CN106701816A - Method for producing recombinant corn protein from kelp female gametophyte - Google Patents

Abstract

The invention relates to a method for producing recombinant zein by using kelp female gametophytes. The steps include: transformation of kelp female gametophyte cells; expression of recombinant zein z108β in kelp female gametophytes; extraction and purification of recombinant zein z108β. In the present invention, for the first time, recombinant zein with a purity of 99.9% is obtained through mass culture of Laminaria gametophytic cells, and the expression of recombinant zein z108β in Laminaria gametophytic cells makes Laminaria female gametophytic cells a bioreactor capable of producing biopesticides. The present invention The recombinant protein of terrestrial plants is expressed in aquatic plants, and the divided female gametophytic cells under large-scale culture conditions have the genetic stability of the target gene.

Description

A method for producing recombinant corn protein by using kelp female gametophyte

technical field

The present invention relates to a method for producing recombinant zein by using kelp female gametophytes, in particular to expressing zein gene z108β in kelp female gametophytes through gene transformation, and obtaining recombinant zein with application value through protein purification and extraction z108 beta.

Background technique

Recombinant zein is a type II ribosome inactivating protein (RIP), which is a defense protein that inhibits the activity of heterologous organisms in plants. Its main function is that the hydrolyzed protein RIP has two peptide chains——α Peptide and β-peptide, wherein α-peptide has N-glycosidase activity, and β-peptide has clusterin-like Lectin activity. The α-peptide can specifically cut the adenosine of ribosomal 28RrRNA 4324, inhibit the combination of elongation factor EF-2 and ribosome, thereby inhibiting the protein synthesis of exogenous organisms. After recombination of the z108 gene obtained from corn germ, the recombinant protein expressed in Escherichia coli has the effect of inhibiting spore germination against various plant pathogens such as Aspergillus flavus (A. flavus), and is a defense protein with antibacterial characteristics; and Its β-peptide chain can independently combine with glycogen or galactose on the cell surface to inhibit the feeding of beet armyworm (S. exigua) and other pests, showing an effect of inhibiting the growth of Lepidoptera insects.

In the past research, the inventors have transferred the ribosome inactivating protein gene z108 of corn into Escherichia coli BL21, extracted, purified and obtained the recombinant antibacterial protein under laboratory conditions. Obtained an invention patent in 2010 (a gene of corn antimicrobial protein and the application of its encoded protein ZL200810014139.6). Kelp (L.japonica) belongs to the Phytophyta and is a perennial macroalgae. The area of kelp cultivation in Yantai City and its surrounding coastal areas is more than 150,000 mu, with an annual output of more than 1.5 million tons of fresh kelp, accounting for 80% of the country's total. Kelp contains a variety of medicinal active ingredients, such as fucoidan sulfate, an anticancer drug that has a direct effect on macrophages and T cells. Laminaria female gametophytic cells cultured in air-filled suspension grow extremely fast, and the biomass can be doubled every week, and the biomass can be increased by 20 times in a month. The recombinant zein z108β gene was expressed in the female gametophyte of Laminaria and carried out large-scale cell culture, and the expression of zein anti-insect function was used as a cheap bioreactor to synthesize biopesticides.

Contents of the invention

The invention expresses the recombinant zein gene z108β in the female gamete of Laminaria kelp, and carries out large-scale cell culture, which helps to use the kelp as a marine bioreactor to produce the recombinant zein.

The recombinant zein z108β is a small peptide chain with a length of 375 bp and a total of 124 amino acids. The nucleotide sequence of the recombinant zein z108β is SEQ ID No.1, and the amino acid sequence is SEQ ID No.2. Recombinant zein z108β is a transformation vector pBA002-z108β constructed in the recombinant plasmid pBA002 vector, and its transformation function is to integrate the z108β gene into the genome of kelp, and let the kelp seedlings express such a gene in the growth of seawater , producing such a protein.

Construction of transformation vector pBA002-z108β: Carrier plasmid pBA002 was digested with XbaI/SacI double enzymes, ligated with the insert sequence z108β gene in pUCm-T-1192 (after XbaI/SacI double enzyme digestion), and the ligated product was transformed into Escherichia coli DH5a , the recombinant plasmid vector pBA002-z108β was obtained and stored at -70°C.

The method of gene transformation in the present invention uses the gene gun bombardment technology, and the cell membrane of the kelp gametophytic cell is hit by gold particles, so that the recombinant plasmid carrier carrying the recombinant zein gene is integrated into the genome of the kelp chromosome, and a genetically stable Express. The invention uses a gene gun to bombard Laminaria gametophytes with gold particles and then adds plasmids for co-cultivation to obtain more female gametophyte transformed cells. The transformation rate of Laminaria gametophyte cells can also be greatly improved through genetic testing.

Technical scheme of the present invention is as follows:

A method for producing recombinant zein by using Laminaria female gametophyte, the specific steps are as follows:

(1) Transformation of Laminaria female gametophyte cells

①Grind the cell mass of Laminaria gametophyte cell line No. 13 in growth to form a Laminaria gametophyte cell mass containing 5-7 cells, with a cell density of 5×10 ⁵ -2×10 ⁶ cells/mL (with a hemocytometer counting plate counts). When transforming, use a 60mm sterile petri dish to evenly spread the female and female gametophyte cells in the middle of the filter paper to form a circular bombardment circle with a diameter of about 2cm.

Preferably, the growing cell mass of Laminaria gametophyte cell line No. 13 is ground to form a Laminaria gametophyte cell mass containing 5-7 cells, with a cell density of 1×10 ⁶ cells/mL.

② DNA of plasmid pBA002-z108β was extracted with a kit for co-cultivation and gene transformation after bombardment.

③Bombard the kelp female gametophytic cell cluster: turn on the power of the PDS-1000\He gene gun, open the valve of the helium cylinder, and rotate the helium adjustment lever to make the air pressure about 200psi higher than the selected splitting membrane pressure, that is, 1500psi; When the distance between the net and the material is 3cm, the bombardment pressure is 1100psi;

④ Add pBA002-z108β plasmid DNA to the bombarded Laminaria gametophyte cell mass, culture at 11-13°C, light at 800 Lux, and co-cultivate for 48 hours;

Preferably, pBA002-z108β plasmid DNA is added to the bombarded Laminaria gametophyte cell mass, the culture temperature is 12° C., and the light is 800 Lux. Co-cultivate for 48 hours.

⑤ Select the transformed female gametophytic cells at the concentration of glufosinate 40mg/L.

(2) Expression of recombinant zein z108β in Laminaria female gametophytic cells

The transformed female gametophytic cells were cultured for 28-35 days, and clones were picked and cultured separately. Laminaria gametocytes were evenly spread on a petri dish with filter paper, brown transformants were picked out with a capillary under a dissecting microscope, and culture medium was added for mass cultivation. The nutrient solution for cultivating Laminaria female gametophytic cells is: NaNO ₃ with a concentration of 10.0g/L and KH ₂ PO ₄ with a concentration of 1.0g/L are added to sterilized seawater, wherein the amount of NaNO ₃ solution added is 1.0-10.0mL; the amount of KH ₂ PO ₄ solution added is 0.4-1.0mL per liter of seawater. And change the nutrient solution once a day, the culture temperature is 11-13 ℃, every day is light culture 14 hours, dark culture 10 hours, the light is 800Lux; The transformant is placed in the glufosinate that the concentration is 40mg/L, and selection is still The brown transformants were cultured for 5-10 days.

Preferably, the transformed female gametophytic cells are cultured for 30 days; the amount of NaNO solution added is 5.0 mL per liter of seawater, and the amount of KH ₂ PO ₄ solution added is 0.7 mL per liter of sea water _; the culture conditions of the female gametophytic cells of Laminaria are as follows: : The culture temperature is 12°C, 14 hours of light culture and 10 hours of dark culture every day, and the light is 800 Lux; the transformants are placed in glufosinate with a concentration of 40 mg/L, and the transformants that are still brown are selected and cultured for 8 days.

Large-scale culture of the screened Laminaria gametophyte cells is carried out, PCR detection is carried out continuously, the transgenic method of Laminaria gametophytes is optimized, the transformation rate and the survival rate of transformants are improved, and a complete Laminaria gametophyte cell asexual reproduction system is established.

After two weeks of culture, the female gametophyte cells of Laminaria remained brown under the concentration of glufosinate 40mg/L, and were not killed by glufosinate, and some gametophytes of Laminaria turned green and faded, which were untransformed and did not contain recombinant zein z108β of female gametophytic cells.

(3) Extraction and purification of recombinant zein z108β

①Cracking and separation of transformed female gametophyte cells of Laminaria

Take 0.1g sample and grind it in liquid nitrogen, add 1× sample buffer (sample buffer) 200μL (1.0Mtris-HCL (pH 6.8) 1mL, 10% SDS 6.0mL, β-mercaptoethanol 0.2mL, ddH ₂ O 2.8 mL), boil for 5 minutes after mixing, and centrifuge at 12000rpm for 10 minutes;

②Extract the supernatant and identify it by SDS-PAGE electrophoresis

Take 20 μL supernatant for spotting test; SDS-PAGE electrophoresis uses 10% separation gel (deionized water 9.9mL, 30% acrylamide 8.3mL, Tris-HCL (pH 8.8) 6.3mL, 10% SDS 0.25 mL, 10% ammonium persulfate (AP) 0.25mL, TEMED 0.01mL) and stacking gel (deionized water 5.5mL, 30% acrylamide 3.5mL, Tris-HCL (pH 8.8) 1.5mL, 10% SDS 0.08mL , 10% AP 0.08mL, TEMED 0.008mL).

Protein samples were loaded, 20 μL per well, electrophoresed at 80V for 30 minutes, and then electrophoresed at 120V for 60-80 minutes.

③ Western blot verification and purification of recombinant zein z108β

The verification of Laminaria female gametophytic cell transformants was performed using Bio-Rad's standard wet transfer device for PVDF membrane transfer with a current of 90mA for 100min. Immediately after transferring the membrane, place the protein membrane in the pre-prepared deionized water and rinse for 1 minute to wash away the transfer solution on the membrane. Put it into the blocking solution and block it on a shaker for 2 hours. After washing, soak the membrane in the diluted primary antibody (the ratio of Z108 antiserum of New Zealand white rabbit to 5% skimmed milk powder is 1:1000), incubate at 37°C for 1-2h; wash the membrane with 10mL TBST three times; then wash the membrane Soak in the diluted secondary antibody (the ratio of HRP-labeled goat anti-rabbit IgG to 5% skimmed milk powder is 1:2000), incubate at 37°C for 1 hour; wash the membrane three times with 10 mL TBST and once with 10 mL TBS; enhanced HRP- DAB substrate chromogenic kit color development and photography.

Preferably, after washing, the membrane is immersed in the diluted primary antibody and incubated at 37°C for 1.5h.

The obtained recombinant zein was dialyzed to desalt with an 8kD regenerated cotton linter cellulose dialysis membrane (product number F128056-0001, Shanghai Sangong), and centrifuged at 12000rpm for 20 minutes to precipitate.

④Concentration and preservation of recombinant protein

The centrifuged purified protein was collected centrally, and the centrifuged purified recombinant zein z108β was stored at -20°C.

According to the electrophoresis protein standard test, the content of recombinant zein z108β reached 0.2 μg/μL; the purity reached 99.9%.

In the present invention, unless otherwise specified, "%" means mass concentration.

Compared with the prior art, the present invention has the following advantages:

1. For the first time, the present invention obtains recombinant zein with a purity of 99.9% through mass culture of Laminaria gametophytic cells. This invention enables the expression of the protein encoded by the target gene to meet the requirements of practical applications under artificial conditions;

2. Recombinant zein z108β has the effect of inhibiting the development of Lepidoptera insects. The expression of this gene in Laminaria gametophyte cells makes Laminaria gametophyte cells a bioreactor capable of producing biopesticides.

3. The conditions for mass culture of kelp gametocytes must meet:

(1) NaNO ₃ with a final concentration of 10mg/L and KH ₂ PO ₄ with a final concentration of 1.0mg/L were added to sterilized seawater as nutrients for growth, and the nutrient solution was changed once a day, and the culture temperature was 11-12°C. The light is 800Lux. The cultures were cultivated for 14 hours in the light and 10 hours in the dark every day.

(2) Continue to use 40mg/L glufosinate as the screening pressure for large-scale culture of female gametophytic cells to maintain recombinant zein expression. Laminaria gametophytic cells have the ability to express the target protein.

4. Express the recombinant protein of terrestrial plants in aquatic plants, and the divided female gametophytic cells under large-scale culture conditions have the genetic stability of the target gene.

Description of drawings

Fig. 1 is the enzyme digestion electrophoresis verification diagram of the recombinant zein gene z108β; the fragment length after double digestion with XbaI/SacI is 389bp.

Figure 2 is a picture of Laminaria female gametophyte cells expressing recombinant zein in large-scale culture; Figure A is brown Laminaria gametophyte cells that survived under the microscope after screening; Figure B is the morphology of Laminaria female gametophytes expressing recombinant zein; Figure C is Laminaria female gametophyte cells that do not express turn green and gradually turn white and apoptotic.

Fig. 3 is a gel electrophoresis image of the PCR amplification product of recombinant zein z108β; the size of the DNA fragment is 375bp.

Fig. 4 is an SDS-PAGE electrophoresis map showing recombinant zein isolated from Laminaria gametophytic cells; the protein size is 20.5 kDa.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, the examples are merely exemplary and do not limit the scope of the present invention in any way. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

The seawater used in the specific embodiment of the present invention is taken from the coast of Yantai City, Shandong Province, and seawater suitable for the growth of kelp is suitable for the present invention.

Embodiment 1 A kind of method utilizing Laminaria female gametophyte to produce recombinant zein, the specific steps are as follows:

(1) Transformation of Laminaria female gametophyte cells

① The cell mass of the growing Laminaria gametophyte cell line No. 13 (from the Laboratory of Algae Biology, Institute of Oceanology, Chinese Academy of Sciences) was ground to form a Laminaria gametophyte cell mass containing 5-7 cells, and the cell density was 1 About ×10 ⁶ cells/mL (counted with a hemocytometer). When transforming, use a 60mm sterile petri dish to evenly spread the female and female gametophyte cells in the middle of the filter paper to form a circular bombardment circle with a diameter of about 2cm.

② DNA of plasmid pBA002-z108β was extracted with a kit for co-cultivation and gene transformation after bombardment.

Construction of transformation vector pBA002-z108β: Carrier plasmid pBA002 was digested with XbaI/SacI double enzymes, ligated with the insert sequence z108β gene in pUCm-T-1192 (after XbaI/SacI double enzyme digestion), and the ligated product was transformed into Escherichia coli DH5a , the recombinant plasmid vector pBA002-z108β was obtained and stored at -70°C. The electrophoresis verification diagram of the restriction enzyme digestion of recombinant zein z108β with XbaI/SacI is shown in Figure 1.

③Bombard the kelp female gametophytic cell cluster: turn on the power of the PDS-1000\He gene gun, open the valve of the helium cylinder, and rotate the helium adjustment lever to make the air pressure about 200psi higher than the selected splitting membrane pressure, that is, 1500psi; When the distance between the net and the material is 3cm, the bombardment pressure is 1100psi;

④ Add pBA002-z108β plasmid DNA to the bombarded Laminaria gametophyte cell mass, culture at 12°C and light at 800 Lux. Co-cultivate for 48 hours.

⑤ Select the transformed female gametophytic cells at the concentration of glufosinate 40mg/L.

(2) Expression of recombinant zein z108β in Laminaria female gametophytic cells

The transformed female gametophytic cells were cultured for 30 days, and clones were picked and cultured separately. Laminaria gametocytes were evenly spread on a petri dish with filter paper, brown transformants were picked out with a capillary under a dissecting microscope, and culture medium was added for mass cultivation. The nutrient solution for cultivating Laminaria female gametophytic cells is: NaNO ₃ with a concentration of 10.0g/L and KH ₂ PO ₄ with a concentration of 1.0g/L are added to sterilized seawater, wherein the amount of NaNO ₃ solution added is 5.0mL; the amount of KH ₂ PO ₄ solution added is 0.7mL per liter of seawater. And the nutrient solution was replaced once a day, and the culture temperature was 12°C. The light is 800Lux. The cultures were cultivated for 14 hours in the light and 10 hours in the dark every day. The transformants were placed in glufosinate at a concentration of 40 mg/L, and the transformants that were still brown were selected and cultured for 8 days.

Large-scale culture of the screened Laminaria gametophyte cells is carried out, PCR detection is carried out continuously, the transgenic method of Laminaria gametophytes is optimized, the transformation rate and the survival rate of transformants are improved, and a complete Laminaria gametophyte cell asexual reproduction system is established. The transformation methods were adding plasmids after bombardment and adding plasmids during bombardment, and the average cell transformation rates of the two reached 39.87% and 23.89%, respectively, as shown in Table 1:

Table 1 Test of gene gun transformation efficiency

After two weeks of culture, the female gametophyte cells of Laminaria remained brown under the concentration of glufosinate 40mg/L, and were not killed by glufosinate, and some gametophytes of Laminaria turned green and faded, which were untransformed and did not contain recombinant zein z108β of female gametophytic cells.

Figure 2 shows the large-scale cultured Laminaria female gametophytic cells expressing recombinant zein; Figure A is the brown Laminaria female gametophyte cells that survived under the microscope after screening; Figure B is the morphology of Laminaria female gametophytic cells expressing recombinant zein; C: Laminaria gametophytic cells without expression turned green and gradually turned white and apoptotic.

(3) Extraction and purification of recombinant zein z108β

①Cracking and isolation of transformants of Laminaria female gametophytes

Take 0.1g sample and grind it in liquid nitrogen, add 1× sample buffer (sample buffer) 200μL (1.0Mtris-HCL (pH 6.8) 1mL, 10% SDS 6.0mL, β-mercaptoethanol 0.2mL, ddH ₂ O 2.8 mL), boil for 5 minutes after mixing, and centrifuge at 12000rpm for 10 minutes;

②Extract the supernatant and identify it by SDS-PAGE electrophoresis

Take 20 μL supernatant for spotting test; SDS-PAGE electrophoresis uses 10% separation gel (deionized water 9.9mL, 30% acrylamide 8.3mL, Tris-HCL (pH 8.8) 6.3mL, 10% SDS 0.25 mL, 10% ammonium persulfate (AP) 0.25mL, TEMED 0.01mL) and stacking gel (deionized water 5.5mL, 30% acrylamide 3.5mL, Tris-HCL (pH 8.8) 1.5mL, 10% SDS 0.08mL , 10% AP 0.08mL, TEMED 0.008mL).

Load the protein sample, 20uL per well, electrophoresis at 80V for 30 minutes, and then electrophoresis at 120V for 60-80 minutes. The result is shown in Figure 3. The protein size is 20.5KD, which is similar to a ribosome inactivating protein produced in corn endosperm The β-peptide chains of z108 were of the same size.

③ Western blot verification and purification of recombinant zein z108β

The verification of Laminaria female gametophytic cell transformants was performed using Bio-Rad's standard wet transfer device for PVDF transfer with a current of 90mA and transfer for 1h40min. Immediately after transferring the membrane, place the protein membrane in the pre-prepared deionized water and rinse for 1 minute to wash away the transfer solution on the membrane. Put it into the blocking solution and block it on a shaker for 2 hours. After washing, soak the membrane in the diluted primary antibody (the ratio of Z108 antiserum of New Zealand white rabbit to 5% skimmed milk powder is 1:1000), incubate at 37°C for 1.5h; wash the membrane with 10mL TBST for 3 times; then soak the membrane Incubate in the diluted secondary antibody (the ratio of HRP-labeled goat anti-rabbit IgG to 5% skimmed milk powder is 1:2000) at 37°C for 1 hour; wash the membrane three times with 10 mL TBST and once with 10 mL TBS; enhanced HRP-DAB Substrate Chromogenic Kit for color development and photographing.

The obtained recombinant zein was dialyzed to desalt with an 8kD regenerated cotton linter cellulose dialysis membrane (product number F128056-0001, Shanghai Sangong), and centrifuged at 12000rpm for 20 minutes to precipitate.

④Concentration and preservation of recombinant protein

The centrifuged purified protein was collected centrally, and the centrifuged purified recombinant zein z108β was stored at -20°C.

According to the electrophoresis protein standard test, the content of recombinant zein z108β reached 0.2 μg/μL; the purity reached 99.9%.

Test Example 1 Extraction and PCR Verification of Laminaria Female Gametophyte Transformant

(1) DNA extraction: Take Laminaria female gametophyte transformed cells under the condition of 40mg/L herbicide and put them into liquid nitrogen for grinding, add 200 μL DNA extraction solution (1.0MTris-HCL (PH 6.8) 1mL, CTAB 200mM 2mL, ddH ₂ (7.mL, appropriate amount of mercaptoethanol), incubate at 65°C for 20 minutes, and centrifuge at 12000rpm for 10min;

Take a centrifuge tube and add twice the volume of pre-cooled isopropanol and 1/10 volume of NaAC, add the supernatant after centrifugation, store in a -70°C refrigerator for 20 minutes, and fully precipitate. Centrifuge at 12000 rpm for 10 min, discard the supernatant; add pre-cooled 75% ethanol to the precipitate, repeat washing twice and then centrifuge. After drying, dissolve the DNA with TE, add 1 μL RNase and incubate at 37°C for 1 hour, then perform 1% agarose gel electrophoresis.

(2) PCR reaction system: 40 μL ddH ₂ O, 5 μL 10×PCR Buffer, 1 μL 10 mM dNTP, 1 μL template, 1 μL Pfu, 1 μL each of upstream and downstream primers. The primer sequences are:

Jpri-1191(204)F ATGGCCGAGATAACCCTAGAGCCGAGT

Jpri-1191(204)R TCAGGCCTCGTCGTCGTCGTTGTCAGCACT

(3) PCR reaction program: pre-denaturation at 95°C for 10 minutes; then 95°C, 30S; 55°C, 50S; 72°C, 80S, for 34 cycles; finally, extension at 72°C for 10 minutes. Store at 4°C. 0.8% agarose gel electrophoresis spotting, TAC buffer, 90V, 40 minutes, the size of the amplified fragment detected under ultraviolet light was 375bp, as shown in Figure 4.

<110> Yantai Research Institute of China Agricultural University, Qingdao Agricultural University

〈120〉A method for producing recombinant zein by using Laminaria female gametophyte

<130> 16-1-1166

<160>2

<170> Patent In Version 3.5

<210>1

<211>375 bp

<212>DNA

<213> recombinant zein gene z108β

<400>1

atggccgaga taaccctaga gccgagtgat cttgacccac aggccgacac gaagagcaag 60

ctggtgaagc tggtggtcat ggtgtgcgag gggctgcggt tcaacaccgt gtcccgcacg 120

gtggacgcgg ggttcaacag ccagcacggg gtgaccttga ccgtgacgca ggggaagcag 180

gtgcagaagt gggacaggat ctccaaggcg gccttcgagt gggctgacca ccccaccgct 240

gtgatccccg acatgcagaa gcttggcatc aaggataaga acgaagcagc gaggatcgtt 300

gcgctcgtta agaatcaaac tactgccgct gccgctactg ctgccagtgc tgacaacgac 360

gacgacgagg cctga 375

<210>2

<211>124AA

<212> amino acid

<213> recombinant zein z108β

<400>2

MET Ala Glu Ile Thr Leu Glu Pro Ser Asp Leu Asp Pro Gln Ala Asp Thr Lys Ser Lys 20

Leu Val Lys Leu Val Val MET Val Cys Glu Gly Leu Arg Phe Asn Thr Val Ser Arg Thr 40

Val Asp Ala Gly Phe Asn Ser Gln His Gly Val Thr Leu Thr Val Thr Gln Gly Lys Gln 60

Val Gln Lys Trp Asp Arg Ile Ser Lys Ala Ala Phe Glu Trp Ala Asp His Pro Thr Ala 80

Val Ile Pro Asp MET Gln Lys Leu Gly Ile Lys Asp Lys Asn Glu Ala Ala Arg Ile Val 100

Ala Leu Val Lys Asn Gln Thr Thr Ala Ala Ala Ala Thr Ala Ala Ser Ala Asp Asn Asp 120

Asp Asp Glu Ala * 124

Claims (8)

1. a kind of method of utilization Female Gametophytes of Laminaria Japonica production restructuring zein, comprises the following steps that：

(1) Female Gametophytes of Laminaria Japonica transformation

The cell mass of the Female Gametophytes of Laminaria Japonica cell strain 13 in 1. growing, grinding is allowed into and contains the 5-7 sea-tangle of cell Egagametophyte cell mass, cell density is 5 × 10⁵-2×10⁶Individual/mL, with the sterile petri dish of 60mm during conversion, matches somebody with somebody female The evenly laid out filter paper middle part of daughter cell, forms the circular bombardment circle of diameter 2cm or so；

2. the DNA of plasmid pBA002-z108 β is extracted with kit, is used to the co-cultivation after bombarding and genetic transformation；

3. Female Gametophytes of Laminaria Japonica cell mass is bombarded：Bombarded when backstop and storeroom are away from 3cm, bombarding pressure is 1100psi；

4. to pBA002-z108 β DNAs are added in the kelp gametophyte cell mass after bombardment, cultivation temperature is 11-13 DEG C, Illumination is 800Lux, is co-cultured 48 hours；

5. the egagametophyte cell of conversion is screened under the concentration of careless fourth phosphine 40mg/L；

(2) expression of the restructuring zein z108 β in Female Gametophytes of Laminaria Japonica cell

Egagametophyte cell culture 28-35d after conversion, picked clones are individually cultivated, and kelp gametophyte is uniformly coated on into band Have in the culture dish of filter paper, with the transformant of capillary picking brown under anatomical lens, add nutrient solution mass propgation, culture sea Nutrient solution with egagametophyte cell is：It is the NaNO of 10.0g/L that concentration is added in sterilizing seawater₃It is 1.0g/L's with concentration KH₂PO₄, wherein, NaNO₃The addition of solution is every liter of seawater addition 1.0-10.0mL；KH₂PO₄The addition of solution is every liter Seawater adds 0.4-1mL, and changes one time of nutrition liquid daily, and cultivation temperature is 11-13 DEG C, daily for illumination cultivation 14 is small When, light culture 10 hours, illumination is 800Lux；Transformant is placed in the careless fourth phosphine that concentration is 40mg/L, it be still brown to choose Transformant culture 5-10d；

(3) extraction and purifying of restructuring zein z108 β

1. the broken and separation of Female Gametophytes of Laminaria Japonica transformant cell

Take during the kelp gametophyte transformant sample after 0.1g conversion cultures is put into liquid nitrogen and grind, add 1 × sample buffer 200 μ L (1.0M tris-HCL (pH 6.8) 1mL, 10%SDS 6.0mL, beta -mercaptoethanol 0.2mL, ddH₂O 2.8mL), after mixing Boil 5min, 12000rpm centrifugations 10min；

2. extract supernatant and utilize SDS-PAGE electroresis appraisals

20 μ L of supernatant liquid are taken, for point sample inspection；SDS-PAGE electrophoresis using 10% separation gel (deionized water 9.9mL, 30% Acrylamide 8.3mL, Tris-HCL (pH 8.8) 6.3mL, 10%SDS0.25mL, 10% ammonium persulfate (AP) 0.25mL, TEMED 0.01mL) and spacer gel (deionized water 5.5mL, 30% acrylamide 3.5mL, Tris-HCL (pH 8.8) 1.5mL, 10%SDS0.08mL, 10%AP 0.08mL, TEMED 0.008mL)；

Protein sample loading, after 30 minutes voltage 120V electrophoresis 60-80 minutes for electrophoresis under μ L, the 80V voltages of every hole 20；

3. the Western blotting checking and purifying of restructuring zein z108 β

The checking of Female Gametophytes of Laminaria Japonica cell transformation carries out PVDF transferring films using the standard wet type membrane-transferring device of Bio-Rad, electricity Stream 90mA, transferring film 100min, after transferring film is finished, are placed into protein film in preprepared deionized water immediately, rinse 1 point Clock, to wash away the transferring film liquid on film, is put into confining liquid, is closed 2 hours on shaking table；Film is dipped in the primary antibody for having diluted after cleaning (the Z108 antiserums of new zealand white rabbit and the ratio of 5% skimmed milk power are 1：1000) in, 37 DEG C of incubation 1-2h；With 10mLTBST washes film 3 times；Film is dipped in secondary antibody (goat anti-rabbit igg of HRP marks and the ratio of 5% skimmed milk power for having diluted again It is 1：2000) in, 37 DEG C of incubation 1h；Film is washed with 10mLTBST 3 times, washed once with 10mLTBS；Enhanced HRP-DAB substrates show The colour developing of color reagent box is taken pictures；

The regeneration lint cellulose dialysis membrane dialysis desalination for recombinating zein 8kD of acquisition, and 12000rpm is centrifuged 20 points Clock collects precipitation；

4. the concentration and preservation of recombinant protein

The albumen centralized collection of centrifugal purification, the restructuring zein z108 β after centrifugal purification are preserved under the conditions of -20 DEG C.

2. method according to claim 1, it is characterised in that the Female Gametophytes of Laminaria Japonica in the step (1) in growth is thin The cell mass that born of the same parents' strain 13, grinding is allowed into containing the 5-7 Female Gametophytes of Laminaria Japonica cell mass of cell, cell density for 1 × 10⁶Individual/mL.

3. method according to claim 1, it is characterised in that thin to the kelp gametophyte after bombardment in the step (1) PBA002-z108 β DNAs are added in born of the same parents group, cultivation temperature is 12 DEG C, and illumination is 800Lux, co-cultured 48 hours.

4. method according to claim 1, it is characterised in that the egagametophyte cell training in the step (2) after conversion Support 30d.

5. method according to claim 1, it is characterised in that NaNO in the step (2)₃The addition of solution is every liter Seawater adds 5.0mL, KH₂PO₄The addition of solution is every liter of seawater addition 0.7mL.

6. method according to claim 1, it is characterised in that the culture of Female Gametophytes of Laminaria Japonica cell in the step (2) Condition is：Cultivation temperature is 12 DEG C, is daily illumination cultivation 14 hours, light culture 10 hours, and illumination is 800Lux.

7. method according to claim 1, it is characterised in that transformant is placed in concentration for 40mg/ in the step (2) In the careless fourth phosphine of L, it be still the transformant culture 8d of brown to choose.

8. method according to claim 1, it is characterised in that after transferring film is finished in the step (3), immediately protein film It is placed into preprepared deionized water, rinses 1 minute, to wash away the transferring film liquid on film, is put into confining liquid, on shaking table Closing 2 hours；Film is dipped in after cleaning in the primary antibody for having diluted, 37 DEG C of incubation 1.5h.