Maize gene GRMZM2G038988 and its application in regulating yield

Abstract

The present invention relates to the field of plant molecular biology technology, and specifically to a corn gene GRMZM2G038988 and its application in regulating yield. The CDS sequence of the corn gene GRMZM2G038988 is shown in SEQ ID No. 2, and the amino acid sequence of the protein encoded by it is shown in SEQ ID No. 1. The corn gene GRMZM2G038988 identified by the present invention encodes a protein that binds to calmodulin and regulates calcium signal transduction, and the CDS length of the gene is 1290 bp. At present, there are no research reports on the gene and its homologous genes in plant seed size, grain weight and yield, 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 invention relates to the technical field of plant molecular biology, and in particular to a corn gene GRMZM2G038988 and an application thereof in regulating yield. Background Art

[0002] Nowadays, the yield of major food crops such as corn and rice has reached its limit, and it has become increasingly difficult to significantly increase yields by relying on traditional breeding methods. Grain shape has an important impact on crop yield. Grain shape usually includes parameters such as grain length, grain width, grain thickness and thousand-grain weight. These characteristics not only affect the appearance quality of crop seeds, but are also closely related to yield. They are traits that need to be considered in crop breeding and cultivation. In the process of breeding and cultivation, optimizing grain shape can not only increase yield, but also improve seed quality and stability. Therefore, the study and improvement of grain shape is an important way to achieve high and stable crop yields. At present, several genes that regulate grain size, such as GW2, GS2, and GS3, have been cloned, but the genetic factors of crop grain size are complex. Continuing to clone more regulatory genes for grain size is of great significance to global food security and sustainable agricultural development. Summary of the invention

[0003] The purpose of the present invention is to provide a corn gene GRMZM2G038988 and its application in regulating yield.

[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 GRMZM2G038988 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] The protein is any of the following:

[0024] A1) a protein whose amino acid sequence is SEQ ID No. 1;

[0025] 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;

[0026] 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).

[0027] 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.

[0028] Those skilled in the art can easily mutate the nucleotide sequence encoding the protein of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the protein isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the aforementioned protein and have the aforementioned protein function.

[0029] In the above applications, the substance is a biological material, and the biological material is any of the following:

[0030] B1), an RNA molecule that inhibits, reduces or down-regulates the expression of the gene encoding the protein or an RNA molecule that inhibits, reduces or down-regulates the activity or content of the protein;

[0031] B2), the coding gene expressing the RNA molecule of B1);

[0032] B3), an expression cassette containing the gene of B2);

[0033] B4), a recombinant vector containing the gene of B2), or a recombinant vector containing the expression cassette of B3);

[0034] 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);

[0035] 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);

[0036] B7), transgenic plant tissue containing B2) gene, or transgenic plant tissue containing B3) expression cassette, or transgenic plant tissue containing B4) recombinant vector;

[0037] 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);

[0038] B9), a nucleic acid molecule encoding the aforementioned protein;

[0039] B10), an expression cassette containing the nucleic acid molecule of B9);

[0040] B11), a recombinant vector containing a nucleic acid molecule of B9), or a recombinant vector containing an expression cassette of B10);

[0041] 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);

[0042] 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);

[0043] B14), transgenic plant tissue containing the nucleic acid molecule of B9), or transgenic plant tissue containing the expression cassette of B10);

[0044] B15), transgenic plant organs containing the nucleic acid molecule of B9), or transgenic plant organs containing the expression cassette of B10).

[0045] Furthermore, the recombinant vector was obtained by replacing the SmaI and XbaI sites of the pCam23A vector with the nucleotides shown in SEQ ID No. 2.

[0046] 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.

[0047] 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.

[0048] In the above application, the B1) RNA molecule targets the mRNA transcribed from the gene of the aforementioned protein.

[0049] 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.

[0050] In the above applications, the vector may be a plasmid, cosmid, phage or virus vector.

[0051] In the above applications, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0052] In the above applications, transgenic plant cell lines do not include propagation materials.

[0053] 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.

[0054] In the above application, the plant is any of the following:

[0055] G1) Monocots;

[0056] G2) Gramineae;

[0057] G3) Oryza plants;

[0058] G4) Rice plants;

[0059] G5) Rice.

[0060] The beneficial effects of the present invention are:

[0061] The corn gene GRMZM2G038988 identified by the present invention encodes a protein that binds to calmodulin and regulates calcium signal transduction, and the CDS length of the gene is 1290bp. At present, there are no research reports on the gene and its homologous genes in plant grain size, grain weight and yield, 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

[0062] Figure 1 is the relative expression level of maize GRMZM2G038988 gene in the control CK, OE-1, OE-2 and OE-3 overexpression lines;

[0063] Figure 2 Seed phenotypes of transgenic lines were obtained for control CK and overexpressing maize gene GRMZM2G038988;

[0064] Figure 3 Figure 1 is the statistical analysis result of the phenotype of the control CK and the strain overexpressing the corn gene GRMZM2G038988; A is the statistical analysis result of the grain length of the control CK and the transgenic strain; B is the statistical analysis result of the grain width of the control CK and the transgenic strain; C is the statistical analysis result of the thousand-grain weight of the control CK and the transgenic strain; D is the statistical analysis result of the single plant yield of the control CK and the transgenic strain. DETAILED DESCRIPTION

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Agrobacterium AGL1: purchased from Beijing Zhuangmeng International Bio-Gene Technology Co., Ltd., catalog number ZK296.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Example 1: Cloning and vector construction of corn gene GRMZM2G038988

[0073] 1. Extraction of RNA from Maize

[0074] 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:

[0075] (1) Grind 100 mg of sample under liquid nitrogen cryogenic conditions, then add 500 μL of lysis buffer SL. Add β-mercaptoethanol before use and vortex vigorously to mix.

[0076] (2) Centrifugation at 12500 rpm for 5 min;

[0077] (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.

[0078] (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.

[0079] (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.

[0080] (6) Preparation of DNase I working solution: Take 10 μL of DNase I stock solution, add 70 μL RDD solution, and flick to mix.

[0081] (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.

[0082] (8) Add 350 μL of RW1 to CR3, centrifuge at 12,500 rpm for 30 sec, discard the waste liquid, and return CR3 to the tube.

[0083] (9) Add 500 μL RW to CR3, centrifuge at 12500 rpm for 30 seconds, discard the waste liquid, and return CR3 to the tube.

[0084] (10) Repeat step 9.

[0085] (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.

[0086] 2. RNA Reverse Transcription

[0087] 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:

[0088] (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;

[0089] (2) Incubate the RNA sample at 65°C for 3 min in a PCR instrument and then immediately transfer to 55°C;

[0090] (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.

[0091] (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;

[0092] (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.

[0093] 3. Construction of gene GRMZM2G038988CDS overexpression vector

[0094] Primers GRMZM2G038988-CDS-F and GRMZM2G038988-CDS-R were designed using the primer design software DNAMAN:

[0095] GRMZM2G038988-CDS-F:

[0096] 5'-GTAGAAGAGGTACCCGGGATGGGCATCTCATCCAAGTGGA-3';

[0097] GRMZM2G038988-CDS-R:

[0098] 5'-GCAGGTCGACTCTAGATCAGGCCTCTAACTCATCACC-3'.

[0099] The cDNA obtained above was used as a template and PCR amplification was performed with primers GRMZM2G038988-CDS-F and GRMZM2G038988-CDS-R (high-fidelity KOD enzyme was used in the amplification system) to obtain a PCR product.

[0100] 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-GRMZM2G038988 was obtained by replacing the SmaI and XbaI sites of the pCam23A vector with the nucleotides shown in SEQ ID No.2.

[0101] Example 2: Application of corn gene GRMZM2G038988 in improving rice grain shape, grain weight and yield

[0102] 1. Preparation of transgenic rice Nipponbare overexpressing maize gene GRMZM2G038988

[0103] 1. Genetic transformation of pCam23A-GRMZM2G038988 overexpression vector

[0104] (1) The recombinant vector pCam23A-GRMZM2G038988 prepared above was introduced into Agrobacterium AGL1 to obtain the recombinant bacteria AGL1 / pCam23A-GRMZM2G038988, and positive recombinant bacteria were obtained after enzyme digestion verification.

[0105] (2) The recombinant bacteria AGL1 / pCam23A-GRMZM2G038988 were transformed into rice Nipponbare by rice genetic transformation to obtain a T0 generation rice strain that overexpressed the corn gene GRMZM2G038988 in rice Nipponbare. The specific steps are as follows:

[0106] S1, seed sterilization and callus induction stage:

[0107] Mature seeds were shelled, 300 plump and intact shelled rice seeds were selected and placed in a 50 mL sterilized centrifuge tube, and the seeds were washed three times with sterilized ultrapure water. Then, the surface was disinfected with 40 mL 75% ethanol for 5 min, and sterilized with 40 mL 50% sodium hypochlorite for 5 min, and the sterilization was repeated once. Finally, the seeds were washed 10 times with sterile water until the water was clear. The sterilized seeds were blotted dry with sterile filter paper and placed on MS+ medium, 20 seeds / dish, and cultured at 28°C in the dark for 28 days until callus the size of a fallen millet grain was grown.

[0108] S2. Subculture and pre-culture stage of callus:

[0109] Transfer embryonic calli in good condition to MS medium. Place about 100 calli per dish and culture at 28℃ in the dark for 7 days.

[0110] The picked calli can be put back for further culture. A batch of induced calli can be picked 3-4 times. At the same time, large pieces of calli that have not fallen off can be placed on new MS+ culture medium, made to fall off again, and picked again.

[0111] S3. Preparation of Agrobacterium:

[0112] One day in advance, culture Agrobacterium on YEP (or LB medium) with corresponding resistance screening, kanamycin + rifampicin or spectinomycin + rifampicin; scrape the Agrobacterium colonies on the original plate with a spreading stick, and spread them evenly on the new medium. If it is a bacterial liquid, pour the bacterial liquid into the medium, a small amount, and spread it evenly with a spreading stick, mark the carrier, place it upside down, and incubate it in a 28°C incubator overnight.

[0113] Without activation treatment, the transformed colonies can be directly cultured for 4-5 days. Before shaking the colonies, use a sterilized spoon to evenly spread all the colonies, and then scrape an appropriate amount of bacteria and shake the colonies. (Those stored at 4℃ must be activated)

[0114] S4. OD value adjustment:

[0115] Scrape off the Agrobacterium with a key, put the bacteria into AAM liquid culture medium, and culture it in a 28°C shaker at 200 rpm for 2 hours. Then adjust the OD value of the bacterial solution to 0.12-0.15 with AAM.

[0116] S5. Infection and co-cultivation:

[0117] Collect about 100 embryonic calli into a 100mL triangular flask; pour the adjusted concentration of Agrobacterium into the conical flask for infection, shake it with a shaker at 100g for 20 minutes. After the shaking, pour out the infection solution, absorb the callus with filter paper, move the infected callus to the co-cultivation medium, cover it with sterile filter paper, and ensure that all calli are in contact with the filter paper surface. Culture in the dark at 22℃ for 4 days.

[0118] S6. Resistance screening of transformed callus:

[0119] Collect the callus tissue that has completed the co-cultivation stage into a 50mL sterilized centrifuge tube, rinse the callus with sterile water 10 times until the washing liquid is clear. Then pour in 1ml washing liquid of NBL+Tim200 mg / mL, shake it with a shaker at 100g for 1h, pour out the filtrate after the shaking is completed, and absorb the water with filter paper. Transfer the callus to the selection medium (G418 concentration is 50mg / L), and use tweezers to place the callus particles evenly to prevent contact inhibition and large-scale bacterial infection. 2-3 dishes of one carrier can be screened. Culture in the dark at 28℃ for 2 weeks. This process is called one screening. During this period, pay attention to observe whether there is any contamination. After two weeks, subculture again on the same culture medium, double the number of screening culture dishes, and 4-6 dishes per carrier are enough. Select for about 4 weeks in total. This process is called the second screening. When the callus tissue has obvious yellow round solid particles the size of millet grains falling off, you can proceed to the next stage.

[0120] S7, pre-differentiation stage:

[0121] Transfer the fresh, creamy yellow, round, dense embryonic callus grown from the transformed old callus to the pre-differentiation medium. When picking, pick the new callus that has fallen off from the top, bottom, left, and right sides of a callus. Do not pick repeatedly. Each dish can pick about 50 seeds. Pick pre-differentiation according to the situation, 3-4 dishes per carrier. 28℃, 7-10d, dark culture. The pre-differentiation stage can be appropriately extended depending on the growth of the callus. If the callus is too small, it is not easy to differentiate. It can be differentiated when it grows to the size of a soybean, hard, and yellowish. (The pre-differentiation medium also needs to distinguish between hygromycin HYG and G418 resistance. Dry the moisture before use. Pay attention to the numbering and do not mix them up).

[0122] S8, Differentiation and Rooting:

[0123] Select the white and dense callus and transfer it to the differentiation medium. Before using the differentiation medium, be sure to dry the water vapor. Each dish can hold 20 seeds. Try not to put them on the edge of the culture dish, as they are easy to contact with water. Culture under light at 28℃ for 3-4 weeks. Note that the materials must be placed in layers. Prevent the lower layer of the material from being burned by light and heat, which will affect the differentiation ability. It is best to place it on the bottom layer of the shelf in the tissue culture room to prevent the generation of water vapor and affect the differentiation of the callus. Then subculture it once on the same culture medium. Be careful to handle it with care to prevent water droplets on the lid from dripping onto the callus. The callus will no longer differentiate when it comes into contact with water. The callus that has just been subcultured to the differentiation medium needs to be placed for two days or shaded with a black plastic bag to prevent the callus from overheating and browning.

[0124] S9, strong seedlings:

[0125] If relatively strong seedlings appear, transfer them to 1 / 2MS seedling medium. Culture them under light at 28℃ for 2-3 weeks. Leave the seedlings just transferred to the seedling medium for two days before light treatment.

[0126] S10, transplanting tissue culture seedlings:

[0127] The residual culture medium on the roots was washed off, and the seedlings with good root systems were transferred to the greenhouse. At the same time, the soil was kept moist in the first few days. The regenerated seedlings obtained were the T0 generation transgenic GRMZM2G038988 overexpressing rice lines in rice Nipponbare. The T0 generation plants were self-pollinated to obtain the T1 generation GRMZM2G038988 overexpressing plants, which were used for subsequent analysis.

[0128] S11, the formulas of each culture medium and mother solution are as follows:

[0129] (I) MS vitamins and organic (1000×): niacin 0.5 g / L, thiamine-HCl 0.5 g / L, pyridoxine-HCl 0.5 g / L, glycocine 2.0 g / L.

[0130] (II) Vitamin B5 and organic (1000×): Niacin 1g / L, Thiamine-HCl 10g / L, Pyridoxine-HCl

[0131] 1g / L.

[0132] (III) N6 vitamins and organic (1000×): niacin 0.5 g / L, thiamine-HCl 1.0 g / L, pyridoxine-HCl 0.5 g / L, glycocine 2.0 g / L.

[0133] (IV) MS Micro (1000×): MnSO4·4H2O 22.7 g / L, ZnSO4·7H2O 8.97 g / L, CuSO4·5H2O 0.055 g / L, Na2MoO4·2H2O 0.55 g / L, KI 0.76 g / L, H3BO3 7.3 g / L.

[0134] (V) N6 Micro (1000×): MnSO4·4H2O 5.6 g / L, ZnSO4·7H2O 1.7 g / L, CuSO4·5H2O

[0135] 0.033 g / L, CoCl2·6H2O 0.031 g / L, Na2MoO4·2H2O 0.23 g / L, KI 0.78 g / L, H3BO3

[0136] 1.53 g / L.

[0137] (VI) B5 Micro (1000×): MnSO4·H2O 7.58 g / L, ZnSO4·7H2O 3.11 g / L, CuSO4·5H2O

[0138] 0.032 g / L, CoCl2·6H2O 0.023 g / L, Na2MoO4·2H2O 0.24 g / L, KI 0.82 g / L, H3BO3

[0139] 2.9 g / L.

[0140] (VII) Fe-EDTA: FeSO4·7H2O 4.66 g / L, Na2-EDTA 6.87 g / L.

[0141] (VIII) N6 Macro (20×): KNO3 57.77 g / L, NH4NO3 8.78 g / L, MgSO4·7H2O 4.32 g / L, KH2PO4 7 g / L CaCl2·2H2O 3.32 g / L.

[0142] (IX) AA Macro (1000×): KCl 59 g / L, MgSO4·7H2O 5 g / L, Ca(H2PO4)2 3.4 g / L, CaCl2·2H2O 3.0 g / L.

[0143] (X) AA Micro (1000×): H3BO3 2 g / L, MnSO4·4H2O 9 g / L, ZnSO4·7H2O 1.55 g / L, CuSO4·5H2O 0.032 g / L, CoCl2·6H2O 0.034 g / L, Na2MoO4·2H2O 0.33 g / L, KI

[0144] 0.65g / L.

[0145] (XI)MS bulk (20×): KNO3 38g / L, KH2PO4 4.01g / L, MgSO4.7H2O 8.0g / L, NH4NO331.9g / L, CaCl2.2H2O 9.1g / L.

[0146] (XII) 2,4D (2 mg / mL): Dissolve 2,4D in 5 ml-10 ml of 1N KOH by heating in a microwave oven.

[0147] Add ultrapure water to volume. Store at room temperature.

[0148] (XIII) 6-BA (3 mg / mL): Weigh 150 mg of BA, dissolve it in 5 ml of 1N KOH, and then dilute it to 50 ml with sterile water. Sterilize by filtration.

[0149] (XIV) AS (40 mg / mL): AS was dissolved in dimethyl sulfoxide (DMSO).

[0150] (XV) TIM (200 mg / mL): Dissolve in sterile water and filter sterilize.

[0151] (XVI) Hygromycin HYG (50 mg / mL): Dissolve in sterile water and filter sterilize.

[0152] (XVII) G418 (150 mg / mL): Dissolve in sterile water and filter sterilize.

[0153] (XVIII) ABA (5 mg / mL): ABA was dissolved in 95% analytical grade ethanol.

[0154] (XIX) Rifampicin RIF (25 mg / mL): Rifampicin (rif) was first dissolved in 1N NaOH and then fixed to volume with methanol. (Alternatively, DMSO was directly used to fix the volume and sterilized by filtration).

[0155] (XX) NAA (2 mg / mL): Dissolve in 1N KOH, make up to volume with distilled water, and filter sterilize.

[0156] 2. Identification of plants overexpressing the GRMZM2G038988 gene

[0157] RNA was extracted from young leaves of T1 transgenic GRMZM2G038988-overexpressing rice, and cDNA was obtained by reverse transcription as a template. A pair of primers GRMZM2G038988-QRT1 and GRMZM2G038988-QRT2 were designed from the CDS region for quantitative analysis of transformed seedlings. Quantitative PCR amplification was performed using GRMZM2G038988-QRT1 primer and GRMZM2G038988-QRT2 primer.

[0158] GRMZM2G038988-QRT1: 5'-GGTGTGGCAGTATTGGTTCC-3';

[0159] GRMZM2G038988-QRT2: 5'-CATTGCCCTCTCACGCTTAG-3'.

[0160] 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.

[0161] Reaction system 25μL: 2×Mix 12.5μl, Primer1 1μl, Primer2 1μl, cDNA 3μl, ddH2O 7.5μl.

[0162] 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.

[0163] The internal reference primers used were actin-F and actin-R:

[0164] actin-F: 5'-TGCTATGTACGTCGCCATCCAG-3';

[0165] actin-R: 5'-AATGAGTAACCACGCTCCGTCA-3'

[0166] 2. Preparation of Empty Vector Transgenic Rice Plants

[0167] Recombinant Agrobacterium AGL1 / pCam23A was used to replace recombinant Agrobacterium AGL1 / pCam23A-GRMZM2G038988, and the operation was carried out according to step 1 to obtain empty vector rice plants, which were subsequently referred to as control CK.

[0168] 3. Analysis of agronomic traits of plants overexpressing gene GRMZM2G038988 in rice Nipponbare

[0169] Transgenic GRMZM2G038988 overexpressing rice materials were planted in the field and the phenotypes were observed. Empty vector plants were used as controls (i.e., CK in the figure).

[0170] Each system counted 15 rice plants. Figure 1 and Figure 2 As shown, CK is the empty vector control, OE-1, OE-2, and OE-3 are three rice T1 lines overexpressing transgenic GRMZM2G038988 in rice Nipponbare. It can be seen that compared with the control CK, the expression levels of gene GRMZM2G038988 in OE-1, OE-2, and OE-3 were significantly increased, indicating that the overexpression was successful ( Figure 1 ) and is expressed as grain length ( Figure 2 and Figure 3 Middle A), grain width ( Figure 2 and Figure 3 Middle B), thousand-grain weight ( Figure 3 C) and single plant yield ( Figure 3 D) were significantly increased, indicating that overexpression of the gene GRMZM2G038988 in the crop rice can significantly increase the grain length, grain width, grain weight and yield of rice, and it is a newly discovered high-yield gene.

[0171] Therefore, it can be concluded that overexpression of the protein sequence (SEQ ID No. 1) expressed by the maize gene GRMZM2G038988 (SEQ ID No. 2) can increase traits such as grain length, grain width, grain weight and yield of crops such as rice.

[0172] 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 GRMZM2G038988 identified in this application, the transgenic plants obtained by overexpressing GRMZM2G038988 in the wild type rice Nipponbare, have significantly larger grains, significantly increased thousand-grain weight, and significantly increased single plant yield compared to the wild type Nipponbare. At present, there are no research reports on this gene and its homologous genes in plant grain size and grain weight, and there is no application of it in increasing crop yield. This shows that the gene GRMZM2G038988 (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.

[0173] 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.

[0174] 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 GRMZM2G038988, characterized in that: The grain length of rice can be increased by overexpressing the gene GRMZM2G038988.

2. Application of corn gene GRMZM2G038988, characterized in that: The grain width of rice can be increased by overexpressing the gene GRMZM2G038988.

3. Application of corn gene GRMZM2G038988, characterized in that: The grain weight of rice can be increased by overexpressing the gene GRMZM2G038988.

4. Application of corn gene GRMZM2G038988, characterized in that: Rice yield can be increased by overexpressing the gene GRMZM2G038988.

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