Application of ACR11 protein in improving vegetable protein content and urea utilization efficiency

Abstract

The invention provides an application of ACR11 protein in improving the content of plant protein and the utilization efficiency of urea, and provides an application of the ACR11 protein in improving the yield of the plant protein and / or improving the utilization efficiency of the plant to a urea nitrogen fertilizer, and the ACR11 protein can improve the yield of the plant protein and improve the utilization efficiency of the plant to the urea nitrogen fertilizer. The ACR11 protein disclosed by the invention can also improve the utilization efficiency of a plant on urea nitrogen fertilizer, and is specifically characterized in that a transgenic plant formed by transferring the coding gene of the duckweed ACR11 protein disclosed by the invention into a receptor plant is cultured under the condition of taking urea as a nitrogen source; compared with a receptor plant cultured under the same condition, the transgenic plant has higher biomass, higher protein content, higher protein yield and higher protein production efficiency under the same urea nitrogen fertilizer input. The invention provides a new way for improving the utilization efficiency of urea by crops and improving the protein yield of the crops in agriculture, and is beneficial to promoting the sustainable development of green agriculture.

Description

Technical Field

[0001] This application relates to the field of plant genetic engineering, and in particular to the application of ACR11 protein in improving plant protein content and urea utilization efficiency. Background Technology

[0002] The world's population is projected to reach approximately 9 billion by 2050, requiring a protein supply increase of over 50% to meet the demands of this future population growth. Protein, as an essential macronutrient, plays a crucial role in maintaining a healthy diet. Plant-based protein, derived from primary producers, offers nutritional advantages such as low cholesterol, low levels of saturated fat, and high dietary fiber content, contributing to improved dietary structures and representing a significant future source of increased food protein. Currently, edible plant protein primarily comes from common crops such as soybeans, wheat, peas, and corn. However, the yield and protein content of these crops are not only affected by land resources and environmental climate change but are also constrained by low nitrogen fertilizer utilization rates.

[0003] Nitrogen is one of the most important nutrients for plant growth and development. Plants can utilize nitrogen fertilizers including nitrate nitrogen, ammonium nitrogen, and urea, which are used for protein synthesis and various life activities. Ammonium nitrogen can be directly absorbed by plants, converted from other forms of nitrogen fertilizer, or recycled from nitrogen-containing compounds within the plant. Ammonium nitrogen is mainly synthesized into glutamine and glutamate via the glutamine synthase (GS) / glutamate synthase (GOGAT) pathway. Subsequently, glutamine and glutamate further synthesize other amino acids through transamination and deamination. Therefore, GS plays a crucial role in plant nitrogen assimilation, amino acid synthesis, and protein accumulation.

[0004] ACT domain repeats (ACRs) are a family of proteins containing ACT aspartate kinase, chorionic filament mutase, and benzoic acid dehydrogenase domains, which act as amino acid binding sites in the feedback regulation of amino acid metabolic enzymes.

[0005] In existing technologies, the protein yield of crops is easily constrained by the low utilization efficiency of urea nitrogen fertilizer. Summary of the Invention

[0006] In view of this, the purpose of this application is to provide the application of ACR11 protein in improving plant protein content and urea utilization efficiency, so as to solve the problem that the protein yield of crops is easily restricted by the low utilization efficiency of urea nitrogen fertilizer in the prior art.

[0007] The technical solution adopted in this application to solve the above-mentioned technical problems is as follows:

[0008] The embodiments of the present invention provide the application of ACR11 protein in increasing plant protein yield and / or improving the efficiency of plant utilization of urea nitrogen fertilizer.

[0009] In some embodiments, the plants include one or more of duckweed, tobacco, Arabidopsis thaliana, rice, alfalfa, Sudan grass, ryegrass, woolly grass, corn, wheat, sorghum, tea tree, and sugar beet.

[0010] In some embodiments, the ACR11 protein includes duckweed ACR11 protein, the amino acid sequence of which is selected from:

[0011] (a) The amino acid sequence as shown in SEQ ID No. 1; or,

[0012] (b) The amino acid sequence defined by (a) has at least 69% similarity and encodes an amino acid sequence of the same function.

[0013] In some embodiments, the nucleotide sequence of the gene encoding the duckweed ACR11 protein is selected from:

[0014] (c) The nucleotide sequence as shown in SEQ ID NO.2; or,

[0015] (d) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1; or,

[0016] (e) A nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (c) or (d) and encodes a protein with the same function.

[0017] In some embodiments, the coding gene of the duckweed ACR11 protein is recombined into a plant overexpression vector driven by a strong promoter, and the resulting recombinant expression vector is introduced into a recipient plant to overexpress the coding gene of the duckweed ACR11 protein, thereby obtaining a transgenic plant. The transgenic plant is cultured under conditions where the nitrogen source contains urea, and the transgenic plant has higher protein yield and urea nitrogen fertilizer utilization efficiency compared to the recipient plant.

[0018] In some embodiments, the transgenic plant has higher protein yield and urea nitrogen fertilizer utilization efficiency than the recipient plant, which is manifested in that the transgenic plant has higher amino acid content and protein content than the recipient plant under the same urea nitrogen fertilizer input, and the transgenic plant has significantly higher urea utilization efficiency than the recipient plant.

[0019] In some embodiments, the recipient plant includes one or more of duckweed, tobacco, Arabidopsis thaliana, rice, alfalfa, Sudan grass, ryegrass, woolly grass, corn, wheat, sorghum, tea tree, and sugar beet.

[0020] In some embodiments, a method for detecting whether the transgenic plant contains the gene encoding the duckweed ACR11 protein includes at least one of the following methods: 1) designing primers based on the sequence of the gene encoding the duckweed ACR11 protein and detecting it using PCR; 2) extracting DNA from the plant and performing Southern hybridization; 3) extracting protein from the plant and performing Western blot analysis; 4) extracting RNA from the plant and performing real-time quantitative PCR to detect gene expression quantification.

[0021] The ACR11 protein of this application can increase plant protein yield and improve the utilization efficiency of urea nitrogen fertilizer in plants. Specifically, transgenic plants formed by transferring the duckweed ACR11 protein encoding gene of this invention into recipient plants and then cultivating them under urea as a nitrogen source exhibit higher biomass, protein content, protein yield, and protein production efficiency with the same urea nitrogen fertilizer input compared to recipient plants cultivated under the same conditions. This invention provides a new approach to improving crop urea utilization efficiency and increasing crop protein yield in agriculture. Increased crop urea utilization efficiency means that comparable or better results can be achieved with a smaller urea application rate. This solves the problem that crop protein yield is easily constrained by low urea nitrogen fertilizer utilization efficiency in existing technologies, and is conducive to promoting the sustainable development of green agriculture. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings of the embodiments will be briefly described below. Obviously, the drawings described below only relate to some embodiments of this application and are not intended to limit this application, wherein:

[0023] Figure 1 This is a positive PCR detection result for the ACR11 overexpressing transgenic duckweed ZH0403 plant.

[0024] Figure 2 The results show the expression levels, growth rate, and protein content of the ACR11 gene in wild-type duckweed ZH0403 and transgenic duckweed ZH0403.

[0025] Figure 3 The results show the protein yield and protein production efficiency of wild-type duckweed ZH0403 and transgenic duckweed ZH0403.

[0026] Figure 4 This is a positive PCR detection result from ACR11 overexpressing transgenic tobacco plants.

[0027] Figure 5These are the morphologies of wild-type tobacco and genetically modified tobacco after three weeks of cultivation.

[0028] Figure 6 These are the results of tests on the biomass, protein content, and protein yield of wild-type tobacco and genetically modified tobacco.

[0029] Figure 7 This is a positive PCR detection result for ACR11 overexpressing transgenic rice plants.

[0030] Figure 8 It is the morphology of wild-type rice Zhonghua 11 and transgenic rice Zhonghua 11.

[0031] Figure 9 The results show the biomass, protein content, and protein yield of wild-type rice Zhonghua 11 and transgenic rice Zhonghua 11 after four weeks of cultivation. Detailed Implementation

[0032] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Furthermore, it should be understood that the specific embodiments described herein are only for illustration and explanation of this application and are not intended to limit this application.

[0033] In this application, "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0034] The embodiments of the present invention provide the application of ACR11 protein in increasing plant protein yield.

[0035] In some embodiments, the ACR11 protein is used to improve the efficiency of plant utilization of urea nitrogen fertilizer.

[0036] This application discovers a novel use for the ACR11 protein: it can increase plant protein yield and improve the efficiency of plant utilization of urea nitrogen fertilizer. Specifically, transgenic plants formed by transferring the duckweed ACR11 protein encoding gene of this invention into recipient plants and then cultivating them under urea-based nitrogen source conditions exhibit higher biomass, protein content, protein yield, and protein production efficiency with the same urea nitrogen fertilizer input compared to recipient plants cultivated under the same conditions. This invention provides a new approach to improving crop urea utilization efficiency and increasing crop protein yield in agriculture. Increased crop urea utilization efficiency means that comparable or better results can be achieved with smaller urea application rates, solving the problem that crop protein yield is easily constrained by low urea nitrogen fertilizer utilization efficiency in existing technologies, and promoting the sustainable development of green agriculture.

[0037] Furthermore, the plants include one or more of duckweed, tobacco, Arabidopsis thaliana, rice, alfalfa, Sudan grass, ryegrass, woolly grass, corn, wheat, sorghum, tea tree, and sugar beet.

[0038] Furthermore, the ACR11 protein includes the duckweed ACR11 protein, the amino acid sequence of which is selected from:

[0039] (a) The amino acid sequence as shown in SEQ ID No. 1; or,

[0040] (b) The amino acid sequence defined by (a) has at least 69% similarity and encodes an amino acid sequence of the same function.

[0041] It is understood that (b) is an amino acid sequence defined by (a) that has at least 69% similarity and encodes an amino acid sequence of the same function, wherein (b) is an amino acid sequence defined by (a) that has at least 69%, 70%, 73%, 75%, 76%, 78%, 80%, 81%, 83%, 85%, 87%, 88%, 89%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% similarity and encodes an amino acid sequence of the same function.

[0042] Furthermore, the nucleotide sequence of the gene encoding the duckweed ACR11 protein is selected from:

[0043] (c) The nucleotide sequence as shown in SEQ ID NO.2; or,

[0044] (d) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1; or,

[0045] (e) A nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (c) or (d) and encodes a protein with the same function.

[0046] It is understood that (e) has at least 69% similarity to the nucleotide sequence defined in (c) or (d) and encodes a nucleotide sequence that has at least 69%, 70%, 73%, 75%, 76%, 78%, 80%, 81%, 83%, 85%, 87%, 88%, 89%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% similarity to the nucleotide sequence defined in (c) or (d) and encodes a nucleotide sequence that has the same function.

[0047] Furthermore,

[0048] SEQ ID NO.1:

[0049] MVLAMATGSVGICGRLKSFGGEHQLCPAGGSSRGDLRLVLSNSSRKSGSLLAINLVVAALPATTVDGGSYNDTEEVPTPKVFIDQDSDPDATIVEITFGDRLGALLDTMRALKSLGLNVVKANVFLDSSGKHNTFAITKASTG RKVDDPELLEAIRLTIINNLIEYHPESSSRLAMGATFGVEPPSEVVDIDIATHVHVYDDGPDRSLLVVETADRPGLLVDIVKVVTDISVAIESGEFDTEGLLAKAKFHVSYRGKAIIKPLQQVLANSLRYFLRRPFTEEASF*

[0050] SEQ ID No. 2:

[0051] ATGGTACTGGCCATGGCGACCGGAAGCGTTGGAATCTGCGGCCGCTTAAAGTCTTTTGGCGGCGAGCACCAGTTATGCCCCGCTGGCGGCAGTTCTCGCGGCGATCTGCGGCTCGTGCTTTCGAATAGCTCGAGAAAAAGTGGATCTCTCTTGGCCATCAATCTCGTCGTGGCGGCGCTACCGGCTACTACTGTGGATGGGGGGAGCTATAACGATACCGAAGAGGTTCCCACTCCGAAAGTCTTCATCGATCAGGATTCCGATCCCGACGCAACCATAGTCGAGATCACTTTCGGCGATCGTCTCGGGGCGCTTCTGGACACAATGAGAGCGCTGAAGAGCCTCGGGCTGAACGTGGTCAAGGCGAACGTGTTCCTCGACTCATCCGGCAAGCACAACACATTCGCCATCACGAAAGCGTCCACTGGTCGTAAGGTCGACGATCCCGAGCTGCTCGAAGCTATACGCTTGACGATCATCAATAATCTGATCGAGTACCATCCGGAATCCAGTTCGCGGTTGGCAATGGGGGCAACCTTCGGAGTTGAGCCTCCGTCAGAGGTGGTTGACATCGATATAGCTACCCATGTCCACGTGTACGACGATGGCCCGGATCGCAGTTTGCTGGTGGTAGAGACAGCAGACCGCCCCGGGCTTCTCGTTGACATCGTCAAGGTGGTCACCGACATTAGCGTGGCCATTGAGTCAGGGGAATTCGACACCGAGGGGCTCCTCGCCAAGGCGAAGTTCCACGTCAGCTACCGGGGGAAAGCCATCATCAAGCCCCTGCAACAGGTGTTGGCTAACAGCCTGCGTTATTTCCTGAGGAGGCCCTTCACAGAGGAAGCCAGCTTCTGA

[0052] Furthermore, the coding gene of the duckweed ACR11 protein is recombined into a plant overexpression vector driven by a strong promoter. The resulting recombinant expression vector is introduced into a recipient plant to overexpress the coding gene of the duckweed ACR11 protein, resulting in a transgenic plant. The transgenic plant is cultured under conditions where urea is present as a nitrogen source. The transgenic plant exhibits higher protein yield and urea nitrogen fertilizer utilization efficiency compared to the recipient plant.

[0053] Furthermore, the transgenic plant has higher protein yield and urea nitrogen fertilizer utilization efficiency compared to the recipient plant. This is manifested in that the transgenic plant has higher amino acid and protein content than the recipient plant under the same urea nitrogen fertilizer input, and the transgenic plant has significantly higher urea utilization efficiency compared to the recipient plant.

[0054] For example, the transgenic plant has higher biomass, protein content, protein yield, and protein production efficiency compared to the recipient plant.

[0055] Furthermore, the recipient plants include one or more of the following: duckweed, tobacco, Arabidopsis thaliana, rice, alfalfa, Sudan grass, ryegrass, woolly grass, corn, wheat, sorghum, tea tree, and sugar beet.

[0056] Furthermore, the method for detecting whether the transgenic plant contains the encoding gene of the duckweed ACR11 protein includes at least one of the following methods: 1) designing primers based on the encoding gene sequence of the duckweed ACR11 protein and detecting it using PCR; 2) extracting DNA from the plant and performing Southern hybridization; 3) extracting protein from the plant and performing Western blot analysis; 4) extracting RNA from the plant and performing real-time quantitative PCR to detect gene expression quantification analysis.

[0057] In this application, when cultured under urea as the nitrogen source, the transgenic plant exhibits a higher ACR11 gene expression level compared to the recipient plant, which is beneficial for increasing biomass and protein content. For example, when cultured under urea as the nitrogen source, the transgenic duckweed ZH0403, formed by incorporating the duckweed ACR11 protein-coding gene, shows a 14-fold increase in ACR11 gene expression compared to the wild-type duckweed ZH0403. Simultaneously, the biomass and protein content of the transgenic duckweed ZH0403 plant are 12% and 25% higher than those of the wild-type duckweed ZH0403 plant, respectively. Compared to the wild-type duckweed ZH0403 plant, the protein yield of the transgenic duckweed ZH0403 plant is increased by approximately 50%, and the protein production efficiency under the same urea nitrogen fertilizer input is increased by approximately 29%. Transforming the duckweed ACR11 protein-coding gene into other plants, such as tobacco, into transgenic plants cultured under urea as the nitrogen source can also produce similar effects to the aforementioned transgenic duckweed. For example, transgenic tobacco plants cultured with urea as the nitrogen source, incorporating the ACR11 protein-coding gene from duckweed, exhibited higher biomass and protein content than wild-type tobacco. This indicates that, under the same nitrogen supply, the urea utilization rate of transgenic plants increased, meaning that urea nitrogen fertilizer utilization efficiency was higher. Based on these experimental results, in practical applications, the ACR11 protein-coding gene from duckweed can be transferred into more other plants to increase their biomass and protein content, thereby improving the utilization efficiency of urea nitrogen fertilizer.

[0058] The following examples further illustrate the duckweed ACR11 protein, its encoding gene, and its application in improving plant protein yield and urea utilization efficiency provided by the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in this technical field. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] In the following embodiments, the duckweed ZH0104 and duckweed ZH0403 are derived from the duckweed germplasm resource bank of the Chengdu Institute of Biology, Chinese Academy of Sciences.

[0060] The duckweed ZH0104 is Landoltia punctata ZH0104;

[0061] The duckweed ZH0403 is Lemna minor ZH0403.

[0062] In the following examples, the formulation of the modified nitrogen-free Hoagland medium is as follows: 1 mM CaCl2, 3 mM KCl, 1 mM KH2PO4, 0.14 mM HCl, 4 μM Tartaric acid, 4 μM FeCl3·6H2O, 0.4 mM MgSO4·7H2O, 9.25 μM M3BO3, 0.15 μM ZnSO4·7H2O, 0.1 μM Na2MoO4·2H2O, 0.64 μM CuSO4·5H2O, 3.65 μM MnCl2·4H2O, 6 μM EDTA, and 0.01 mM KOH.

[0063] Example 1: Cloning of the full-length CDS of the duckweed ACR11 gene

[0064] First, the ACR11 gene coding region sequence published in Arabidopsis thaliana was compared with the reference genome sequence of *Lemna minor* ZH0104 to obtain the homologous *Lemna minor* ACR11 gene coding region sequence. Then, primers were designed based on the *Lemna minor* ACR11 gene coding region sequence: forward primer 5'ATGGTACTGGCCATGGCG3' and reverse primer 5'TCAGAAGCTGGCTTCCTCTGT3'.

[0065] Total RNA was extracted from the *Lemna minor* strain ZH0104 using the Promega East Super Total RNA Extraction Kit according to the manufacturer's instructions. The RNA was then reverse transcribed into cDNA using the Prime Script™ RT Reagent kit (Takara). Using the reverse-transcribed cDNA as a template, the full-length CDS sequence of the *Lemna minor* ACR11 gene was amplified by PCR using the aforementioned forward and reverse primers, and then sequenced. Its nucleotide sequence is shown in SEQ ID NO.1 of the sequence listing. The nucleotide sequence shown in SEQ ID NO.1 encodes the *Lemna minor* ACR11 protein, whose amino acid sequence is shown in SEQ ID NO.2.

[0066] PCR reaction system:

[0067]

[0068] PCR amplification procedure:

[0069]

[0070] Example 2: Construction of ACR11 transgenic expression vector

[0071] Recombinant primers (5'GAGCTCGGTACCGGATCCATGGTACTGGCCATGG3' and 5'CATAGATCTGCCAAGCTTGAAGCT GGCTTCCTCTGTGA3') containing BamH1 and HindIII restriction sites were designed. PCR amplification of the ACR11 coding region was performed using cDNA from the duckweed strain ZH0104 as a template, following the same PCR amplification system and procedure as in Example 1. The amplified fragment was then recombined with the pCAMBIA2301 vector, which had been double-digested with BamH1 and HindIII, to obtain a fusion overexpression vector.

[0072] Example 3: Obtaining and detecting ACR11-overexpressing transgenic duckweed plants

[0073] Using Agrobacterium GV3101-mediated genetic transformation, the fusion overexpression vector obtained in Example 2 was introduced into the wild-type duckweed ZH0403 line, followed by screening with 100 mg / L G418 antibiotic. Genomic DNA of the overexpressing transgenic duckweed ZH0403 was extracted and amplified by PCR. The primers for detection were the primers for the vector's resistance gene (5'CTGGGAACTACTCACACATT3' and 5'GAAAGCTCGAGAGAGATA3'), and the PCR amplification system and procedure were the same as in Example 1. The identified and obtained positive transgenic duckweed ZH0403 was cultured under urea nitrogen fertilizer, and its growth rate, protein content, and protein yield were measured.

[0074] To compare the differences between transgenic duckweed ZH0403 and wild-type duckweed ZH0403 under urea nitrogen fertilizer cultivation, transgenic and wild-type duckweed ZH0403 plants were hydroponically cultured in a modified nitrogen-free Hoagland medium supplemented with 2.5 mM urea. After one week of cultivation, the transgenic and wild-type duckweed ZH0403 plants were harvested, and the expression level of ACR11 gene, growth rate, protein content, biomass, protein yield, and protein production efficiency (g protein yield / g N input, abbreviated as g / g N) were measured. All treatments were performed in at least three biological replicates.

[0075] Figure 1 This is a positive PCR detection result for the ACR11 overexpressing transgenic duckweed ZH0403 plant. Figure 2 The results show the expression level, growth rate, protein content, and biomass of the ACR11 gene in wild-type duckweed ZH0403 and transgenic duckweed ZH0403. Figure 3 The results show the protein yield and protein production efficiency of wild-type duckweed ZH0403 and transgenic duckweed ZH0403. Figures 2-3In the text, WT and OE-ACR11 represent wild-type duckweed ZH0403 and transgenic duckweed ZH0403, respectively.

[0076] Depend on Figure 2 It can be seen that, compared with the wild-type duckweed ZH0403, the ACR11 gene expression level of the transgenic duckweed ZH0403 is significantly increased; the growth rate and protein content of the transgenic duckweed ZH0403 plants are increased by approximately 12% and 25% respectively compared with the wild-type duckweed ZH0403 plants; and the biomass of the transgenic duckweed ZH0403 plants is increased by approximately 10% compared with the wild-type duckweed ZH0403 plants. Since nitrogen use efficiency is defined as the biomass or grain yield harvested under a certain amount of nitrogen supply, an increase in the biomass or yield harvested under a certain amount of nitrogen supply means an increase in nitrogen use efficiency. In this embodiment, urea is used as the nitrogen source. Under a certain amount of urea, the harvested biomass increases, which means an increase in urea use efficiency. The simultaneous increase in protein content and biomass means an increase in protein yield. Figure 3 It can be seen that, compared with the wild-type duckweed ZH0403 plant, the transgenic duckweed ZH0403 plant has increased protein yield and protein production efficiency per unit nitrogen input by about 50% and 29%, respectively.

[0077] Example 4: Obtaining and detecting ACR11 overexpressing transgenic tobacco

[0078] The Agrobacterium GV3101-mediated genetic transformation method was used to introduce the fusion overexpression vector obtained in Example 2 into wild-type tobacco, followed by screening with G418 antibiotic. Genomic DNA was extracted from the overexpressing transgenic tobacco and subjected to PCR amplification. The detection primers were the primers for the vector's resistance gene (5'CTGGGAACTACTCACACATT3' and 5'GAAAGCTCGAGAGAGATA3'). The PCR amplification system and procedure were the same as in Example 1. Figure 4 As shown. The identified and obtained positive transgenic tobacco plants were cultured under urea nitrogen fertilizer and their biomass and protein content were tested.

[0079] To compare the differences between transgenic and wild-type tobacco under urea nitrogen fertilizer cultivation, transgenic and wild-type tobacco seedlings were hydroponically cultured in a modified nitrogen-free Hoagland medium supplemented with 2.5 mM urea, with the culture medium changed weekly. After three weeks of cultivation, the biomass and protein content of transgenic and wild-type tobacco were measured. Six biological replicates were set up for all treatments.

[0080] Figure 4 This is a positive PCR detection result from ACR11 overexpressing transgenic tobacco plants. Figure 5These are photos of wild-type tobacco and genetically modified tobacco after three weeks of cultivation. Figure 5 In the text, WT and OE-ACR11 represent wild-type tobacco and genetically modified tobacco, respectively. Figure 6 These are the results of tests on the biomass, protein content, and protein yield of wild-type tobacco and genetically modified tobacco.

[0081] Depend on Figure 6 It was found that, after three weeks of cultivation in a modified nitrogen-free Hoagland medium supplemented with 2.5 mM urea, the transgenic tobacco grew better than wild-type tobacco. Compared to wild-type tobacco, the biomass and protein content of transgenic tobacco increased by approximately 18% and 28%, respectively. The increase in biomass indicates increased urea utilization efficiency, while the simultaneous increase in both biomass and protein content indicates increased protein yield. Considering protein yield, the protein yield of transgenic tobacco increased by 29% compared to wild-type tobacco.

[0082] Example 5: Obtaining and detecting ACR11 overexpressing transgenic rice

[0083] The Agrobacterium GV3101-mediated genetic transformation method was used to introduce the fusion overexpression vector obtained in Example 2 into wild-type rice Zhonghua 11, followed by screening with the antibiotic G418. Genomic DNA of the overexpressing transgenic rice was extracted and amplified by PCR. The primers for detection were the primers for the vector's resistance gene (5'CTGGGAACTACTCACACATT3' and 5'GAAAGCTCGAGAGAGATA3'). The PCR amplification system and procedure were the same as in Example 1. The identified and obtained positive transgenic rice seedlings were cultured under urea nitrogen fertilizer, and the biomass and protein content of the plants were measured.

[0084] To examine the phenotypic changes in transgenic rice cultured with urea as the sole nitrogen source, seedlings of transgenic rice and wild-type rice Zhonghua 11 were hydroponically cultured in a modified nitrogen-free Hoagland medium supplemented with 2.5 mM urea, with the medium changed weekly. After four weeks of culture, biomass and protein content of both transgenic and wild-type Zhonghua 11 rice were measured. All treatments were performed in at least six biological replicates.

[0085] Figure 7 The test result is positive for ACR11 overexpressing transgenic rice. Figure 8 These are photos of wild-type rice Zhonghua 11 and genetically modified rice after four weeks of cultivation. Figure 9 (A), (B), and (C) are the results of the detection of biomass, protein content, and protein yield of wild-type rice Zhonghua 11 and transgenic rice, respectively. Figures 8-9 In the text, WT and OE-ACR11 represent wild-type rice Zhonghua 11 and genetically modified rice, respectively.

[0086] Depend on Figure 8 It can be seen that, after four weeks of cultivation in a modified nitrogen-free Hoagland medium supplemented with 2.5 mM urea, the transgenic rice showed better growth than the wild-type rice Zhonghua 11; for example... Figure 9 As shown, compared to the wild-type rice Zhonghua 11, the biomass and protein content of transgenic rice increased by approximately 12.2% and 19.4%, respectively. Considering protein yield, compared to wild-type rice plants, the protein yield of transgenic rice plants increased by 31.12%, meaning that under the same nitrogen fertilizer input, the urea utilization efficiency of transgenic rice was significantly increased.

[0087] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this application. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this application. Such modifications, improvements, and corrections are suggested in this application, and therefore remain within the spirit and scope of the exemplary embodiments of this application.

Claims

1. Application of ACR11 protein in increasing plant protein yield and / or improving plant utilization efficiency of urea nitrogen fertilizer.

2. The application of the ACR11 protein according to claim 1 in increasing plant protein yield and / or improving the plant's utilization efficiency of urea nitrogen fertilizer, characterized in that: The plants include one or more of the following: duckweed, tobacco, Arabidopsis thaliana, rice, alfalfa, Sudan grass, ryegrass, woolly grass, corn, wheat, sorghum, tea tree, and sugar beet.

3. The application of the ACR11 protein according to claim 1 in increasing plant protein yield and / or improving the plant's utilization efficiency of urea nitrogen fertilizer, characterized in that: The ACR11 protein includes the duckweed ACR11 protein, the amino acid sequence of which is selected from: (a) The amino acid sequence as shown in SEQ ID No. 1; or, (b) The amino acid sequence defined by (a) has at least 69% similarity and encodes an amino acid sequence of the same function.

4. The application of the ACR11 protein according to claim 3 in increasing plant protein yield and / or improving the efficiency of plant utilization of urea nitrogen fertilizer, characterized in that: The nucleotide sequence of the gene encoding the duckweed ACR11 protein is selected from: (c) The nucleotide sequence as shown in SEQ ID NO.2; or, (d) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID No. 1; or, (e) A nucleotide sequence that has at least 69% similarity to the nucleotide sequence defined in (c) or (d) and encodes a protein with the same function.

5. The application of the ACR11 protein according to claim 3 in increasing plant protein yield and / or improving the plant's utilization efficiency of urea nitrogen fertilizer, characterized in that: The coding gene of the duckweed ACR11 protein was recombined into a plant overexpression vector driven by a strong promoter. The resulting recombinant expression vector was introduced into a recipient plant to overexpress the coding gene of the duckweed ACR11 protein, resulting in a transgenic plant. The transgenic plant was cultured under conditions where urea was present as a nitrogen source. The transgenic plant exhibited higher protein yield and urea nitrogen fertilizer utilization efficiency compared to the recipient plant.

6. The application of the ACR11 protein according to claim 5 in increasing plant protein yield and / or improving the efficiency of plant utilization of urea nitrogen fertilizer, characterized in that: The transgenic plant has higher protein yield and urea nitrogen fertilizer utilization efficiency than the recipient plant. This is manifested in that the transgenic plant has higher amino acid and protein content than the recipient plant under the same urea nitrogen fertilizer input, and the transgenic plant has significantly higher urea utilization efficiency than the recipient plant.

7. The application of the ACR11 protein according to claim 5 or 6 in increasing plant protein yield and / or improving the efficiency of plant utilization of urea nitrogen fertilizer, characterized in that: The recipient plants include one or more of the following: duckweed, tobacco, Arabidopsis thaliana, rice, alfalfa, Sudan grass, ryegrass, woolly grass, corn, wheat, sorghum, tea tree, and sugar beet.

8. The application of the ACR11 protein according to claim 5 in increasing plant protein yield and / or improving the efficiency of plant utilization of urea nitrogen fertilizer, characterized in that: A method for detecting whether the transgenic plant contains the gene encoding the duckweed ACR11 protein includes at least one of the following methods: 1) designing primers based on the gene sequence encoding the duckweed ACR11 protein and detecting it using PCR; 2) extracting DNA from the plant and performing Southern hybridization; 3) extracting protein from the plant and performing Western blot analysis; 4) extracting RNA from the plant and performing real-time quantitative PCR to detect gene expression quantification.

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