Application of AP1 gene

Abstract

The invention provides application of an AP1 gene, and relates to the technical field of agricultural molecular breeding. The AP1 gene or the encoded protein thereof is used for regulating and controlling agronomic traits of plants or preparing inhibitors or compositions for regulating and controlling the agronomic traits of the plants; wherein the regulation of the agronomic traits of the plants specifically comprises the steps of improving the photosynthetic rate of plant leaves under salt stress, increasing the chlorophyll content, promoting the germination rate and / or the total area of single plant leaves and the like. The AP1 gene can be used as a target for regulating and controlling plant traits, is applied to plant breeding, and can effectively regulate and control plant fluctuation light utilization efficiency, improve photosynthetic efficiency and Rubisco protein content under high salt stress, and increase resistance to adverse situations such as high salt conditions.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural molecular breeding, and in particular to a use of an AP1 gene. Background Art

[0002] Soybean is an important food and oil crop in my country. At present, while increasing soybean yield, we should also focus on stabilizing production and increasing efficiency, especially increasing yield under adverse conditions. However, my country's land resources are scarce, especially the marginal land is seriously impoverished. Therefore, increasing soybean yield on marginal land is of great significance to ensuring my country's food security. Marginal land mainly includes various soil types such as soda saline-alkali soil in Northeast China, saline-alkali soil in Northwest China, coastal saline-alkali soil, and red soil in southern mountainous hills. The total area of ​​marginal land in my country is about 1.17 billion mu, including 850 million mu of undeveloped arable land reserve resources and 320 million mu of existing low-grade arable land. Marginal land generally faces various unfavorable factors such as shallow soil layer, high salinity, low organic matter, poor nutrients and lack of irrigation, which makes it impossible for staple food crops to grow normally in unimproved marginal land. Green and efficient improvement and utilization of marginal land is a major problem that needs to be solved urgently. In recent years, with the support of the National Key Basic Research and Development Program (973 Program), the Major Transgenic Project, the National Key R&D Program, the Strategic Priority Research Program of the Chinese Academy of Sciences, and provincial and ministerial projects, my country's soybean has made a series of breakthroughs in nitrogen fixation efficiency, seed dormancy, photoperiod, oil content and fatty acid composition, herbicide resistance and disease resistance. However, there are relatively few studies on soybean salt tolerance.

[0003] Aspartic proteinases (APs; EC3.4.23) are a class of proteolytic enzymes with aspartic acid residues as catalytic active sites. When newly synthesized, these proteases exist in the form of single-chain zymogens, and are processed to form catalytically active protease monomers or heterodimers. APs are widely present in various organisms, including vertebrates, plants, yeast, nematodes, green algae and viruses. Many important proteases belong to this class of proteases, such as pepsin, renin, cathepsin D, and HIV-1 proteinase. In the MEROPS database, based on the homology of APs amino acid sequences, evolutionary relationships, and tertiary structures of proteins, these proteases are divided into 6 groups and 16 families. Among them, plant APs are mainly distributed in the A1 and A11 families in the AA group, and a few are in the AC and AD groups. Plant APs have been identified in multiple species and tissues. Similar to animal APs, plant APs are also active in acidic pH, and most of them can be specifically inhibited by pepsin inhibitors. Compared with animals, research on plant APs is relatively lagging. However, since the end of the 20th century, more and more plant APs have been identified and recognized, and people have found that plant APs also have some of their own specific structures and functions.

[0004] Typical plant APs precursors almost all have a protein sequence of about 100 amino acids, called the plant-specific insertion fragment PSI. This fragment is located at the C-terminus of the plant APs precursor. During the maturation of the protease, PSI is cleaved and removed to form a dimer. Some APs also retain PSI to form monomeric APs. PSI has multiple functions, including directing proteins to be localized in vesicles, cleaving cell membranes, and participating in defense responses. In stress responses, plants may be stressed by pathogens, drought, floods, salinity, and other biological and abiotic factors during their growth and development. Experiments have shown that plant APs play an important role in both stress responses. For example, overexpression of CDR1 (Constitutive disease resistance) in Arabidopsis and rice can increase the resistance of plants to pathogens, while AED1 in Arabidopsis has the opposite function to CDR1 and inhibits systemic acquired disease resistance. StAP1 and StAP3 in potatoes are toxic to pathogens and can also cause apoptosis of human Jurkat T cells, making them potentially useful for cancer treatment. ASPG1 in Arabidopsis is a typical example of plant APs involved in abiotic stress. ASPG1 is only expressed in guard cells. When plants are subjected to drought stress, ABA induces ASPG1 expression, and ASPG1 closes stomata to prevent water loss and activates antiperoxidase to protect Arabidopsis from peroxide damage. In addition, some aspartic proteases that respond to abiotic stress have been identified in species such as beans, pineapples, and buckwheat, proving that this type of enzyme helps plants actively cope with possible adverse stresses. The CND41 gene in tobacco encodes an atypical AP without PSI. This protein is localized in plastids and can degrade Rubisco in leaves, and the degradation products are provided to new tissues as a nitrogen source. Mutations in CND41 delay senescence of tobacco leaves, so it is speculated that CND41 may participate in the senescence process of plants by degrading proteins in plastids. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a use of an AP1 gene, which can effectively regulate the efficiency of plant fluctuating light utilization, improve photosynthetic efficiency and Rubisco protein content under high salt stress, and increase resistance to adversity such as high salt conditions.

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

[0007] A use of an AP1 gene, wherein the AP1 gene is used to regulate agronomic traits of plants to prepare transgenic plants or the AP1 gene-encoded protein is used to prepare an inhibitor or composition for regulating agronomic traits of plants; 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) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2;

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

[0011] (4) a polynucleotide sequence having 1 to 60 nucleotides 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 the nucleotide sequence described in any one of (1) to (4).

[0013] Preferably, the plant is selected from the following group or the AP1 gene or its homolog is from the following group: Poaceae, Cruciferae, Bromeliaceae, Orchidaceae, Palmaceae, Nymphaeaceae, Papaveraceae, Myrtaceae, Rubiaceae, Solanaceae, Sterculiaceae, Leguminosae, or Zosteraceae.

[0014] Specifically, the plant is a plant having AP1 or its homolog, preferably, the plant includes but is not limited to: Gramineae, such as rice (Oryza sativa), millet (Setaria italica), foxtail grass (Setaria viridis), Panicum hallii var. hallii, millet (Panicum miliaceum), Dichanthelium oligosanthes, corn (Zea mays), sorghum (Sorghumbicolor), teff (Eragrostis curvula), barley (Hordeum vulgare), Brachypodium distachyon, wheat (Triticum aestivum); Cruciferae, such as Arabidopsis thaliana; Bromeliaceae, such as pineapple (Ananas comosus); Orchidaceae, such as Dendrobium catenatum; Palmaceae, such as date palm (Phoenix dactylifera, oil palm (Elaeis guineensis); Nymphaeaceae, such as lotus (Nelumbo nucifera); Poppyaceae, such as Macleaya cordata; Myrtaceae, such as Syzygium oleosum; Rubiaceae, such as Coffeacanephora; Solanaceae, such as potato (Solanum tuberosum), tobacco (Nicotiana tabacum), Nicotiana sylvestris, Gapsinam chilense; Sterculiaceae, such as cocoa tree (Theobromacacao); Leguminosae, such as peanut (Arachis hypogaea); or Zosteraceae, 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) Promote germination rate;

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

[0020] Preferably, the amino acid sequence of the polypeptide of the protein encoded by the AP1 gene is selected from the following group:

[0021] (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2;

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

[0023] (3) A polypeptide having an amino acid sequence with a homology of ≥85% with the amino acid sequence shown in SEQ ID NO: 2 and having the function of regulating the trait;

[0024] (4) an active fragment of the polypeptide having 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-terminus of the polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or by adding a signal peptide sequence to its N-terminus.

[0026] Preferably, the inhibitor is any one of antisense nucleic acid, antibody, small molecule compound, CRISPR reagent, siRNA, shRNA, miRNA, small molecule ligand, down-regulating 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 agronomic traits of plants is to improve the plants by reducing or increasing the expression of AP1 in the plants.

[0028] Preferably, the method for preparing a transgenic plant comprises the following steps:

[0029] (1) transferring an exogenous AP1 encoding polynucleotide into a plant tissue, organ or tissue to obtain a plant tissue, organ or seed transformed with the AP1 encoding polynucleotide;

[0030] (2) Regenerating the plant tissue, organ or seed obtained in (1) into a plant into which the exogenous AP1 encoding polynucleotide has been transferred.

[0031] The present invention provides a use of an AP1 gene, which has the following advantages compared with the prior art:

[0032] (1) The present invention determines and identifies the traits of plants by analyzing the expression level of AP1 gene in plants; if the expression level of AP1 gene in the tested plant is equal to or lower than the average expression level of AP1 gene in the plants of this type, then: the photosynthetic rate of plant leaves under salt stress is normal or high, the chlorophyll content is normal or high, the germination rate is normal or high, and / or the total leaf area per plant is normal or high; if the expression level of AP1 gene in the tested plant is equal to or higher than the average expression level of AP1 gene in the plants of this type, then the traits are undesirable; wherein the AP1 gene includes its homologs;

[0033] (2) After determining the function of AP1, the present invention can use a variety of methods well known to those skilled in the art to increase the expression of AP1, such as delivering an expression unit (such as an expression vector or virus, etc.) carrying the AP1 gene to the target site through a pathway known to those skilled in the art, and causing it to express active AP1 protein; other methods for reducing the expression of the AP1 gene or its homologous gene are well known in the art; for example, the GNAT7 gene can be knocked out or the expression of its homologous gene can be inhibited by using CRISPR / CAS9 technology, and any appropriate conventional means, including reagents, temperature, pressure conditions, etc., can be used to implement the method;

[0034] (3) The present invention screened for the first time an aspartyl protease family protein (AP1) gene, which encodes an aspartyl 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, the plant traits can be significantly improved, including: increasing the photosynthetic rate of plant leaves under salt stress, increasing the chlorophyll content, promoting the 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 to plant breeding. It can increase resistance to adversity such as high salt conditions by improving photosynthetic efficiency and Rubisco protein content, and has an important role in cultivating soybean varieties that respond to the impoverishment and salinization of marginal land in my country. At the same time, the regulation does not affect the growth and development of plants under normal conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1The figure shows the construction of AP1 gene structure and mutant materials; Figure A shows the AP1 gene structure, showing the guide RNA recognition position in the three mutants (CR-1, CR-2, and CR-3) edited by CRISPR / CAS9; Figure B shows the mutation information of the three AP1 mutants, including the mutation site, mutation type, and the early termination position information of the protein after the amino acid frameshift mutation. Figure C shows the phenotype of the CR mutant and three overexpression (OE) after 20 days of cultivation under 200mM hogland nutrient solution with Dongnong 50 (DN50) as the background; Figure D shows the comparative analysis of the expression level of AP1 gene in the leaves of the corresponding CR mutant and overexpression OE after 20 days of high salt stress; Figure E shows the germination rate of CR mutants and OE overexpression materials after different high salt treatments for different days; Figure F shows the total leaf area statistics of individual plants of CR mutants and OE overexpression materials;

[0036] Figure 2 Figure 1 is a spatiotemporal analysis of AP1 gene expression specificity; Figure A is a subcellular localization map of 35S AP1-overexpressing tobacco leaves; Figure B is a comparative analysis of AP1 gene expression in different tissues and organs, including roots, cotyledons, hypocotyls, leaves, stems, stem tips and flower buds; Figure C is the gene expression dynamics of AP1 gene at different growth stages after germination; Figure D is an analysis of AP1 gene expression after 7 days of salt stress treatment with different concentrations; Figure E is a comparative analysis of gene expression within 200mM NaCl treatment for 20 hours;

[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 phenotype of the AP1 mutant CR1 and two overexpression strains treated with high salt stress for 20 days, in which Molk is an eGFP empty vector transformed Dongnong 50 as a control; Figure B is an immunoprecipitation-mass spectrometry combined analysis, using overexpressed OE1 leaves as materials, extracting total protein, and incubating and enriching with eGFP magnetic beads, and then performing protein spectrum analysis, in which the left side shows the identification of rbcL protein interacting with AP1 after enrichment through Rubisco large subunit antibody, and the right side shows the protein spectrum identification results of interacting with AP1; Figure C shows the control (no salt treatment) or 200mM Comparative analysis of AP1 protein expression level and rbcL expression in leaves after 20 days of NaCl treatment, with actin protein as control; Figure D shows the degradation of rbcL after AP1 protein was purified in vitro and different AP1 protein contents were added to the mixture of total protein (extracted from mutants) and DMSO or pepstatin (AP1 inhibitor), with actin as control; Figures EG show comparative analysis of chlorophyll a, chlorophyll b and photosynthetic efficiency of AP1 mutants and overexpressed materials after high salt treatment;

[0038] Figure 4 Cluster analysis of AP1 gene encoding protein sequences in different species. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The "AP1 protein (polypeptide)" used in the present invention may be a protein (polypeptide) having an amino acid sequence as shown in SEQ ID NO: 2, and a gene encoding the same may have a nucleotide sequence as shown in SEQ ID NO: 1, and also includes homologs thereof.

[0041] The present invention also includes fragments, derivatives and analogs of the AP1 protein. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the AP1 protein of the present invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or more (e.g., 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-3, 1-2) conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substitution group 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) a polypeptide formed by the fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein). According to the definition of the present invention, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0042] Any biologically active fragment of AP1 can be applied to the present invention. Here, the biologically active fragment of AP1 means a polypeptide that can still maintain all or part of the functions of the full-length AP1. Generally, the biologically active fragment maintains at least 50% of the activity of the full-length AP1. Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length AP1.

[0043] In the present invention, AP1 also includes variant forms of the sequence of SEQ ID NO: 2 that have the same function as AP1. These variant forms include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8, 1-5) amino acids, and addition or deletion of one or several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8, 1-5) amino acids at the C-terminus and / or N-terminus (especially the N-terminus). For example, in the art, when substitution is performed with amino acids with similar or similar properties, the function of the protein is usually not changed. For another example, the addition or deletion of one or several amino acids at the C-terminus and / or N-terminus (especially the N-terminus) usually does not change the function of the protein.

[0044] A variety of proteins with high homology to AP1 (e.g., 60% or higher, 70% or higher, 80% or higher homology to the sequence shown in SEQ ID NO: 1; preferably, 85% or higher homology; more preferably, 90% or higher homology, such as 95%, 98% or 99% homology) and having the same function as AP1 are also included in the present 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 the present invention, variants of genes can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutagenesis, etc.

[0045] A variety of proteins with high homology to AP1 (e.g., 60% or higher, 70% or higher, 80% or higher homology to the sequence shown in SEQ ID NO: 1; preferably, 85% or higher homology; more preferably, 90% or higher homology, such as 95%, 98% or 99% homology) and having the same function as AP1 are also included in the present 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 the present invention, variants of genes can be obtained by inserting or deleting regulatory regions, performing random or site-directed mutagenesis, etc.

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

[0047] The present invention also relates to a polynucleotide sequence encoding the AP1 of the present invention or its conservative variant polypeptide. The polynucleotide may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or may be a degenerate variant. As used in the present invention, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding a protein having SEQ ID NO: 2 but differing from the coding region sequence shown in SEQ ID NO: 1. Due to the degeneracy of codons, even if the base sequence identity with SEQ ID NO: 1 is low, the amino acid sequence shown in SEQ ID NO: 2 can be basically encoded.

[0048] The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: a coding sequence encoding 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 non-coding sequences.

[0049] The term "polynucleotide encoding a polypeptide" may be a polynucleotide comprising the polypeptide, and may also be a polynucleotide further comprising additional coding and / or non-coding sequences.

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

[0051] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. Plant transformation can be carried out using methods such as Agrobacterium transformation or gene gun transformation, such as spraying method, leaf disc method, young embryo transformation method, etc.

[0052] Those skilled in the art can use any of the various techniques known in the art or being developed to analyze nucleic acid sequences, and these techniques can all be included in the present invention. Methods include, but are not limited to: sequencing, PCR amplification, probe method, hybridization, restriction enzyme analysis, allele polymorphism analysis (such as melting curve method) to identify nucleic acid sequences, etc. If necessary, those skilled in the art can design primers for identifying molecular markers.

[0053] In addition, the present invention also relates to using AP1 or its encoding gene as a tracking marker for the offspring of a gene-transformed plant. The present invention also relates to using AP1 or its encoding gene as a molecular marker to identify the traits of a plant by detecting the expression of AP1 in the plant. When evaluating the plant to be tested, the expression level or mRNA level of AP1 can be measured to understand whether the expression or mRNA level in the plant to be tested is higher than the average value of such plants. If it is significantly lower, it has improved traits.

[0054] After knowing the molecular mechanism of the present invention and the genes or proteins involved in the molecular mechanism, the substances that can be used to improve plant traits can be screened based on the new discovery. The method of using a protein or a specific region thereon as a target to screen the substance acting on the target is well known to those skilled in the art, and these methods can be used in the present invention. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules and nucleic acid sequences, etc. According to the type of substance to be screened, those skilled in the art know how to choose an applicable screening method. The interaction between proteins and the strength of the interaction can be detected by a variety of techniques well known to those skilled in the art, such as GST-Pull Down, bimolecular fluorescence complementation experiment, yeast two-hybrid system or immunoprecipitation technology, etc.

[0055] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0056] Embodiment 1:

[0057] 1. High salt stress treatment:

[0058] The control (distilled water) and salt treatment (100mM NaCl solution for initial screening and 200mM NaCl solution for further screening) were set up, with 3 replicates each. 30 soybean seeds (DN50) were used for each replicate. The soybean seeds were placed in a plastic culture dish with a diameter of 9cm and covered with 2 layers of sterilized filter paper. 15mL of distilled water was added to the control group, and 15mL of salt solution of corresponding concentration was added to the salt treatment group. Then the culture dish was placed in a 25℃ constant temperature plant incubator for germination for 7 days. The germination potential was counted on the 3rd day of germination, and the germination rate was counted on the 7th day of germination. The germination standard was that the radicle length was greater than or equal to 1 / 2 of the seed length. Then the samples with consistent germination were sown in the seedling tray, and the Hoagland's nutrient solution was replaced every 3 days until the salt stress treatment for 20 days. The phenotype was observed, and the total leaf area, photosynthetic efficiency and gene expression of the single plant were measured.

[0059] 2. Net photosynthetic efficiency and total leaf area and determination

[0060] When measuring the net photosynthetic efficiency, the top leaves of each canopy were selected and a portable photosynthetic meter (LICOR-6800) was used. The light intensity was 800 μmol m-2s-1PPFD, the leaf chamber temperature was 27°C, the CO2 was 400 ppm, and the flow rate was 500 CO2flux / min. The data were recorded automatically every 2 seconds to obtain the dynamic recovery rate data of each photosynthetic efficiency. Each strain had 4 biological replicates.

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

[0062] 3. Gene expression analysis

[0063] In order to compare the differences in gene expression between different strains, the same leaf position as that for photosynthetic efficiency determination was selected for gene expression analysis. For the analysis of expression differences in different growth periods and different tissues and organs, another group of leaf samples were selected for collection. Leaf samples treated with salt stress were quickly stored in liquid nitrogen. RNA was extracted using the TRIzol Plus RNA purification kit (Invitrogen Life Sciences) according to the standard procedure in the instructions. Reverse transcription of cDNA was performed using the SuperScriptVILO cDNA reverse transcription kit (Invitrogen Life Sciences). 2ug of total RNA was used for reverse transcription of cDNA. Quantitative PCR was achieved using the SYBR Green PCR reaction system (Applied Biosystems, USA) and the ABI quantitative PCR instrument (StepOnePlus). The amplification reaction program was: 95°C 10s, 55°C 20s, and 72°C 20s. The housekeeping gene is actin. Three biological replicates and three technical replicates. The newly developed primer sequences are as shown in Table 1:

[0064] Table 1

[0065]

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

[0067] The codon-optimized hSpCas9 and soybean ubiquitin promoter (UBI) were co-linked to the pCAMBIA1300 binary vector (purchased from NTCC Type Culture Collection Center-Biovector Plasmid Vector Bacteria Cell Protein Antibody Gene Collection Center). The vector backbone contains a hygromycin selection marker (HPT).

[0068] The primer screening sequences are: F, AGCTGCGCCGATGGTTTCTACAA; R, ATCGCCTCGCTCCAGTC AATG. To construct the complete CRISPR / Cas9 binary vector pBGK032, it is necessary to additionally introduce the OsU6 promoter, the selection marker gene ccdB, the restriction enzyme site with BsaI and the sgRNA sequence derived from pX260. The specific sequence recognizing the CDS region of the GNAT7 gene was completed by artificial synthesis. Finally, 10ng of the digested pBGK032 vector and 0.05mM oligo binder were connected in a 10μl reaction system. After sequencing confirmed that no base mutation occurred, the next step was carried out, including Escherichia coli expression plasmid, Agrobacterium tumefaciens-mediated soybean transformation and callus regeneration system.

[0069] 5. Agrobacterium-mediated transgenic and mutant detection

[0070] The constructed CRISPR / Cas9 plasmid was expressed in the Agrobacterium tumefaciens strain EHA105 (purchased from the NTCC Typical Culture Collection Center-Biovector Plasmid Vector Bacteria Cell Protein Antibody Gene Collection Center) by heat shock method. The selection of transformation receptors is generally Dongnong 50 (DN50) (purchased from Weimi Hainan Biological Co., Ltd.) seed mature embryos to induce callus tissue. After 2 weeks of induction medium increase and decrease, the embryos are cut off and cultured for 1 week. The vigorously growing callus is selected as the transformation receptor. The conventional Agrobacterium-mediated genetic transformation method is used to infect rice callus with the EHA105 strain containing the above two plasmid vectors. After co-cultivation for 3 days in the dark and 25°C, it is cultured on a screening medium containing 120mg / L G418. The resistant callus is cultured on a pre-differentiation medium containing 120mg / L for about 10 days. The pre-differentiated callus is transferred to the differentiation medium and cultured under light conditions. Resistant transgenic plants are obtained in about one month.

[0071] The specific results of the above tests can be found in Figure 1-Figure 3 ;

[0072] 6. The AP1 gene identified by the present invention is annotated as aspartyl protease, and the AP1 protein sequence is shown in SEQ ID NO: 1:

[0073] MGLGRGPISFTSQLARKLSNTKTKNTFSYCLLDYTLSPPPTSYLTIGPTPNDVVSRNSFTYTPLLTNPFSPSFYYISIQSVSVDGVRLPISESVFRIDANGNGGTVVDSGTTLSFL AEPAYGKILAAFRRRVRLPAVESAAALGFDLCVNVSGVARPKLPRLRFRLAGKAVLSPPVGNYFIEPAEGVKCLAVQPVRPDSGFSVIGNLMQQGYLFEFDLDRRSRIGFTRHGCAVR

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

[0075] .

[0076] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A use of an AP1 gene, characterized in that: The AP1 gene is used to regulate the agronomic traits of plants to prepare transgenic plants or the AP1 gene-encoded protein is used to prepare inhibitors or compositions for regulating the agronomic traits of plants; the nucleotide sequence of the AP1 gene is selected from the following group: (1) the polynucleotide sequence shown in SEQ ID NO: 1; (2) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO: 2; (3) a polynucleotide sequence having a homology of ≥ 90% with the sequence shown in SEQ ID NO: 1; (4) a polynucleotide sequence having 1 to 60 nucleotides truncated or added to the 5' end and / or 3' end of the polynucleotide shown in SEQ ID NO: 1; (5) A nucleotide sequence complementary to the nucleotide sequence described in any one of (1) to (4).

2. The use according to claim 1, characterized in that: The plant is selected from the following group or the AP1 gene or its homolog is from the following group: Gramineae, Cruciferae, Bromeliaceae, Orchidaceae, Palmaceae, Nymphaeaceae, Papaveraceae, Myrtaceae, Rubiaceae, Solanaceae, Sterculiaceae, Fabaceae, or Zosteraceae.

3. The use according to claim 1, characterized in that: The plant agronomic traits include any one or more of the following: (1) Increase the photosynthetic rate of plant leaves under salt stress; (2) Increase chlorophyll content; (3) Promote germination rate; (4) Increase the total leaf area of ​​a single plant.

4. The use according to claim 1, characterized in that: The amino acid sequence of the polypeptide of the protein encoded by the AP1 gene is selected from the following group: (1) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2; (2) A polypeptide derived from (1) having the function of regulating the trait, formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2; (3) A polypeptide having an amino acid sequence with a homology of ≥85% with the amino acid sequence shown in SEQ ID NO: 2 and having the function of regulating the trait; (4) an active fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 2; (5) A polypeptide formed by adding a tag sequence to the N- or C-terminus of the polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or by adding a signal peptide sequence to its N-terminus.

5. The use according to claim 1, characterized in that: The inhibitor is any one of antisense nucleic acid, antibody, small molecule compound, CRISPR reagent, siRNA, shRNA, miRNA, small molecule ligand, down-regulating molecule that interacts with AP1 to reduce its expression or activity, or a combination thereof.

6. The use according to claim 1, characterized in that: The method for applying the AP1 gene to regulate agronomic traits of plants is to improve the plants by reducing or increasing the expression of AP1 in the plants.

7. The use according to claim 1, characterized in that: The method for preparing a transgenic plant comprises the following steps: (1) transferring an exogenous AP1 encoding polynucleotide into a plant tissue, organ or tissue to obtain a plant tissue, organ or seed transformed with the AP1 encoding polynucleotide; (2) Regenerating the plant tissue, organ or seed obtained in (1) into a plant into which the exogenous AP1 encoding polynucleotide has been transferred.

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