A method for producing human serum albumin using corn seeds

Abstract

The application discloses a method for producing human serum albumin by using corn seeds, and the method comprises the following steps: introducing a human serum albumin gene into corn by a DNA recombination technology, obtaining corn with high expression of human serum albumin, and efficiently purifying human serum albumin in the corn seeds, so as to produce human serum albumin more efficiently and at a lower cost. On a unit area, more human serum albumin can be produced by planting the transgenic corn. The application discloses, for the first time, that human serum albumin is expressed by using corn seeds through the DNA recombination technology, wherein the expression level of the high-expression transformant is at least 1% of the weight of the corn seeds, that is, the yield of human serum albumin per kilogram of corn seeds is more than 10 grams. The transgenic corn obtained by using the application can produce human serum albumin more efficiently and at a lower cost: the photosynthesis efficiency of the corn is relatively high, the yield per unit area is significantly higher than that of rice, and the yield of human serum albumin of the high-expression corn transformant is higher than that of the rice transformant.

Description

(I) Technical Field

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

[0002] Human serum albumin (HSA) is the most abundant plasma protein in blood. It is a monomeric, multi-domain macromolecule and a non-glycosylated protein. HSA possesses exceptional ligand-binding properties, providing a storage site and transport mechanism for both 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 requiring glycemic regulation. Furthermore, HSA is widely used in the clinical treatment of various diseases, including hypovolemia, shock, burns, surgical blood loss, trauma, massive hemorrhage, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, acute liver failure, chronic liver disease, nutritional support, resuscitation, and hypoalbuminemia. Human serum albumin is an important medically used protein with high demand.

[0003] Traditionally, HSA is mainly extracted and isolated from human plasma. However, this process carries certain risks, such as limited plasma sources (restricted blood donation) and potential problems with the blood itself (potentially containing dangerous infectious pathogens or posing a high risk of viral transmission). Over the past few decades, various expression systems have been used for exogenous HSA expression, including Escherichia coli, yeast, transgenic animals, and transgenic plants. Transgenic plants include apoplasts of potato tubers, chloroplasts of tobacco leaves, rice cells, and rice seed endosperm. Plant expression of rHSA was first achieved in tobacco leaves and potato tubers; however, its expression level was very low, only 0.02% of total soluble protein (PCSijmons, BMDekker, B. Schrammeijer, et al., Production of correctly processed human serum albumin in transgenic plants, Biotechnology (NY) 8 (1990) 217-221.). Seed expression systems have significant advantages for producing recombinant proteins, mainly in the following aspects: plant seeds have similar protein processing and post-translational modifications as animals and humans; they can avoid contamination by pathogens and viruses; they are easy to produce on a large scale; they are easy to store and extract; and the production cost of recombinant proteins is low. Seed expression systems mainly include rice endosperm, barley, corn seeds, and oil bodies. Currently, transgenic rice is used to produce recombinant human serum albumin on a large scale (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. The method involves using promoters and signal peptides specifically expressed in rice endosperm to mediate the entry of rHSA into the inner membrane system of rice endosperm cells and its storage in the protein bodies of rice endosperm, thereby enabling rHSA to accumulate in rice seeds. The resulting rHSA expression level can reach 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 transgenic rice plants produce normal seeds.

[0004] Although producing human serum albumin from rice holds promise for significantly reducing costs compared to extraction from human plasma, rice yields per acre are lower than crops like corn, and rice expressing human serum albumin suffers from reduced grain weight, lower seed setting rate, and lower overall yield. Albumin is a protein requiring a large supply, making it urgent to increase the yield per unit area of ​​recombinant human serum albumin and further reduce its cost. Corn, a C4 crop, has high photosynthetic efficiency and a significantly higher yield per unit area than C3 rice. Therefore, producing human serum albumin using genetically modified corn could be more efficient and cost-effective. However, to date, there are no reports on producing human serum albumin from corn. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing human serum albumin using maize seeds. By introducing the human serum albumin gene into maize through DNA recombination technology, maize with high expression of human serum albumin can be obtained and human serum albumin in maize seeds can be purified efficiently. This method enables more efficient and lower-cost production of human serum albumin. More human serum albumin can be produced per unit area by planting genetically modified maize.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a method for producing human serum albumin using maize seeds. The method involves introducing at least one expression cassette of human serum albumin into maize seeds to obtain maize seeds that highly express human serum albumin.

[0008] Furthermore, the expression cassette of human serum albumin is functionally linked 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 endosperm-specific expression promoters of the cereal seeds include endosperm-specific expression promoters from grasses, such as maize, rice, wheat, barley, and millet.

[0010] The promoters used in this invention are cereal endosperm-specific promoters, such as the globulin promoter, glutelin promoter, prolamine promoter, USP promoter, napin promoter, GBSS I promoter, SBE I promoter, AGP small subunit gene promoter, and FAE I promoter. Their characteristic is that they can initiate the specific expression of the target gene in the cereal endosperm. We have found that endosperm-specific promoters in rice can initiate the specific expression of the controlled protein in maize endosperm; therefore, endosperm-specific promoters from various grasses can be used to control the expression of human serum albumin in maize endosperm.

[0011] Furthermore, the cereal seed endosperm-specific expression promoter includes one of SEQ ID NO:1 to 10.

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

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

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

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

[0016] Furthermore, the method involves introducing two expression cassettes for human serum albumin into maize. One expression cassette codon-optimized the nucleotide sequence encoding human serum albumin for maize expression and added a GT13 signal peptide coding sequence to the 5' end. The nucleotide sequence encoding human serum albumin and linking the signal peptide is SEQ ID NO: 13. The GT1 promoter (SEQ ID NO: 1) is linked to the 5' end of the DNA sequence encoding human serum albumin and linking the signal peptide, and the Tzein-Hsp terminator (SEQ ID NO: 14) is linked to the 3' end. The other expression cassette changed the promoter to the ZmZ27 promoter (SEQ ID NO: 2) and was constructed using the same terminator and signal peptide.

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

[0018] Furthermore, the expression cassettes of the human serum albumin are linked with enhancers, which include the FMV enhancer (SEQ NOID:18), the Peanut chlorotic streakcaulimovirus enhancer (SEQ NO ID:19), and the MMV enhancer (SEQ NO ID:20).

[0019] This invention provides a method for extracting human serum albumin from genetically modified corn kernels, the method comprising the following steps: 1) separating corn endosperm from the kernels by physical methods; 2) further extracting human serum albumin from the separated corn endosperm.

[0020] Expressing a protein in plants is a well-established and known technique. Typically, a functional promoter in plants is functionally linked to the DNA encoding the target protein, and a functional terminator in plants is attached to the 3' end, forming a heterologous gene expression cassette that can be expressed in plants. This expression cassette can be further linked to a selection gene expression cassette, constructing a T-DNA. The T-DNA is then introduced into the genome of the target plant using an Agrobacterium-mediated vector method. The selection 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, many different nucleic acid sequences can encode the same amino acid protein. Generating other nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art; therefore, the human serum albumin gene described in this 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) This invention discloses for the first time the expression of human serum albumin by using maize seeds through DNA recombination technology, wherein the expression level of the high-expression transformant reaches at least 1% of the weight of the maize seed, that is, the human serum albumin yield per kilogram of maize seed reaches more than 10 grams.

[0024] (2) The production of human serum albumin using the transgenic maize obtained by the present invention is more efficient and lower cost: maize has a higher photosynthetic efficiency and a significantly higher yield per unit area than rice. The output of human serum albumin with high expression of the transgenic maize reaches 1373 g / mu.

[0025] (3) Corn starch has a larger particle size than rice starch, making it easier to precipitate and separate, and giving it an advantage in subsequent purification processes. (iv) Description of the attached drawings

[0026] Figure 1 The carrier structure diagram of HAS.

[0027] Figure 2 Electrophoresis images of human serum albumin maize ZmHSA: 1: ZmHSA-26; 2: ZmHSA-1; 3: ZmHSA-35; 4: ZmHSA-3; 5: Mark protein molecular weight standard; 6: Positive pHSA protein. (V) Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0029] Those skilled in the art will understand that the techniques disclosed in the following embodiments represent methods that the inventors have found to work well in practicing the invention, and therefore can be considered as constituting preferred embodiments for carrying out the invention. However, those skilled in the art will understand that many changes can be made to the specific embodiments disclosed according to the present invention, and similar or analogous results can still be obtained without departing from the spirit and scope of the invention.

[0030] The molecular biology and biochemical methods used in the following embodiments of the present invention are all known techniques. They are described in detail in publications such as *Current Protocols in Molecular Biology* by Ausubel, published by John Wiley and Sons, and *Molecular Cloning: A Laboratory Manual*, 3rd ed., published by Cold Spring Harbor Laboratory Press (2001), by J. Sambrook et al.

[0031] Example 1: Vector Construction and Acquisition

[0032] Reference Figure 1 The specific steps for constructing the HSA transformation vector are as follows:

[0033] (1) Human serum albumin (HSA) gene expression cassette ①: The amino acid sequence of human serum albumin is SEQ ID NO: 11; for plant transformation, the nucleotide sequence encoding human serum albumin was codon-optimized for maize expression and a GT13 signal peptide coding sequence was added to the 5' end. The optimized nucleotide sequence encoding human serum albumin for maize transformation after linking the signal peptide is SEQ ID NO: 13. The GT1 promoter (SEQ ID NO: 1) was linked to the 5' end of the DNA sequence of human serum albumin after linking the signal peptide, and the Tzein-Hsp terminator (SEQ ID NO: 14) was linked to the 3' end. The pGT1-HSA-Tzein-Hsp fragment was artificially synthesized to form the 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 the human serum albumin. The nucleotide sequence encoding human serum albumin after optimizing the linkage of the signal peptide for maize transformation is SEQ ID NO: 13. The 5' end of the human serum albumin DNA sequence was linked to the ZmZ27 promoter (SEQ ID NO: 2), and the 3' end was linked to the Tzein-Hsp terminator (SEQ ID NO: 14). The pZmZ27-HSA-Tzein-Hsp fragment was artificially synthesized to form the human serum albumin gene expression cassette.

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

[0036] (3) Transformation vector: The vector pCambia1300 (NCBI sequence number AF234296) was digested with HindIII / XhoI, and the 6783bp fragment was recovered. The three gene expression frames were recombined by seamless cloning. The human serum albumin (HSA) gene expression frame ① and the human serum albumin (HSA) gene expression frame ② were sequentially linked with the FMV enhancer (SEQ ID NO:18), the Peanut chlorotic streakcaulimovirus enhancer (SEQ ID NO:19), and the MMV enhancer (SEQ ID NO:20). The vector was then transformed into Escherichia coli TG1 strain, and the results were confirmed by sequencing to obtain the transformation vector HSA.

[0037] (4) Agrobacterium transformation: plasmid HSA was electroporated into Agrobacterium LBA4404 competent cells. After enzyme digestion and identification, the cells were preserved with glycerol to obtain recombinant Agrobacterium, which was then used for crop transformation.

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

[0039] 1. Maize plant transformation

[0040] Maize genetic transformation was performed using Agrobacterium-mediated transformation, specifically following the method and culture medium formulation reported by Frame et al. (Plant Physiol, 2002, 129:13-22). Glyphosate was used as the screening reagent. The steps are 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 concentration OD was adjusted. 660 A concentration of 0.5-0.6 indicates an 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 them stand at room temperature for 5 minutes, take out the embryos, absorb the liquid, place them with the embryos face down on the co-culture medium, and culture at 22℃ for 3-5 days.

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

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

[0045] (5) Transfer the surviving embryonic tissue from step (4) to a regeneration medium and culture it in the dark at 28°C for 10-14 days. Then transfer it to a fresh regeneration medium and culture it in the light at 26°C for 10-14 days.

[0046] (6) Select the fully developed plants from step (5) and place them on a rooting medium. Incubate them under light at 26°C until the roots are fully developed. Transplant the regenerated seedlings after rooting to a greenhouse for growth and cultivation. Obtain transgenic maize (ZmHSA) expressing human serum albumin. All T0 generations of this transgenic maize produced seeds normally.

[0047] 2. Rice plant transformation

[0048] This embodiment was conducted according to existing literature and experimental methods (Lu Xiongbin & Gong Zuxun, 1998 Life Sciences 10: 125-131; Liu Fan et al., 2003 Molecular Plant Breeding 1: 108-115). Mature and plump rice seeds were selected, hulled, and callus tissue was induced to form the transformation material. The callus tissue to be transformed was placed in the infection solution containing Agrobacterium obtained in step 1 and cultured at 28°C and 220 rpm for 30 min to allow Agrobacterium to bind to the surface of the callus tissue. Then, the callus tissue was transferred to NBDC co-culture medium and cultured in the dark at 28°C for 3 days. The co-cultured rice callus was picked into a sterile Erlenmeyer flask and washed with water 8-10 times until the water was clear to remove excess Agrobacterium from the surface of the callus. Add 100 ml of sterile water containing 200 mg / L Timentin (tecarcillin sodium clavulanate potassium), shake at 28°C and 120 rpm for 1-2 hours, then transfer to selection medium containing 200 mg / L glyphosate antibiotic, and culture in the dark at 28°C for two months (with one subculture in between). Callus with good growth viability is transferred to pre-differentiation medium and cultured in the dark at 28°C for about 20 days. Then, the pre-differentiated callus tissue is transferred to differentiation medium and germinated under 14 hours of light. After 2-3 weeks, the resistant regenerated plants are transferred to rooting medium (200 mg / L glyphosate) to promote seedling growth and root development. Finally, the regenerated plants are washed off the agar and transplanted into a greenhouse. Transgenic rice expressing human serum albumin (OsHSA) was obtained, of which only 47% of the T0 transformants yielded seeds.

[0049] 3. Transformation of cowpea plants

[0050] According to existing technology, genetically modified cowpeas are obtained by Agrobacterium tumefaciens-mediated transformation (Chinese Patent CN113106118B).

[0051] Scrape Agrobacterium from Example 1 into B5 liquid culture medium, suspend and mix well. Use B5 liquid culture medium as a 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 OD 600nm value is 0.6. Then add 2 mg / L 6-BA (6-benzylaminopurine), 1 mg / L GA3 (gibberellin), and 5 mg / L AS (acetylsylsyringone) to obtain the Agrobacterium infection solution.

[0052] Sterilized non-GMO cowpea seeds were cultured on MS solid-state substrates and germinated in the dark at 26°C for 2 days. The outer shell was removed, and the seeds were divided in half along the hypocotyl to obtain two explants. One hundred explants were immersed in 100 ml of Agrobacterium infection solution and incubated at 26°C for 45 min.

[0053] The explants infected in the above steps were placed on a co-culture medium and cultured for 4 days under constant temperature / light / dark conditions at 27℃, 5000Lx, and 16h / 8h for 16h;

[0054] The explants co-cultured in the above steps were transferred to the bud induction and selection medium with the hypocotyl end facing down, and cultured for 28 days under constant temperature / light / dark conditions of 27℃, 5000Lx, and 16h / 8h to obtain explants with new buds.

[0055] The explants with new shoots from the above steps were transferred to the shoot elongation selection medium and cultured for 28 days under constant temperature / light / dark conditions of 27℃, 5000Lx, and 16 / 8h to elongate positive shoots and obtain explants with positive shoots.

[0056] The explants with positive buds from the above steps were transferred to rooting medium and cultured under constant temperature and light / dark conditions of 27℃, 5000Lx, and 16 / 8h until roots emerged. After culturing for another 5 days, the seedlings were hardened off and transplanted to a greenhouse.

[0057] A transgenic cowpea (VuHSA) expressing human serum albumin was obtained, and seeds were harvested from 85% of the T0 transformants.

[0058] This shows that the introduction of exogenous human serum albumin into corn, rice, and cowpea affected the seed setting rate of rice and cowpea to varying degrees, with rice being most affected, while corn remained unaffected.

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

[0060] T1 generation seeds from the transgenic maize (ZmHSA), transgenic rice (OsHSA), and transgenic cowpea (VuHSA) of Example 2 were collected. Approximately 200 mg of seed powder was weighed from each transgenic plant, ground into powder, and mixed with 2000 μL of PBS buffer. The mixture was shaken for 3 min, allowed to stand for 2 hours, and then centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was collected as the protein sample. The HSA content of the supernatant protein sample was detected using a human serum albumin-linked immunosorbent assay (ELISA) kit (Shanghai Sangon Biotech, D711424).

[0061] The seven best-expressing transformants were selected, and the specific HSA quantification results are shown in Table 1 below.

[0062] Table 1. Quantitative Results of HSA

[0063]

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

[0065] Example 4: Electrophoretic detection of human serum albumin from maize ZmHSA

[0066] SDS-PAGE detection of human serum albumin in transgenic maize seeds. The detection method is as follows: Take maize seeds containing human serum albumin obtained by the method in Example 2, grind each seed thoroughly, weigh 10 mg of each seed, add 1000 μL of PBS buffer, vortex to mix for 3 min, centrifuge at 12000 rpm for 10 min at 4℃, and collect the supernatant as the protein sample. Take 40 μL of the protein sample, add 10 μL of 5× protein loading buffer, mix well, boil in water for 10 min, centrifuge at 12000 rpm for 5 min, and collect the supernatant for electrophoresis. Use GenScript SurePAGE precast gel (4-20%), load the sample, and electrophores at 200V for 30 min. After electrophoresis, stain using GenScript eBlot™ L1 rapid wet transfer instrument.

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

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

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

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

[0071] (1) Statistics on yield traits: The survey object was mature corn harvested in the field, and the weight of a single ear and the weight of 100 kernels were recorded.

[0072] ① Single ear weight: 20 replicates of mature and dried corn ears were randomly selected and their weights were measured;

[0073] ② Weight per 100 kernels: Randomly selected mature, dried corn kernels were threshed, and 100 kernels were randomly selected (20 replicates) and weighed. The obtained data were statistically analyzed using an Excel spreadsheet.

[0074] Twenty mature transgenic maize plants (ZmHSA-3, ZmHSA-26, and ZmHSA-35) and non-transgenic maize plants were randomly selected from the field. The weight of each ear was measured, and the seeds were dried. 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 maize

[0076]

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

[0078] (2) Yield per mu of human serum albumin-containing maize ZmHSA-3, ZmHSA-26 and ZmHSA-35

[0079] Planting was carried out using equal row spacing. Each plot was 4.8m long and 4.2m wide, with a net area of ​​20m². 2 Three plots were repeated. The planting method was open-field sowing in raised beds, with a plant spacing of 0.4m × 0.77m. Each plot had 5 rows and 13 beds per row, for a total of 65 beds, equivalent to 667m². 2 Planting density: 2166 ponds; two plants per pond, 130 plants per plot, 667m² 2 4332 basic seedlings. Standard fertilization, NPK recommended formula (nutrient content: N: 20, P: 6, K: 5 kg / 667 m³). 2 ).

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

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

[0082]

[0083] The results showed that the highest yield per mu (approximately 0.067 hectares) for human serum albumin-treated maize ZmHSA-3, ZmHSA-26, and ZmHSA-35 was approximately 747 kg.

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

[0085] (1) Statistics on yield traits: The survey object was mature rice harvested in the field, and the number of grains per panicle and the weight of a thousand grains were recorded.

[0086] ① Number of grains per panicle: Randomly select 20 replicates of mature rice panicles and count the number of grains in normal grain filling.

[0087] ② 1000-grain weight: Mature, dried rice was randomly selected and threshed. 1000 seeds were randomly selected, with 20 replicates, and their weight was measured. The obtained data were statistically analyzed using an Excel spreadsheet.

[0088] Twenty mature transgenic rice plants (OsHSA-7) and non-transgenic rice plants were randomly selected from the field, and 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 1000-grain weight (g) 24.712 20.462 Human serum albumin expression level (mg / g) 0 1.06

[0091] The results showed that, compared with non-GMO rice, GMO rice had a reduced number of grains per panicle and a lower thousand-grain weight to varying degrees, and its yield tended to decrease.

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

[0093] Planting was carried out using equal row spacing. Each plot was 4.8m long and 4.2m wide, with a net area of ​​20m². 2 Three plots were repeated. After soaking, the seeds were sown in seedbeds and transplanted to the field one month later. The planting spacing was 0.18m × 0.25m, with 19 rows per plot and 23 holes per row, for a total of 437 holes, equivalent to 667m². 2 Planting density was 14,574 ponds; three plants were left per pond, resulting in 1,311 plants per plot, maintaining a basic seedling density of approximately 43,700 plants per 667m². 2 For conventional fertilization, the recommended NPK formula is (Nutrient content: N: 13, P: 6, K: 6 kg / 667 m³). 2 ).

[0094] After harvesting, all mature ears obtained from the plot were dried, threshed, and weighed. The average yield and actual grain yield of the plot were investigated, and 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 )]]> Yield per 667 m² (kg) OsHSA-7 12.76 425.43

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

[0098] In summary, compared to non-GMO maize, the human serum albumin maize varieties ZmHSA-3, ZmHSA-26, and ZmHSA-35 did not show a significant reduction in single ear weight and 100-grain weight, with a maximum yield of approximately 747 kg / mu. GMO rice OsHSA-7 showed varying degrees of reduction in single ear grain number and 1000-grain weight, with a yield of approximately 425 kg / mu. The human serum albumin maize variety ZmHSA-26 is expected to produce 1373 g / mu of human serum albumin, nearly three times that of rice. Therefore, we can conclude that the GMO maize obtained in this invention is more efficient and lower cost-effective for producing human serum albumin.

Claims

1. A method for producing human serum albumin using corn seeds, characterized in that, The method involves introducing two expression cassettes for human serum albumin into maize. One cassette codon-optimized the nucleotide sequence encoding human serum albumin for maize expression and added a GT13 signal peptide coding sequence to the 5' end. The nucleotide sequence encoding human serum albumin and linking the signal peptide is SEQ ID NO:

13. The GT1 promoter is linked to the 5' end of the DNA sequence encoding human serum albumin and linking the signal peptide to the 3' end of the GT1 promoter, and the Tzein-Hsp terminator is linked to the 3' end. The other expression cassette uses the ZmZ27 promoter instead of the GT1 promoter and is constructed using the same terminator and signal peptide. The nucleotide sequence of the GT1 promoter is shown in SEQ ID NO: 1, the nucleotide sequence of the ZmZ27 promoter is shown in SEQ ID NO: 2, and the nucleotide sequence of the Tzein-Hsp terminator is shown in SEQ ID NO: 14.

Images