Application of high-protein duckweed mutant mutant-47

Abstract

The invention discloses application of a high-protein duckweed mutant mutant-47, and belongs to the technical field of plant breeding. The total protein content of the novel duckweed variety mutant-47 provided by the invention reaches 50% of the dry weight, and the total protein content is increased by about 12% compared with that of wild duckweed; the duckweed mutant mutant-47 provided by the invention is rich in eight amino acids necessary for a human body, compared with a wild type, the content of free lysine is increased by 1.7 times, the content of acetyl-L-lysine is increased by 2.3 times, valine, threonine, phenylalanine, methionine, isoleucine and leucine are up-regulated and accumulated to different degrees, and tryptophan is down-regulated and accumulated; key enzyme genes of an amino acid metabolic pathway of the provided duckweed mutant mutant-47 are remarkably enriched; the duckweed mutant mutant-47 provided by the invention can be used as a raw material for preparing high-protein animal feed, realizes partial replacement of animal feed soybean protein, and relieves the problem that soybean consumption in China seriously depends on import.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to an application of a high-protein duckweed mutant mutant-47. Background Art

[0002] Food security is at the core of the Sustainable Development Goals, and many studies have focused on providing adequate food supply for the growing world population while protecting the environment. The rise and development of alternative proteins will greatly alleviate the problems of traditional food protein production methods. Duckweed has a total protein content of 30%-40% of dry weight and is rich in 8 essential amino acids for the human body, comparable to egg white and milk protein, and has been recognized by the U.S. Food and Drug Administration (FDA) as a new alternative protein.

[0003] Duckweed (Lemna minor L.) is an aquatic floating plant of the subfamily Lemnoideae of the family Araceae, hence the name duckweed. Duckweed, as a biological resource with fast growth and high biomass, has good application prospects in starch and protein production, synthesis of functional active ingredients, water pollution control, carbon sink reduction and many other aspects. Duckweed is rich in nutrients such as protein, sugars, trace elements, vitamins, and bioactive substances such as flavonoids, terpenes, and polysaccharides. It has a positive effect on improving animal growth performance, immune function, and product quality. It has a low production cost and can be used as a high-quality plant protein source or feed additive in animal feed. Fresh duckweed can be used directly as feed, and duckweed dry powder can also be used to partially replace traditional feeds such as fish meal and soybean meal to feed livestock and poultry. It has the effects of promoting animal growth, body coloration, anti-infection, anti-inflammatory, anti-oxidation, and immune regulation. It is beneficial to shorten the fattening cycle of chickens, ducks, fish, pigs, sheep and other animals, greatly reduce the cost of feed production, and improve economic benefits. At present, duckweed is a good feed for pigs, ducks and grass carp. Grass carp fed with duckweed are less likely to suffer from hepatobiliary syndrome. In addition, the rich lutein and tyrosine in duckweed also help improve the body color of fish and affect the deposition of melanin in the body. Fish meal is comprehensive in nutrition and has good palatability. It is a high-quality protein source in aquatic feed and is also the main component of aquatic feed.

[0004] The amino acids that make up duckweed protein are complete and rich in 8 essential amino acids for the human body. Its nutritional value is comparable to that of eggs and milk. Essential amino acids have the following characteristics: 1) They have important physiological functions. Essential amino acids are the basic units that make up proteins and are essential for cell growth, repair and maintenance. The human body needs these amino acids to synthesize various enzymes, hormones, muscle tissue and other important molecules; many essential amino acids participate in the body's metabolic process, helping to regulate energy metabolism, immune function and nerve conduction, etc. 2) The human body cannot synthesize or synthesize in insufficient quantities and can only obtain them through food. One is that the human body lacks enzymes or metabolic pathways to synthesize essential amino acids, such as the synthesis pathways of lysine, tryptophan, phenylalanine, etc. in the human body are incomplete. Another is that some amino acids can be synthesized in small amounts in the human body, but their synthesis speed and amount are far from meeting physiological needs. 3) Deficiency can lead to specific diseases. For example, a lack of essential amino acids can lead to blocked protein synthesis, causing a series of health problems such as growth retardation, decreased immune function, and muscle atrophy.

[0005] The amino acid pattern of duckweed is close to the standard amino acid pattern advocated by the World Health Organization, so it is easy to be digested and absorbed by livestock and poultry. Lysine is the first type of limiting amino acid required for the growth of livestock and poultry. It cannot be synthesized in the body and must rely on exogenous intake. The main sources of lysine are animal protein and soy foods, and the lysine content in cereal foods is relatively low.

[0006] The physiological functions of lysine mainly include promoting protein synthesis. As the main raw material for protein synthesis, lysine participates in the synthesis of important proteins in organisms. In addition, it participates in regulating energy metabolism. Lysine is an important component of carnitine, which is an essential cofactor for fatty acid metabolism. Lysine can also enhance human immune function. Lysine is the first limiting amino acid in a typical pig diet. It affects cell proliferation and metabolism, and has a regulatory effect on the growth and development of pigs and meat quality. Studies have shown that lysine restriction at different feeding stages has a significant effect on pig growth performance, meat quality traits, etc. Pigs fed high-lysine diets have better growth performance and feed conversion than pigs fed low-lysine diets. Summary of the invention

[0007] The purpose of the present invention is to provide an application of a high-protein duckweed mutant mutant-47, so as to provide a duckweed mutant with high protein and high lysine content for preparing animal feed and provide a new material for extracting lysine.

[0008] To achieve the above purpose, the present invention provides an application of a Lemna minor mutant mutant-47 in preparing animal feed, wherein the total protein content of the Lemna minor mutant mutant-47 is significantly higher than that of the wild type Lemna minor, wherein the lysine content is increased by 1.7 times, and the acetyl-L-lysine content is increased by 2.3 times.

[0009] Preferably, the preparation method of the duckweed mutant mutant-47 is: subjecting duckweed callus to carbon ion irradiation, placing the irradiated callus in a differentiation medium, culturing in the dark at 25°C for 3 weeks, and then transferring to light for differentiation culture, transferring the differentiated duckweed to SH solid culture medium for culture, determining the total protein content of the duckweed mutant, and screening plants with high essential amino acid content from high-protein mutant plants.

[0010] Preferably, the method for obtaining the D. nobilis callus is as follows: placing D. nobilis fronds on an induction medium, culturing at room temperature in the dark, and replacing the induction medium once a month until callus grows.

[0011] Preferably, the components of the induction medium are: MS 4.43 g / L, 2,4-D 1.0 mg / L, TDZ 0.1 mg / L, sucrose 30 g / L, agar 7.9 g / L, and the pH is adjusted to 5.8 using NaOH and HCl and then sterilized at 121° C. for 15 min.

[0012] Preferably, the conditions for carbon ion irradiation are: 80 MeV / μ, an average LET value of 31 keV / μm, and a series of radiation doses including 10, 25, 50, and 75 Gy.

[0013] Preferably, the components of the differentiation medium are: B53.2g / L+KT 1mg / L+IAA 4.5mg / L+sucrose 10g / L+agar 7.9g / L, pH is 5.8, sterilized at 121°C for 15min, cooled to room temperature, and TMT 400mg / L+hygromycin 10mg / L was added in an ultra-clean workbench (extraction and UV sterilization for 30min); the components of the SH solid culture medium are: SH 3.2g / L+sucrose 10g / L+agar 8g / L, adjusted to pH 5.8, and sterilized at 121°C for 15min.

[0014] Preferably, the illumination condition is a light intensity of 100 μmol×m-2×s -1 Lx, 16h light / 8h dark.

[0015] A use of the above-mentioned duckweed mutant mutant-47 in the preparation of a lysine product.

[0016] Therefore, the application of a high-protein duckweed mutant mutant-47 provided by the present invention has the following specific technical effects:

[0017] (1) The total protein content of the duckweed mutant mutant-47 provided by the present invention reaches 50% of the dry weight, and the total protein content is increased by about 12% compared with the wild-type duckweed;

[0018] (2) The mutant mutant-47 provided by the present invention is rich in 8 essential amino acids for the human body. Compared with the wild type, the free lysine content is increased by 1.7 times, the acetyl-L-lysine content is increased by 2.3 times, and the accumulation of valine, threonine, phenylalanine, methionine, isoleucine and leucine are all up-regulated to varying degrees, while the accumulation of tryptophan is down-regulated;

[0019] (3) The duckweed mutant mutant-47 provided by the present invention can be used as a raw material for preparing high-protein animal feed, thereby partially replacing soybeans in animal feed and alleviating the problem that my country's soybean consumption is heavily dependent on imports. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0021] Figure 1 is the measurement result of the protein content of the duckweed mutant mutant-47 and WT in Example 1 of the present invention; WT is the wild-type duckweed, and ** represents a significant difference;

[0022] Figure 2 It is a leaf photo of the duckweed mutant mutant-47 and WT in Example 1 of the present invention, wherein WT is the wild-type duckweed, the scale bar in WT is 300 μm, and the scale bar in duckweed mutant mutant-47 is 500 μm;

[0023] Figure 3 is a growth curve diagram of the duckweed mutant mutant-47 and WT in Example 1 of the present invention;

[0024] Figure 4 is the heat map analysis result of differentially accumulated free amino acids between the duckweed mutant mutant-47 and WT in Example 2 of the present invention;

[0025] Figure 5 is the PCA analysis result of the transcriptome of the duckweed mutant mutant-47 and WT in Example 3 of the present invention;

[0026] Figure 6 This is the result of differentially expressed gene analysis between the transcriptomes of the duckweed mutant mutant-47 and WT in Example 3 of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0029] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0030] Embodiment 1

[0031] The heavy ion radiation mutagenesis method was used to obtain a high-protein duckweed variety. The specific steps are as follows:

[0032] S1. Prepare Lemna minor callus.

[0033] First prepare the induction medium, MS 4.43g / L, 2,4-D 1.0mg / L, TDZ 0.1mg / L, sucrose 30g / L, agar 7.9g / L, adjust the pH to 5.8 and sterilize at 121℃ for 30min.

[0034] Place the duckweed leaves on the induction culture medium and culture them in the dark at room temperature. Replace the induction culture medium once a month until callus tissue grows.

[0035] S2. Heavy ion radiation mutagenesis and mutant collection.

[0036] Three-week-old Lemna callus was irradiated with carbon ions at 80 MeV / μ, with an average LET value of 31 keV / μm, and a series of radiation doses including 10, 25, 50, and 75 Gy.

[0037] The irradiated callus was transferred to differentiation medium and cultured in the dark at 25°C for 3 weeks before being transferred to light for differentiation. The light intensity was 100 μmol×m-2×s -1 Lx, 16h light / 8h dark, after the leaves grow out, the regenerated leaves are transferred to SH solid medium for growth.

[0038] Differentiation medium: B53.2g / L+KT 1mg / L+IAA 4.5mg / L+sucrose 10g / L+agar 7.9g / L, pH 5.8, sterilized at 121°C for 15min, cooled to room temperature and added TMT400mg / L+hygromycin 10mg / L in an ultra-clean workbench (extraction and UV sterilization for 30min).

[0039] SH solid culture medium: 3.2g / L+sucrose 10g / L+agar 8g / L, adjust pH to 5.8, sterilize at 121℃ for 15min.

[0040] The newly grown thallus was propagated on SH solid medium, and after 2 months of regeneration, collection and propagation, a heavy ion-induced duckweed mutant library was established.

[0041] S3. A total protein extraction kit (produced by Beijing Aidelai Biotechnology Co., Ltd.) was used to extract the total protein of the mutants in the heavy ion mutagenesis duckweed mutant library according to the attached instructions, and a BCA protein quantification kit (produced by Beijing Aidelai Biotechnology Co., Ltd.) was used to quantitatively analyze the obtained protein. A mutant with a significantly increased protein content compared with the wild-type duckweed (WT) was obtained, with a protein content of about 50%, named mutant-47. The measurement results of the protein content of mutant-47 and WT are shown in the figure. Figure 1 The photos of mutant-47 and WT are shown in Figure 2 As shown, the leaves of the duckweed mutant mutant-47 became smaller and thinner.

[0042] Select mutant-47 leaves and wild-type duckweed leaves and plant them in SH liquid culture medium, with 10 leaves in each bottle, 5 bottles of each kind, and the planting day is recorded as day 0. The number of duckweed leaves in the SH liquid culture medium is detected on the 1st, 4th, 7th, and 10th days, respectively. Each detection is repeated 10 times, and the growth curves of duckweed mutant mutant-47 and WT are drawn as shown in the figure below. Figure 3 As shown, the results showed that the growth rate of the duckweed mutant mutant-47 was significantly lower than that of the WT.

[0043] Embodiment 2

[0044] The mutant duckweed mutant-47 and WT plants obtained in Example 1 were sent to the company for metabolomics testing. The results of the heat map analysis of differentially accumulated free amino acids were as follows: Figure 4As shown, different degrees of red represent different degrees of up-regulation, and different degrees of blue represent different degrees of down-regulation. The types of differentially accumulated free amino acids and the difference fold information are shown in Table 1. Compared with WT, there are 18 up-regulated amino acids in the mutant-47 mutant duckweed, including 6 essential amino acids (leucine, lysine, valine, isoleucine, phenylalanine, and threonine), and 2 down-regulated amino acids. The up-regulated ones are N6-acetyl-L-lysine, citrulline, N-acetylaspartic acid, proline, arginine, leucine, valine, lysine, serine, N-acetyl-L-tyrosine, isoleucine, Nα-acetyl-L-arginine, asparagine, phenylalanine, alanine, ornithine, threonine, and glycine. The down-regulated amino acids are tryptophan and histidine.

[0045] The test results showed that compared with the wild type, the free lysine content of the mutant-47 of Alpinia quinata increased by 1.7 times, and the acetyl-L-lysine content increased by 2.3 times.

[0046] Table 1

[0047]

[0048]

[0049] Embodiment 3

[0050] The mutant Lemna mutant-47 and WT plants obtained in Example 1 were sent to the company for transcriptome analysis. The sequencing of the sample generated a total of 15.95G of Rawdata, and the filtered Cleandata was 15.79G, and the data volume was sufficient. Q20 was 96.81%, Q30 was 91.63%, and the sequencing quality was qualified. The GC content was 44.07%, and the distribution was normal. In summary, the sample library was successfully constructed and sequenced. The reference genome size is 434,452,052bp, the sample alignment rate is 99.47%, the average coverage depth of the reference genome (excluding the N region) is 31.89X, and the 1X coverage (coverage of at least one base) is 98.74%. The alignment results are normal and can be used for subsequent variation detection and related analysis. The PCA analysis results are as follows Figure 5 As shown, M_47 is the mutant Lemna mutant-47, and WT is the wild-type Lemna.

[0051] The genes detected in transcription were screened, and the screening criterion was |log2FoldChange|≥1. Figure 6As shown in the figure, a total of 634 differentially expressed genes were detected in the mutant mutant-47, of which 344 genes were significantly down-regulated and 290 genes were significantly up-regulated. The enrichment analysis of the differentially expressed gene signal pathways found that the amino acid synthesis metabolism pathway was significantly enriched, of which 7 genes were significantly up-regulated and 8 genes were significantly down-regulated. The up-regulated genes were Chr04_441, Chr02_1427, Chr11_606, Chr05_893, Chr01_881, Chr05_359, and Chr15_592. The down-regulated genes were Chr21_618, Chr02_1418, Chr14_229, Chr01_930, Chr19_103, Chr21_614, Chr21_59, and Chr01_933.

[0052] Therefore, the total protein content of the duckweed mutant mutant-47 provided by the present invention reaches 50% of the dry weight, and the total protein content is increased by about 12% compared with the wild-type duckweed; the duckweed mutant mutant-47 provided is rich in 8 essential amino acids for the human body, and compared with the wild-type, the free lysine content is increased by 1.7 times, the acetyl-L-lysine content is increased by 2.3 times, valine, threonine, phenylalanine, methionine, isoleucine and leucine are all up-regulated and accumulated to varying degrees, while tryptophan is down-regulated and accumulated; the duckweed mutant mutant-47 provided is significantly enriched in key enzyme genes of the amino acid metabolic pathway; the duckweed mutant mutant-47 provided can be used as a raw material for preparing high-protein animal feed, realize partial substitution of animal feed soybean, and alleviate the problem that my country's soybean consumption is seriously dependent on imports.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of a Lemna minor mutant mutant-47 in preparing animal feed, characterized in that: The total protein content of the duckweed mutant mutant-47 was 12% higher than that of the duckweed wild type, of which the lysine content was 1.7 times higher and the acetyl-L-lysine content was 2.3 times higher.

2. The use of a Lemna mutant mutant-47 according to claim 1 in preparing animal feed, characterized in that: The preparation method of the duckweed mutant mutant-47 is as follows: subjecting duckweed callus to carbon ion irradiation, transferring the irradiated callus to a differentiation medium, culturing in the dark at 25°C for 3 weeks, then transferring to light for differentiation culture, transferring the differentiated duckweed to SH solid culture medium for culture, determining the total protein content of the duckweed mutant, and selecting plants with high essential amino acid content from high-protein mutant plants.

3. The use of a Lemna mutant mutant-47 according to claim 2 in preparing animal feed, characterized in that: The method for obtaining the callus tissue of Lemna odorata is as follows: placing the Lemna odorata fronds on an induction culture medium, culturing them in the dark at room temperature, and replacing the induction culture medium once a month until the callus tissue grows out.

4. The use of a Lemna mutant mutant-47 according to claim 3 in preparing animal feed, characterized in that: The components of the induction medium are: MS 4.43 g / L, 2,4-D 1.0 mg / L, TDZ 0.1 mg / L, sucrose 30 g / L, agar 7.9 g / L. The pH is adjusted to 5.8 with NaOH and then sterilized at 121°C for 15 min.

5. The use of a Lemna minor mutant mutant-47 in preparing animal feed according to claim 2, characterized in that: The conditions of carbon ion irradiation were: 80 MeV / μ, average LET value was 31 keV / μm, and a series of radiation doses included 10, 25, 50, and 75 Gy.

6. The use of a Lemna mutant mutant-47 according to claim 2 in preparing animal feed, characterized in that: The components of the differentiation medium are: B5 3.2 g / L + KT 1 mg / L + IAA 4.5 mg / L + sucrose 10 g / L + agar 7.9 g / L, pH 5.8, sterilized at 121 ° C for 15 min, cooled to room temperature, add TMT 400 mg / L + hygromycin 10 mg / L in an ultra-clean workbench; the components of the SH solid culture medium are: SH 3.2 g / L + sucrose 10 g / L + agar 8 g / L, adjusted to pH 5.8, sterilized at 121 ° C for 15 min.

7. The use of a Lemna mutant mutant-47 according to claim 2 in preparing animal feed, characterized in that: The light intensity was 100 μmol×m -2 ×s -1 Lx, 16h light / 8h dark.

8. Use of the Lemna mutant mutant-47 as claimed in any one of claims 1 to 7 in the preparation of a lysine product.

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