Recombinant III-type human-like collagen based on lettuce chassis expression as well as coding gene and expression method of recombinant III-type human-like collagen

Abstract

The invention belongs to the technical field of genes, and particularly relates to a recombinant III type human-like collagen based on lettuce chassis expression as well as a coding gene and an expression method thereof, the amino acid sequence of the obtained recombinant III type human-like collagen is as shown in SEQ ID NO.3, and the gene sequence is as shown in SEQ ID NO.1. The gene sequence is obtained by repeating a human type III collagen conserved region peptide fragment gene sequence for eight times, optimizing according to the codon characteristics of a receptor plant and inserting ASCI and XbaI double enzyme cutting sites at the two ends of the gene at the same time. The recombinant III-type human-like collagen provided by the invention has good activity and stability, the amino acid sequence of the recombinant III-type human-like collagen is 100% identical to that of a corresponding part of natural collagen, and the recombinant III-type human-like collagen is applied to a human body to avoid allergy and immunological rejection and has important practical significance and market value.

Description

Technical Field

[0001] The present invention belongs to the field of gene technology, and in particular, relates to a recombinant type III human-like collagen expressed based on lettuce chassis, a coding gene thereof and an expression method thereof. Background Art

[0002] Collagen is an extremely important structural protein in connective tissue. Its biological activity enables it to participate in cell migration, differentiation and reproduction, giving connective tissue mechanical strength. It can also promote cell growth and has properties such as hemostasis, biocompatibility, and biodegradability. Collagen is widely used in medical materials and drugs, beauty cosmetics, health products, and various practical industries.

[0003] So far, more than 30 coding genes for collagen chains have been discovered, which can form more than 16 types of collagen molecules. The common types are I, II, III, V and XI. Type III collagen is mainly found in infants' skin or vascular endothelium, intestines, and adult cartilage, vitreous body, intervertebral discs, etc. Type III collagen (CLO3A1) has a strong synthesis ability and can differentiate stem cells from bone marrow into fibroblasts that help repair in wound repair. Therefore, in biomedical engineering, type III collagen is more widely used in repairing tissue wounds, supporting cell reorganization, improving skin condition, and enhancing skin elasticity and tenderness.

[0004] At present, the method for preparing collagen in industry is mainly the traditional extraction method, that is, using animal tissues such as pig skin, cowhide, donkey skin, etc. to treat with acid, alkali, salt, and enzyme to obtain collagen. Although the collagen prepared by animal sources is simple in operation method and has high recovery rate, there are safety hazards in application. The process fails to completely remove non-collagen donor proteins and biological factors. The extracted products also have the risk of carrying animal-derived viruses. Direct application to the human body will produce immune rejection reactions, which greatly limits its application range in the biomedical field. At present, plant expression systems have been regarded as promising alternatives to animal cells and microbial cultures for large-scale production of recombinant proteins. The production method of using plants as a chassis to produce recombinant proteins can be found in, for example, Chinese patents CN107827975 and CN106554971.

[0005] However, there are no reports on the use of lettuce to express recombinant human-like type III collagen. Therefore, providing an expression method that can be mass-produced and the expressed recombinant collagen has good biological activity and can be used in the human body without producing immune rejection has important practical significance and market value. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a method for expressing recombinant type III human-like collagen based on lettuce chassis.

[0007] To solve the above problems, the technical solution adopted by the present invention is: The present invention provides a recombinant type III human-like collagen protein expressed based on lettuce chassis, and the amino acid sequence of the recombinant type III human-like collagen protein is shown in SEQ ID NO.3.

[0008] Another aspect of the present invention provides an AGⅢC8 gene encoding the recombinant type III human-like collagen, and the gene sequence is shown in SEQ ID NO.1.

[0009] Preferably, the recipient plant is lettuce.

[0010] In another aspect, the present invention provides a method for expressing recombinant type III human-like collagen based on lettuce chassis, comprising the following steps: S1: using the gene with the sequence as shown in SEQ ID NO.1 according to claim 2, amplifying and purifying the gene fragment of recombinant type III human collagen AGⅢC8 by PCR; S2: After double restriction digestion and ligation of the gene fragment and plasmid obtained in S1, they were transformed into Escherichia coli, the transformed Escherichia coli were screened and expanded, and the recombinant type III human collagen expression vector was obtained through plasmid extraction; S3: transforming the recombinant human-like collagen type III expression vector obtained in S2 into Agrobacterium, screening, expanding the culture and preserving it; S4: Transiently infect lettuce leaves with Agrobacterium containing the recombinant type III human collagen expression vector; S5: The lettuce leaves obtained in S4 were quickly frozen with liquid nitrogen and then ground, the extract was added, mixed evenly and then centrifuged, the supernatant was collected and purified by affinity chromatography to obtain recombinant type III human-like collagen AGⅢC8.

[0011] Preferably, the plasmid in S2 is pBINPLUS.

[0012] Preferably, the restriction endonucleases used in the double digestion in S2 are ASCI and XbaI.

[0013] Preferably, the Agrobacterium in S3 is Agrobacterium EHA105.

[0014] Preferably, the transient infection in S4 is as follows: transferring the overnight cultured Agrobacterium to LB liquid culture medium, shaking the culture medium until the OD value of the culture medium is 600 The value is 1.5~2.0, collect the bacteria by centrifugation, resuspend the bacteria with resuspension solution, and dilute the bacteria to the corresponding OD 600 The concentration of 0.4~0.6 was set at room temperature for 2~5 hours. The plants were immersed in the mixed infection solution of Agrobacterium and placed under the condition of -0.1~-0.05MPa pressure for 5~6 minutes.

[0015] Preferably, the resuspension consists of 8-12 mM magnesium chloride, 8-12 mM MES and 100-200 μM acetosyringone.

[0016] Preferably, the ratio of the extract in S5 to the lettuce leaves is 1-3 mL:1 g.

[0017] The beneficial effects of adopting the above technical solution are: 1. The present invention adopts plant expression, avoiding the risk of viral infection extracted by animals and the expression of endogenous proteins by bacteria; using lettuce as the base to produce collagen improves the purity of the target protein, and at the same time, the production cycle is short, the production cost is low, and large-scale production is easy to achieve.

[0018] 2. The recombinant type III human-like collagen prepared by the present invention has good activity and stability. After being stored for 90 days, the obtained recombinant type III human-like collagen has no obvious degradation.

[0019] 3. The amino acid composition of the recombinant type III human-like collagen prepared by the present invention is 100% identical to the amino acid sequence of the corresponding part of natural collagen. Applying it to the human body can avoid allergic reactions and immune rejection, and can be widely used in the biopharmaceutical and cosmetic industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the electrophoresis verification result of the PCR product of gene AGⅢC8 in Example 1 of the present invention; Figure 2 is a schematic diagram of the structure of plasmid pBINPLUS-AGⅢC8 of Example 1 of the present invention; Figure 3 This is a diagram showing the electrophoresis verification result of the PCR product of the Escherichia coli DH5α colony after the plasmid pBINPLUS-AGⅢC8 was transformed in Example 1 of the present invention; Figure 4 This is a PAGE gel electrophoresis detection diagram of the centrifuged supernatant of the lettuce protein extract obtained in Example 1 of the present invention; Figure 5 1 is a PAGE gel electrophoresis result diagram of the purified recombinant type III human-like collagen sample obtained in Example 1 of the present invention; wherein (a) and (b) are purified recombinant type III human-like collagen samples at 0 days; (c) and (d) are purified recombinant type III human-like collagen samples after being stored at -20°C for 90 days; Figure 6 This is a data graph of the cell adhesion rate of the recombinant type III human-like collagen obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the invention is described clearly and completely in conjunction with specific embodiments below, which is only for the convenience of describing the present invention and cannot be understood as limiting the present invention. The test methods and operations in this embodiment are conventional methods and conventional operations unless otherwise specified; the raw materials, reagents, etc. used are commercially available products unless otherwise specified.

[0022] It should be noted that the plasmid extraction kit was purchased from Sangon Biotechnology; Agrobacterium EHA105 was purchased from Weidi Biotechnology; BCA protein concentration determination kit (enhanced) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., model number 20201ES76; mouse fibroblast 3T3 was purchased from Shanghai Jihe Biotechnology Co., Ltd., catalog number JH-M2003; human collagen standard was purchased from Shanghai Sangon Biotechnology Technology Service Co., Ltd., catalog number A001654; cell adhesion assay The kit was purchased from Beibo Biotechnology with the product number BB-48120; the SDS-PAGE gel preparation kit was purchased from Biotech with the product number P0012A; and Coomassie Brilliant Blue R-250 was purchased from Biotech with the product number P0017. ALB solid culture medium: 40.0 g / L, Sangon Biotech (Shanghai) Co., Ltd., with the product number A507003; LB liquid culture medium: 25.0 g / L, Sangon Biotech (Shanghai) Co., Ltd., with the product number A507002.

[0023] Example 1 S1. Obtaining the recombinant type III human collagen AGⅢC8 gene: (1) A conserved peptide segment of human type III collagen was selected, and its amino acid sequence was shown in SEQ ID NO.2. The sequence was repeated 8 times to obtain the amino acid sequence of recombinant type III human-like collagen as shown in SEQ ID NO.3. The sequence was optimized according to the codon characteristics of lettuce, and ASCI and XbaI restriction sites were added at both ends of the gene to obtain the nucleotide sequence as shown in SEQ ID NO.1, which was recorded as gene AGⅢC8 and synthesized by GenScript Biotech Co., Ltd.; (2) Amplify the AGⅢC8 gene using high-fidelity PCR technology: Mix 2× PrimeSTAR GC Buffer (Mg 2+ plus) 10uL, dNTP Mixture (2.5mM each) 1.6uL, Primer F (sequence as shown in SEQ ID NO.4) 0.4uL, Primer R (sequence as shown in SEQ ID NO.5) 0.4uL, PrimeSTAR HS DNA Polymerase (2.5U / μL) 0.1uL and gene AGⅢC8 DNA 1uL, and finally add ddH 20 to 20 μL, placed in a PCR instrument, set the reaction conditions, and the reaction was completed to obtain the PCR product of gene AGⅢC8; wherein the reaction conditions are shown in Table 1 below: Table 1 High-fidelity PCR reaction conditions for gene AGⅢC8 fragment The PCR product of gene AGⅢC8 was verified by electrophoresis. The results of electrophoresis verification were as follows: Figure 1 As shown; (3) Purify the PCR product: Use the DiaSpin column DNA gel recovery kit to perform agarose gel electrophoresis on the PCR product, cut out the agarose gel containing the target fragment under ultraviolet light, and weigh it; add Buffer B2 3 to 6 times the weight of the gel block, and sol in a 50℃ water bath for 10 minutes. Transfer the sol solution to the adsorption column, centrifuge at 8000g for 30s, and pour out the liquid in the collection tube; add 500uL Wash Solution, centrifuge at 9000g for 30s, pour out the liquid in the collection tube, add 500uL Wash Solution again, centrifuge at 9000g for 30s, and pour out the liquid in the collection tube; centrifuge the empty adsorption column at 9000g for 1min, place the adsorption column in a clean 1.5mL centrifuge tube, add 40uL Elution Buffer in the center of the adsorption membrane, let it stand at room temperature for 1min, and centrifuge for 1min to obtain the purified gene AGⅢC8 fragment; S2. Construction of recombinant expression vector pBINPLUS-AGⅢC8: (1) Construction of plasmid pBINPLUS-AGⅢC8: The AGⅢC8 PCR product was double-digested with ASCI and XbaI: 1ug of AGⅢC8 DNA, 5uL of 10×NEBuffer and 1uL of Restriction Enzyme were added, and finally Nuclease-free water was added to 50uL, incubated at 37°C for 15min and 80°C for 20min to obtain the fragment COL1446 (NB); at the same time, the pBINPLUS empty vector was double-digested with ASCI and XbaI: 1ug of plasmid pBINPLUS, 5uL of 10×NEBuffer and 1uL of Restriction Enzyme were added, and finally Nuclease-free water was added to 50uL, incubated at 37°C for 15min and 80°C for 20min to obtain the fragment pBINPLUS (NB); Mix fragment pBINPLUS (NB) 50ng, fragment COL1446 (NB) 150ng, rapid ligation buffer (2×) 5uL, Rapid T4 DNA ligase 0.5uL, and finally add ddH2 0 to 10uL, ligated at 25℃ for 4 hours to obtain the recombinant plasmid pBINPLUS-AGⅢC8; (2) Obtaining the recombinant plasmid pBINPLUS-AGⅢC8: 100uL of E. coli DH5α competent cells were taken out from the -80℃ storage environment and quickly inserted into ice. After 5 minutes, when the bacterial block melted, 10uL of recombinant plasmid pBINPLUS-AGⅢC8 was added and gently mixed by hand on the bottom of the EP tube, and then allowed to stand in ice for 25 minutes; heat-shocked in a 42℃ water bath for 45 seconds, quickly put back on ice and allowed to stand for 2 minutes; 700μL of sterile LB liquid culture medium without antibiotics was added to the tube, mixed and revived at 37℃ and 200rpm for 60 minutes; centrifuged at 5000rpm for 1 minute to collect the bacteria, and about 100μL of supernatant was retained to gently blow and resuspend the bacterial block and spread on LB solid culture medium containing 50μg / mL kanamycin; the culture medium was inverted and placed in a 37℃ incubator for overnight culture; Single colony clones were selected using LB solid medium containing 50 μg / mL kanamycin, PCR was performed, and the PCR products were subjected to electrophoresis to identify positive clones. The identification results were as follows: Figure 3 As shown; positive clones were selected for sequencing to determine whether the recombinant plasmid successfully constructed the recombinant expression vector pBINPLUS-AGⅢC8; The correct positive clone colony was inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C. The plasmid was extracted using a plasmid extraction kit to obtain the recombinant plasmid pBINPLUS-AGⅢC8; S3. Agrobacterium transformation of recombinant type III human-like collagen vector: (1) Take the competent cells of Agrobacterium tumefaciens EHA105 stored at -80℃ and hold them in the palm of your hand for a while to partially melt. When they are in an ice-water mixing state, insert them into ice; add 0.5μg of recombinant plasmid pBINPLUS-AGⅢC8 DNA for every 100μL competent cells, stir the bottom of the tube by hand to mix, and let it stand on ice for 5 minutes, liquid nitrogen for 5 minutes, 37℃ water bath for 5 minutes, and ice bath for 5 minutes in sequence; add 700μL of antibiotic-free LB liquid culture medium and shake culture at 28℃ for 3 hours; centrifuge at 6000rpm for 1min, collect the bacteria, keep about 100μL of supernatant, gently blow, resuspend the bacteria block and spread it on LB solid culture medium containing 50μg / mL kanamycin and 20μg / mL rifampicin, and place it upside down in a 28℃ incubator for 3 days; (2) Small shaking: screen and pick positive clones and place them into 1 mL of LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampicin, and shake at 30°C and 200 rpm for 48 h; (3) Large shaking: Take a small shaking culture solution and inoculate 2% of the culture solution into 20 mL of LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampicin, and shake the culture at 30°C and 200 rpm for 48 h; (4) The obtained bacterial liquid was inoculated into LB solid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampicin, and cultured at 28°C for 60 h to screen positive clones; the positive colonies were cultured in 5 mL LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL rifampicin at 28°C overnight, transferred to LB liquid medium at a volume ratio of 1:100, and cultured in a shake flask at 28°C until OD600 was 0.6, cultured at 28°C for 60 h, centrifuged at 5000 g for 5 minutes, collected the bacterial precipitate, and stored at -80°C; S4. Transient infection of lettuce leaves with Agrobacterium containing recombinant type III human collagen vector: Resuspension solution: 10 mM MgCl2, 10 mM MES, and 150 μM acetosyringone; The inoculation loop was used to pick up a single colony of Agrobacterium EHA105 transformed with the recombinant plasmid pBINPLUS-AGⅢC8 obtained in S3 and placed in 5 mL LB liquid medium. The culture was shaken at 28°C and 200 rpm overnight. 1 mL of overnight cultured Agrobacterium was transferred to 50 mL LB liquid medium and shaken at 28°C and 200 rpm until the OD value of the culture solution was monitored. 600 The value was 1.8, and the bacteria were collected by centrifugation at 5000g, 15min, and 4℃. The bacteria were resuspended with resuspension solution and diluted to the corresponding OD 600 0.4, and let it stand at room temperature for 3 hours; immerse the lettuce in the mixed infection solution of Agrobacterium, place it under the condition of -0.09MPa, and treat it under negative pressure for 6 minutes to complete the instantaneous infection of the lettuce; S5. Extraction and purification of recombinant type III human collagen AGⅢC8: Extraction solution: 20 mM Tris-HCl (pH = 7.5), 150 mM potassium chloride, 7 mM magnesium acetate, 150 mM glucose, 30 mM mercaptoethanol, 1 mM EDTA, 100 μM PMSF, and 10% glycerol; After infection, the culture was carried out at 50% humidity and 28°C for 14 h, and at 50% humidity and 18°C ​​for 10 h. After 7 days of cycle culture, the lettuce leaves were taken, quick-frozen in liquid nitrogen, and then ground. 2 mL of the extract was added to every 1 g of lettuce leaves, mixed evenly, and centrifuged twice at 15000g, 4°C, and 30 min. The supernatant was collected and subjected to PAGE gel electrophoresis to detect the protein size. The results are as follows: Figure 4 As shown, the supernatant contains a large amount of recombinant type III human collagen AGⅢC8; Protein buffer: 20 mM NaH2 PO 4 , 500mM NaCl, adjust the pH to 8.0 with NaOH solution, and filter with 0.45um filter membrane; wash the nickel ion affinity column with protein buffer, and then gently shake the affinity column and the supernatant on ice for 30 minutes and then load them onto the column, wash the impurities with 5mL of PBS solution containing 5mM imidazole, and finally release the AGⅢC8 protein from the column with 10mL of PBS solution containing 200mM imidazole, collect the eluate and dialyze overnight to obtain purified recombinant type III human collagen AGⅢC8.

[0024] Test Example 1: SDS-PAGE protein gel detection: Referring to the method of J. Sambrook et al., Molecular Cloning Experiment Guide (3rd Edition), the purified recombinant type III human-like collagen sample obtained in Example 1 was sealed and stored at -20°C, and PAGE gel electrophoresis was performed at 0d and 90d to detect the protein size and purity; (1) Sample treatment: Add recombinant type III human collagen AGⅢC8 to the loading buffer and mix well. Boil in a boiling water bath for 10 min, cool naturally, and set aside. (2) Use an SDS-PAGE gel preparation kit to prepare a gel with a concentration of 10%, load the sample, and run electrophoresis at 80V for 30min and 120V for 50min until the bromophenol blue band reaches the bottom of the gel; (3) Staining: After electrophoresis is completed, pry open the gel sheet to remove the gel, and then place it in a staining container containing 100 mL of staining solution; place it on a horizontal shaker and shake it slowly, and stain it at room temperature for 1 hour; pour out the staining solution, add an appropriate amount of Coomassie Brilliant Blue staining destaining solution, and ensure that the destaining solution can fully cover the gel; pour out the destaining solution, add new destaining solution, and destain at room temperature for 24 hours, during which time the destaining solution is replaced 3 times, until the blue background is basically completely removed and the background is clear; The results are as follows Figure 5 As shown: there is a single, clear target protein band close to the 55KDa marker; and there are almost no mixed bands in the test results, which proves that the obtained recombinant type III human-like collagen has a high purity; the obtained purified recombinant type III human-like collagen sample was tested after being stored for 90 days, and no mixed bands appeared, indicating that the obtained recombinant type III human-like collagen sample had no obvious degradation and had good stability.

[0025] Test Example 2: Activity detection of recombinant type III human collagen AGⅢC8: (1) Protein concentration detection: The protein concentration was determined using the BCA Protein Quantification Kit (enhanced). BCA working solution: Prepare BCA working solution with BCA reagent A and BCA reagent B in a volume ratio of 50:1 and mix thoroughly; The purified recombinant type III human-like collagen AGⅢC8 obtained in Example 1 was prepared into a 0.5 mg / mL sample with PBS, 20 μL of the sample was taken into the sample wells of a 96-well plate, 200 μL of BCA working solution was added to each well, and the plate was placed at 37° C. for 30 minutes, and A562 was measured; it was calculated that the concentration of recombinant type III human-like collagen in the sample was 2 mg / mL; Protein concentration = (A562 value - 0.16) / 0.712.

[0026] (2) Cell adhesion test: Culture of 3T3 cells: Mouse fibroblast 3T3 cells were inoculated into DMEM medium containing 10% FBS and cultured at 37°C for 6 hours to observe the cell adhesion. The culture medium was changed every two days until the cells grew to 80%, and 3T3 cells were obtained. The purified recombinant type III human-like collagen AGⅢC8 and human collagen standard prepared in Example 1 were prepared into 0.5 mg / mL protein solutions using PBS; Add 100 μL of 0.5 mg / mL recombinant type III human collagen AGⅢC8 solution and human collagen standard solution to a 96-well plate as test cells and control cells, respectively, with PBS as a blank control, and let stand at room temperature for 60 minutes. Add 105 3T3 cells to each well and incubate at 37°C for 60 minutes; wash each well 4 times with PBS; use a cell adhesion detection kit to detect the absorbance at OD450nm; calculate the degree of cell adhesion based on the value of the blank control; the cell adhesion rate can reflect the activity of collagen: the higher the activity of the protein, the better it can provide cells with a high-quality external environment in a short time to help cells adhere to the wall; Taking the adhesion rate of human collagen standard as 1, the relative cell adhesion activity of AGⅢC8 sample can be calculated. The results are as follows: Figure 6 As shown, compared with the 40% cell adhesion efficiency of human collagen, the cell adhesion efficiency of recombinant type III human-like collagen AGⅢC8 can reach 92%.

[0027] Cell adhesion rate%=((test cell OD-blank OD) / (control cell OD-blank OD))*100.

[0028] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant type III human-like collagen expressed based on lettuce chassis, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. An AGⅢC8 gene encoding the recombinant type III human-like collagen according to claim 1, characterized in that: The gene sequence is shown in SEQ ID NO.

1.

3. The AGⅢC8 gene encoding the recombinant type III human-like collagen according to claim 2, characterized in that: The recipient plant is lettuce.

4. A method for expressing recombinant type III human-like collagen based on lettuce chassis, characterized in that: Here are the steps: S1: using the gene with the sequence as shown in SEQ ID NO.1 according to claim 2, amplifying and purifying the gene fragment of recombinant type III human collagen AGⅢC8 by PCR; S2: After double restriction digestion and ligation of the gene fragment and plasmid obtained in S1, they were transformed into Escherichia coli, the transformed Escherichia coli were screened and expanded, and the recombinant type III human collagen expression vector was obtained through plasmid extraction; S3: transforming the recombinant human-like collagen type III expression vector obtained in S2 into Agrobacterium, screening, expanding the culture and preserving it; S4: Transiently infect lettuce leaves with Agrobacterium containing the recombinant type III human collagen expression vector; S5: The lettuce leaves obtained in S4 were quickly frozen with liquid nitrogen and then ground, the extract was added, mixed evenly and then centrifuged, the supernatant was collected and purified by affinity chromatography to obtain recombinant type III human-like collagen AGⅢC8.

5. The expression method according to claim 4, characterized in that: S2 The plasmid is pBINPLUS.

6. The expression method according to claim 4, characterized in that: The restriction endonucleases used in the double digestion in S2 are ASCI and XbaI.

7. The expression method according to claim 4, characterized in that: The Agrobacterium described in S3 is Agrobacterium EHA105.

8. The expression method according to claim 4, characterized in that: The transient infection in S4 is as follows: transfer the overnight cultured Agrobacterium to LB liquid medium, shake the culture medium, and monitor the OD of the culture medium. 600 The value is 1.5~2.0, collect the bacteria by centrifugation, resuspend the bacteria with resuspension solution, and dilute the bacteria to the corresponding OD 600 The concentration of 0.4~0.6 was set at room temperature for 2~5 hours. The plants were immersed in the mixed infection solution of Agrobacterium and placed under the condition of -0.1~-0.05MPa pressure for 5~6 minutes.

9. The expression method according to claim 8, characterized in that: The resuspension solution consists of 8-12 mM magnesium chloride, 8-12 mM MES and 100-200 μM acetosyringone.

10. The expression method according to claim 4, characterized in that: The ratio of the extract in S5 to the lettuce leaves is 1-3 mL:1 g.

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