claim 5, is characterized in that, described step (2) comprises utilizing kit to extract rice genome, specifically comprises the following steps: 21). Take 100 mg of fresh rice leaves, add liquid nitrogen to the mortar and grind to a fine powder, add 400 μL buffer LP1 and 4 μL RNase A (10 mg/ml), vortex for 1 min, and place at room temperature for 10 min; 22). Add 130 μL buffer LP2, mix up and down, vortex for 1 minute, centrifuge at 12,000 rpm for 6 minutes, and absorb the supernatant; 23). Add 1.5 times the volume of LP3 in the supernatant, and mix by pipetting; 24). Put the mixture obtained in step 23) into the adsorption column CB3, put the adsorption column into the collection tube and centrifuge at 12,000 rpm for 60 seconds, discard the waste liquid in the collection tube, and put the adsorption column CB3 back into the collection tube; 25). Add 600 μL of washing solution WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste liquid; 26). Repeat step 5; 27). Put the adsorption column CB3 back into the empty collection tube, centrifuge at 13,000rpm for 2 minutes, pour off the waste liquid; place the adsorption column at room temperature for 3 minutes, and remove the rinse solution; 28). Take out the adsorption column CB3, put it into a clean centrifuge tube, add 50-200μL ddH2O to the middle part of the adsorption membrane, let it stand at room temperature for 4 minutes, centrifuge at 13,000rpm for 2 minutes, collect the solution into the centrifuge tube, and place in- Store in a refrigerator at 20°C. 8. the method utilizing rice eukaryotic expression system to prepare lunasin polypeptide according to claim 1, is characterized in that, described step (3) comprises the following steps: (31) The grains of the transgenic rice were ground in a pre-cooled mortar and pestle, then dissolved in PBS buffer, and extracted by shaking at 4°C for 48 hours. (32) The homogenate was centrifuged at 12000g at 4°C for 15 minutes, and the supernatant was collected. (33) Purify by ultrafiltration at 4°C, pass through columns with filter membrane pore sizes of 10KD and 1KD respectively, and lunasin polypeptide with a molecular weight cut-off of 5.5KD. (34) The concentrated solution is subjected to vacuum freeze-drying to obtain highly pure lunasin polypeptide. 9. An application of the highly pure lunasin polypeptide prepared by the method for preparing the lunasin polypeptide using the rice eukaryotic expression system according to any one of claims 1-8 in making functional food.