Method for producing lysozyme and application

Abstract

The invention discloses a method for producing lysozyme and application of the lysozyme, and the method comprises the following steps: transferring an exogenous lysozyme gene into a corn genome, and screening transgenic corn expressing high-enzyme-activity lysozyme. According to the method, the exogenous lysozyme gene is introduced into the corn through a DNA recombination technology, the influence on the seed activity and the storage resistance is small while the lysozyme protein corn is highly expressed, preservation and propagation are easier, and low-cost large-scale production of the lysozyme is achieved. The transgenic corn obtained by the invention can be directly used as a feed or a feed additive for breeding without additionally extracting the lysozyme manually and adding the lysozyme into the feed, so that the feeding efficiency is greatly improved and the economic cost is reduced. The obtained transgenic corn lysozyme is high in activity, the yield is not obviously different from that of non-transgenic corn, the seed vigor is not affected, and the seeds are resistant to storage and easy to propagate and have excellent application prospects and economic values.

Description

(I) Technical field

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

[0002] Lysozyme is widely found in nature, such as human tissue, egg white of birds and poultry, etc. Lysozyme is a type of hydrolase, also known as muramidase, which can selectively hydrolyze the β-1,4 glycosidic bonds of peptidoglycan on the bacterial cell wall, thereby causing cell dissolution and death. It is a natural, highly safe biocide and preservative. Lysozyme mainly acts on Gram-positive bacteria (G+), because the cell wall of Gram-positive bacteria (G+) is almost entirely composed of peptidoglycan; Gram-negative bacteria (G-) only have the inner wall layer of peptidoglycan, so lysozyme is more effective in destroying the cell wall of G+ bacteria than that of G- bacteria. Lysozyme can be divided into three categories based on the source: animal lysozyme, plant lysozyme, and microbial lysozyme; based on the amino acid sequence and protein structure, it can be divided into chicken-type (C type), phage-type (T4 type), and goose-type (G type). Among them, C-type lysozyme is widely distributed, and egg white lysozyme is a typical representative of this type of lysozyme. In terms of content, egg white lysozyme accounts for about 3.5% of all protein in egg white. At present, lysozyme is generally separated and purified from egg white in industry. However, in terms of enzyme activity, the lysozyme activity in human milk, tears, and saliva is about 3 times that of egg white lysozyme.

[0003] Lysozyme has good biocompatibility and is non-irritating and non-toxic to body tissues. Adding lysozyme to feed can not only extend the shelf life of feed, but also balance the intestinal flora of animals, prevent diarrhea, and effectively improve the economic benefits of breeding. Adding a small amount of lysozyme to poultry feed can not only reduce the incidence of poultry, but also increase egg production; adding lysozyme to the feed of livestock such as pigs, cattle, and sheep can effectively prevent and control the incidence of diarrhea. In addition, adding 1% lysozyme to dairy cow feed can effectively inhibit the incidence of mastitis, increase dairy cow milk production and the quality of fresh milk. Especially after 2020, my country will completely ban the addition of antibiotics to feed, and reducing the impact of feed antibiotics on the breeding industry is an urgent problem that the breeding and feed industries need to solve. The application of lysozyme in feed has important industrial significance for improving animal nutrition and health care. At present, lysozyme can be extracted from egg white in industry, and lysozyme can also be produced on a large scale by using animal, plant or microbial expression systems through DNA recombination technology. However, animal cell culture and transgenic animals cannot overcome problems such as animal pathogen contamination. For example, lysozyme produced from egg white contains allergens, and its activity is 3.5 times lower than that of human lysozyme. Therefore, lysozyme derived from egg white cannot be used as a food additive. Extraction of lysozyme by microbial expression system has become the mainstream, but a series of extraction and purification operations are required after microbial expression of lysozyme. The extraction process is complicated, large-scale production is difficult and costly. The high production cost is the main reason why lysozyme is difficult to be used on a large scale in feed.

[0004] Based on the significant advantages of high expression level, low production cost and simple processing, the seed expression system is one of the more mature expression systems for large-scale production. Seeds can provide a relatively stable internal environment, so that the expression of recombinant proteins accumulates to a high level; at the same time, seeds are the best "natural container". Recombinant proteins can be stored at room temperature for 2-3 years without losing biological activity, and because the recombinant proteins are stored in a very small space, the product transportation is very convenient. Chinese patent CN101979591B discloses a method for producing human lysozyme using rice as a bioreactor, using rice storage prolamin Gt13a as the promoter and signal peptide for endosperm-specific expression, increasing the expression of lysozyme in rice endosperm by more than 40%, but only 50-60% of the transgenic plants of the T0 generation are normally fruited.

[0005] Since high expression of exogenous genes often seriously affects seed vitality, resulting in low seed germination rate, poor storage resistance, easy inactivation and other problems, it is necessary to find a method that can highly express exogenous lysozyme without affecting plant seed yield, vitality and storage. (III) Summary of the invention

[0006] The purpose of the present invention is to provide a method for producing lysozyme using corn and its application. Exogenous lysozyme genes are introduced into corn by DNA recombination technology to obtain corn with high expression of lysozyme protein without affecting seed yield and vitality. Transgenic corn plants and seeds can be directly used as feed, eliminating the steps of artificially extracting lysozyme and adding lysozyme to feed, saving costs and greatly improving feeding efficiency.

[0007] The technical solution adopted by the present invention is:

[0008] The invention provides a method for producing lysozyme. The method comprises the steps of transferring an exogenous lysozyme gene into a corn genome and screening transgenic corn expressing lysozyme with high enzyme activity.

[0009] Furthermore, the lysozyme is chicken lysozyme or human lysozyme.

[0010] Furthermore, the amino acid sequence of chicken lysozyme is shown in SEQ ID NO:1; and the amino acid sequence of human lysozyme is shown in SEQ ID NO:2.

[0011] 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. 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.

[0012] Due to the degeneracy of the genetic code, there are a variety of different nucleic acid sequences that can encode the same amino acid protein. It is within the capabilities of those of ordinary skill in the art to generate other nucleic acid sequences encoding the same protein, so the lysozyme gene of the present invention encompasses nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code.

[0013] Furthermore, the transcribable DNA sequence encoding the lysozyme protein is a sequence optimized by corn codons and connected to a signal peptide. The nucleotide sequence encoding chicken lysozyme is shown in SEQ ID NO:3, and the nucleotide sequence encoding human lysozyme is shown in SEQ ID NO:4.

[0014] The invention provides a recombinant DNA molecule for expressing exogenous lysozyme in corn. The recombinant DNA molecule comprises a transcribable DNA sequence encoding lysozyme protein operably connected with a functional promoter.

[0015] The present invention provides a corn cell, which comprises the above-mentioned recombinant DNA molecule.

[0016] The invention provides an application of transgenic corn containing lysozyme prepared by the method as feed or feed additive. The feed or feed additive can be used for preventing or treating diarrhea.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] (1) The present invention introduces exogenous lysozyme gene into corn through DNA recombination technology, and the corn highly expresses lysozyme protein while having little effect on seed vitality and storage resistance, making it easier to preserve and propagate, thereby realizing low-cost large-scale production of lysozyme.

[0019] (2) The transgenic corn obtained by the present invention can be directly used in breeding as feed or feed additive, without the need for artificial extraction of lysozyme and addition of lysozyme to feed, which greatly improves feeding efficiency and reduces economic costs.

[0020] (3) The transgenic corn obtained by the present invention has high lysozyme activity, and its yield is not significantly different from that of non-transgenic corn. The seed vitality is not affected, and the seeds are resistant to storage and easy to propagate, thus having excellent application prospects and economic value. (IV) Description of the drawings

[0021] Figure 1 , Enzyme activity diagram of lysozyme from chicken lysozyme corn (ZLZM), human lysozyme corn (ZLYZ), chicken lysozyme soybean (SLZM) and chicken lysozyme rice (RLZM) in Example 3.

[0022] Figure 2 , SDS-PAGE electrophoresis of chicken lysozyme corn ZLZM7 in Example 4.

[0023] Figure 3 , SDS-PAGE electrophoresis of human lysozyme corn ZLYZ4 in Example 4. (V) Specific implementation methods

[0024] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0025] 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.

[0026] 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).

[0027] Example 1. Vector construction and acquisition

[0028] 1. Obtaining the plasmid vector containing chicken lysozyme

[0029] (1) Chicken lysozyme (LZM) gene expression cassette: The amino acid sequence of chicken lysozyme is SEQ ID NO: 1; for plant transformation, the nucleotide sequence encoding chicken lysozyme is codon-optimized for corn expression and a GT1 signal peptide is added to the 5' end. After the optimized signal peptide connection, the nucleotide sequence encoding chicken lysozyme for corn transformation is SEQ ID NO: 3. The 5' end of the chicken lysozyme DNA sequence is connected to the GT1 promoter (SEQ ID NO: 5), and the 3' end is connected to the Tnos terminator (SEQ ID NO: 6), and the pGT1-LZM-Tnos fragment is artificially synthesized to form a chicken lysozyme gene expression cassette.

[0030] (2) Screening gene expression cassette: The screening gene G10-evo (SEQ ID NO: 7) was connected to the CaMV35S promoter (SEQ NO ID: 8) at the 5' end and the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ NO ID: 9) at the 3' end to artificially synthesize the p35S-G10-evo-TCaMV35S fragment.

[0031] (3) Transformation vector: The vector pCambia1300 (NCBI sequence number AF234296) was double-digested with HindⅢ / XhoⅠ, and a fragment of 6783 bp was recovered. The above two gene expression frames were recombined by seamless cloning and transformed into Escherichia coli TG1 strain. After sequencing confirmation, the transformation vector was obtained.

[0032] (4) Agrobacterium transformation: The plasmid LZM 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.

[0033] 2. Obtaining plasmid vector containing human lysozyme

[0034] (1) Human lysozyme (LYZ) gene expression cassette: The amino acid sequence of human lysozyme is SEQ ID NO: 2; for plant transformation, the nucleotide sequence encoding human lysozyme is codon-optimized for corn expression and a GT1 signal peptide is added to the 5' end. After the optimization and connection of the signal peptide, the nucleotide sequence encoding human lysozyme used for corn transformation is SEQ ID NO: 4. The 5' end of the DNA sequence of human lysozyme is connected to the GT1 promoter (SEQ ID NO: 5), and the 3' end is connected to the Tnos terminator (SEQ ID NO: 6), and the p GT1-LYZ-Tnos fragment is artificially synthesized to form a human lysozyme gene expression cassette.

[0035] (2) Screening gene expression cassette: The screening gene G10-evo (SEQ ID NO: 7) was connected to the CaMV35S promoter (SEQ NO ID: 8) at the 5' end and the cauliflower mosaic virus 35s terminator TCaMV35S (SEQ NO ID: 9) at the 3' end to artificially synthesize the p35S-G10-evo-TCaMV35S fragment.

[0036] (3) Transformation vector: The vector pCambia1300 (NCBI sequence number AF234296) was double-digested with HindⅢ / XhoⅠ, and a fragment of 6783 bp was recovered. The above two gene expression frames were recombined by seamless cloning and transformed into Escherichia coli TG1 strain. After sequencing confirmation, the transformation vector was obtained.

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

[0038] Example 2: Transformation of corn, soybean and rice 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 Agrobacterium (LZM and LYZ) containing the transformation vector obtained in Example 1 was mixed evenly in the infection medium, and the bacterial solution concentration OD660 was adjusted to 0.5-0.6, that 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 from step (5) were selected and placed on a rooting medium and cultured under light at 26° C. until the roots were fully developed. The rooted regenerated seedlings were transplanted to a greenhouse for growth and cultivation.

[0047] Transgenic maize expressing chicken lysozyme (ZLZM) and transgenic maize expressing human lysozyme (ZLYZ) were obtained, and 97% of the plants in the T0 generation were harvested into T1 generation seeds.

[0048] 2. Soybean plant transformation

[0049] The soybean transformation process and culture medium formulation mainly refer to the Agrobacterium-mediated half-seed transformation method of Paz (PazMM, et al. Plant Cell Reports, 2006, 25(3):206-213). Glyphosate was used as the screening agent, as follows:

[0050] (1) Sterilization of soybean seeds: Select high-quality soybean seeds, place them in a desiccator, and sterilize them with chlorine gas for 16 hours. The chlorine gas is generated by slowly adding 8 mL of concentrated hydrochloric acid to a beaker containing 100 mL of 30% (v / v) sodium hypochlorite aqueous solution in the desiccator.

[0051] (2) Seed germination: The sterilized soybean seeds in step (1) are soaked in sterile distilled water at room temperature for 16 hours to obtain germinated soybean seeds.

[0052] (3) Preparation of Agrobacterium culture medium: Streak the Agrobacterium (LZM) containing the transformant prepared in Example 1 on a YEP solid medium containing 50 mg / L Kan (kanamycin) and 25 mg / L rifampicin, and culture at 28°C for 2 days. Scrape the colonies on the YEP culture plate, evenly suspend them in the infection medium, and adjust the concentration to OD650 = 0.5-0.6, i.e., the Agrobacterium culture medium. The YEP solid medium composition: 10 g of peptone, 10 g of yeast extract, 5 g of NaCl, 8 g of agar, and add water to make the volume to 1 L.

[0053] (4) Preparation and infection of explants: The soybean seeds germinated in step (2) were cut longitudinally along the hilum to separate them into two lobes. The lob with the hypocotyl and embryo was removed from the seed coat and quickly placed in the Agrobacterium solution prepared in step (3). The lobes were infected at room temperature for 30 min. The lobes were taken out and placed in a sterile culture dish lined with filter paper. After sealing, the lobes were cultured in the dark at 22°C for 3 days.

[0054] (5) Bud induction culture: The explants after co-cultivation in step (4) were transferred to a bud induction medium containing 2 mM glyphosate and cultured at 26°C, 16 h light / 8 h dark conditions for 3 weeks. The bud induction medium composition: 3.21 g B5 salt (Phytotech, item number: G768), 1 mL B5 vitamin (Phytotech, item number G249-100ML), 30 g sucrose, 0.64 g 4-morpholineethanesulfonic acid, 8 g agar, water was added to make up to 1 L and the pH was adjusted to 5.6, and 200 mg timentin and 1.67 mg 6-benzyladenine were added after sterilization.

[0055] (6) Bud elongation and screening: The cotyledons of the explants induced in step (5) were cut off, and the part with clustered buds was transferred to a bud elongation medium containing 2 mM glyphosate and cultured at 26°C, 16 h light / 8 h dark conditions for 21 days. The bud elongation medium composition: MS salt (Phytotech, item number: M524) 4.33 g, B5 vitamin (Phytotech, item number G249-100ML) 1 mL, sucrose 30 g, 4-morpholineethanesulfonic acid 0.64 g, asparagine 50 mg, glutamine 50 mg, agar 8 g, water was added to make up to 1 L and the pH was adjusted to 5.6, and after sterilization, 0.5 mg gibberellin, 0.1 mg indole-3-acetic acid, 1 mg zeatin riboside, and 200 mg timentin were added.

[0056] (7) Rooting and transplanting: Transfer the elongated buds in step (6) to the rooting medium and culture them under 26°C, 16h light / 8h dark conditions. When the roots grow out, wash off the culture medium attached to the roots and transplant the seedlings into the matrix for culture until the T0 generation seeds are harvested. Plant the T0 generation seeds and collect leaf samples for analysis of gene editing results. Rooting medium composition: MS salt 4.33g, B5 vitamin 1mL, sucrose 20g, 4-morpholineethanesulfonic acid 0.64g, agar 8g, add water to make up to 1L and adjust the pH to 5.6. After sterilization, add 200mg of timentin.

[0057] Transgenic soybean (SLZM) expressing chicken lysozyme was obtained, and 95% of the plants in the T0 generation were harvested into T1 generation seeds.

[0058] 3. Rice plant transformation

[0059] 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 Agrobacterium bacterial solution (LZM) obtained by the method in step 2, 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 callus surface is removed. Add 100ml sterile water containing 200mg / L Timentin (Timentin, Ticarcillin Sodium Clavulanate Potassium), shake at 28℃, 120rpm for 1-2h, transfer to the screening medium containing 200mg / L glyphosate antibiotic, and culture in the dark at 28℃ for two months (subculture once in the middle). Calli with good growth activity are transferred to the pre-differentiation medium and cultured in the dark at 28℃ for about 20 days, and then the pre-differentiated callus is transferred to the differentiation medium, and differentiated and germinated for 14 hours of light. After 2-3 weeks, the resistant regenerated plants are transferred to the rooting medium (200mg / L glyphosate) for strong seedlings and rooting, and finally the regenerated plants are washed off the agar and transplanted to the greenhouse.

[0060] Transgenic rice expressing chicken lysozyme (RLZM) was obtained, of which 40-50% of the transformants could produce T1 generation seeds.

[0061] It can be seen from this that when the exogenous gene lysozyme was simultaneously transferred into corn, soybeans and rice, only the fruit set rate of rice was affected.

[0062] Example 3: Turbidimetric quantitative detection of lysozyme activity in transgenic plants

[0063] The lysozyme activity of the chicken lysozyme corn (ZLZM), human lysozyme corn (ZLYZ), chicken lysozyme soybean (SLZM) and chicken lysozyme rice (RLZM) obtained in Example 2 was detected.

[0064] In a turbid bacterial solution of a certain concentration, lysozyme can hydrolyze the peptidoglycan on the bacterial cell wall to lyse the bacteria, thereby reducing the concentration and increasing the transmittance. Therefore, the content of lysozyme can be inferred based on the change in transmittance. Therefore, a lysozyme test kit (purchased from Shanghai Shenggong, item number: NO.D799854) was used to determine the lysozyme activity of transgenic seeds by turbidimetry. The specific operation is as follows:

[0065] 1. Reagent formula

[0066] 1) Sample: Take 1g of transgenic seeds and freeze them with liquid nitrogen for 3 minutes, then grind them with a tissue grinder at 45Hz for 45s, then add 1ml of PBS buffer and stir at 220rpm for 10min to get the sample. Prepare it before use and shake it well. Seal it at 2-8℃ and it can be stored for one week.

[0067] 2) Bacterial solution: Micrococcus lysodeikticus, which is included in the test kit and does not need to be prepared. It can be sterilized in a microwave oven for 20 seconds at medium temperature every month and sealed at 2-8℃.

[0068] 3) Standard solution: Add 1.0 ml of distilled water to accurately prepare lysozyme into a 2 mg / mL standard solution, then dilute it with distilled water at a ratio of 1:799 (volume ratio) to a 2.5 ug / mL standard solution and store it in a dry place at 2-8°C.

[0069] 2. Reaction steps

[0070] Add each reagent in sequence according to the operation table shown in Table 1 (in an ice water bath)

[0071] Table 1 Operation table

[0072]

[0073] 3. Activity calculation

[0074] 1) Lysozyme content (U / mL) = (Ttest-Tblank) ÷ (Tstandard-Tblank) × Cstandard × sample dilution factor before test

[0075] 2) C standard: standard solution concentration, 2.5ug / mL or 200U / mL.

[0076] 3) According to the measurement results ( Figure 1), the highest activity of chicken lysozyme in corn (ZLZM7) reached 720,000 U / g; the highest activity of human lysozyme in corn (ZLYZ4) reached 650,000 U / g; the highest activity of chicken lysozyme in soybean (SLZM6) reached 361,000 U / g; the highest activity of chicken lysozyme in rice (RLZM9) reached 670,000 U / g; it can be seen that the enzyme activity of all chicken lysozyme soybean transformants is low, therefore, soybean is not suitable for the expression of exogenous lysozyme.

[0077] Example 4: Western blot hybridization of chicken lysozyme corn ZLZM7 and human lysozyme corn ZLYZ4

[0078] SDS-PAGE detection of lysozyme in transgenic corn seeds. The detection method is as follows: Take the seeds of chicken lysozyme corn ZLZM7 and human lysozyme corn ZLYZ4 obtained by the method of Example 2, and take another non-transgenic seed. 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 electrophoresed at 200V for 30 minutes. After the electrophoresis, use GenScript eBlotTM L1 fast wet transfer instrument for staining.

[0079] The results are as follows Figure 2 and Figure 3 As shown, Figure 2 The target protein of chicken lysozyme corn ZLZM7, Figure 3 It is the target protein of human lysozyme maize ZLYZ4.

[0080] Example 5: Analysis of yield traits and seed storage resistance of transgenic corn and transgenic rice

[0081] The transformant with the highest chicken lysozyme corn activity (ZLZM7), the transformant with the highest human lysozyme corn activity (ZLYZ4), and the transformant with the highest chicken lysozyme rice activity (RLZM9) in Example 2 were selected for breeding and their yield traits and seed storage resistance were analyzed.

[0082] 1. Yield trait analysis and seed storage resistance analysis of chicken lysozyme corn ZLZM7 and human lysozyme corn ZLYZ4

[0083] (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.

[0084] ① Single ear weight: Randomly select 20 replicates of mature, dried single ears of corn and weigh them;

[0085] 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 spreadsheets and T-test methods.

[0086] 20 mature transgenic corn ZLZM7, transgenic corn ZLYZ4 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.

[0087] Table 2. Agronomic traits of corn

[0088] Non-GMO corn Chicken Lysozyme Corn ZLZM7 Human Lysozyme Corn ZLYZ4 Single ear weight (g) 95.313 94.981 95.243 100-grain weight (g) 22.161 21.352 22.236 Enzyme activity (U / g) 0 720000 650000

[0089] 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.

[0090] (2) The storage resistance of corn seeds is measured by germination rate, germination potential and storage resistance index.

[0091] ① Germination rate: the percentage between the final number of seeds germinated and the total number of seeds within a specified time;

[0092] ② Germination potential: It is the percentage of the total number of germinated seeds to the number of test seeds when the number of germinated seeds reaches the highest during the germination experiment, which reflects the uniformity of seed germination;

[0093] ③ Storage resistance index: The formula is:

[0094] Storage resistance index (%) = 100 - [(germination rate of unaged seeds - germination rate of aged seeds) / germination rate of unaged seeds] * 100

[0095] Artificial accelerated aging treatment of seeds: After the corn seeds mature and are harvested, they are then manually threshed, placed in seed bags, and placed in an oven at 42°C for 7 days to break dormancy, and then placed in plastic bags for storage. 200 plump, disease-free, and uniformly mature seeds are placed in a seed bag, and then the batch of seeds is uniformly placed in a plastic bottle (without a lid), and then the plastic bottle is placed in a glass bottle containing 100 mL of water, sealed tightly, and placed in an oven at 42°C for artificial accelerated aging for 10 days. After the accelerated aging treatment, the seeds are taken out of the oven, dried at room temperature for 3 days, and used directly in the experiment or bagged and stored in a -80°C refrigerator for later use.

[0096] 50 seeds of unaged and aged seeds with full grains, uniform maturity and no mold were selected and placed in 50 mL enzyme-free tubes, and then 40 mL dH2O (distilled dH2O) was added. The seeds were germinated at 37°C in the dark. The number of seeds that germinated was counted after 7 days of germination. The number of seeds that germinated was considered to be germinated when the embryo or radicle broke through the seed coat by 2 mm. The batches were repeated for 20 times. The results are shown in Table 3 below.

[0097] Table 3. Germination rate, germination potential and storage resistance index of corn seeds

[0098] Non-GMO corn Chicken Lysozyme Corn ZLZM7 Human Lysozyme Corn ZLYZ4 Germination rate (%) 95.64 94.38 95.82 Germination potential (%) 84.97 88.21 87.62 Storage resistance index (%) 98.27 95.67 97.64 Enzyme activity (U / g) 0 720000 650000

[0099] The results showed that there was no significant difference in germination rate, germination potential and storage resistance index between transgenic corn and non-transgenic corn.

[0100] 2. Analysis of yield traits and seed storage resistance of chicken lysozyme rice RLZM9

[0101] (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.

[0102] ① Number of grains per panicle: Randomly select 20 replicates of mature rice panicles and count the number of grains that are normally filled;

[0103] 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 data obtained are statistically analyzed using Excel spreadsheets and T-test methods.

[0104] 20 mature transgenic rice RLZM9 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.

[0105] Table 4. Agronomic traits of rice

[0106] Non-GMO Rice Chicken Lysozyme Rice RLZM9 Number of grains per ear 175.8 152.6 Thousand-grain weight (g) 24.674 20.152 Enzyme activity (U / g) 0 670000

[0107] 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, and the yield tended to decrease.

[0108] (2) The storage resistance of rice seeds is measured by germination rate, germination potential and storage resistance index.

[0109] ① Germination rate: the percentage between the final number of seeds germinated and the total number of seeds within a specified time;

[0110] ② Germination potential: It is the percentage of the total number of germinated seeds to the number of test seeds when the number of germinated seeds reaches the highest during the germination experiment, which reflects the uniformity of seed germination;

[0111] ③ Storage resistance index: The formula is:

[0112] Storage resistance index (%) = 100 - [(germination rate of unaged seeds - germination rate of aged seeds) / germination rate of unaged seeds] * 100

[0113] Artificial accelerated aging treatment of seeds: rice seeds were harvested 40 days after heading, then threshed manually, put into seed bags and placed in an oven at 42°C for 7 days to break dormancy, and then placed in plastic bags for storage. 200 plump, disease-free and mature seeds were placed in a seed bag, and then the batch of seeds was uniformly placed in a plastic bottle (without a lid), and then the plastic bottle was placed in a glass bottle containing 100 mL of water, sealed and placed in an oven at 42°C for 10 days of artificial accelerated aging. After the accelerated aging treatment, the seeds were taken out of the oven, dried at room temperature for 3 days, and used directly in the experiment or bagged and stored in a -80°C refrigerator for later use.

[0114] 50 seeds of unaged and aged seeds with full grains, uniform maturity and no mold were selected and placed in a 50 mL enzyme-free tube, and then 40 mL dH2O (distilled dH2O) was added. The seeds were germinated at 37°C in the dark. The number of seeds that germinated was counted after 7 days of germination. The number of seeds that germinated was considered to be germinated when the embryo or radicle broke through the seed coat by 2 mm. The batches were repeated for 20 times. The results are shown in Table 5 below.

[0115] Table 5. Germination rate, germination potential and storage resistance index of rice seeds

[0116] Non-GMO Rice Chicken Lysozyme Rice RLZM9 Germination rate (%) 67.29 26.91 Germination potential (%) 56.32 20.54 Storage resistance index (%) 89.31 51.95 Enzyme activity (U / g) 0 670000

[0117] The results showed that the germination rate, germination potential and storage index of transgenic rice were significantly lower than those of non-transgenic rice, and there was a significant difference. Therefore, rice is not suitable for lysozyme production.

[0118] Example 6: Application of transgenic corn feed

[0119] According to the principle of similar body weight and half male and half female, 80 weaned piglets were randomly selected and divided into 4 groups (non-transgenic corn, chicken lysozyme corn, human lysozyme corn, lysozyme), with 20 pigs in each group. The basic diet formula and nutritional level fed during the experiment are shown in Table 6 below.

[0120] Table 6. Basic diet formula and nutritional level

[0121]

[0122] Note: The premix contains minerals (calcium, phosphorus, salt, copper, iron, manganese, zinc, selenium, iodine, cobalt), multiple vitamins (vitamin A, vitamin D, vitamin B1, vitamin B2, vitamin B12, biotin, folic acid, pantothenic acid, niacin, vitamin K, vitamin E) and some amino acids (lysine, methionine, threonine, tryptophan), and does not contain antibiotics.

[0123] The corn in Table 6 are:

[0124] (1) Experimental group 1: corn was ordinary non-GMO corn;

[0125] (2) Experimental group 2: corn was transgenic chicken lysozyme corn (lysozyme content: 720,000 U / g);

[0126] (3) Experimental group 3: corn was transgenic corn containing human lysozyme (lysozyme content: 650,000 U / g);

[0127] (4) Experimental group 4: ordinary non-GMO corn + lysozyme (buy lysozyme from Chaojing mixed feed additive, added at 0.6g / KG)

[0128] The experimental pigs were free to eat and drink, and were fed in separate meals 4 times a day (at 7:00, 11:00, 15:00, and 19:00, respectively). The immunization procedure was carried out in accordance with the pig farm feeding and management procedures. If diarrhea occurred, neomycin sulfate and multivitamins were immediately used for treatment with oral rehydration salts. The entire experimental period was 40 days (30 days to 70 days of age). The occurrence of diarrhea in each group of piglets was observed and recorded in detail every day, and the diarrhea rate was calculated. Diarrhea rate (%) = total number of diarrhea in piglets during the experimental period / (number of experimental days × number of piglets) × 100%.

[0129] Table 7. Diarrhea rate in each group

[0130] Experimental Group Diarrhea rate 1 7.5％ 2 2.8％ 3 3.6％ 4 5.1％

[0131] The test results show that compared with test group 1, test groups 2 and 3 with the addition of corn with high lysozyme expression have significant effects on preventing and treating piglet diarrhea; compared with test group 4 with the additional addition of lysozyme, the effect of the lysozyme transgenic corn of the present invention is better.

[0132] The above embodiments are only preferred implementations of the present invention. It should be pointed out that a person skilled in the art may make several improvements without departing from the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing lysozyme, characterized in that: The method comprises the following steps: transferring the exogenous lysozyme gene into the corn genome, and screening the transgenic corn expressing the lysozyme with high enzyme activity.

2. The method according to claim 1, characterized in that The lysozyme is chicken lysozyme or human lysozyme.

3. The method according to claim 2, characterized in that The amino acid sequence of chicken lysozyme is shown in SEQ ID NO: 1; the amino acid sequence of human lysozyme is shown in SEQ ID NO:

2.

4. A recombinant DNA molecule for expressing exogenous lysozyme in corn according to claim 1, characterized in that: The recombinant DNA molecule comprises a transcribable DNA sequence encoding a lysozyme protein operably linked to a functional promoter.

5. The recombinant DNA molecule according to claim 4, characterized in that The transcribable DNA sequence encoding the lysozyme protein is shown in SEQ ID NO:3 or SEQ ID NO:

4.

6. A corn cell containing the recombinant DNA molecule of claim 4.

7. Use of transgenic corn containing lysozyme prepared by the method of claim 1 as feed or feed additive.

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