Use of an ap1 gene

Abstract

The application provides a use of an AP1 gene, and relates to the technical field of agricultural molecular breeding. The AP1 gene or a coded protein thereof is used for regulating the agronomic traits of plants or preparing an inhibitor or a composition for regulating the agronomic traits of plants; the regulation of the agronomic traits of plants specifically comprises: increasing the photosynthetic rate of plant leaves under salt stress, increasing the chlorophyll content, promoting the germination rate and / or total leaf area per plant, and the like. The AP1 gene can be used as a target for regulating the traits of plants and applied to plant breeding. The AP1 gene can effectively regulate the fluctuating light use efficiency of plants, increase the photosynthetic efficiency and Rubisco protein content under high-salt stress, and increase the resistance to adversity such as high-salt conditions.

Description

Technical Field

[0001] This invention relates to the field of agricultural molecular breeding technology, specifically to the use of an AP1 gene. Background Technology

[0002] Soybeans are an important food and oilseed crop in my country. Currently, while increasing soybean yield per unit area, it is even more important to focus on stabilizing production and increasing efficiency, especially increasing yield per unit area under adverse environmental stress. However, my country's land resources are scarce, especially the severe infertility of marginal lands. Therefore, increasing soybean yields on marginal lands is of great significance to ensuring my country's food security. Marginal lands mainly include various soil types such as soda saline-alkali soil in Northeast China, inland saline-alkali soil in Northwest China, coastal saline-alkali soil, and red soil in the mountains and hills of Southern China. The total area of ​​marginal land in my country is approximately 1.17 billion mu (approximately 78 million hectares), including 850 million mu (approximately 50 million hectares) of undeveloped reserve arable land and 320 million mu (approximately 21 million hectares) of existing low-grade arable land. Marginal lands generally face various unfavorable factors such as shallow soil layers, high salinity and alkalinity, low organic matter, poor nutrient content, and lack of irrigation, making it impossible for staple crops to grow normally in unimproved marginal lands. The green and efficient improvement and utilization of marginal lands is a major problem that urgently needs to be solved. In recent years, with the support of the National Key Basic Research and Development Program (973 Program), the Transgenic Major Project, the National Key Research and Development Program, the Strategic Priority Research Program of the Chinese Academy of Sciences, and provincial and ministerial projects, my country has made a series of breakthroughs in soybeans in terms of nitrogen fixation efficiency, seed dormancy, photoperiod, oil content and fatty acid composition, herbicide resistance and disease resistance. However, there is relatively little research on the salt tolerance of soybeans.

[0003] Aspartic proteinases (APs; EC3.4.23) are a class of proteolytic enzymes that use aspartic acid residues as their catalytic active sites. These proteases are synthesized as single-stranded zymogens and undergo processing to form catalytically active protease monomers or heterodimers. APs are widely found in various organisms, including vertebrates, plants, yeast, nematodes, green algae, and viruses. Many important proteases belong to this class, such as pepsin, renin, cathepsin D, and HIV-1 proteinase. In the MEROPS database, based on amino acid sequence homology, evolutionary relationships, and tertiary structure, these proteases are divided into 6 groups and 16 families. Plant APs are mainly distributed in the A1 and A11 families within the AA group, with a few in the AC and AD groups. Plant APs have been identified in multiple species and tissues. Similar to animal active peptides (APs), plant APs are also active in acidic pH environments, and most can be specifically inhibited by pepsin inhibitors. Compared to animals, research on plant APs has lagged behind; however, since the late 20th century, more and more plant APs have been identified and recognized, and researchers have discovered that plant APs also possess some unique structures and functions.

[0004] Typical plant active protein (AP) precursors almost always contain a protein sequence of approximately 100 amino acids, called the plant-specific insertion fragment (PSI). This fragment is located at the C-terminus of the plant AP precursor. During protease maturation, the PSI is cleaved and removed to form a dimer, while some APs retain the PSI to form monomeric APs. The PSI has multiple functions, including directing protein localization to vesicles, cleaving cell membranes, and participating in defense responses. In stress responses, plants may be subjected to various biotic and abiotic factors such as pathogens, drought, flooding, and salinity during growth and development. Experiments have shown that plant APs play an important role in both types of stress responses. For example, overexpression of CDR1 (Constitutive Disease Resistance) in Arabidopsis and rice can enhance plant resistance to pathogens, while AED1 in Arabidopsis has the opposite function to CDR1, inhibiting systemic acquired disease resistance. StAP1 and StAP3 in potatoes are toxic to pathogens and can also induce apoptosis in human Jurkat T cells, making them potentially useful for cancer treatment. ASPG1 in Arabidopsis thaliana is a typical example of plant abiotic proteins (APs) participating in abiotic stress. ASPG1 is expressed only in guard cells. When plants are under drought stress, ABA induces ASPG1 expression. ASPG1 closes stomata to prevent water loss and simultaneously activates antiperoxidases to protect Arabidopsis thaliana from peroxide damage. In addition, aspartic proteases responding to abiotic stress have been identified in species such as common bean, pineapple, and buckwheat, demonstrating that these enzymes help plants actively cope with potential abiotic stresses. The CND41 gene in tobacco encodes an atypical AP without PSI. This protein is located in the plastid and can degrade Rubisco in leaves, with the degradation products providing nitrogen to new tissues. Mutations in CND41 delay senescence in tobacco leaves, suggesting that CND41 may participate in plant senescence by degrading proteins in the plastid. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a use for the AP1 gene, which can effectively regulate the light utilization efficiency of plants, improve photosynthetic efficiency and Rubisco protein content under high salt stress, and increase resistance to adverse conditions such as high salt conditions.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solution:

[0007] One use of the AP1 gene, wherein the AP1 gene is used to regulate agronomic traits of plants to produce transgenic plants or the AP1 gene-encoded protein is used to prepare inhibitors or compositions for regulating plant agronomic traits; the nucleotide sequence of the AP1 gene is selected from the following group:

[0008] (1) The polynucleotide sequence shown in SEQ ID NO:1;

[0009] (2) The nucleotide sequence encoding the polypeptide shown in SEQ ID NO:2;

[0010] (3) A polynucleotide sequence with ≥90% homology to the sequence shown in SEQ ID NO:1;

[0011] (4) A polynucleotide sequence in which 1-60 nucleotides are truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO:1;

[0012] (5) A nucleotide sequence complementary to any of the nucleotides described in (1)-(4).

[0013] Preferably, the plant is selected from the group below or the AP1 gene or its homologs are from the group below: grasses, cruciferous plants, bromeliads, orchids, palms, water lilies, poppies, myrtaceae, Rubiaceae, solanaceae, sterculiaceae, legumes, or phycophytes.

[0014] Specifically, the plant is a plant containing AP1 or its homologs. Preferably, the plant includes, but is not limited to: grasses such as rice (Oryza sativa), millet (Setaria italica), foxtail grass (Setaria viridis), Panicum hallii var. hallii, millet (Panicum miliaceum), Dichanthelium oligosanthes, maize (Zea mays), sorghum (Sorghum bicolor), dandelion (Eragrostis curvula), barley (Hordeum vulgare), brachypodium distachyon, and wheat (Triticum aestivum); cruciferous plants such as Arabidopsis thaliana; bromeliads such as pineapple (Ananas comosus); orchids such as dendrobium (Dendrobium catenatum); and palms such as date palm (Phoenix). Plants such as *Dactylifera*, oil palm (*Elaeis guineensis*); water lily family plants, such as lotus (*Nelumbo nucifera*); poppy family plants, such as *Macleaya cordata*; myrtaceae family plants, such as *Syzygium oleosum*; Rubiaceae family plants, such as *Coffeacanephora*; Solanaceae family plants, such as potato (*Solanum tuberosum*), tobacco (*Nicotiana atabacum*), beautiful tobacco (*Nicotiana sylvestris*), Gapsinam chilense; Sterculiaceae family plants, such as cacao (*Theobromacacao*); legume family plants, such as peanut (*Arachis hypogaea*); or phycophyceae family plants, such as zosteramarina.

[0015] Preferably, the plant agronomic traits include any one or more of the following:

[0016] (1) Increase the photosynthetic rate of plant leaves under salt stress;

[0017] (2) Increase chlorophyll content;

[0018] (3) Promotes germination rate;

[0019] (4) Increase the total leaf area of ​​a single plant.

[0020] Preferably, the amino acid sequence of the polypeptide encoding the AP1 gene is selected from the group consisting of:

[0021] (1) A polypeptide having the amino acid sequence shown in SEQ ID NO:2;

[0022] (2) A polypeptide derived from (1) having the regulatory trait function formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2;

[0023] (3) A polypeptide whose amino acid sequence has ≥85% homology with the amino acid sequence shown in SEQ ID NO:2 and has the aforementioned regulatory trait function;

[0024] (4) The active fragment of the polypeptide with the amino acid sequence shown in SEQ ID NO:2;

[0025] (5) A polypeptide formed by adding a tag sequence to the N or C end of the polypeptide with the amino acid sequence shown in SEQ ID NO:2, or by adding a signal peptide sequence to its N end.

[0026] Preferably, the inhibitor is any one of antisense nucleic acid, antibody, small molecule compound, CRISPR reagent, siRNA, shRNA, miRNA, small molecule ligand, downregulating molecule that interacts with AP1 to reduce its expression or activity, or a combination thereof.

[0027] Preferably, the method of applying the AP1 gene to regulate the agronomic traits of plants is to improve it by reducing or increasing the expression of AP1 in plants.

[0028] Preferably, the method for preparing transgenic plants includes the following steps:

[0029] (1) Transform the polynucleotide encoded by exogenous AP1 into plant tissues, organs or seeds to obtain plant tissues, organs or seeds that have been transformed into the polynucleotide encoded by AP1.

[0030] (2) Regenerate plant plants from plant tissues, organs or seeds that have been transfected with exogenous AP1 encoding polynucleotides obtained in (1).

[0031] This invention provides a use for the AP1 gene, which has the following advantages compared to existing technologies:

[0032] (1) This invention determines the characteristics of plants by analyzing the expression level of the AP1 gene in plants; if the expression level of the AP1 gene in the plant to be tested is equal to or lower than the average expression level of the AP1 gene in that type of plant, then the plant has: normal or high photosynthetic rate of leaves under salt stress, normal or high chlorophyll content, normal or high germination rate, and / or normal or high total leaf area per plant; if the expression level of the AP1 gene in the plant to be tested is equal to or higher than the average expression level of the AP1 gene in that type of plant, then its characteristics are not ideal; wherein, the AP1 gene includes its homologs.

[0033] (2) After determining the function of AP1, the present invention can use a variety of methods known to those skilled in the art to enhance the expression of AP1. For example, an expression unit carrying the AP1 gene (such as an expression vector or virus) can be delivered to the target site through methods known to those skilled in the art, and the active AP1 protein can be expressed. Other methods for reducing the expression of the AP1 gene or its homologous gene are known to those skilled in the art. For example, the GNAT7 gene can be knocked out or its homologous gene can be suppressed by using CRISPR / CAS9 technology. Any appropriate conventional means, including reagents, temperature, pressure conditions, etc., can be used to implement the method.

[0034] (3) This invention has for the first time screened an aspartyl protease family protein (AP1) gene, which encodes an aspartic acid protease. Through genome-wide association analysis, the inventors found that the AP1 gene is a key gene controlling photosynthetic efficiency under high salt stress. When the expression of the AP1 gene is reduced, plant traits can be significantly improved, including: increasing the photosynthetic rate of plant leaves under salt stress, increasing chlorophyll content, promoting germination rate, and / or the total leaf area per plant. Therefore, the AP1 gene can be used as a target for regulating plant traits and applied in plant breeding. It can increase resistance to adverse conditions such as high salt by increasing photosynthetic efficiency and Rubisco protein content, which plays an important role in breeding soybean varieties that can cope with the background of salinization and barrenness in my country's marginal lands. At the same time, the regulation does not affect the growth and development of plants under normal conditions. Attached image description:

[0035] Figure 1The following figures illustrate the construction of the AP1 gene structure and mutant materials: Figure A shows the AP1 gene structure, displaying the guide RNA recognition location in three CRISPR / CAS9 edited mutants (CR-1, CR-2, CR-3); Figure B shows the mutation information of the three AP1 mutants, including mutation sites, mutation types, and the premature termination location of the protein after the amino acid frameshift mutation; Figure C shows the phenotypes of the CR mutant and three overexpression (OE) strains after 20 days of high-salt cultivation in 200 mM Hogland nutrient solution against the background of Dongnong 50 (DN50); Figure D compares the expression levels of the AP1 gene in the leaves of the corresponding CR mutant and OE overexpression strains after 20 days of high-salt stress; Figure E shows the germination rate of the CR mutant and OE overexpression materials after different high-salt treatments for different numbers of days; Figure F shows the total leaf area statistics of individual plants of the CR mutant and OE overexpression materials.

[0036] Figure 2 Figure A shows the spatiotemporal expression specificity analysis of the AP1 gene; Figure B shows the subcellular localization of tobacco leaves transformed with 35S AP1 overexpression; Figure C shows the expression comparison of the AP1 gene in different tissues and organs, including roots, cotyledons, hypocotyls, leaves, stems, shoot tips, and flower buds; Figure D shows the gene expression dynamics of the AP1 gene at different growth stages after germination; Figure E shows the expression of the AP1 gene after 7 days of treatment with different concentrations of salt stress; Figure E shows the comparative analysis of gene expression within 20 hours of treatment with 200mM NaCl.

[0037] Figure 3 The AP1 gene interacts with the Rubisco large subunit and promotes the degradation of the Rubisco large subunit (rbcL). Figure A shows the phenotypes of the AP1 mutant CR1 and two overexpression lines after 20 days of high salt stress treatment, with Molk being a control (eGFP empty vector transformed into Dongnong 50). Figure B shows the combined immunoprecipitation-mass spectrometry analysis, using leaves overexpressing OE1 as material, extracting total protein, enriching it with eGFP magnetic beads, and then performing protein proteometry analysis. The left side shows the rbcL protein interacting with AP1 identified by the Rubisco large subunit antibody after enrichment, and the right side shows the protein proteometry identification results interacting with AP1. Figure C shows the results of the control (salt-free treatment) or 200mM... Comparative analysis of AP1 protein expression levels and rbcL expression in leaves after 20 days of NaCl treatment, with actin protein as a control; Figure D shows the degradation of rbcL after adding different amounts of AP1 protein to a mixture of total protein (extracted from mutants) and DMSO or pepstatin (AP1 inhibitor) after in vitro purification of AP1 protein, with actin as a control; Figure EG shows a comparative analysis of chlorophyll a, chlorophyll b, and photosynthetic efficiency in AP1 mutants and overexpression materials after high salt treatment;

[0038] Figure 4 Cluster analysis of the AP1 gene-encoded protein sequence in different species. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The “AP1 protein (polypeptide)” used in this invention can be a protein (polypeptide) having the amino acid sequence shown in SEQ ID NO: 2, and the gene encoding it can have the nucleotide sequence shown in SEQ ID NO: 1, and also includes its homologs.

[0041] This invention also includes fragments, derivatives, and analogs of the AP1 protein. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the AP1 protein of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides having one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-3, 1-2) conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituents in one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-3, 1-2) amino acid residues; or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., leader sequence or secretory sequence or sequence used to purify this polypeptide or proteogenic sequence, or fusion protein). According to the definitions of the present invention, these fragments, derivatives and analogues are within the scope known to those skilled in the art.

[0042] Any bioactive fragment of AP1 can be used in this invention. Here, "bioactive fragment of AP1" means a polypeptide that retains all or part of the function of the full-length AP1. Typically, the bioactive fragment retains at least 50% of the activity of the full-length AP1. Under more preferred conditions, the active fragment can retain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length AP1.

[0043] In this invention, AP1 also includes variants of the sequence SEQ ID NO: 2 having the same function as AP1. These variants include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5); and the addition or deletion of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8 or 1-5) at the C-terminus and / or N-terminus (especially the N-terminus). For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition or deletion of one or more amino acids at the C-terminus and / or N-terminus (especially the N-terminus) generally does not alter the function of the protein.

[0044] Proteins with high homology to AP1 (e.g., 60%, 70%, 80%, or more homology to the sequence shown in SEQ ID NO:1; preferably 85% or more homology; more preferably 90% or more homology, such as 95%, 98%, or 99% homology) and having the same function as AP1 are also included in this invention. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids or polypeptides according to the percentage of identical positions. In this invention, gene variants can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, etc.

[0045] Proteins with high homology to AP1 (e.g., 60%, 70%, 80%, or more homology to the sequence shown in SEQ ID NO:1; preferably 85% or more homology; more preferably 90% or more homology, such as 95%, 98%, or 99% homology) and having the same function as AP1 are also included in this invention. "Homology" refers to the level of similarity (i.e., sequence similarity or identity) between two or more nucleic acids or polypeptides according to the percentage of identical positions. In this invention, gene variants can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, etc.

[0046] According to the phylogenetic analysis of the AP1 gene in different species by the present inventors, the protein sequence encoded by the AP1 gene is widely present and highly conserved in different species. Figure 4Therefore, it should be understood that although AP1 of the present invention is preferably obtained from rice, other polypeptides or genes obtained from other plants (especially plants belonging to the same family or genus as soybean) that are highly homologous to AP1 in rice (e.g., having more than 80%, such as 85%, 90%, 95%, 98%, or even 99% sequence identity) are also within the scope of the present invention, provided that those skilled in the art can easily isolate such polypeptides or genes from other plants based on the information provided in this application after reading it. These polypeptides or genes are also referred to as "homophores" of AP1. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0047] This invention also relates to a polynucleotide sequence encoding the AP1 of this invention or a conserved variant thereof. The polynucleotide can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to or a degenerate variant of the coding region sequence shown in SEQ ID NO: 1. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes the protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 1. Due to codon degeneracy, even with low base sequence identity to SEQ ID NO: 1, the amino acid sequence shown in SEQ ID NO: 2 can still be substantially encoded.

[0048] The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0049] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes a polypeptide or a polynucleotide that also includes additional coding and / or non-coding sequences.

[0050] The present invention also relates to vectors containing polynucleotides, and host cells using vectors or AP1 coding sequences generated by genetic engineering.

[0051] Transforming host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. Plant transformation can be performed using methods such as Agrobacterium-mediated transformation or gene gun transformation, including spraying, leaf disc transformation, and embryo transformation.

[0052] Those skilled in the art can employ any of the well-known or developing techniques to analyze nucleic acid sequences, and these techniques are all included in this invention. Methods include, but are not limited to: sequencing, PCR amplification, probe methods, hybridization, restriction enzyme digestion analysis, allele polymorphism analysis (such as melting curve analysis), etc., for nucleic acid sequence identification. If necessary, those skilled in the art can design primers for identifying molecular markers.

[0053] Furthermore, this invention relates to using AP1 or its encoding gene as a tracking marker for the progeny of gene-transformed plants. This invention also relates to using AP1 or its encoding gene as a molecular marker to identify plant traits by detecting AP1 expression in plants. When evaluating plants to be tested, the expression level or mRNA level of AP1 can be measured to determine whether the expression or mRNA level in the tested plant is higher than the average for such plants; if it is significantly lower, then it possesses improved traits.

[0054] Having learned the molecular mechanism of this invention and the genes or proteins involved in it, substances that can be used to improve plant traits can be screened based on this new discovery. Methods for screening substances that act on proteins or specific regions thereof as targets are well known to those skilled in the art, and these methods can all be used in this invention. Candidate substances can be selected from: peptides, polymeric peptides, peptide-like substances, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, and nucleic acid sequences, etc. Depending on the type of substance to be screened, those skilled in the art know how to select an appropriate screening method. Detecting protein-protein interactions and their strength can be done using various techniques well known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation assays, yeast two-hybrid systems, or immunoprecipitation techniques, etc.

[0055] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0056] Example 1:

[0057] 1. Treatment of high salt stress:

[0058] A control group (distilled water) and a salt treatment group (initial screening with 100mM NaCl solution, further screening with 200mM NaCl solution) were set up, each with three replicates. Each replicate used 30 soybean seeds (DN50). The soybean seeds were placed in 9cm diameter plastic petri dishes lined with two layers of sterile filter paper. The control group received 15mL of distilled water, and the salt treatment group received 15mL of the corresponding salt solution. The petri dishes were then placed in a 25℃ constant temperature plant culture incubator for 7 days of germination. Germination potential was measured on day 3 of germination, and germination rate was measured on day 7. A radicle length greater than or equal to half the seed length was used as the germination standard. Samples with consistent germination were then sown in seedling trays, with Hoagland's nutrient solution changed every 3 days until 20 days of salt stress treatment. Phenotypic characteristics were observed, and relevant indicators such as total leaf area, photosynthetic efficiency, and gene expression were measured.

[0059] 2. Measurement of net photosynthetic efficiency and total leaf area

[0060] Net photosynthetic efficiency was measured using the uppermost leaves of each plant canopy, employing a portable photosynthesis system (LICOR-6800). The light intensity was 800 μmol m⁻² s⁻¹ PPFD, the leaf chamber temperature was 27℃, CO₂ was 400 ppm, and the flow rate was 500 CO₂ flux / min. An automated program recorded data every 2 seconds to obtain the dynamic recovery rate of photosynthetic efficiency for each strain. Four biological replicates were performed for each strain.

[0061] Five wild-type, five mutant, and five overexpressing plants were selected. After leaf removal, the total leaf area was measured using a handheld leaf area meter (produced by Hangzhou Daji Company).

[0062] 3. Gene expression analysis

[0063] To compare gene expression differences among different strains, the same leaf position as that used for photosynthetic efficiency determination was selected for gene expression analysis. For the analysis of expression differences at different growth stages and in different tissues and organs, another group of leaf samples was collected. Leaf samples subjected to salt stress were rapidly preserved in liquid nitrogen. RNA extraction was performed using the TRIzol Plus RNA purification kit (Invitrogen Life Sciences, Inc.) according to the standard procedure in the manufacturer's instructions. Reverse transcription of cDNA was performed using the SuperScriptVILO cDNA reverse transcription kit (Invitrogen Life Sciences, Inc.). 2 μg of total RNA was used for reverse transcription of cDNA. Quantitative PCR was performed using the SYBR Green PCR reaction system (Applied Biosystems, Inc.) and an ABI quantitative PCR instrument (StepOnePlus). The amplification program was: 95℃ for 10 s, 55℃ for 20 s, and 72℃ for 20 s. The housekeeping gene was actin. Three biological replicates and three technical replicates were performed. The newly developed primer sequences are shown in Table 1 below.

[0064] Table 1

[0065]

[0066] 4. Construction of the CRISPR-CAS9 vector system

[0067] The codon-optimized hSpCas9 gene was co-linked with the soybean ubiquitin promoter (UBI) into the pCAMBIA1300 binary vector (purchased from the NTCC Type Culture Collection - Biovector Plasmid Vector Strain Cell Protein Antibody Gene Collection). The vector backbone contains the hygromycin selection marker (HPT).

[0068] The primer selection sequences were: F, AGCTGCGCCGATGGTTTCTACAA; R, ATCGCCTCGCTCCAGTC AATG. To construct the complete CRISPR / Cas9 binary vector pBGK032, an OsU6 promoter was introduced, along with the marker gene ccdB, a BsaI restriction enzyme site, and an sgRNA sequence derived from pX260. A specific sequence recognizing the CDS region of the GNAT7 gene was synthesized artificially. Finally, 10 ng of the digested pBGK032 vector was ligated to a 0.05 mM oligo binder in a 10 μl reaction volume. After sequencing confirmed the absence of base mutations, further operations were performed, including E. coli expression plasmids, Agrobacterium tumefaciens-mediated soybean transformation, and a callus regeneration system.

[0069] 5. Agrobacterium-mediated detection of transgenes and mutants

[0070] The constructed CRISPR / Cas9 plasmid was expressed in Agrobacterium tumefaciens strain EHA105 (purchased from the NTCC Type Culture Collection Center - Biovector Plasmid Vector Strain Cell Protein Antibody Gene Collection Center) via heat shock. The recipient was typically selected from mature embryos of Dongnong 50 (DN50) seeds (purchased from Weimi Hainan Biotechnology Co., Ltd.) to induce callus tissue. After two weeks of induction culture with adjustments to the medium, the embryos were cut off and cultured for another week. Vigorously growing callus was selected as the recipient for transformation. Using conventional Agrobacterium-mediated genetic transformation, EHA105 strain containing the two plasmid vectors described above was used to infect rice callus. After three days of co-culturing in the dark at 25°C, the callus was cultured on selection medium containing 120 mg / L G418. Resistant callus was screened and cultured on pre-differentiation medium containing 120 mg / L for approximately 10 days. The pre-differentiated callus was then transferred to differentiation medium and cultured under light. Resistant transgenic plants were obtained after approximately one month.

[0071] For detailed test results, please see [link / reference]. Figures 1-3 ;

[0072] 6. The AP1 gene identified in this invention is annotated as an aspartic acid protease, and the AP1 protein sequence is shown in SEQ ID NO:1 below:

[0073] MGLGRGPISFTSQLARKLSNTKTKNTFSYCLLDYTLSPPPTSYLTIGPTPNDVVSRNSFTYTPLLTNPFSPSFYYISIQSVSVDGVRLPISESVFRIDANGNGGTVVDSGTTLSFL AEPAYGKILAAFRRRVRLPAVESAAALGFDLCVNVSGVARPKLPRLRFRLAGKAVLSPPVGNYFIEPAEGVKCLAVQPVRPDSGFSVIGNLMQQGYLFEFDLDRSRIGFTRHGCAVR

[0074] The coding region sequence of the AP1 gene is shown in SEQ ID NO:2 below:

[0075] .

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of inhibiting AP1 gene in improving chlorophyll content, total leaf area per plant, photosynthetic rate of soybean leaf under salt stress, and promoting germination rate; the nucleotide sequence of the AP1 gene is a nucleotide sequence encoding a polypeptide as shown in SEQ ID NO:

2.

2. Use according to claim 1, characterized in that: The nucleotide sequence of the AP1 gene is a polynucleotide sequence as shown in SEQ ID NO:

1.

3. Use according to any of claims 1-2, characterized in that: The method for inhibiting the AP1 gene is to use an inhibitor, and the inhibitor is any one of antisense nucleic acid, Crispr reagent, siRNA, shRNA, and miRNA.

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