Ap2 gene or its encoded protein and its use in increasing plant yield or promoting plant growth

Abstract

This invention discloses AP2 Genes or their encoded proteins and their uses in increasing plant yield or promoting plant growth. This invention relates to genes identified in maize. ZmAP2 The gene was overexpressed in rice, and compared with the control (CK), the overexpressing plants showed a significant increase in panicle grain number, grain length, thousand-grain weight, biomass, and plant height; this invention further uses its homologous rice OsAP2 Gene overexpression in rice ， Compared to the control (CK), the overexpressed rice plants showed a significant increase in the number of grains per panicle, significantly longer grains, significantly increased thousand-grain weight, and significantly higher yield per plant. This invention has potential applications in promoting crop growth, increasing crop yield, and breeding high-yield crop varieties.

Description

Technical Field

[0001] This invention relates to growth-related genes isolated from grasses and their uses, particularly to genes isolated from grasses. AP2 Genes and their uses in promoting plant growth belong to AP2 Genes and their applications. Background Technology

[0002] Rice yield is a complex agronomic trait, primarily determined by three yield factors: grains per panicle, number of effective panicles per plant, and grain weight. These three components of rice yield are typical quantitative traits, as well as other yield-related traits, and are controlled by quantitative trait loci (QTLs). The number of panicles per rice plant depends on its tillering ability, a fundamental trait that determines the number of panicles and rice yield, including primary, secondary, and tertiary tillers. The number of grains per panicle depends on the two daughter components and the number of florets, primarily determined by the number of primary and secondary branches and the floret's seed setting rate. The number of grains per panicle is largely determined by panicle morphology, which is mainly related to panicle branching and development. The development of the rice panicle is often influenced by endogenous hormones, making the number of grains per panicle a complex trait regulated by multiple factors. Grain weight is mainly determined by thousand-grain weight and grain size, which is determined by three factors (length, width, and thickness) and grain filling. Grain length and width are key factors affecting yield, while grain size is mainly related to genetic factors. Therefore, cloning genes that increase the number of grains per panicle, the number of effective tillers per plant, and grain weight can lay the foundation for breeding high-yielding rice varieties.

[0003] Proteins encoding the AP2 domain in plants (i.e., the AP2 / ERF transcription factor family) are a class of DNA-binding proteins with highly conserved AP2 / ERF domains. These domains contain two key elements, YRG and RAYD, forming a three-dimensional structure of α-helix and β-sheet, specifically recognizing cis-acting elements in DNA. The AP2 / ERF family regulates plant growth and development and abiotic stress responses by integrating hormone signals such as ethylene, jasmonic acid, and abscisic acid, as well as stress responses. Therefore, cloning new AP2 family genes that regulate crop yield has significant application value for breeding stress-resistant and high-yielding crop varieties. Summary of the Invention

[0004] One of the objectives of this invention is to provide a product isolated from corn. ZmAP2 Gene or its encoded protein;

[0005] A second objective of this invention is to provide a method for isolating from rice. OsAP2 Gene or its encoded protein;

[0006] A third objective of this invention is to provide a product containing the aforementioned... ZmAP2 Gene or OsAP2 A recombinant expression vector of a gene or a recombinant host cell containing the recombinant expression vector;

[0007] The fourth objective of this invention is to... ZmAP2 Gene or OsAP2 Genes are used to increase crop yields or promote crop growth.

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] One aspect of the present invention is to provide a method for separating from corn ZmAP2 The gene, whose nucleotide sequence is shown in SEQ ID NO. 1; ZmAP2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0010] Another aspect of the present invention provides a method for isolating from rice. OsAP2 The gene, whose nucleotide sequence is shown in SEQ ID NO.3; OsAP2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4.

[0011] In addition, those skilled in the art can optimize the nucleotides shown in SEQ ID NO.1 and SEQ ID NO.3 to enhance their expression efficiency in plants.

[0012] Those skilled in the art can readily employ known methods, such as directed evolution or point mutation, to... ZmAP2 Gene or OsAP2 The nucleotide sequence of a gene is mutated. Those that are artificially modified have the same... ZmAP2 Gene or OsAP2 Nucleotides with 75% or higher nucleotide sequence identity to a gene, provided that the encoded protein has the function of regulating plant growth, are derived from and are equivalent to the nucleotide sequence of this invention.

[0013] In addition, the nucleotide sequence described in this invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; or it can be RNA, such as mRNA or hnRNA.

[0014] Another aspect of the present invention is to provide a product containing the aforementioned ZmAP2 Gene or OsAP2 Gene expression cassettes or recombinant expression vectors; wherein the recombinant expression vector is preferably a recombinant plant expression vector. Those skilled in the art can construct such cassettes using conventional techniques. ZmAP2 Gene or OsAP2 Plant recombinant expression vector.

[0015] For reference, the present invention provides ZmAP2 Gene or OsAP2 Gene recombinant expression vectors for plants, including: ZmAP2 Gene or OsAP2 A recombinant plant expression vector is obtained by linking a gene with an expression regulatory element. This recombinant plant expression vector may consist of a 5′ untranslated region, the nucleotides shown in SEQ ID NO.1 or SEQ ID NO.3, and a 3′ untranslated region. The 5′ untranslated region may include a promoter sequence, an enhancer sequence, and / or a translational enhancement sequence. The promoter may be a constitutive promoter, an inducible promoter, or a tissue- or organ-specific promoter. The 3′ untranslated region may contain a terminator sequence, an mRNA cleavage sequence, etc. A suitable terminator sequence can be obtained from the Ti-plasmid of Agrobacterium tumefaciens, such as the terminator regions of octopine synthase and carmine synthase.

[0016] The recombinant plant expression vector may also contain selective marker genes for selecting transformed cells or tissues. These marker genes include genes encoding antibiotic resistance and genes conferring resistance to herbicides. Furthermore, the marker genes may also include phenotypic markers, such as β-galactosidase and fluorescent proteins.

[0017] The transformation scheme and the scheme for introducing the polynucleotide or polypeptide into the plant can vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the polynucleotide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-velocity ballistic bombardment. In certain embodiments, various transient transformation methods can be used to... ZmAP2 Gene or OsAP2 Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0018] The present invention also provides a product containing the aforementioned ZmAP2 Gene or OsAP2 Recombinant host cells of gene expression cassettes or recombinant expression vectors.

[0019] Another aspect of the present invention is to ZmAP2 Gene, OsAP2 Genes, containing ZmAP2 Gene or OsAP2 Recombinant plant expression vectors are used to increase plant yield or promote plant growth.

[0020] In a preferred embodiment of the present invention, the increase in plant yield includes increasing the number of grains per spike, grain length, thousand-grain weight, and / or biomass.

[0021] In a preferred embodiment of the present invention, promoting plant growth includes increasing plant height.

[0022] This invention further provides a method for promoting the cultivation of high-yielding plant varieties or increasing plant yield and promoting plant growth, comprising: taking... ZmAP2 Gene or OsAP2 Genes are overexpressed in plants, increasing ZmAP2 Gene or OsAP2 The expression level or activity of genes in plants, thereby increasing plant yield or promoting plant growth, including increasing the number of grains per spike, grain length, thousand-grain weight, biomass and / or plant height.

[0023] The plant described in this invention is preferably a grass, and more preferably, the grass is rice or corn.

[0024] In the early stages of this invention, transcriptome sequencing analysis was performed on different cell tissues of the third leaf stage of three-leaf rice (Nipponbare) and maize B73. Transcription factors were screened for those expressed in maize leaves but not in rice leaves, those with strong expression in maize leaves but weak expression in rice leaves, and those specifically expressed only in maize bundle sheath cells. A total of 870 transcription factors belonging to 44 families, including ARF, bHLH, GRAS, MYB, and NAC, were obtained. Overexpression vectors were constructed for each gene and transformed into rice, creating a maize transcription factor overexpression mutant library. Large-scale agronomic trait surveys of the mutant library plants revealed that overexpression of more than twenty genes individually significantly increased plant yield, indicating novel genes with high-yield application potential. Among these, the maize gene identified in this application... ZmAP2 It is an AP2 transcription factor family protein with a CDS length of 717 bp, and is overexpressed in wild-type Nipponbare rice. ZmAP2 The transgenic plants obtained showed significantly increased panicle length, grain length, thousand-grain weight, biomass, and plant height compared to the control (CK). This invention further describes the rice homolog of this gene. OsAP2 Using the same method, it was isolated from rice. OsAP2 The gene was overexpressed in wild-type Nipponbare rice. OsAP2 Gene ， The transgenic plants obtained showed a significant increase in ear grain number, grain length, thousand-grain weight, and yield per plant compared to the control (CK), while the plant height remained largely unchanged. This demonstrates that the maize gene provided by this invention... ZmAP2 and rice OsAP2The genes and their encoded protein sequences are of great theoretical and practical significance for breeding new high-yield crop varieties with increased ear grain number, larger grain size, and reduced plant height.

[0025] Definitions of terms involved in this invention

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] The terms "polynucleotide" or "nucleotide" mean deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and their polymers, in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, including PNAs (peptide nucleic acids), DNA analogs (phosphate thioesters, phosphoramidites, etc.) used in antisense techniques. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses variants of its conserved modifications (including (but not limited to) degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue.

[0028] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. That is, the description of a polypeptide is equally applicable to the description of a peptide and the description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms cover amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked by covalent peptide bonds.

[0029] The term "recombinant host cell" refers to a cell containing the polynucleotides of the present invention, regardless of the method used for insertion to produce the recombinant host cell, such as direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotides may remain as, for example, non-integrating vectors of plasmids or may be integrated into the host genome. The host cell may be a prokaryotic or eukaryotic cell, and may also be a monocotyledonous or dicotyledonous plant cell.

[0030] The term "operable connection" refers to a functional connection between two or more elements, which may be adjacent or non-adjacent.

[0031] The term "recombinant plant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, along with vectors containing helper plasmids, are commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: the cis-acting sequence required for T-DNA transfer, an engineered selection marker for expression in plant cells, and the heterologous DNA sequence to be transcribed.

[0032] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.

[0033] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in plant cells. Attached Figure Description

[0034] Figure 1 For corn ZmAP2 Results of agronomic traits of rice lines overexpressing the gene; where A is a comparison of plant architecture between the control (CK) and transgenic lines; B is a comparison of grain size between the control (CK) and transgenic lines; C is a comparison of maize in the control (CK) and transgenic lines. ZmAP2 Gene expression level analysis results; D represents the statistical analysis results of the number of grains per ear in the control CK and transgenic lines; E represents the statistical analysis results of the grain length in the control CK and transgenic lines; F represents the statistical analysis results of the thousand-grain weight in the control CK and transgenic lines; G represents the statistical analysis results of the biomass in the control CK and transgenic lines; H represents the statistical analysis results of the plant height in the control CK and transgenic lines.

[0035] Figure 2 For rice OsAP2 Agronomic traits of rice lines overexpressing the gene; where A is a comparison of plant architecture between the control (CK) and transgenic lines; B is a comparison of grain size between the control (CK) and transgenic lines; C is a comparison of rice grain size between the control (CK) and transgenic lines. OsAP2 Gene expression level analysis results; D represents the statistical analysis results of the number of grains per ear in the control CK and transgenic lines; E represents the statistical analysis results of the grain length in the control CK and transgenic lines; F represents the statistical analysis results of the thousand-grain weight in the control CK and transgenic lines; G represents the statistical analysis results of the biomass in the control CK and transgenic lines. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments or test examples, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments or test examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples or test cases 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 and reagents used in the following examples are commercially available.

[0038] Vectors, strains and experimental materials

[0039] pCam23A vector: described in "Xuean Cui, Zhiguo Zhang, Yanwei Wang, Jinxia Wu, Xiao Han, Xiaofeng Gu, Tiegang Lu. TWI1 regulates cell-to-cell movement of OSH15 to control leaf cell fate. New Phytol. 2019 Jan;221(1):326-340.", is available to the public from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. This biomaterial is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0040] Agrobacterium tumefaciens AGL1: Purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., catalog number ZK296.

[0041] The wild type of rice is Nipponbare rice (Nipponbare rice) Oryza sativa L. ssp. Japonica variety Nipponbare (Hereinafter also referred to as wild-type rice WT), preserved by the Crop High Light Efficiency Functional Genomics Team of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, and planted in the experimental fields of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0042] The pTEAY-T1 Cloning Kit, Taq enzyme, Trans5α competent cells, and related reagent kits were all purchased from Beijing TransGen Biotech Co., Ltd.; restriction endonucleases SmaI and XbaI, infusion recombinase, and T4 ligase were all purchased from Dalian TaKaRa Co., Ltd.; antibiotics were purchased from SIGMA, USA; and all other reagents were domestically produced analytical grade.

[0043] Data processing

[0044] Data were processed using GraphPad Prism 8 statistical software. Experimental results are expressed as mean ± standard deviation. Student's... tThe test is performed using ns, where ns indicates no significant difference, P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0045] Example 1: Maize Genes ZmAP2 ( GRMZM2G009598 Cloning and construction of overexpression vectors for )

[0046] 1. Extraction of maize RNA

[0047] Total RNA was extracted from maize plants using the Plant Total RNA Kit (ZP405) from Beijing Zhuangmeng International Biotechnology Co., Ltd. Field samples were collected, and the plant tissues to be tested were wrapped in aluminum foil, flash-frozen in liquid nitrogen, and then transported back to the Beijing laboratory for storage in an ultra-low temperature freezer. The specific steps of RNA extraction are as follows:

[0048] (1) Take 30-80 mg of young plant tissue, grind it thoroughly with liquid nitrogen, and transfer it into a 1.5 ml centrifuge tube containing 1 ml of plant lysis buffer. Vortex to mix. (2) Centrifuge at 12000 rpm for 10 min at 4 °C. Carefully transfer the supernatant into a new RNase-free centrifuge tube. (3) Add 0.2 ml of chloroform to each 1 ml of plant lysis buffer. Tightly cap the sample tube, shake vigorously for 10 s, and incubate at room temperature for 5 min. (4) Centrifuge at 12000 rpm for 10 min at 4 °C. The sample will separate into three layers: a lower organic phase, a middle phase, and an upper colorless aqueous phase. RNA is present in the aqueous phase. Transfer the aqueous phase to a new tube. (5) Add 0.5 times the volume of anhydrous ethanol and 100 μL of binding buffer. Invert to mix. The obtained solution and any possible precipitate are transferred together into the adsorption column, which is then placed inside the collection tube; (6) Centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column back into the collection tube; (7) Add 500 μL of wash buffer RW, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid; (8) Repeat step 7; (9) Place the adsorption column back into the empty collection tube, centrifuge at 13000 rpm for 2 minutes to remove as much wash buffer as possible to prevent residual ethanol in the wash buffer from inhibiting the downstream reaction; (10) Take out the adsorption column and place it in a new RNase-free centrifuge tube. Add 60-100 μL of RNase-free water to the middle of the adsorption membrane according to the expected RNA yield, place at room temperature for 2 minutes, and centrifuge at 12000 rpm for 1 minute to obtain the RNA solution.

[0049] 2RNA reverse transcription

[0050] First-strand cDNA synthesis was performed using the Novizan HiScript III All-in-one RT SuperMixPerfect for qPCR kit. The specific method is as follows:

[0051] (1) Preparation of reaction system: 1 pg - 1 μg total RNA; 4 μL, 5 × All-in-one qRTSuperMix; 1 μL, Enzyme Mix; RNase-Free ddH2O to 20 μL; (2) Gently pipette 8 - 10 times until fully mixed, and briefly centrifuge to collect to the bottom of the tube; (3) Incubate the RNA sample at 50℃ for 15 min in the PCR instrument, and then incubate at 85℃ for 15 s to terminate the reaction.

[0052] The reverse transcription product can be used immediately for qPCR reactions, or stored at -30 to -15°C and used within six months; for long-term storage, it is recommended to aliquot and store at -85 to -65°C, and cDNA should avoid repeated freeze-thaw cycles.

[0053] 3 genes ZmAP1 Cloning and construction of overexpression vectors

[0054] Primers ZmAP2-CDS-F and ZmAP2-CDS-R were designed using the primer design software DNAMAN:

[0055] ZmAP2-CDS-F:

[0056] 5'-AGGTAGAAGAGGTACCCGGGATGGGTACAAACCCTCACCT-3' (SEQ ID No. 5);

[0057] ZmAP2-CDS-R:

[0058] 5'-GCCTGCAGGTCGACTCTAGAGCCACCGCCAACCTCCTCCA-3' (SEQ ID No. 6).

[0059] Using the cDNA obtained above as a template, PCR amplification was performed using primers ZmAP2-CDS-F and ZmAP2-CDS-R (using high-fidelity KOD enzyme in the amplification system) to obtain PCR products; the PCR products obtained above were then subjected to gel electrophoresis and gel recovery to obtain the recovered products. ZmAP2The gene, after sequencing, has a nucleotide sequence as shown in SEQ ID No. 1, and its encoded protein amino acid sequence is shown in SEQ ID No. 2. The recovered product was recombined with the pCam23A vector, which had undergone the same enzyme digestion, using infusion recombinase to obtain the recombinant vector pCam23A-ZmAP2. After sequencing, the recombinant vector pCam23A-ZmAP2 is a recombinant expression vector obtained by replacing the SmaI and XbaI sites of the pCam23A vector with the nucleotides of the ZmAP2 gene shown in SEQ ID No. 1.

[0060] Example 2 Rice OsAP2 Gene cloning and construction of overexpression vectors

[0061] 1. Extraction of RNA from rice

[0062] Using the same methods as in Examples 1 and 2, RNA was extracted from Nipponbare rice and reverse transcribed into cDNA, which was then used as a template to amplify Os. AP2 CDS sequence.

[0063] 2 Rice Os AP2 Cloning of the CDS gene and construction of overexpression vectors

[0064] Primers Os were designed using the primer design software DNAMAN. AP2 -OE-F and Os AP2 -OE-R:

[0065] OsAP2-OE-F:

[0066] 5'-AGGTAGAAGAGGTACCCGGGATGGGTACAAACCCTAGCCT-3' (SEQ ID No. 7);

[0067] OsAP2-OE-R:

[0068] 5'-GCCTGCAGGTCGACTCTAGAGGCCCCGCCAACTTCTTCCA-3' (SEQ ID No. 8);

[0069] Using the obtained rice Nipponbare cDNA as a template, and OsAP2-OE-F and OsAP2-OE-R as primers, PCR amplification was performed (using high-fidelity KOD enzyme in the amplification system) to obtain the PCR product. The obtained PCR product was then subjected to gel electrophoresis and gel recovery to obtain the recovered product, which was OsAP2-OE-F. AP2CDS, after sequencing, has the nucleotide sequence shown in SEQ ID No. 3, and the amino acid sequence of its encoded protein shown in SEQ ID No. 4. The recovered product was then recombined with the pCam23A vector, which had undergone the same enzyme digestion, using Infusion recombinase to obtain the recombinant vector pCam23A-Os. AP2 After sequencing, the recombinant vector pCam23A-Os AP2 The recombinant expression vector was obtained by replacing the SmaI and XbaI sites of the pCam23A vector with nucleotides of the ZmAP2 gene shown in SEQ ID No. 2.

[0070] Experimental Example 1: Maize Genes ZmAP2 Application trial of overexpression in Nipponbare rice to enhance grain number and yield in rice.

[0071] 1. Experimental Methods

[0072] 1.1 Maize Genes ZmAP2 Preparation of rice plants overexpressing Nipponbare rice

[0073] 1.1.1 Genetic transformation of pCam23A-ZmAP2 overexpression vector

[0074] (1) The recombinant vector pCam23A-ZmAP2 prepared in Example 1 was introduced into Agrobacterium AGL1 to obtain recombinant bacteria AGL1 / pCam23A-ZmAP2. After enzyme digestion verification, positive recombinant bacteria were obtained.

[0075] (2) The recombinant strain AGL1 / pCam23A-ZmAP2 was transformed into Nipponbare rice using rice genetic transformation to obtain T0 generation rice lines overexpressing the maize gene ZmAP2 in Nipponbare rice. The specific operation steps are as follows:

[0076] S1. Rice callus induction

[0077] The wild type of rice is Nipponbare rice (preserved by the High Photoefficiency Crop Team of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences; the public can obtain it from the High Photoefficiency Crop Team of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, but must promise that the biological material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes). The optimal G418 concentration for screening resistant callus is 50 mg / L.

[0078] Dehull mature seeds and select about 150 well-developed seeds to place in a 50ml Erlenmeyer flask. Rinse the flask 1-2 times with sterile water to remove surface dust and impurities. Then, surface sterilize with 30mL of 70% ethanol for 1 minute, followed by simmering in 30mL of 50% sodium hypochlorite for 9 minutes. Repeat once. Finally, rinse 5-7 times with sterile water until the water is clear. Blot the sterilized seeds dry with sterile filter paper and place them on MS+ medium at 12-15 seeds / plate (90*20mm). Incubate in the dark at 28-30℃ for 28 days until callus the size of a small grain of rice grows.

[0079] S2. Callus subculture

[0080] Transfer the dense, round, milky-yellow embryogenic callus that has grown and detached from the mature embryo to MS medium. Subculture approximately 100 callus per dish. Incubate in the dark at 28°C for 7 days. The picked callus can be returned to the culture medium for further induction. Each batch of induction can be picked 3-4 times. At the same time, large pieces of callus that have not detached can be placed on fresh MS+ medium to detach again and picked again.

[0081] S3. Prepare Agrobacterium culture medium

[0082] One day in advance, prepare YEP (or LB medium) with the corresponding selection resistance, kanamycin + rifampin or spectinomycin + rifampin, and spread Agrobacterium AGL1 on the medium. Use a spreader to scrape Agrobacterium colonies from the original plate and spread them evenly on the new medium. If it is a bacterial culture, pour the bacterial culture into the medium in a small amount and spread it evenly with a spreader. Label the carriers, invert the plate, and incubate overnight at 28°C.

[0083] Without activation treatment, the transformed colonies can be cultured directly for 4-5 days. Before shaking, use a sterilized spoon to spread all the colonies evenly, and then scrape off an appropriate amount of colony for shaking.

[0084] S4. Agrobacterium infection and co-culture

[0085] Scrape off the Agrobacterium using a key and place it in AAM liquid medium containing 40 mg AS. Shake at 28°C and 200 rpm for 1 hour. Then adjust the OD value to 0.12-0.15 using the same AAM containing AS. Note that the control AAM used for measuring the OD value is the same as the AAM used for conditioning the bacteria.

[0086] Collect the pre-cultured, milky-yellow, round, dense callus cells into 100ml Erlenmeyer flasks (approximately one plate per size); pour in the prepared Agrobacterium tumefaciens, inoculate, and shake at 90 rpm for 30 minutes. After 30 minutes, discard the inoculum, blot the callus cells dry with filter paper, and ensure they are completely dry before transferring them to NBCO+AS co-culture medium (place sterile small filter papers on the medium, or directly place filter papers of appropriate size to the culture dish, one dish per size), ensuring all callus cells are in contact with the filter paper surface. (Each carrier is divided into 2 dishes for co-culture with small filter papers). Incubate in the dark at 22℃ for 3-4 days.

[0087] S5. Callus screening and culture

[0088] Collect the co-cultured callus tissue into an Erlenmeyer flask (or 50ml centrifuge tube) using forceps. Rinse the callus several times (5-7 times) with sterile water until the washings are clear. Then add 1ml of NBL + Tim 200mg / ml washing solution and gently shake on a shaker at 100-120 rpm for 1 hour. Afterward, discard the washings, blot dry with filter paper, and transfer the callus to selective medium with a spoon. Use forceps to evenly distribute the callus particles to prevent contact inhibition and large-scale contamination. 2-3 plates per vector are sufficient for screening. Incubate in the dark at 28℃ for 2 weeks; this process constitutes the first screening. During this period, observe for any signs of contamination. Two weeks later, subculture again on the same medium, doubling the number of screening plates to 4-6 plates per vector. Select for approximately 4 weeks in total. This process constitutes the second screening. The next stage is complete when small, round, yellow granules (about the size of millet grains) detach from the callus tissue.

[0089] S6. Callus differentiation and rooting culture

[0090] Select dense, white callus and transfer it to differentiation medium. Ensure the medium is completely dry before use. Each dish can hold 20 callus cells, taking care to avoid placing them on the edge of the dish to prevent water contact. Incubate at 28°C under light for 3-4 weeks. Subculture again on the same medium. Newly subcultured callus should be left to stand for two days or protected from light by covering with a black plastic bag to prevent overheating and browning. (Change the medium every two weeks.)

[0091] S7. Transplanting of tissue culture seedlings

[0092] If robust seedlings appear, transfer them to 1 / 2 MS seedling culture medium. Incubate at 28°C under light for 2-3 weeks. Place the seedlings on the medium for two days before applying light. Wash away any residual culture medium from the roots, and transfer the seedlings with good root systems to a greenhouse. Keep the soil moist for the first few days. The resulting regenerated seedlings are the T0 generation transgenic plants. Use this T0 material for self-pollination to obtain the T1 generation transgenic rice, which will be used for subsequent analysis.

[0093] 1.1.2 ZmAP2 Identification of gene overexpression plants

[0094] Extracting T0 generation transgenic material ZmAP2 RNA from rice seedlings was overexpressed, and cDNA was obtained by reverse transcription. A pair of primers, ZmAP2-QRT1 and ZmAP2-QRT2, were designed from the CDS region for quantitative analysis of the transformed seedlings. Quantitative PCR amplification was performed using primers ZmAP2-QRT1 and ZmAP2-QRT2.

[0095] ZmAP2-QRT1: 5'-AGCCAGCGAAGCCTATCATC-3' (SEQ ID No. 9);

[0096] ZmAP2-QRT2: 5'-CACGTAGATGGAGGCAGCAT-3' (SEQ ID No. 10).

[0097] Quantitative PCR was performed on an iQ5 Muticolor Real-Time PCR Detection System (Bio-Rad) using SYBR Green Mix as the quantitative PCR reagent; each sample was independently replicated three times.

[0098] The reaction system consisted of 25 μL of: 12.5 μL of 2×Mix, 1 μL of Primier1, 1 μL of Primier2, 3 μL of cDNA, and 7.5 μL of ddH2O.

[0099] Reaction procedure: collect fluorescence at 95℃ for 5 min, 95℃ for 10 s, 60℃ for 20 s, 72℃ for 30 s, and 80℃ for 10 s. Increase the temperature at 72℃ for 5 min, then increase the temperature from 55℃ to 95℃ at a rate of 0.5℃ / s to create a melting curve. Repeat steps 2-5 for 40 cycles.

[0100] The internal reference primers used were actin-F and actin-R:

[0101] actin-F: 5'-TGCTATGTACGTCGCCATCCAG-3' (SEQ ID No. 11);

[0102] actin-R: 5'-AATGAGTAACCACGCTCCGTCA-3' (SEQ ID No. 12);

[0103] ZmAP2-1, ZmAP2-2, and ZmAP2-3 are T1 generation transgenic strains. ZmAP2 Three random lines overexpressing the gene in Nipponbare rice showed the following results: Figure 1 As shown in C, genes ZmAP2The expression level increased significantly in the transgenic material, indicating that the overexpression was successful.

[0104] 1.2 Preparation of rice plants transfected with empty vectors

[0105] Recombinant Agrobacterium AGL1 / pCam23A was used instead of recombinant Agrobacterium AGL1 / pCam23A- ZmAP2 Following the steps in 1.1 above, empty vector rice plants were obtained, which were subsequently referred to as the control CK.

[0106] 2. Experimental Results

[0107] Gene ZmAP2 Agronomic trait analysis of rice plants overexpressing the Nipponbare transgene: Rice materials were planted in the field and phenotypic observations and statistical analyses were performed. The control group (CK) consisted of 15 rice plants per system. Phenotypic observation results are as follows: Figure 1 As shown, Figure 1 A is a comparison diagram of plant type between the control (CK) and the transgenic line; Figure 1 B shows a comparison of grain count between the control (CK) and the transgenic lines; compared to the control (CK), the ZmAP2-1, ZmAP2-2, and ZmAP2-3 lines all showed a significant increase in the number of grains per ear. Figure 1 D) Significant increase in grain length ( Figure 1 E) Significant increase in thousand-grain weight ( Figure 1 F) Significantly increased biomass per plant ( Figure 1 G) and a significant increase in plant height ( Figure 1 H) indicates that the gene is overexpressed in the crop rice. ZmAP2 It can significantly increase the number of grains per panicle, grain length, thousand-grain weight, biomass, and plant height in rice. Therefore, it can be concluded that maize genes... ZmAP2 Overexpression in rice can increase rice biomass, grain length, panicle length, plant height, thousand-grain weight, and number of grains per panicle.

[0108] Experimental Example 2: Rice gene Os AP2 Application of expression in Nipponbare rice to enhance grain number and yield

[0109] 1. Experimental Methods

[0110] 1.1 OsAP2 Preparation of rice plants overexpressing Nipponbare rice

[0111] 1.1.1 Genetic transformation of pCam23A-OsAP2 overexpression vector

[0112] Using the same method as in Experiment 1, the constructed rice OsAP2 The gene overexpression vector pCam23A-OsAP2 was transferred into wild-type rice Nipponbare using Agrobacterium tumefaciens, resulting in multiple rice varieties. OsAP2 The gene was overexpressed in the T0 generation of Nipponbare rice, and the T1 generation was obtained by self-pollination of the T0 generation plants. OsAP2 Overexpressing plants, OsAP2#1-1, OsAP2#2-2 and OsAP2#3-4 were randomly selected for subsequent analysis.

[0113] 1.1.2 Os AP2 Identification of gene overexpression plants

[0114] Extracting T0 generation transgenic material OsAP2 RNA from rice seedlings was overexpressed, and cDNA was obtained by reverse transcription. Using cDNA as a template, a pair of primers, OsAP2-QRT1 and OsAP2-QRT2, were designed from the CDS region for quantitative analysis of the transformed seedlings. Quantitative PCR amplification was performed using primers OsAP2-QRT1 and OsAP2-QRT2.

[0115] OsAP2-QRT1: 5'- CAGCTAAGCCGACCCTTCTT-3' (SEQ ID No. 13);

[0116] OsAP2-QRT2: 5'-ATCGCTTCTTACGCTTGCCT-3' (SEQ ID No. 14).

[0117] Quantitative PCR was performed on an iQ5 Muticolor Real-Time PCR Detection System (Bio-Rad) using SYBR Green Mix as the quantitative PCR reagent; each sample was independently replicated three times.

[0118] The reaction system consisted of 25 μL of: 12.5 μL of 2×Mix, 1 μL of Primier1, 1 μL of Primier2, 3 μL of cDNA, and 7.5 μL of ddH2O.

[0119] Reaction procedure: collect fluorescence at 95℃ for 5 min, 95℃ for 10 s, 60℃ for 20 s, 72℃ for 30 s, and 80℃ for 10 s. Increase the temperature at 72℃ for 5 min, then increase the temperature from 55℃ to 95℃ at a rate of 0.5℃ / s to create a melting curve. Repeat steps 2-5 for 40 cycles.

[0120] The internal reference primers used were actin-F and actin-R:

[0121] actin-F: 5'-TGCTATGTACGTCGCCATCCAG-3' (SEQ ID No. 15);

[0122] actin-R: 5'-AATGAGTAACCACGCTCCGTCA-3' (SEQ ID No. 16);

[0123] OsAP2#1-1, OsAP2#2-2, and OsAP2#3-4 are T1 generation genetically modified organisms. AP2 Three random lines overexpressing the gene in Nipponbare rice showed the following results: Figure 2 As shown in C, genes AP2 The expression level increased significantly in the transgenic material, indicating that the overexpression was successful.

[0124] 1.2 Preparation of rice plants transfected with empty vectors

[0125] Recombinant Agrobacterium AGL1 / pCam23A was used to replace recombinant Agrobacterium AGL1 / pCam23A-OsAP2, and the operation was carried out according to step 1.1 above to obtain rice plants with empty vector, which were referred to as the control CK.

[0126] 2. Experimental Results

[0127] Gene OsAP2 Agronomic trait analysis of rice plants overexpressing the Nipponbare transgene: Rice materials were planted in the field and phenotypic observations and statistical analyses were performed. The control group (CK) consisted of 15 rice plants per system. Phenotypic observation results are as follows: Figure 2 As shown, Figure 2 A is a comparison diagram of plant type between the control (CK) and the transgenic line; Figure 2 B shows a comparison of grain count between the control (CK) and the transgenic lines; compared to the control (CK), the OsAP2#1-1, OsAP2 #2-2, and OsAP2 #3-4 lines all showed a significant increase in the number of grains per ear. Figure 2 D) Significant increase in grain length ( Figure 2 E) Significant increase in thousand-grain weight ( Figure 2 F) and a significant increase in biomass ( Figure 2 G), indicating gene overexpression in the crop rice. OsAP2 It can significantly improve the thousand-grain weight, number of grains per panicle, grain length, plant height, and biomass of rice. Therefore, it can be concluded that genes... OsAP2 Expression in rice increases the thousand-grain weight, number of grains per panicle, grain length, plant height, and biomass.

Claims

1. AP2 Gene or AP2 The uses of gene expression vectors in increasing rice yield or promoting rice growth; including: Will AP2 The gene was overexpressed in rice, increasing AP2 The expression level or activity of genes in rice; the increase in rice yield includes increasing the number of grains per panicle, grain length, thousand-grain weight, or biomass; the promotion of rice growth includes increasing plant height; The aforementioned AP2 The gene was isolated from corn. ZmAP2 The gene, whose nucleotide sequence is shown in SEQ ID No. 1; or the aforementioned AP2 The gene was isolated from rice. OsAP2 The gene, whose nucleotide sequence is shown in SEQ ID No.

3.

2. A method for cultivating high-yield rice varieties or increasing rice yield and promoting rice growth, characterized in that, include: Will AP2 The gene was overexpressed in rice, increasing AP2 The expression level or activity of genes in rice can increase rice yield or promote rice growth; the increase in rice yield includes increasing the number of grains per panicle, grain length, thousand-grain weight, or biomass; the promotion of rice growth includes increasing plant height. The aforementioned AP2 The gene was isolated from corn. ZmAP2 The gene, whose nucleotide sequence is shown in SEQ ID No. 1; or the aforementioned AP2 The gene was isolated from rice. OsAP2 The gene, whose nucleotide sequence is shown in SEQ ID No. 3.

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