Maize gene GRMZM5G845366 and its application in regulating plant height and yield of crops

Abstract

The present invention relates to the field of plant molecular biology technology, and specifically to a corn gene GRMZM5G845366 and its application in regulating crop plant height and yield. The CDS sequence of the corn gene GRMZM5G845366 is shown in SEQ ID No.2, and the protein encoded by it is characterized in that its amino acid sequence is shown in SEQ ID No.1. The corn gene GRMZM5G845366 identified by the present invention encodes a zinc finger protein transcription factor, and the CDS length of the gene is 2244bp. At present, there are no research reports on the gene and its homologous genes in plant seed size, grain weight, yield and plant height, and there are no reports on its application in increasing crop yield, especially in rice genetic breeding or in the corresponding high-yield transgenic crop rice.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular biology, and in particular to a corn gene GRMZM5G845366 and an application thereof in regulating plant height and yield of crops. Background Art

[0002] Grain shape of crops is a complex quantitative trait controlled by multiple genes. It is significantly correlated with yield, appearance quality, processing quality and taste quality, and is one of the most commonly used target traits in breeding. Rice grain shape is composed of grain length, grain width, grain thickness, aspect ratio, etc. In recent years, multiple genes regulating grain shape have been cloned, such as GL7, PGL2, GLW10, WTG1 and GW5L. These genes encode different types of functional proteins, which mainly regulate grain size and shape through ubiquitin proteasome, transcriptional regulatory factors, G protein signaling and crop hormone signaling. Its regulatory network is relatively complex. Therefore, it is necessary to explore more genes related to grain shape regulation and conduct functional studies to help analyze the genetic mechanism of crop grain shape, and at the same time provide useful genetic resources for crop breeding. Summary of the invention

[0003] The purpose of the present invention is to provide a corn gene GRMZM5G845366 and its application in regulating plant height and yield of crops.

[0004] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0005] The present invention provides an application of the protein corn gene GRMZM5G845366 in regulating the grain shape of crop seeds or an application of a substance for regulating the expression of a protein encoding gene or a substance for regulating the activity and / or content of a protein in any of the following:

[0006] D1) Regulate plant yield;

[0007] D2) preparing products for regulating plant yield;

[0008] D3) Breeding plants with increased yield;

[0009] D4) preparing products for breeding plants with increased yield;

[0010] D5) Improving high-yield plants or preparing products of plants with increased yield;

[0011] D6) regulates the grain length of plants;

[0012] D7) preparing a product for regulating seed length of a plant;

[0013] D8) breeding plants with increased grain length;

[0014] D9) preparing a product for breeding plants with increased kernel length;

[0015] D10) regulates the grain width of the plant;

[0016] D11) preparing a product for regulating the grain width of a plant;

[0017] D12) cultivating plants with increased kernel width;

[0018] D13) preparing a product for breeding plants with increased kernel width;

[0019] D14) regulating the thousand-grain weight of the plant;

[0020] D15) preparing a product for regulating thousand-grain weight of a plant;

[0021] D16) cultivating plants with increased thousand-grain weight;

[0022] D17) preparing a product for breeding plants with increased thousand-grain weight;

[0023] D18) regulating plant height;

[0024] D19) preparing a product for regulating plant height;

[0025] D20) cultivating plants with reduced plant height;

[0026] D21) preparing products for growing plants with reduced plant height;

[0027] The protein is any of the following:

[0028] A1) a protein whose amino acid sequence is SEQ ID No. 1;

[0029] A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues;

[0030] A3) A fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid shown in A1) or A2).

[0031] Tagged proteins include, but are not limited to, GST (glutathione sulfhydryltransferase) tagged proteins, His6 tagged proteins (His-tag), MBP (maltose binding protein) tagged proteins, Flag tagged proteins, SUMO tagged proteins, HA tagged proteins, Myc tagged proteins, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tagged proteins.

[0032] In the above applications, the substance is a biological material, and the biological material is any of the following:

[0033] B1), RNA molecules that inhibit, reduce or down-regulate the expression of protein-coding genes or RNA molecules that inhibit, reduce or down-regulate the activity or content of proteins;

[0034] B2), the coding gene expressing the RNA molecule of B1);

[0035] B3), an expression cassette containing the gene of B2);

[0036] B4), a recombinant vector containing the gene of B2), or a recombinant vector containing the expression cassette of B3);

[0037] B5), a recombinant microorganism containing the gene of B2), or a recombinant microorganism containing the expression cassette of B3), or a recombinant microorganism containing the recombinant vector of B4);

[0038] B6), a transgenic plant cell line containing the gene of B2), or a transgenic plant cell line containing the expression cassette of B3), or a transgenic plant cell line containing the recombinant vector of B4);

[0039] B7), transgenic plant tissue containing B2) gene, or transgenic plant tissue containing B3) expression cassette, or transgenic plant tissue containing B4) recombinant vector;

[0040] B8), transgenic plant organs containing the gene of B2), or transgenic plant organs containing the expression cassette of B3), or transgenic plant organs containing the recombinant vector of B4);

[0041] B9), a nucleic acid molecule encoding the aforementioned protein;

[0042] B10), an expression cassette containing the nucleic acid molecule of B9);

[0043] B11), a recombinant vector containing a nucleic acid molecule of B9), or a recombinant vector containing an expression cassette of B10);

[0044] B12), a recombinant microorganism containing a nucleic acid molecule of B9), or a recombinant microorganism containing an expression cassette of B10), or a recombinant microorganism containing a recombinant vector of B3);

[0045] B13), a transgenic plant cell line containing a nucleic acid molecule of B9), or a transgenic plant cell line containing an expression cassette of B10);

[0046] B14), transgenic plant tissue containing the nucleic acid molecule of B9), or transgenic plant tissue containing the expression cassette of B10);

[0047] B15), transgenic plant organs containing the nucleic acid molecule of B9), or transgenic plant organs containing the expression cassette of B10).

[0048] In some specific embodiments of the present invention, the recombinant vector is obtained by replacing the SmaI and XbaI sites of the pCam23A vector with the nucleotides shown in SEQ ID No. 2.

[0049] In the above application, the expression cassette containing the nucleic acid molecule encoding the aforementioned protein (the gene expression cassette of the gene encoding the aforementioned protein) refers to a DNA capable of expressing the gene encoding the aforementioned protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene encoding the aforementioned protein, but also a terminator for terminating transcription of the gene encoding the aforementioned protein. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to, constitutive promoters; tissue, organ and development-specific promoters and inducible promoters. Suitable transcription terminators include, but are not limited to, Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine synthase terminators.

[0050] Existing expression vectors can be used to construct recombinant vectors containing the gene expression cassettes encoding the aforementioned proteins. Plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. Plant expression vectors can also contain the 3'-end non-translated region of the foreign gene, that is, containing polyadenylation signals and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the non-translated region transcribed at the 3' end of the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos) and the plant gene (such as the rice or corn storage protein gene) all have similar functions. When using the gene of the present invention to construct a plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of translation control signals and start codons are extensive and can be natural or synthetic. The translation start region can come from the transcription start region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

[0051] In the above application, the B1) RNA molecule targets the mRNA transcribed from the gene of the aforementioned protein.

[0052] In the above application, B9) the nucleic acid molecule is a cDNA molecule or a DNA molecule whose coding sequence is SEQ ID No.2.

[0053] In the above applications, the vector may be a plasmid, cosmid, phage or virus vector.

[0054] In the above applications, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0055] In the above applications, transgenic plant cell lines do not include propagation materials.

[0056] In the above application, the substance that regulates the expression of the gene encoding the aforementioned protein or the substance that regulates the content or activity of the aforementioned protein is a substance that increases the expression of the gene encoding the aforementioned protein in cells.

[0057] In the above application, the plant is any of the following:

[0058] G1) Monocots;

[0059] G2) Gramineae;

[0060] G3) Oryza plants;

[0061] G4) Rice plants;

[0062] G5) Rice.

[0063] The beneficial effects of the present invention are:

[0064] The maize gene GRMZM5G845366 identified by the present invention encodes a zinc finger protein transcription factor, and the CDS length of the gene is 2244bp. At present, there are no reports on the research of the gene and its homologous genes in plant grain size, grain weight, yield and plant height, and there are no reports on its application in increasing crop yield, especially in rice genetic breeding or in the corresponding high-yield transgenic rice crop. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 is the relative expression level of maize GRMZM5G845366 gene in the control CK, OE-3, OE-7 and OE-12 overexpression lines;

[0066] Figure 2 Seed phenotypes of transgenic lines were obtained for control CK and overexpressing maize gene GRMZM5G845366;

[0067] Figure 3 The statistical analysis results of the phenotypes of the control CK and the lines overexpressing the corn gene GRMZM5G845366; A is the statistical analysis result of the grain length of the control CK and the transgenic lines; B is the statistical analysis result of the grain width of the control CK and the transgenic lines; C is the statistical analysis result of the thousand-grain weight of the control CK and the transgenic lines; D is the statistical analysis result of the single plant yield of the control CK and the transgenic lines; E is the statistical analysis result of the plant height of the control CK and the transgenic lines. DETAILED DESCRIPTION

[0068] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0069] Example

[0070] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0071] pCam23A vector: recorded in "Xuean Cui, Zhiguo Zhang, Yanwei Wang, Jinxia Wu, XiaoHan, 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.", which is available to the public from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0072] Agrobacterium AGL1: purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd., catalog number ZK296.

[0073] The wild type of rice is Nipponbare rice (Oryza sativa L.ssp.Japonica variety Nipponbare; hereinafter also referred to as wild type rice WT), which was 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 field of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0074] The primers used in the following examples were synthesized by Beijing Qingke Biotechnology Co., Ltd., and the sequencing was completed by Beijing Qingke Biotechnology Co., Ltd. pTEAY-T1 Cloning Kit, Taq enzyme, Trans5α competent medium and related kits were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; restriction endonucleases SmaI and XbaI, Infusion recombinase, and T4 ligase were purchased from Dalian TaKaRa Company; antibiotics were purchased from SIGMA Company, USA; and the remaining reagents were all domestic analytical grade.

[0075] Induction medium formula: Based on N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, 0.8 g / L hydrolyzed casein, 0.3 g / L proline, 30 g / L sucrose and 3 g / L phytagel.

[0076] N6 culture medium formula: solutes are potassium nitrate 2830g / L, ammonium sulfate 463g / L, calcium chloride (CaCl2·2H2O) 166g / L, magnesium sulfate (MgSO4·7H2O) 185g / L, potassium dihydrogen phosphate 400g / L, ferrous sulfate (FeSO4·7H2O) 27.8g / L, manganese sulfate (MnSO4·H2O) 4.4g / L, zinc sulfate (ZnSO4·7H2O) 1.6g / L, boric acid 0.8g / L, potassium iodide 1.6g / L, vitamin B1 (thiamine hydrochloride) 1.0g / L, vitamin B6 (pyridoxine hydrochloride) 0.5g / L, niacin 0.5g / L, glycine 2.0g / L, and the solvent is deionized water.

[0077] Suspension medium: N6 medium was supplemented with 2,4-D at a final concentration of 2.5 mg / L, 0.8 g / L hydrolyzed casein, 0.3 g / L proline, 30 g / L sucrose, 10 g / L glucose and 100 μM acetosyringone.

[0078] Co-culture medium: Add 2,4-D with a final concentration of 2.5 mg / L, 0.3 g / L proline, 30 g / L sucrose, 10 g / L glucose, 100 μM acetosyringone and 8 g / L agar powder to N6 medium.

[0079] Selection medium: Based on N6 medium, add 2,4-D with a final concentration of 2.5 mg / L, 0.3 g / L proline, 50 mg / L G418, 200 mg / L timentin, 30 g / L sucrose and 8 g / L agar powder.

[0080] Differentiation medium formula: 2 mg / L KT, 0.2 mg / L NAA, 2 mg / L 6-BA, 0.2 mg / L IAA, 0.8 g / L hydrolyzed casein, 0.3 g / L proline, 30 g / L sucrose and 3 g / L phytagel were added to MS medium, and the balance was sterile water.

[0081] MS culture medium formula: solutes are CaCl2·2H2O 440mg / L, KH2PO4 170mg / L, MgSO4·7H2O370mg / L, NH4NO3 1650mg / L, KNO3 1900mg / L, KI 0.83mg / L, CoCl2·6H2O 0.025mg / L, H3BO46.2mg / L, Na2MoO4·7H2O 0.25mg / L, MnSO4·4H2O22.3mg / L, CuSO4·5H2O 0.025mg / L, ZnSO4·7H2O 8.6mg / L, FeSO4·7H2O 27.8mg / L, Na2EDTA 37.3mg / L, thiamine hydrochloride 0.1mg / L, pyridoxine hydrochloride 0.5mg / L, niacin 0.5mg / L, inositol 100mg / L, glycine 2.0mg / L, sucrose 30000mg / L, agar powder 7000mg / L, and the balance is deionized water.

[0082] Rooting medium formula: Add sucrose with a final concentration of 20g / L and 3g / L phytagel to 1 / 2MS medium.

[0083] 1 / 2MS medium: The final concentration of solutes in MS is halved.

[0084] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. The Student's t test was used for the test. P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated a very significant difference.

[0085] Example 1: Cloning and vector construction of corn gene GRMZM5G845366

[0086] 1. Extraction of RNA from Maize

[0087] The RNAprep pure plant kit from Tiangen Biochemical was used to extract total RNA from corn B73. The tissue samples used for testing were placed in tin foil after being sampled in the field, quickly frozen in liquid nitrogen, and brought back to the Beijing laboratory for storage in an ultra-low temperature refrigerator. The specific steps for extracting RNA are as follows:

[0088] (1) Grind 100 mg of sample under liquid nitrogen low temperature environment, then add 500 μL lysis buffer SL. Add β-mercaptoethanol before use and vortex vigorously to mix;

[0089] (2) Centrifugation at 12500 rpm for 5 min;

[0090] (3) Transfer the supernatant to a CS filter column, centrifuge at 12,500 rpm for 2 min, and transfer the supernatant to a new tube.

[0091] (4) Add 0.4 times the volume of the supernatant liquid anhydrous ethanol, mix well and a precipitate will precipitate. Transfer it to the CR3 adsorption column and centrifuge at 12500 rpm for 30 seconds. Discard the waste liquid and put the CR3 back into the original tube.

[0092] (5) Carefully add 400 μL RW1 to CR3, incubate at 13,000 rpm for 1 min, discard the lower solution, and return CR3 to the tube.

[0093] (6) Preparation of DNase I working solution: Take 10 μL of DNase I stock solution, add 70 μL RDD solution, and flick to mix.

[0094] (7) Add 80 μL of DNase I working solution to the center of the CR3 adsorption column and digest at room temperature for 15 min.

[0095] (8) Add 350 μL of RW1 to CR3, centrifuge at 12,500 rpm for 30 seconds, discard the waste liquid, and return CR3 to the tube.

[0096] (9) Add 500 μL RW to CR3, centrifuge at 12500 rpm for 30 sec, discard the waste liquid, and return CR3 to the tube.

[0097] (10) Repeat step 9.

[0098] (11) Centrifuge at 12500 rpm for 3 min, transfer CR3 into a clean RNase-Free centrifuge tube, add 70 μL RNase-Free ddH2O to the center of the membrane, incubate at room temperature for 5 min, and centrifuge at 13000 rpm for 2 min to obtain the RNA extraction product.

[0099] 2. RNA Reverse Transcription

[0100] First-strand cDNAs were synthesized using a reverse transcription kit IIIFirst Strand Synthesis Kit (Invitrogen, USA) was used, and the high GC reverse transcription method recommended by the kit was used, and Oligo(dT)20 (50 μM) was selected for synthesis. The specific method is as follows:

[0101] (1) Preparation of RNA-primer mixture: 2 μg total RNA: n μL (volume n is determined based on RNA concentration), Oligo(dT)20 (50 μM): 1 μL, 10 mM dNTP mix: 2.5 μL, RNase-Free ddH2O to 25 μL;

[0102] (2) Incubate the RNA sample at 65°C in a PCR machine for 3 min and then immediately transfer to 55°C;

[0103] (3) Preparation of cDNA-Synthesis Mix (25 μL): RNase-Free ddH2O: 3 μL, 10×RT buffer: 5 μL, 25 mM MgCl2: 10 μL, 0.1 M DTT: 5 μL, RNase OUTRecombinant RNaseI inhibitor: 1 μL, Super ScriptIIIRT: 1 μL. Mix the above ingredients and set aside.

[0104] (4) Gently and thoroughly mix the RNA-primer mixture and cDNA-Synthesis Mix, react at 55°C for 1 h, and terminate at 85°C for 3 min on a PCR instrument;

[0105] (5) Add 1-2 μL of RNase H and digest at 37°C for 30 min in a PCR instrument. Store at -20°C until use.

[0106] 3. Construction of gene GRMZM5G845366CDS overexpression vector

[0107] Primers GRMZM5G845366-CDS-F and GRMZM5G845366-CDS-R were designed using the primer design software DNAMAN:

[0108] GRMZM5G845366-CDS-F:

[0109] 5'-GTAGAAGAGGTACCCGGGATGGACGGCACGCCGATCTC-3';

[0110] GRMZM5G845366-CDS-R:

[0111] 5'-GCAGGTCGACTCTAGACTATGTTGCTAGACTACCAATCT-3'.

[0112] The cDNA obtained above was used as a template and PCR amplification was performed with primers GRMZM5G845366-CDS-F and GRMZM5G845366-CDS-R (high-fidelity KOD enzyme was used in the amplification system) to obtain a PCR product.

[0113] The PCR product obtained above was run on a gel and recovered from the gel, and the recovered product was recombined with the pCam23A vector that had been digested with the same enzyme using the Infusion recombinase. After sequencing, the recombinant vector pCam23A-GRMZM5G845366 was obtained by replacing the SmaI and XbaI sites of the pCam23A vector with the nucleotides shown in SEQ ID No. 2.

[0114] Example 2: Application of corn gene GRMZM5G845366 in increasing crop yield and reducing plant height

[0115] 1. Preparation of transgenic rice Nipponbare overexpressing maize gene GRMZM5G845366

[0116] 1. Genetic transformation of pCam23A-GRMZM5G845366 overexpression vector

[0117] (1) The recombinant vector pCam23A-GRMZM5G845366 prepared above was introduced into Agrobacterium AGL1 to obtain the recombinant bacteria AGL1 / pCam23A-GRMZM5G845366, and the positive recombinant bacteria were obtained after enzyme digestion verification.

[0118] (2) The recombinant bacteria AGL1 / pCam23A-GRMZM5G845366 was transformed into rice Nipponbare by rice genetic transformation to obtain a T0 generation rice strain that overexpressed the corn gene GRMZM5G845366 in rice Nipponbare. The specific steps are as follows:

[0119] S1. Rice callus induction

[0120] The wild type of rice is Oryza sativa L.ssp.Japonica variety Nipponbare, which is preserved by the Crop High Photoefficiency Team of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. The public can obtain it from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes. The optimal G418 concentration required for resistant callus screening is 50 mg / L.

[0121] The mature rice Nipponbare seeds were shelled, and then treated with 75% ethanol for 1 minute and 20% sodium hypochlorite for 15 minutes; the seeds were washed with sterile water for 5 times; the seeds were placed on callus induction medium and cultured in the dark for 4 weeks at a culture temperature of 28±1°C.

[0122] S2. Callus subculture

[0123] Bright yellow, compact and relatively dry embryonic callus was selected and placed on a subculture medium (with the same nutrient composition as the induction medium), and cultured in the dark for 3 weeks at a temperature of 28±1°C.

[0124] S3. Preparation of Agrobacterium culture medium

[0125] Agrobacterium AGL1 / CAS9-OsCY2-1 and AGL1 / CAS9-OsCY2-2 were cultured in LA medium (10 g / L Tryptone, 5 g / L Yeast extract, 10 g / L NaCl, 15 g / L agar powder, the rest was deionized water, pH 7.0) containing 50 mg / L kanamycin and rifampicin for 2 days at 28±1°C; Agrobacterium AGL1 / CAS9-OsCY2-1 and AGL1 / CAS9-OsCY2-2 were transferred to suspension culture medium, cultured on a shaker at 28°C, 200 rpm for 2 h to obtain Agrobacterium suspension.

[0126] S4. Agrobacterium infection

[0127] The callus tissue was transferred to a sterilized Erlenmeyer flask; the Agrobacterium suspension was adjusted to an OD600nm value of about 0.3; the callus tissue was soaked in the Agrobacterium suspension for 30 minutes; the callus tissue was transferred to a sterilized filter paper and blotted dry; and then placed on a co-culture medium and cultured for 3 days at a culture temperature of 22°C.

[0128] S5. Callus screening and culture

[0129] The callus tissue after co-cultivation was washed with sterile water for 10 times; then the callus tissue was immersed in sterile water containing 200 mg / L of timentin for 30 minutes; the callus tissue was transferred to a sterilized filter paper and blotted dry; the callus tissue was then transferred to a selective medium for selection culture for 2 times, each culture lasting 2 weeks.

[0130] S6. Callus differentiation culture

[0131] The resistant callus was transferred to differentiation medium and cultured under illumination (2000 Lux) (temperature 28±1°C).

[0132] S7. Rooting culture

[0133] Rooting: Cut off the roots produced during differentiation and transfer them to rooting medium and culture them under 2000 Lux light for 3 weeks (temperature 28 ± 1°C).

[0134] S8. Transplantation of tissue culture seedlings

[0135] The residual culture medium on the roots was washed off, and the seedlings with good root systems were transferred to the greenhouse. Meanwhile, the soil was kept moist in the first few days. The regenerated seedlings obtained were T0 generation transgenic plants. The T0 generation materials were self-pollinated to obtain T1 generation transgenic rice for subsequent analysis.

[0136] 2. Identification of plants overexpressing the GRMZM5G845366 gene

[0137] RNA was extracted from young leaves of T0 generation transgenic GRMZM5G845366-overexpressing rice, and cDNA was obtained by reverse transcription as a template. A pair of primers GRMZM5G845366-QRT1 and GRMZM5G845366-QRT2 were designed from the CDS region for quantitative analysis of transformed seedlings. Quantitative PCR amplification was performed using GRMZM5G845366-QRT1 primer and GRMZM5G845366-QRT2 primer.

[0138] GRMZM5G845366-QRT1: 5'-ATTGGGGCGTAGATGGTGAA-3';

[0139] GRMZM5G845366-QRT2: 5'-ATGCACCCAGGAGAGATCAG-3'.

[0140] Quantitative PCR was performed on the iQ5 Muticolor Real-Time PCR Detection System (Bio-Rad) using SYBR Green Mix as the fluorescent quantitative reagent; each sample was repeated three times independently.

[0141] Reaction system 25μL: 2×Mix 12.5μl, Primer1 1μl, Primer2 1μl, cDNA 3μl, ddH2O 7.5μl.

[0142] Reaction program: 95°C for 5 min, 95°C for 10 s, 60°C for 20 s, 72°C for 30 s, 80°C for 10 s to collect fluorescence, 72°C for 5 min, 55°C to 95°C at 0.5°C / s to make a melting curve, and run steps 2-5 for 40 cycles.

[0143] The internal reference primers used were actin-F and actin-R:

[0144] actin-F: 5'-TGCTATGTACGTCGCCATCCAG-3';

[0145] actin-R: 5'-AATGAGTAACCACGCTCCGTCA-3'

[0146] OE-3, OE-7, and OE-12 are three random lines of T1 transgenic GRMZM5G845366 overexpressed in rice Nipponbare. Figure 1 As shown, the expression level of gene GRMZM5G845366 was significantly increased in transgenic materials, indicating that the overexpression was successful.

[0147] 2. Preparation of Empty Vector Transgenic Rice Plants

[0148] Recombinant Agrobacterium AGL1 / pCam23A was used to replace recombinant Agrobacterium AGL1 / pCam23A-GRMZM5G845366, and the operation was carried out according to step 1 to obtain empty vector rice plants, which were subsequently referred to as control CK.

[0149] 3. Analysis of agronomic traits of plants overexpressing gene GRMZM5G845366 in rice Nipponbare

[0150] The rice materials were planted in the field and phenotypic observation and statistical analysis were performed. CK was the empty vector control, and each plant system counted 15 rice plants. Figure 2 and Figure 3 As shown, the OE-3, OE-7, and OE-12 strains all showed a significant increase in grain length compared to the control CK ( Figure 2 and Figure 3 A), the grain width increased significantly ( Figure 2 and Figure 3 Middle B), thousand-grain weight increased significantly ( Figure 2 and Figure 3 C), the yield per plant increased significantly ( Figure 3 D) and the plant height was significantly reduced ( Figure 3 (E) shows that overexpression of the gene GRMZM5G845366 in the crop rice can significantly increase the grain length, grain width, grain weight and yield of rice, while significantly reducing the plant height.

[0151] Therefore, it can be concluded that overexpression of the protein sequence (SEQ ID No. 1) expressed by the maize gene GRMZM5G845366 (SEQ ID No. 2) can increase grain length, grain width, grain weight and yield of crops such as rice, while reducing plant height.

[0152] In summary, the inventors previously conducted transcriptome sequencing analysis on the third leaf of three-leaf rice Nipponbare and corn B73 in segments, screened for transcription factors that were expressed in corn leaves but not in rice leaves, expressed strongly in corn leaves but weakly in rice leaves, and specifically expressed only in corn bundle sheath cells, and obtained a total of 870 transcription factors distributed in 44 families such as ARF, bHLH, GRAS, MYB, and NAC. Overexpression vectors were constructed for each gene and transformed into rice, creating a corn transcription factor overexpression mutant library. Through large-scale agronomic trait investigation and analysis of mutant library plants, it was found that more than 20 genes, after being overexpressed individually, could significantly increase plant yields, and were new genes with high-yield application potential. Among them, the corn gene GRMZM5G845366 identified in the present application, the transgenic plants obtained by overexpressing GRMZM5G845366 in the wild type Nipponbare rice, compared with the wild type Nipponbare, have significantly larger grains, significantly increased thousand-grain weight, significantly increased single plant yield, and significantly reduced plant height. At present, there are no research reports on this gene and its homologous genes in plant grain size, grain weight and plant height, nor on its application in increasing crop yield, which shows that the gene GRMZM5G845366 (SEQ ID No. 2) and the protein sequence (SEQ ID No. 1) expressed by the present invention have important theoretical and practical significance for cultivating new high-yield crop materials with enlarged grains and reduced plant height.

[0153] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0154] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Application of corn gene GRMZM5G845366, characterized in that: Overexpression of the gene GRMZM5G845366 can increase the grain length of rice.

2. Application of corn gene GRMZM5G845366, characterized in that: The grain width of rice can be increased by overexpressing the gene GRMZM5G845366.

3. Application of corn gene GRMZM5G845366, characterized in that: The grain weight of rice can be increased by overexpressing the gene GRMZM5G845366.

4. Application of corn gene GRMZM5G845366, characterized in that: Rice yield can be increased by overexpressing the gene GRMZM5G845366.

5. Application of corn gene GRMZM5G845366, characterized in that: The plant height of rice can be increased by overexpressing the gene GRMZM5G845366.

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