Method for producing human serum albumin by using corn seeds

Abstract

The invention discloses a method for producing human serum albumin by using corn seeds, which is characterized in that a human serum albumin gene is introduced into corn through a DNA (Deoxyribose Nucleic Acid) recombination technology to obtain corn with high expression of human serum albumin and efficiently purify the human serum albumin in the corn seeds, so that the human serum albumin can be produced more efficiently at low cost, and the human serum albumin can be produced more efficiently in unit area. More human serum albumin can be produced by planting the transgenic corn. The invention discloses the expression of human serum albumin by using corn seeds through a DNA recombination technology for the first time, and the expression level of a high-expression transformant at least reaches more than 1% of the weight of the corn seeds, that is, the yield of the human serum albumin per kilogram of the corn seeds reaches more than 10 grams. The transgenic corn obtained by the invention is more efficient and low in cost when being used for producing human serum albumin, the photosynthesis efficiency of the corn is higher, the yield per unit area is obviously higher than that of rice, and the yield of the human serum albumin of the high-expression corn transformant is higher than that of the rice transformant.

Description

(I) Technical field

[0001] The invention relates to the field of biotechnology, and in particular to a method for producing human serum albumin by utilizing corn seeds. (II) Background technology

[0002] Human serum albumin (HSA) is the most abundant plasma protein in the blood. It is a monomeric multidomain macromolecule and a non-glycosylated protein. HSA has extraordinary ligand binding properties and provides storage and transportation for endogenous and exogenous complexes. HSA is also a valuable biomarker for many diseases, including cancer, rheumatoid arthritis, ischemia, postmenopausal obesity, severe acute graft-versus-host disease, and some diseases that require monitoring of blood glucose regulation. In addition, HSA is also widely used in the clinical treatment of some diseases, including hypovolemia, shock, burns, surgical blood loss, trauma, massive bleeding, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, nutritional support, resuscitation, and hypoproteinemia. Human serum albumin is an important protein for medical use and is in high demand.

[0003] Traditionally, HSA is mainly extracted and separated from human plasma, but there are certain risks, such as the plasma source is limited (blood donation is limited), and the blood itself may also have problems (may contain dangerous infectious pathogens, high risk of virus transmission). In the past few decades, a variety of expression systems have been used to exogenously express HSA, such as Escherichia coli, yeast, transgenic animals and transgenic plants. Among them, transgenic plants include the apoplast of potato tubers, tobacco leaf chloroplasts, rice cells, rice seed endosperm, etc. Plant expression of rHSA was first achieved in tobacco leaves and potato tubers, but its expression level was very low, only 0.02% of the total soluble protein (PC Sijmons, BM Dekker, B. Schrammeijer, et al, Production of correctly processed human serum albumin in transgenic plants, Biotechnology (NY) 8 (1990) 217-221.). Seed expression systems have great advantages in producing recombinant proteins, mainly in that plant seeds have similar protein processing and post-translational modifications as animals and humans, can avoid contamination by pathogens and viruses, are easy to mass produce, store and extract, and have low production costs for recombinant proteins. Seed expression systems mainly include rice endosperm, barley, corn seeds and oil bodies. Currently, transgenic rice is used to mass produce recombinant human serum albumin (Zhang Q, Yu H, Zhang F, et al. Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice [J]. Journal of Zhejiang University SCIENCE B, 2013, 14 (10): 867-874; He Y, Ning T, Xie T, et al. Large-scale production of functional human serum albumin from transgenic rice seeds [J]. Proceedings of the National Academy of Sciences, 2011, 108 (47): 19078-19083.).Chinese patent application 200510019084 discloses a method for producing rHSA using rice endosperm cells as a bioreactor, including using a promoter and signal peptide specifically expressed in rice endosperm to mediate the entry of rHSA into the endosperm system of rice endosperm cells and store it in the protein bodies of rice endosperm, thereby allowing rHSA to accumulate in rice seeds. The resulting rHSA expression level is as high as more than 0.83% of the weight of rice seeds. At the same time, the patent mentions that only 50-60% of the T0 generation of transgenic rice plants bear fruit normally.

[0004] Although the production of human serum albumin from rice is expected to significantly reduce costs compared to separating and extracting it from human plasma, the per-acre yield of rice is lower than that of crops such as corn, and rice expressing human serum albumin has problems such as reduced grain weight, reduced fruit set rate, and reduced yield. Albumin is a protein that requires a large supply, and it is still urgent to increase the yield per unit area of ​​recombinant human serum albumin and further reduce the cost of human serum albumin. Corn is a C4 crop with a relatively high photosynthesis efficiency and a significantly higher yield per unit area than rice, a C3 crop. Therefore, the production of human serum albumin using genetically modified corn may be more efficient and low-cost. However, so far, there are no reports on the production of human serum albumin using corn. (III) Summary of the invention

[0005] The purpose of the present invention is to provide a method for producing human serum albumin using corn seeds, by introducing human serum albumin genes into corn through DNA recombination technology, obtaining corn with high expression of human serum albumin and efficiently purifying human serum albumin in corn seeds, so as to produce human serum albumin more efficiently and at a lower cost. On a unit area, planting transgenic corn can produce more human serum albumin.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides a method for producing human serum albumin by using corn seeds. The method comprises introducing at least one expression frame of human serum albumin into corn to obtain corn seeds highly expressing human serum albumin.

[0008] Furthermore, the expression frame of human serum albumin is functionally connected by a cereal seed endosperm-specific expression promoter, a gene sequence encoding a signal peptide, a DNA sequence of human serum albumin, and a terminator sequence.

[0009] Furthermore, the cereal seed endosperm-specific expression promoter includes an endosperm-specific expression promoter from Gramineae plants, and the Gramineae plants include corn, rice, wheat, barley, millet, and the like.

[0010] The promoter used in the present invention is a cereal endosperm-specific promoter, such as a globulin promoter, a glutelin promoter, a prolamine promoter, a USP promoter, a napin promoter, a gbssⅠ promoter, a SBEⅠ promoter, an AGP small subunit gene promoter, a FAEⅠ promoter, etc. They are characterized by being able to initiate the specific expression of the target gene in the endosperm of cereals. We have found that the endosperm-specific promoter in rice can initiate the specific expression of the controlled protein in the endosperm of corn. Therefore, the endosperm-specific promoters of various grass plants can be used to control the expression of human albumin in the endosperm of corn.

[0011] Furthermore, the cereal seed endosperm-specific expression promoter includes one of SEQ ID NOs: 1 to 10.

[0012] Furthermore, the amino acid sequence of human serum albumin is shown in SEQ ID NO:11, and the nucleotide sequence is shown in SEQ ID NO:12.

[0013] Furthermore, the terminator includes Tzein-Hsp terminator (SEQ ID NO: 14) and cauliflower mosaic virus 35s terminator TCaMV35S (SEQ NO ID: 17).

[0014] Furthermore, the signal peptide includes GT13 signal peptide and AHAS signal peptide.

[0015] Furthermore, the nucleotide sequence of the DNA sequence of human serum albumin with a GT13 signal peptide added to the 5' end is shown in SEQ ID NO:13.

[0016] Furthermore, the method is to introduce two human serum albumin expression frames into corn, wherein one expression frame performs codon optimization on the nucleotide sequence encoding human serum albumin for corn expression and adds a GT13 signal peptide coding sequence at the 5' end, the nucleotide sequence encoding human serum albumin connected to the signal peptide is SEQ ID NO: 13, the 5' end of the DNA sequence encoding human serum albumin connected to the signal peptide is connected to the GT1 promoter (SEQ ID NO: 1), and the 3' end is connected to the Tzein-Hsp terminator (SEQ ID NO: 14); the other expression frame changes the promoter to the ZmZ27 promoter (SEQ ID NO: 2) and is constructed using the same terminator and signal peptide.

[0017] Furthermore, the method also includes introducing a screening gene expression frame into corn, preferably adding an AHAS signal peptide (SEQ ID NO: 15) to the 5' end of the screening gene G10-evo, connecting the 5' end to the CaMV35S-ubi promoter (SEQ NO ID: 16), and connecting the 3' end to the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ NO ID: 17).

[0018] Furthermore, enhancers are connected between the expression frames of human serum albumin, and the enhancers include FMV enhancer (SEQ NOID: 18), Peanut chlorotic streak caulimovirus enhancer (Peanut chlorotic streak caulimovirus enhancer) (SEQ NO ID: 19) and MMV enhancer (SEQ NO ID: 20).

[0019] The invention provides a method for extracting human serum albumin from transgenic corn kernels, which comprises the following steps: 1) separating corn endosperm from kernels by a physical method; and 2) further extracting human serum albumin from the separated corn endosperm.

[0020] Expressing a protein in plants is a well-known mature technology. Usually, a promoter that is functional in plants is functionally connected to the DNA encoding the target protein, and a terminator that is functional in plants is connected at the 3' end to form a heterologous gene expression cassette that can be expressed in plants. This expression cassette can be further connected to a screening gene expression cassette and constructed in a T-DNA. The T-DNA is introduced into the genome of the target plant by an Agrobacterium vector-mediated method. The screening gene can be one of the following: CP4-EPSPS gene, Bar gene, CdP450 gene, G10-EPSPS gene, etc.

[0021] Due to the degeneracy of the genetic code, there are multiple different nucleic acid sequences that can encode the same amino acid protein. It is within the ability of ordinary technicians in this field to generate other nucleic acid sequences encoding the same protein. Therefore, the human serum albumin gene of the present invention covers nucleic acid sequences that encode the same amino acid sequence due to the degeneracy of the genetic code.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] (1) The present invention discloses for the first time the use of DNA recombination technology to express human serum albumin in corn seeds, wherein the expression level of the high-expressing transformant is at least 1% of the weight of the corn seeds, that is, the human serum albumin yield per kilogram of corn seeds reaches more than 10 grams.

[0024] (2) The transgenic corn obtained by the present invention is more efficient and low-cost in producing human serum albumin: corn has a relatively high photosynthesis efficiency, and its yield per unit area is significantly higher than that of rice. The output of human serum albumin from the high-expression transformant reaches 1373 g / mu.

[0025] (3) The particle size of corn starch is larger than that of rice starch. Corn starch is easier to precipitate and separate, and the subsequent purification process is more advantageous. (IV) Description of the drawings

[0026] Figure 1 , diagram of the HAS vector structure.

[0027] Figure 2 , protein electrophoresis of human serum albumin corn ZmHSA, 1: ZmHSA-26; 2: ZmHSA-1; 3: ZmHSA-35; 4: ZmHSA-3; 5: Maker protein molecular mass standard; 6: positive pHSA protein. (V) Specific implementation methods

[0028] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0029] It will be appreciated by those skilled in the art that the disclosed techniques in the following examples represent methods that the inventors have found to work well in practicing the present invention, and therefore can be considered to constitute preferred embodiments for implementing the present invention. However, it will be appreciated by those skilled in the art that many changes can be made to the disclosed specific embodiments according to the present disclosure, and similar or similar results can still be obtained without departing from the spirit and scope of the present invention.

[0030] The molecular biology and biochemical methods used in the following examples of the present invention are all known techniques. Detailed descriptions are provided in documents such as Current Protocols in Molecular Biology, compiled by Ausubel and published by John Wiley and Sons, and Molecular Cloning: A Laboratory Manual, 3rd ED., compiled by J. Sambrook et al. and published by Cold Spring Harbor Laboratory Press (2001).

[0031] Example 1. Vector construction and acquisition

[0032] Reference Figure 1 Construct the transformation vector HSA, the specific steps are as follows:

[0033] (1) Human serum albumin (HSA) gene expression cassette①: The amino acid sequence of human serum albumin is SEQ ID NO: 11; for plant transformation, the nucleotide sequence encoding human serum albumin is codon-optimized for corn expression and the GT13 signal peptide coding sequence is added to the 5' end. After the optimization and connection of the signal peptide, the nucleotide sequence encoding human serum albumin for corn transformation is SEQ ID NO: 13. After the signal peptide is connected, the 5' end of the DNA sequence of human serum albumin is connected to the GT1 promoter (SEQ ID NO: 1), and the 3' end is connected to the Tzein-Hsp terminator (SEQ ID NO: 14), and the pGT1-HSA-Tzein-Hsp fragment is artificially synthesized to form a human serum albumin gene expression cassette.

[0034] (2) Human serum albumin (HSA) gene expression cassette ②: The GT13 signal peptide coding sequence was added to the 5' end of human serum albumin, and the nucleotide sequence encoding human serum albumin for corn transformation after optimizing the signal peptide connection was SEQ ID NO: 13. The 5' end of the DNA sequence of human serum albumin was connected to the ZmZ27 promoter (SEQ ID NO: 2), and the 3' end was connected to the Tzein-Hsp terminator (SEQ ID NO: 14), and the pZmZ27-HSA-Tzein-Hsp fragment was artificially synthesized to form a human serum albumin gene expression cassette.

[0035] (2) Screening gene expression cassette: AHAS signal peptide (SEQ ID NO: 15) was added to the 5' end of the screening gene G10-evo, the 5' end was connected to the CaMV35S-ubi promoter (SEQ ID NO: 16), and the 3' end was connected to the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ ID NO: 17), and the pCaMV35S-ubi-G10-evo-TCaMV35S fragment was artificially synthesized.

[0036] (3) Transformation vector: The vector pCambia1300 (NCBI sequence number AF234296) was double-digested with HindⅢ / XhoⅠ to recover a fragment of 6783 bp, and the above three gene expression cassettes were recombined by seamless cloning, wherein the human serum albumin (HSA) gene expression cassette ① and the human serum albumin (HSA) gene expression cassette ② were sequentially connected with the FMV enhancer (SEQ ID NO: 18), the peanut chlorotic streak caulimovirus enhancer (Peanut chlorotic streak caulimovirus enhancer) (SEQ ID NO: 19) and the MMV enhancer (SEQ ID NO: 20), and then transformed into the Escherichia coli TG1 strain and sequenced to obtain the transformation vector HSA.

[0037] (4) Agrobacterium transformation: The plasmid HSA is electroporated into Agrobacterium LBA4404 competent cells. After enzyme digestion and identification, the cells are preserved in glycerol stock to obtain recombinant Agrobacterium for crop transformation.

[0038] Example 2: Transformation of corn, rice and cowpea plants and acquisition of transgenic crops

[0039] 1. Corn plant transformation

[0040] Maize genetic transformation was carried out by Agrobacterium-mediated transformation, specifically according to the method and medium formulation reported by Frame et al. (Plant Physiol, 2002, 129: 13-22), using glyphosate as a screening agent, and the steps were as follows:

[0041] (1) The recombinant Agrobacterium containing the transformation vector (HSA) obtained in Example 1 was mixed in the infection medium and the bacterial solution concentration OD was adjusted to 660 It is 0.5-0.6, which means it is the infection solution containing Agrobacterium.

[0042] (2) Take corn ears 8-10 days after pollination and collect immature embryos with a size of 1.0-1.5 mm. Immerse the collected immature embryos in the infection solution of step (1), let stand at room temperature for 5 minutes, take out the embryos, absorb the liquid, place the embryos flat side down on the co-culture medium, and culture at 22°C for 3-5 days.

[0043] (3) The immature embryos cultured in step (2) were transferred to a callus induction medium containing a final concentration of 200 mg / L of Timentin antibiotic (GlaxoSmithKline, USA), and cultured in the dark at 28° C. for 10-14 days to kill Agrobacterium.

[0044] (4) All callus tissues after induction culture in step (3) were transferred to a screening medium containing a final concentration of 2 mM glyphosate and cultured in the dark at 28° C. for 2-3 weeks. After induction culture, all callus tissues were transferred to a fresh screening medium containing 2 mM glyphosate and cultured in the dark at 28° C. for 2-3 weeks.

[0045] (5) The embryonic tissue that survived in step (4) was transferred to a regeneration medium, cultured in the dark at 28° C. for 10-14 days, then transferred to a fresh regeneration medium, and cultured in the light at 26° C. for 10-14 days.

[0046] (6) The fully developed plants in step (5) were selected and placed on a rooting medium, cultured at 26°C under light until the roots were fully developed, and the rooted regenerated seedlings were transplanted to a greenhouse for growth and cultivation. Transgenic corn (ZmHSA) expressing human serum albumin was obtained, and all T0 generations of the corn were normally fruited and harvested for seeds.

[0047] 2. Rice plant transformation

[0048] This example is based on existing literature and experimental methods (Lu Xiongbin, Gong Zuxun, 1998 Life Science 10: 125-131; Liu Fan et al., 2003 Molecular Plant Breeding 1: 108-115), and mature and plump rice seeds are selected for shelling to induce callus tissue as transformation material. The callus tissue to be transformed is placed in the infection solution containing Agrobacterium obtained by the method in step 1, and cultured at 28°C and 220rpm for 30 minutes to allow Agrobacterium to bind to the surface of the callus tissue. The callus tissue is then transferred to the NBDC co-culture medium and cultured in the dark at 28°C for 3 days. The co-cultured rice callus is picked into a sterilized triangular flask, washed with water 8-10 times until the water is clear, and excess Agrobacterium on the surface of the callus is removed. Add 100 ml of sterile water containing 200 mg / L Timentin (Timentin, ticarcillin sodium clavulanate potassium), shake at 28°C and 120 rpm for 1-2 hours, transfer to a screening medium containing 200 mg / L glyphosate antibiotic, and culture in the dark at 28°C for two months (subculture once in the middle). Calli with good growth activity are transferred to a pre-differentiation medium and cultured in the dark at 28°C for about 20 days, and then the pre-differentiated callus is transferred to a differentiation medium and differentiated for 14 hours of light for germination. After 2-3 weeks, the resistant regenerated plants are transferred to a rooting medium (200 mg / L glyphosate) for rooting, and finally the regenerated plants are washed off the agar and transplanted in a greenhouse. Transgenic rice expressing human serum albumin (OsHSA) was obtained, of which only 47% of the T0 transformants were harvested as seeds.

[0049] 3. Cowpea plant transformation

[0050] The method for obtaining transgenic cowpea is based on the existing technology, using Agrobacterium tumefaciens-mediated transformation method (Chinese patent CN113106118B).

[0051] Scrape the Agrobacterium of Example 1 into B5 liquid culture medium, suspend and mix, use B5 liquid culture medium as blank control, detect OD 600nm using a spectrophotometer (Eppendorf BioPhotometer D30), adjust the concentration of Agrobacterium with B5 liquid culture medium, if the concentration is low, scrape Agrobacterium again until the measured OD600nm value is 0.6, then add 6-BA (6-benzylaminoadenine) at a final concentration of 2 mg / L, GA3 (gibberellic acid) at a final concentration of 1 mg / L, and AS (acetosyringone) at a final concentration of 5 mg / L to obtain Agrobacterium infection solution;

[0052] Sterilized non-transgenic cowpea seeds were placed on MS solid culture medium, germinated and cultured in the dark at 26°C for 2 days, the husks were removed, and the seeds were divided into two parts along the embryonic axis as two explants. 100 explants were immersed in 100 ml of Agrobacterium infection solution and infected at 26°C for 45 minutes;

[0053] The explants infected in the above step were placed on co-culture medium and cultured for 4 days under constant temperature conditions of 27°C, 5000Lx, and 16h / 8h light / darkness;

[0054] The explants co-cultured in the above step were transferred to a bud induction screening medium, with the hypocotyl end facing downward, and cultured for 28 days under a constant temperature condition of 27°C, 5000Lx, and 16h / 8h light / darkness to obtain explants with new buds;

[0055] The explants with new buds obtained in the above step are transferred to a bud elongation screening medium, and cultured for 28 days under constant temperature conditions of 27°C, 5000Lx, and 16 / 8h light / darkness to elongate positive buds and obtain explants with positive buds;

[0056] The explants with positive buds in the above step were transferred to rooting medium and cultured under constant temperature conditions of 27°C, 5000Lx, and 16 / 8h light / darkness until roots grew out. After culturing for another 5 days, the seedlings were hardened and transplanted to the greenhouse.

[0057] Transgenic cowpea expressing human serum albumin (VuHSA) was obtained, of which 85% of TO transformants were harvested as seeds.

[0058] It can be seen that when the exogenous gene human serum albumin was transferred into corn, rice and cowpea, the fruit setting rate of rice and cowpea was affected to varying degrees, among which rice was most affected, while corn was not affected.

[0059] Example 3: Screening of transgenic plants that highly express human serum albumin

[0060] Take the T1 generation seeds of transgenic corn (ZmHSA), transgenic rice (OsHSA) and transgenic cowpea (VuHSA) in Example 2, weigh about 200 mg of seed powder for each transgenic plant, add 2000 μL PBS buffer, shake and mix for 3 minutes, let stand for 2 hours, centrifuge at 4°C 12000rpm for 10 minutes, and take the supernatant as the protein sample. The supernatant protein sample was detected for HSA content using human serum albumin enzyme-linked immunosorbent ELISA kit (Shanghai Shenggong, D711424).

[0061] The 7 transformants with the best expression were selected, and the specific HSA quantitative results are shown in Table 1 below.

[0062] Table 1. HSA quantitative results

[0063]

[0064] The results showed that four transformants of ZmHSA reached above 1 mg / g, among which the highest expression level of ZmHSA-26 reached 1.86 mg / g, while the highest rice transformant OsHSA-7 expressed 1.06 mg / g. All transformants of cowpea VuHSA were below 0.8 mg / g, and cowpea expressed human serum albumin the least ideally.

[0065] Example 4: Protein electrophoresis detection of human serum albumin and corn ZmHSA

[0066] SDS-PAGE detection of human serum albumin in transgenic corn seeds. The detection method is as follows: Take the seeds of human serum albumin corn obtained by the method of Example 2, grind each seed thoroughly, weigh 10 mg each, add 1000 μL PBS buffer, oscillate and mix for 3 minutes, centrifuge at 4°C 12000rpm for 10 minutes, and take the supernatant, which is the protein sample. Take 40 μL of protein sample, add 10 μL 5× protein loading buffer and mix, boil in boiling water for 10 minutes, centrifuge at 12000rpm for 5 minutes, and take the supernatant for electrophoresis. Use GenScript SurePAGE precast gel (4-20%), load the sample, and perform electrophoresis at 200V for 30 minutes. After the electrophoresis, use GenScript eBlotTM L1 fast wet transfer instrument for staining.

[0067] The results are as follows Figure 2 As shown, the transformants all contain human serum albumin HSA, 1: ZmHSA-26; 2: ZmHSA-1; 3: ZmHSA-35; 4: ZmHSA-3; 5: Maker protein molecular mass standard; 6: positive pHSA protein.

[0068] Example 5. Yield trait analysis and per-acre yield determination of human serum albumin corn ZmHSA-3, ZmHSA-26 and ZmHSA-35 and human serum albumin rice OsHSA-7

[0069] Human serum albumin maize ZmHSA-3, ZmHSA-26 and ZmHSA-35 and human serum albumin rice OsHSA-7 were selected for breeding and their yield traits were analyzed and the yield per mu was measured.

[0070] 1. Yield traits and yield per mu analysis of human serum albumin corn ZmHSA-3, ZmHSA-26 and ZmHSA-35

[0071] (1) The yield trait statistics were based on mature corn harvested from the field, and the weight of a single ear and the weight of 100 kernels were recorded.

[0072] ① Single ear weight: Randomly select 20 replicates of mature, dried single ears of corn and weigh them;

[0073] ② Hundred-grain weight: Randomly select mature, dried corn for threshing, randomly pick out 100 seeds, repeat 20 times, and weigh them. The obtained data are statistically analyzed using Excel tables.

[0074] 20 mature transgenic corn ZmHSA-3, ZmHSA-26 and ZmHSA-35 and non-transgenic corn were randomly selected in the field, and the weight of single ear was measured. The seeds were dried and the weight of 100 seeds was randomly weighed, with 20 replicates. The results are shown in Table 2 below.

[0075] Table 2. Agronomic traits of corn

[0076]

[0077] The results showed that there was no significant difference in single ear weight and 100-grain weight between genetically modified corn and non-genetically modified corn.

[0078] (2) Human serum albumin corn ZmHSA-3, ZmHSA-26 and ZmHSA-35 per mu yield

[0079] The planting area is 4.8m long and 4.2m wide, which is equivalent to a net area of ​​20m 2 Repeat for 3 plots. The planting method is open field sowing, with a row spacing of 0.4m×0.77m, 5 rows per plot, 13 ponds per row, a total of 65 ponds, equivalent to 667m 2 Planting density: 2166 ponds; keep two plants in each pond, 130 plants in each plot, 667m 2 4332 basic seedlings. Conventional fertilization, NPK recommended formula (nutrient content N: 20, P: 6, K: 5kg / 667m 2 ).

[0080] After harvesting, all mature ears obtained from the plot were dried and threshed, and their weights were measured. The average yield of the plot and the actual grain yield of the plot were investigated. The results are shown in Table 3 below.

[0081] Table 3. Average yield and per-acre yield of corn plots

[0082]

[0083] The results showed that the highest per-acre yield of human serum albumin corn ZmHSA-3, ZmHSA-26 and ZmHSA-35 was approximately 747 kg.

[0084] 2. Yield trait analysis and per-acre yield analysis of human serum albumin rice OsHSA-7

[0085] (1) The yield trait statistics were based on mature rice harvested from the field, and the number of grains per ear and thousand-grain weight were recorded.

[0086] ① Number of grains per panicle: Randomly select 20 replicates of mature rice panicles and count the number of grains that are normally filled;

[0087] ② Thousand-grain weight: Randomly select mature and dried rice for threshing, randomly pick out 1,000 seeds, repeat 20 times, and weigh them. The obtained data are statistically analyzed using Excel tables.

[0088] 20 mature transgenic rice OsHSA-7 and non-transgenic rice were randomly selected in the field, the number of grains per panicle was recorded, the seeds were dried, and the weight of 1000 seeds was randomly weighed, with 20 replicates. The results are shown in Table 4 below.

[0089] Table 4. Agronomic traits of rice

[0090] Non-GMO Rice Human serum albumin rice OsHSA-7 Number of grains per ear 180.6 143.7 Thousand-grain weight (g) 24.712 20.462 Human serum albumin expression (mg / g) 0 1.06

[0091] The results showed that compared with non-genetically modified rice, the number of grains per panicle and the thousand-grain weight of genetically modified rice were reduced to varying degrees, and the yield tended to decrease.

[0092] (2) Human serum albumin rice OsHSA-7 per mu yield

[0093] The planting area is 4.8m long and 4.2m wide, which is equivalent to a net area of ​​20m 2 , repeat 3 plots. After soaking, the seeds are sown in the seedbed and transplanted to the field one month later. The planting row spacing is 0.18m×0.25m, 19 rows are planted in each plot, 23 ponds in each row, a total of 437 ponds, equivalent to 667m 2 Planting density: 14,574 ponds; three plants per pond, 1,311 plants per plot, basic seedlings maintained at about 43,700 plants / 667m 2 Conventional fertilization, NPK recommended formula (nutrient content N: 13, P: 6, K: 6kg / 667m 2 ).

[0094] After harvesting, all mature ears obtained from the plot were dried and threshed, and their weights were measured. The average yield of the plot and the actual grain yield of the plot were investigated. The results are shown in Table 5 below.

[0095] Table 5. Average yield and yield per mu of rice plots

[0096] <![CDATA[Average plot yield (kg / 20m 2 )]]> Equivalent to 667m2 output (kg) OsHSA-7 12.76 425.43

[0097] The results showed that the yield of human serum albumin rice OsHSA-7 was approximately 425 kg per mu.

[0098] In summary, compared with non-transgenic corn, human serum albumin corn ZmHSA-3, ZmHSA-26 and ZmHSA-35 have no significant reduction in single ear weight and 100-grain weight, and the highest yield per mu is about 747kg. The number of grains per ear and 1,000-grain weight of transgenic rice OsHSA-7 have different degrees of reduction, and the yield per mu is about 425kg. The output of human serum albumin per mu of human serum albumin corn ZmHSA-26 is expected to reach 1373g / mu, which is nearly 3 times that of rice. For this reason, we can believe that the transgenic corn obtained by the present invention produces human serum albumin more efficiently and at a lower cost.

Claims

1. A method for producing human serum albumin using corn seeds, characterized in that: The method comprises introducing at least one human serum albumin expression frame into corn to obtain corn seeds highly expressing human serum albumin.

2. The method according to claim 1, characterized in that The human serum albumin expression frame is functionally connected by a cereal seed endosperm-specific expression promoter, a gene sequence encoding a signal peptide, a DNA sequence of human serum albumin, and a terminator sequence.

3. The method according to claim 1, characterized in that The cereal seed endosperm-specific expression promoter includes an endosperm-specific expression promoter from Gramineae plants, and the Gramineae plants include corn, rice, wheat, barley, and millet.

4. The method according to claim 1, characterized in that The cereal seed endosperm-specific expression promoter includes one of SEQ ID NOs: 1-10.

5. The method according to claim 2, characterized in that The DNA sequence of human serum albumin is shown in SEQ ID NO:

12.

6. A corn cell highly expressing human serum albumin produced by the method of claim 1.

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