Application of OsFTL1 and coding gene thereof in improving protein content and chlorophyll content

Abstract

The invention discloses an application of OsFTL1 and a coding gene thereof in improving protein content and chlorophyll content. The OsFTL1 disclosed by the invention is derived from rice and is a protein as shown in SEQ ID No. 1. Experiments prove that the OsFTL1 and the coding gene thereof can increase the chlorophyll content and the protein content, and under the normal nitrogen condition, the contents of chlorophyll a, chlorophyll b and carotenoid of leaves of the OsFTL1 transgenic plant are higher than those of wild plants; under the condition of low nitrogen, the leaf protein content of the OsFTL1 transgenic plant is obviously higher than that of a wild type; after the plants growing in the field are harvested, the grain protein content of the OsFTL1 transgenic plants is obviously higher than that of the wild plants. The results show that the OsFTL1 and the coding gene thereof can promote nitrogen assimilation, increase pigment synthesis and increase protein content, and have good application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically the application of OsFTL1 and its encoding gene in increasing protein and chlorophyll content. Background Technology

[0002] Proteins are the primary carriers of life activities, and crop grains and leaves provide direct sources of protein and amino acids for humans and animals. Chlorophyll is a pigment molecule essential for capturing light energy during photosynthesis, converting it into electrical and chemical energy to power the carbon assimilation process. The synthesis of both proteins and chlorophyll depends on the absorption and utilization of nitrogen by crops. Nitrogen is an essential macronutrient for crop growth and development, and a crucial element in the composition of chlorophyll, proteins, and nucleic acids. Applying nitrogen fertilizer can enhance crop photosynthesis, increase chlorophyll content, protein content, tiller number, biomass, and yield. For nearly half a century, the excessive application of nitrogen fertilizer in pursuit of high crop yields has caused serious resource waste and environmental pollution. To address these problems, researchers have cloned many genes regulating crop nitrogen absorption, transport, and assimilation, laying the foundation for improving crop nitrogen use efficiency. However, our understanding of these nitrogen-utilizing genes is still far from complete. Whether these genes affect carbon assimilation and metabolism, their genetic cooperation in crop nitrogen uptake, transport, and assimilation, and the molecular mechanisms by which protein-level modifications alter their function remain unclear. For example, these proteins exhibit numerous post-transcriptional modifications, such as phosphorylation, which play a crucial role in their function. However, how these modifications occur and the mechanisms of protein-level interactions are unclear, limiting the construction of nitrogen-utilizing molecular networks and the breeding of nitrogen-efficient varieties.

[0003] In rice, nitrogen absorption and transport occur in two forms: nitrate nitrogen and ammonium nitrogen. Nitrate nitrogen absorption and transport are accomplished through nitrate transporters (NRTs), which mainly include… OsNRT1 , OsNRT1.1A, OsNRT1.1B , OsNRT2.1 , OsNRT2.2 , OsNRT2.3 , OsNRT2.4 etc., among which OsNRT1.1B There are differences between japonica and indica rice; indica rice... OsNRT1.1B Alleles exhibit higher efficiency in nitrate absorption and transport, resulting in higher nitrogen fertilizer use efficiency in indica rice. Ammonium nitrogen absorption is accomplished through ammonium transporters (AMTs), and rice primarily absorbs and transports ammonium nitrogen via... OsAMT1;1 , OsAMT1;2 , OsAMT1;3 , OsAMT1;4 , OsAMT2;1and OsAMT5 Ammonium transporter completion. Overexpression. OsAMT1.1 It significantly enhances the absorption efficiency of ammonium ions in rice, thus improving nitrogen fertilizer use efficiency and grain yield. During nitrogen assimilation in rice, nitrate is absorbed and then reduced to nitrite by nitrate reductase, which further reduces it to ammonium ions by nitrite reductase. Indica rice nitrate reductase OsNR2 has higher activity than japonica rice, resulting in higher nitrogen fertilizer use efficiency. Overexpression in indica rice... OsNR2 Increase rice tillering and improve rice yield. Glutamine synthetase (GS) plays an important role in nitrogen assimilation, and rice has three cytoplasm-localized glutamine synthetase genes. OsGS1;1 , OsGS1;2 and OsGS1;3 These three substances are mainly expressed in leaves, roots, and spikelets, respectively. OsGS1;1 It plays an important role in the growth, development, and grain filling of rice. OsGS1;1 After gene silencing, the content of free glutamine in roots and leaves decreased, and the growth and grain filling of the mutant were significantly delayed. In addition, rice also has a chloroplast glutamine synthase gene. OsGS2 Decreased expression of wheat in rice leads to reduced leaf pigment content and GS enzyme activity, resulting in shorter plant height and fewer tillers. TaGS1 This gene can increase the activity of glutamine synthase (GS) in various tissues of rice, leading to a 31-40% increase in nitrogen use efficiency and a 15-36% increase in rice yield. Besides glutamine synthase, asparagine synthase is also a key enzyme in nitrogen assimilation, derived from teosinte, the ancestor of maize. THP9 The gene encodes an asparagine synthase 4, which can significantly improve nitrogen use efficiency and grain protein content in cultivated maize. The cloning and functional identification of these nitrogen absorption, translocation, and assimilation genes lay a solid foundation for the genetic improvement of nitrogen efficiency in crops. However, the unclear interaction and regulatory mechanisms of these genes at the protein level limit the construction and application of nitrogen use regulatory networks.

[0004] Therefore, in-depth research is needed on the effects of nitrogen utilization genes on protein content, chlorophyll content, and carbon and nitrogen metabolism, as well as the genetic mechanisms of nitrogen utilization, and the construction of a molecular regulatory network for nitrogen utilization, to provide support for the breeding of nitrogen-efficient rice varieties. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to regulate the protein content and / or chlorophyll content of plants.

[0006] To address the aforementioned technical problems, the present invention first provides any of the following applications of proteins or substances that regulate the content or activity of said proteins: D1) Regulates plant protein content; D2) Prepare products that regulate plant protein content; D3) Regulates plant chlorophyll content; D4) Prepare products that regulate plant chlorophyll content; D5) Cultivating plants with altered protein content; D6) Prepare products from plants with altered protein content; D7) Cultivating plants with altered chlorophyll content; D8) Prepare products from plants with altered chlorophyll content; The protein is derived from rice and is named OsFTL1, which is represented by A1), A2), A3), or A4). A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) A protein that has the same function as the amino acid sequence shown in SEQ ID No. 1 in the sequence listing, but with one or more amino acid residues substituted and / or deleted and / or added; A3) A protein derived from rice, corn, sorghum, millet, goatgrass, two-stalked short-stalked grass or wheat that has 75% or more identity with SEQ ID No. 1 and has the same function as the protein described in A1); A4) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1), A2), or A3).

[0007] The OsFTL1 protein in A2) and A3) above is a protein with 75% or more amino acid sequence identity and the same function as the protein shown in SEQ ID No. 1. Identity refers to the similarity of the amino acid sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained. The phrase "having 75% or more of the sameness" means having 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sameness.

[0008] The OsFTL1 protein in A2) and A3) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0009] The gene encoding the OsFTL1 protein in A2) and A3) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID No. 2, and / or by performing a missense mutation of one or more base pairs, and / or by attaching a tag coding sequence to its 5′ and / or 3′ ends. The DNA molecule shown in SEQ ID No. 2 encodes the OsFTL1 protein shown in SEQ ID No. 1.

[0010] The tag described in A4) can be a polypeptide or protein fused with the target protein using in vitro DNA recombination technology, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be Poly-Arg, Poly-His, FLAG, Strep-tag II, c-myc, MBP tag, HA tag, GST tag, and / or SUMO tag, etc.

[0011] In the above applications, the substance that regulates the content or activity of OsFTL1 is any one of the following B1) to B7): B1) Nucleic acid molecules encoding OsFTL1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

[0012] In the above applications, the nucleic acid molecule described in B1) may be as follows: (b11), (b12), (b13), (b14), or (b15) b11) The coding sequence is the cDNA molecule or DNA molecule of SEQ ID No. 2 in the sequence listing; b12) The DNA molecule shown in SEQ ID No. 2 of the sequence listing; b13) The DNA molecule shown in SEQ ID No. 3 of the sequence listing; b14) has 75% or more identity with the nucleotide sequence defined by b11) or b12) or b13) and encodes a DNA molecule that encodes OsFTL1; b15) hybridizes under strict conditions with a nucleotide sequence defined by b11) or b12) or b13) or b14) and encodes a DNA molecule of OsFTL1.

[0013] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0014] Those skilled in the art can readily mutate the nucleotide sequence encoding the OsFTL1 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence of the OsFTL1 protein isolated in this invention, provided they encode and function the OsFTL1 protein, are derived from and equivalent to the nucleotide sequence of this invention.

[0015] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the protein of SEQ ID No. 1 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0016] In the above applications, the stringent conditions may be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO4 and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS.

[0017] The aforementioned 75% or higher identity can be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0018] In the above application, the expression cassette (OsFTL1 gene expression cassette) containing a nucleic acid molecule encoding the OsFTL1 protein described in B2) refers to DNA capable of expressing the OsFTL1 protein in host cells. This DNA may include not only a promoter to initiate OsFTL1 gene transcription but also a terminator to terminate OsFTL1 gene transcription. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; wound-inducible promoters from tomatoes, leucine aminopeptidase ("LAP", Chao et al. (1999) PlantPhysiol 120: 979-992); chemically induced promoters from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both induced by methyl jasmonic acid); heat shock promoters (US Patent 5,187,267); tetracycline-inducible promoters (US Patent 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)); and promoters specific to seed storage proteins (e.g., beta-carotene, napin, etc.). The promoters of oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J.4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are cited in full.Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627.

[0019] Recombinant vectors containing the OsFTL1 gene expression cassette can be constructed using existing expression vectors. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA). The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as Agrobacterium crown gall tumor inducing (Ti) plasmid genes (e.g., carmine synthase genes). NosThe untranslated regions transcribed at the 3' end of plant genes (such as soybean storage protein genes) have similar functions. When constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (e.g., those encoding enzymes that produce color changes). GUS Genes, luciferase genes, etc.), antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics). nptII Genes that confer resistance to the herbicide phosphinic acid bar Genes that confer resistance to the antibiotic hygromycin hph Genes, and the genes that confer resistance to methotrexate dhfr Genes such as EPSPS genes (which confer resistance to glyphosate) or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose, can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.

[0020] In the above applications, the vector can be a plasmid, granule, bacteriophage, or viral vector. Specifically, the plasmid can be the pTCK303 vector.

[0021] B3) The recombinant vector may specifically be pTCK303- OsFTL1 The pTCK303- OsFTL1 The DNA fragment between the BamHI and SacI recognition sequences of the pTCK303 vector is replaced with the one shown in SEQ ID No. 2. OsFTL1 The recombinant vector obtained from the gene. pTCK303- OsFTL1 It can be expressed under the drive of the maize Ubiqutin (UBI) promoter. OsFTL1 The protein encoded by the gene (i.e., the OsFTL1 protein shown in SEQ ID No. 1).

[0022] In the above applications, the microorganisms can be yeast, bacteria, algae, or fungi. Among them, bacteria can be Agrobacterium, such as Agrobacterium EHA105.

[0023] In the above applications, the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs do not include propagation material.

[0024] In the above applications, the substance that regulates the content or activity of OsFTL1 can be a substance that increases the content or activity of OsFTL1, the substance that regulates the plant protein content can be a substance that increases the plant protein content, the substance that regulates the plant chlorophyll content can be a substance that increases the plant chlorophyll content, the substance that changes the protein content can be a substance that increases the protein content, and the substance that changes the chlorophyll content can be a substance that increases the chlorophyll content.

[0025] The present invention also provides any of the following methods: X1) A method for cultivating plants with increased protein content and / or increased chlorophyll content, including increasing the content or activity of OsFTL1 in a recipient plant to obtain a target plant with increased protein content and / or increased chlorophyll content compared to the recipient plant. X2) A method for reducing the protein content and / or chlorophyll content of plants, including reducing the content or activity of OsFTL1 in a recipient plant to obtain a target plant with increased protein content and / or increased chlorophyll content compared to the recipient plant, thereby achieving an increase in the protein content and / or chlorophyll content of plants.

[0026] The methods described in X1) and X2) can be implemented by introducing the encoding gene of OsFTL1 into the recipient plant and expressing the encoding gene.

[0027] In the above method, the encoding gene can be the nucleic acid molecule described in B1).

[0028] In the above method, the OsFTL1 encoding gene can be modified as follows before being introduced into the recipient plant to achieve better expression: 1) Modify and optimize according to actual needs to enable efficient gene expression; for example, the codons of the encoding gene of OsFTL1 described in this invention can be changed to conform to plant preference while maintaining the amino acid sequence of the gene. During the optimization process, it is best to maintain a certain GC content in the optimized coding sequence to achieve the best high-level expression of the introduced gene in the plant. The GC content can be 35%, more than 45%, more than 50%, or more than about 60%. 2) Modify the gene sequence adjacent to the initiation methionine to enable efficient translation initiation; for example, by using a sequence known to be effective in plants. 3) Linked to promoters of various plant expression to facilitate their expression in plants; the promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-selective, and tissue-specific promoters; the selection of promoters will vary with the time and space requirements of expression, and also depends on the target species; for example, tissue or organ-specific expression promoters, depending on the stage of development of the target receptor; although it has been shown that many promoters derived from dicotyledons are functional in monocotyledons and vice versa, ideally, dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters are selected for expression in monocotyledons; 4) Linking with a suitable transcription terminator can also improve the expression efficiency of the gene of the present invention; for example, tml from CaMV, E9 from rbcS; any available terminator known to function in plants can be linked with the gene of the present invention. 5) Introduce enhancer sequences, such as intron sequences (e.g., derived from Adhl and Bronzel) and viral leader sequences (e.g., derived from TMV, MCMV, and AMV).

[0029] The OsFTL1 coding gene can be introduced into recipient plants using a recombinant expression vector containing the OsFTL1 coding gene. Specifically, the recombinant expression vector can be the pTCK303- OsFTL1 .

[0030] The recombinant expression vector can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition)).

[0031] The target plant is understood to include not only first-generation plants containing overexpression of the OsFTL1 protein or its encoding gene, but also its progeny. For the target plant, the gene can be propagated within the species, or it can be transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. The target plant includes seeds, callus tissue, intact plants, and cells.

[0032] OsFTL1 or substances that regulate the content or activity of OsFTL1 are also within the scope of protection of this invention.

[0033] In this invention, the plant may be M1, M2, or M3. M1) Monocotyledons or dicotyledons; M2) Gramineae plants; M3) Rice (Oryza sativa), maize (Zea mays), sorghum (Sorghum bicolor), millet (Setaria italica), goat grass (Aegilopstauschii), two-spike short-stalked grass (Brachypodium distachyon), or wheat (Triticumaestivum).

[0034] In this invention, the protein content can be the protein content in plant tissues or organs. Further, the plant tissues or organs can be leaves or seeds. The protein content can be the total protein content.

[0035] The chlorophyll content may be the chlorophyll content in plant leaves. Further, the chlorophyll may be chlorophyll a, chlorophyll b, and / or carotenoids.

[0036] The chlorophyll content can refer to the chlorophyll content of plants under low nitrogen levels. Low nitrogen levels mean that the nitrogen content in the plant's growth environment (such as nutrient solution or soil) is lower than the nitrogen content required for normal growth.

[0037] Experiments have shown that the OsFTL1 and its encoding gene of this invention can increase chlorophyll content and improve protein content. Under normal nitrogen conditions, it was found that the transgenic... OsFTL1 The content of chlorophyll a, chlorophyll b, and carotenoids in the leaves of genetically modified plants was significantly higher than that in wild-type plants; under low nitrogen conditions, the content of chlorophyll a, chlorophyll b, and carotenoids in the leaves of genetically modified plants was significantly higher than that in wild-type plants; OsFTL1 The leaves of genetically modified plants have significantly higher protein content than wild-type plants; after harvesting, the protein content of field-grown plants... OsFTL1 The seed protein content of the genetically modified plant was significantly higher than that of the wild type. These results indicate that... OsFTL1 Genes and their encoded proteins can promote nitrogen assimilation, increase pigment synthesis, and improve protein content, showing great promise for applications.

[0038] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0039] Figure 1 This is the sequence alignment result.

[0040] Figure 2The results show the relative expression level of the OsFTL1 gene in transgenic OsFTL1 rice. Detailed Implementation

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are all commercially available. All quantitative experiments in the following examples were performed in at least three replicates, and the results were averaged. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0042] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. Two-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0043] The pTCK303 vector in the following examples (Zhang, H., Zhang, J., Yan, J., Gou, F., Mao, Y., Tang, G., Botella, JR, & Zhu, JK (2017). Short tandem targetmimic rice lines uncover functions of miRNAs in regulating important agronomic traits. Proceedings of the National Academy of Sciences of the United States of America, 114(20), 5277–5282. https: / / doi.org / 10.1073 / pnas.1703752114) is a biological material that is publicly available from the applicant. This biological material is only for repeating the relevant experiments of the present invention and shall not be used for any other purpose.

[0044] Example 1: OsFTL1 can increase the protein and chlorophyll content of rice. This embodiment provides a protein derived from Nipponbare rice that can increase the protein and chlorophyll content of rice. The protein is named OsFTL1, and its sequence is SEQ ID No. 1 in the sequence listing. In Nipponbare rice, the coding gene sequence of OsFTL1 is SEQ ID No. 2, and the genome sequence is SEQ ID No. 3.

[0045] Sequence alignment of rice OsFTL1 with homologous proteins from other plants revealed sequence similarities of 94.15%, 93.57%, 93.57%, 92.40%, 92.40%, and 92.98% with those from maize, sorghum, millet, Aegilops spp., wheat, and Brachypodium distichum, respectively. Figure 1 ).

[0046] 1. Construction of recombinant vectors The sequence shown in the artificially synthesized sequence listing is SEQ ID No. 2. OsFTL1 The gene, by replacing the DNA fragment between the BamHI and SacI recognition sequences of the pTCK303 vector with the one shown in SEQ ID No. 2. OsFTL1 The gene was obtained as a recombinant vector and named pTCK303- OsFTL1 pTCK303- OsFTL1 It can be expressed under the drive of the maize Ubiqutin (UBI) promoter. OsFTL1 The protein encoded by the gene (i.e., the OsFTL1 protein shown in SEQ ID No. 1).

[0047] 2. Construction of transgenic plants After sterilization, mature seeds of the japonica rice variety Nipponbare were induced to produce embryogenic callus. The pTCK303- obtained in step 1 was then... OsFTL1 After being introduced into Agrobacterium EHA105, callus tissue was infected and co-cultured using Agrobacterium-mediated rice genetic transformation. Transgenic plants were obtained through resistance selection, and the selected transgenic rice is the genetically modified rice. OsFTL1 Genetically modified rice.

[0048] Using the japonica rice variety Nipponbare (WT) as a control, the qRT-PCR method was used to detect... OsFTL1 In genetically modified rice OsFTL1 The relative expression levels of the gene at the RNA level were determined using primers: 5′-TACACCCTGGTGATGGTGGAT-3′, 5′-AGAGACTCCTGTGGTAGCCG-3′; the internal reference gene was the rice Ubiqutin gene, and the primers for the internal reference gene were: 5′-AAGAAGCTGAAGCATCCAGC-3′, 5′-CCAGGACAAGATGATCTGCC-3′.

[0049] The results showed that OsFTL1 Three lines of genetically modified rice ( OsFTL1 -OE1、 OsFTL1 -OE2 and OsFTL1 -OE3) OsFTL1 The relative expression levels of the genes were significantly higher than those of the wild type (WT), and all three lines were overexpressing the genes. OsFTL1 Rice materials ( Figure 2 ).

[0050] 3. Turn OsFTL1 Genetically modified rice has increased protein and chlorophyll content. The protein and chlorophyll content of rice was measured. The rice variety tested was wild-type Nipponbare rice (WT), overexpressing... OsFTL1 Rice ( OsFTL1 -OE1、 OsFTL1 -OE2 and OsFTL1 -OE3).

[0051] Rice was cultured using nutrient solutions with normal nitrogen levels (2 mM) and low nitrogen levels (0.1 mM). Samples were taken from the leaves of each rice variety four weeks after sowing to determine the protein and chlorophyll content. The protein and chlorophyll content were then determined using a spectrophotometer colorimetric method.

[0052] Leaf protein content determination: Harvest leaves of hydroponically grown rice seedlings (approximately 3-5 cm in length), flash-freeze and grind them in liquid nitrogen, add 200 μL of protein extraction buffer, and place the EP tube containing the extracted protein sample horizontally in an ice box with shaking for 30 minutes. Then, transfer to a centrifuge at 12800 rpm for 20 minutes at 4 °C, and collect the supernatant for storage. Mix 999 μL of Quick Start Bradford 1×Dye Reagent reaction solution with 1 μL of supernatant and react for 5 minutes. Measure the absorbance at 595 nm using a UV spectrophotometer, and calculate the protein concentration using a standard curve.

[0053] Standard curve creation: Using the absorbance at 595 nm as the ordinate and the standard protein concentration as the abscissa (six points were set at 10 μg / μl, 20 μg / μl, 30 μg / μl, 40 μg / μl, 50 μg / μl, and 60 μg / μl), the absorbance of BSA at A595 nm at the above concentrations was measured using a spectrophotometer. A standard curve was plotted on the coordinate axis, and the regression linear equation was determined based on the standard curve data.

[0054] Protein extraction solution formulation: 20 mM Tris (pH 7.5), 100 mM NaCl, 2.5 mM MgCl2, 1 mM MEGTA, 1 mM DTT and protease inhibitor cocktail (1:50) (Roche).

[0055] For the determination of chlorophyll content in rice, please refer to the determination method (LICHTENTHALER HK, 1987. Chlorophylls and carotenoids pigments of photosynthetic biomembranes. Methods in Enzymology, 148: 350-382. DOI: 10.1016 / 0076-6879(87)48036-1.). Weigh 0.05 g of fresh rice leaves, crush the sample with steel beads (quickly freeze with liquid nitrogen), add 1 mL of 100% acetone to each tube, remove the beads and mix well (vibrate vigorously), centrifuge at 5000 g for 15 min at 4 °C, collect the supernatant and add 100% acetone to 10 mL (judged by color, absorbance value should not exceed 1), and then use a quartz cuvette to measure the absorbance values ​​at 661.6 nm, 644.8 nm and 470 nm on a UV spectrophotometer. The calculation formula is as follows: C a = 11.24×A661.6-2.04×A644.8; C b = 20.13×A644.8-4.19×A661.6; C car =(1000×A470-1.90×C a -63.14×C b ) / 214.

[0056] Final calculation formula: Chlorophyll a content (mg / g) = C a (mg / L) × Total extract volume (L) × Dilution factor / Fresh weight of material (g); Chlorophyll b content (mg / g) = C b (mg / L) × Total extract volume (L) × Dilution factor / Fresh weight of material (g); Carotenoid content (mg / g) = C car (mg / L) × Total extract volume (L) × Dilution factor / Fresh weight of material (g).

[0057] The nutrient solution with normal nitrogen level (2 mM) consists of: 0.5 mM (NH4)2SO4, 0.54 mM MgSO4·7H2O, 1 mM KNO3, 0.3 mM CaCl2, 0.18 mM KH2PO4, 0.09 mM K2SO4, 16 µM Na2SiO3·9H2O, 9.14 µM MnCl2·4H2O, 46.2 µM Na2MoO4·2H2O, 0.76 µM ZnSO4·7H2O, 0.32 µM CuSO4·5H2O, and 40 µM Fe(II)-EDTA (pH=5.8). Nutrient solution with low nitrogen level (0.1 mM): 0.025 mM (NH4)2SO4, 0.54 mM MgSO4·7H2O, 0.05 mM KNO3, 0.3 mM CaCl2, 0.18 mM KH2PO4, 0.09 mM K2SO4, 16 µM Na2SiO3·9H2O, 9.14 µM MnCl2·4H2O, 46.2 µM Na2MoO4·2H2O, 0.76 µM ZnSO4·7H2O, 0.32 µM CuSO4·5H2O, 40 µM Fe(II)-EDTA (pH=5.8).

[0058] Rice was grown under field conditions in Beijing, and the protein content of mature grains was tested. The method for determining the protein content of grains was performed in accordance with the People's Republic of China National Standard GB / T 6432-2018dz.

[0059] The results showed that rice cultured with nutrient solutions at normal nitrogen levels (2 mM) and low nitrogen levels (0.1 mM) exhibited improved transformation. OsFTL1 The contents of chlorophyll a, chlorophyll b, and carotenoids in the leaves of genetically modified rice were significantly higher than those in the wild type (Table 1). Furthermore, under low nitrogen levels (0.1 mM), the content of chlorophyll a, chlorophyll b, and carotenoids in the leaves of genetically modified rice was significantly higher than that in the wild type (Table 1). OsFTL1 The leaf protein content of genetically modified rice was significantly higher than that of wild-type rice (Table 2). After harvesting rice grown in a field in Beijing, seed protein content analysis revealed that the genetically modified rice... OsFTL1 Genetically modified rice grains had significantly higher protein content than wild-type rice (Table 3). These results indicate that... OsFTL1 Genes can promote nitrogen assimilation, increase pigment synthesis, and improve protein content.

[0060] Table 1. Average chlorophyll content (mg·g) of wild-type and transgenic rice -1 FW)

[0061] In Table 1, normal nitrogen was 2 mM and low nitrogen was 0.1 mM. WT, OsFTL1-OE1, OsFTL1-OE2, and OsFTL1-OE3 each had 5 biological replicates, with FW representing fresh weight.

[0062] Table 2. Protein content (mg·g) in leaves of wild-type and transgenic rice. -1 FW)

[0063] In Table 2, normal nitrogen was 2 mM and low nitrogen was 0.1 mM. WT, OsFTL1-OE1, OsFTL1-OE2, and OsFTL1-OE3 each had 3 biological replicates, and FW represents fresh weight.

[0064] Table 3. Protein content of wild-type and transgenic rice grains

[0065] In Table 3, WT, OsFTL1-OE1, OsFTL1-OE2, and OsFTL1-OE3 each had 3 biological replicates.

[0066] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Any of the following applications of proteins or substances that regulate the content or activity of said proteins: D1) Regulates plant protein content; D2) Prepare products that regulate plant protein content; D3) Regulates plant chlorophyll content; D4) Prepare products that regulate plant chlorophyll content; D5) Cultivating plants with altered protein content; D6) Prepare products from plants with altered protein content; D7) Cultivating plants with altered chlorophyll content; D8) Prepare products from plants with altered chlorophyll content; The protein is either A1), A2), A3), or A4. A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) A protein that has the same function as the amino acid sequence shown in SEQ ID No. 1 in the sequence listing, but with one or more amino acid residues substituted and / or deleted and / or added; A3) A protein derived from rice, corn, sorghum, millet, goatgrass, two-stalked short-stalked grass or wheat that has 75% or more identity with SEQ ID No. 1 and has the same function as the protein described in A1); A4) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of A1), A2), or A3).

2. The application according to claim 1, characterized in that: The substance that regulates the content or activity of the protein is any one of the following B1) to B7): B1) The nucleic acid molecule that encodes the protein; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2).

3. The application according to claim 2, characterized in that: B1) The nucleic acid molecule described is as follows: (b11) or (b12) or (b13) or (b14) or (b15) b11) The coding sequence is the cDNA molecule or DNA molecule of SEQ ID No. 2 in the sequence listing; b12) The DNA molecule shown in SEQ ID No. 2 of the sequence listing; b13) The DNA molecule shown in SEQ ID No. 3 of the sequence listing; b14) has 75% or more identity with the nucleotide sequence defined by b11) or b12) or b13) and is a DNA molecule encoding the protein; b15) hybridizes under stringent conditions to a nucleotide sequence defined by b11) or b12) or b13) or b14) and to a DNA molecule encoding the protein thereon.

4. The application according to any one of claims 1-3, characterized in that: The substance that regulates the protein content or activity is a substance that increases the protein content or activity; the regulation of plant protein content is to increase plant protein content; the regulation of plant chlorophyll content is to increase plant chlorophyll content; the change in protein content is an increase in protein content; and the change in chlorophyll content is an increase in chlorophyll content.

5. The application according to any one of claims 1-4, characterized in that: The protein content refers to the protein content in plant tissues or organs; further, the plant tissues or organs are leaves or seeds. The chlorophyll content refers to the chlorophyll content in plant leaves; further, the chlorophyll is chlorophyll a, chlorophyll b, and / or carotenoids.

6. The application according to any one of claims 1-5, characterized in that: The chlorophyll content refers to the chlorophyll content of plants under low nitrogen levels.

7. Any of the following methods: X1) A method for cultivating plants with increased protein content and / or increased chlorophyll content, comprising increasing the content or activity of the protein described in claim 1 in a recipient plant to obtain a target plant with increased protein content and / or increased chlorophyll content compared to the recipient plant; X2) A method for reducing the protein content and / or chlorophyll content of plants, comprising reducing the content or activity of the protein described in claim 1 in the recipient plant, obtaining a target plant with increased protein content and / or increased chlorophyll content compared to the recipient plant, thereby achieving an increase in the protein content and / or chlorophyll content of plants.

8. The method according to claim 7, characterized in that: The methods described in X1) and X2) are implemented by introducing the gene encoding the protein of claim 1 into the recipient plant and expressing the gene.

9. The application according to any one of claims 1-6, or the method according to claim 7 or 8, characterized in that: The plant is M1, M2, or M3. M1) Monocotyledons or dicotyledons; M2) Gramineae plants; M3) Rice, corn, sorghum, millet, goat grass, two-stalked short-stalked grass or wheat.

10. The protein of claim 1 or any of the substances in claims 1-3 that regulate the content or activity of the protein.

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