ZmHPE3 gene based on multi-omics intelligent design and application thereof

Abstract

The application relates to the technical field of plant molecular biology, in particular to a ZmHPE3 gene based on multi-omics intelligent design and application thereof. The CDS sequence of the ZmHPE3 gene is shown as SEQ ID No. 2, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 1. After overexpression of the gene ZmHPE3 in crops such as rice, the plant photosynthetic efficiency and the number of grains per panicle can be increased, the plant height can be moderately reduced, and the yield can be significantly increased, so that gene resources and material resources are provided for creating crops with high light efficiency and high yield.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant molecular biology, and in particular to ZmHPE3 gene based on multi-omics intelligent design and its application in regulating photosynthetic efficiency, ear grain number, plant height and yield. BACKGROUND

[0002] Photosynthesis is the basis of crop yield formation, and more than 90% of dry matter comes from photosynthesis. The light energy utilization efficiency of current major crops is only about 1%, and it is even lower under adverse conditions such as drought, high / low temperature. For crops, photosynthesis is not only the main way to obtain energy, but also one of the key factors to determine yield. Improving photosynthetic efficiency is one of the effective ways to increase crop yield. C4 plants, such as corn, sorghum and sugarcane, have a photosynthetic efficiency 50% higher than C3 plants. Introducing C4 pathway high-efficiency system into C3 crops such as rice and wheat to improve the inherent genetic basis of C3 crop photosynthesis and significantly improve photosynthetic efficiency to achieve biological yield increase is a frontier topic in plant biological science research. Cloning genes regulating crop photosynthetic efficiency and yield through multi-omics data joint analysis and exploring the practical application value of high-efficiency genes in high yield have important significance for cultivating high-efficiency and high-yield crops. SUMMARY

[0003] The purpose of the present application is to provide ZmHPE3 gene based on multi-omics intelligent design and its application in regulating photosynthetic efficiency, ear grain number, plant height and yield.

[0004] In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is:

[0005] The application provides a corn gene ZmHPE3 or a protein coded by the corn gene ZmHPE3 or a substance for regulating expression of a coding gene of the protein or a substance for regulating activity and / or content of the protein in any one of the following:

[0006] D1) regulating yield of a plant;

[0007] D2) preparing a product for regulating yield of a plant;

[0008] D3) cultivating a plant with increased yield;

[0009] D4) preparing a product for cultivating a plant with increased yield;

[0010] D5) regulating photosynthetic efficiency of a plant;

[0011] D6) preparing a product for regulating photosynthetic efficiency of a plant;

[0012] D7) cultivating a plant with increased photosynthetic efficiency;

[0013] D8) a product for producing a plant with increased photosynthetic efficiency;

[0014] D9) modulating plant height;

[0015] D10) a product for modulating plant height;

[0016] D11) producing a plant with reduced plant height;

[0017] D12) a product for producing a plant with reduced plant height;

[0018] D13) modulating grain number per spike;

[0019] D14) a product for modulating grain number per spike;

[0020] D15) producing a plant with increased grain number per spike;

[0021] D16) a product for producing a plant with increased grain number per spike.

[0022] The protein is any one of:

[0023] A1) a protein having an amino acid sequence of SEQ ID No. 1;

[0024] A2) a protein having 80% or more identity to the protein shown in A1) and having the same function, obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in A1);

[0025] A3) a fusion protein having the same function, obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid shown in A1) or A2).

[0026] The tag protein includes, but is not limited to, a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0027] A person of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein of the present application using known methods, such as methods of directed evolution or point mutation. Those nucleotides artificially modified to have 75% or more identity to the nucleotide sequence of the protein isolated from the present application, as long as they encode the aforementioned protein and have the function of the aforementioned protein, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[0028] In the above applications, the substance is a biological material, and the biological material is any one of the following:

[0029] B1) an RNA molecule that inhibits or reduces or down-regulates the expression of a gene encoding a protein, or an RNA molecule that inhibits or reduces or down-regulates the activity or content of a protein;

[0030] B2) a gene encoding the RNA molecule of B1);

[0031] B3) an expression cassette containing the gene of B2);

[0032] B4) a recombinant vector containing the gene of B2), or a recombinant vector containing the expression cassette of B3);

[0033] 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);

[0034] 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);

[0035] B7) a transgenic plant tissue containing the gene of B2), or a transgenic plant tissue containing the expression cassette of B3), or a transgenic plant tissue containing the recombinant vector of B4);

[0036] B8) a transgenic plant organ containing the gene of B2), or a transgenic plant organ containing the expression cassette of B3), or a transgenic plant organ containing the recombinant vector of B4);

[0037] B9) a nucleic acid molecule encoding the aforementioned protein;

[0038] B10) an expression cassette containing the nucleic acid molecule of B9);

[0039] B11) a recombinant vector containing the nucleic acid molecule of B9), or a recombinant vector containing the expression cassette of B10);

[0040] B12) a recombinant microorganism containing the nucleic acid molecule of B9), or a recombinant microorganism containing the expression cassette of B10), or a recombinant microorganism containing the recombinant vector of B3);

[0041] B13) a transgenic plant cell line containing the nucleic acid molecule of B9), or a transgenic plant cell line containing the expression cassette of B10);

[0042] B14) a transgenic plant tissue containing the nucleic acid molecule of B9), or a transgenic plant tissue containing the expression cassette of B10);

[0043] B15) a transgenic plant organ comprising the nucleic acid molecule of B9), or a transgenic plant organ comprising the expression cassette of B10).

[0044] Further, the recombinant vector is obtained by replacing the nucleotide shown in SEQ ID No. 2 to the Smal and Xbal sites of pCam23A vector.

[0045] In the above-mentioned applications, 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, which can include not only a promoter that initiates transcription of the gene encoding the aforementioned protein, but also a terminator that terminates transcription of the gene encoding the aforementioned protein. Further, the expression cassette can further include an enhancer sequence. The promoters that can be used in the present application 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, the Agrobacterium nopaline synthase terminator (NOS terminator), the CaMV 35S terminator of the Cauliflower Mosaic Virus, the tml terminator, the pea rbcS E9 terminator, and the nopaline and opine synthase terminator.

[0046] The recombinant vector containing the gene expression cassette of the aforementioned protein coding gene can be constructed using the existing expression vector. The plant expression vector includes binary Agrobacterium vector and vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corporation), etc. The plant expression vector can also contain the 3' untranslated region of the exogenous gene, that is, it contains a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as the 3' untranslated region of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nos nopaline synthase gene), the 3' untranslated region of the plant gene (such as the rice or corn storage protein gene). When using the gene of the present application to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent regions of start codons, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation 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 to produce color-changing enzymes or luminescent compounds (GUS gene, luciferase gene, etc.), marker genes of antibiotics (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to herbicide phosphine, hph gene conferring resistance to antibiotic hygromycin, and dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate), or anti-chemical agent marker genes (such as herbicide-resistant genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0047] In the above application, B1) the RNA molecule targets the mRNA of the gene transcription of the aforementioned protein.

[0048] 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.

[0049] In the above application, the vector can be a plasmid, a cosmid, a bacteriophage or a viral vector.

[0050] In the above use, the microorganism can be a yeast, a bacterium, an algae or a fungus, such as Agrobacterium.

[0051] In the above use, the transgenic plant cell lines do not include propagation material.

[0052] In the above use, 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 the cell.

[0053] In the above use, the plant is any one of the following:

[0054] G1) a monocotyledonous plant;

[0055] G2) a plant of the family Poaceae;

[0056] G3) a plant of the genus Oryza;

[0057] G4) a plant of the species Oryza sativa;

[0058] G5) rice.

[0059] The present application has the following beneficial effects:

[0060] The corn gene ZmHPE3 provided by the present application encodes a transcriptional repressor protein, the length of the CDS of the gene is 1149 bp, and no research report about the gene and its homologous genes in the aspects of plant photosynthetic efficiency, panicle grain number, plant height and yield has been found, and no application about the genes in improving crop yield has been found. After overexpression of the gene ZmHPE3 in rice, the photosynthetic efficiency and panicle grain number of the plant are significantly improved, the plant height is reduced, and the yield is significantly increased, and the purpose of crop yield increase is achieved by optimizing the source-sink-flow of plant photosynthetic products, and gene resources and material resources for breeding new varieties of high light efficiency and high yield crops are provided. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 It is a schematic diagram of the relative expression amount of the corn ZmHPE3 gene in the control CK and the overexpression lines 5-155-1, 5-155-2 and 5-155-3 in Example 3;

[0062] Figure 2 It is a plant type diagram of the control CK and the transgenic lines obtained by overexpression of the corn gene ZmHPE3 in Example 3;

[0063] Figure 3 It is a panicle type diagram of the control CK and the transgenic lines obtained by overexpression of the corn gene ZmHPE3 in Example 3;

[0064] Figure 4 It is a grain type diagram of the control CK and the transgenic lines obtained by overexpression of the corn gene ZmHPE3 in Example 3;

[0065] Figure 5 The statistical analysis results of the agronomic traits of the control CK and the corn gene ZmHPE3 overexpression strain in Example 3: A is the statistical analysis result of plant height; B is the statistical analysis result of single plant yield; C is the statistical analysis result of kernel length; D is the statistical analysis result of kernel width; E is the statistical analysis result of thousand kernel weight; F is the statistical analysis result of spike kernel number. DETAILED DESCRIPTION

[0066] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0067] The experimental methods in the following examples are all routine methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0068] Determination of plant height: at the mature stage, the height from the ground surface to the top of the highest ear per plant (not including awn). The average value of 15 plant heights in the strain is taken.

[0069] Determination of photosynthetic efficiency parameters: one week after heading, sunny days with air temperature above 30 degrees are selected, and photosynthetic rate determination of the flag leaf of selected rice plants is started at 9:00 am every day, the determination site is the front 1 / 3 of the flag leaf blade; each determination data is determined for 1-5 minutes; each group of materials is determined within 1 hour; the determination in the morning is repeated at 13:00 on the same day; the determination is repeated for 2 more times in the next week; the photosynthetic determination system is a single Li-cor6800 portable photosynthetic rate determination instrument produced by the United States, which can automatically determine the net photosynthetic rate, light respiration rate and intercellular CO2 concentration.

[0070] 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.", publicly available from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, and the biological material is only used for the relevant experiments of the present application, and is not used for other purposes.

[0071] Kernel type statistical analysis: The determination of kernel length, kernel width and 1000-grain weight was carried out by using the automatic kernel analysis and 1000-grain weight instrument SC-G type, and the specific method was referred to the instruction manual.

[0072] The wild type of rice was Nipponbare (Oryza sativa L. ssp. Japonica variety Nipponbare; hereinafter also referred to as wild type rice WT), which was preserved by the Crop High-efficiency Functional Genome Team of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, and was planted in the experimental field of the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.

[0073] The primers used in the following examples were synthesized by Beijing Genesee Biotech Co., Ltd., and the sequencing was completed in Beijing Genesee Biotech Co., Ltd. Restriction endonuclease SmaI and XbaI, Infusion recombinase, T4 ligase were purchased from Dalian Liuhetong Company (Takara); pTEAY-T1 Cloning Kit, Taq enzyme, Trans5α competent cells and related kits were purchased from Beijing Quansijin Biotechnology Co., Ltd.; RNA extraction and reverse transcription kit was purchased from Tiangeng Biochemical Technology (Beijing) Co., Ltd.; Agrobacterium AGL1: purchased from Beijing Zhuangmeng International Biological Gene Technology Co., Ltd., item number ZK296; antibiotics were purchased from SIGMA Company, USA; the rest of the reagents were domestic analytical pure.

[0074] The reagent formula used is as follows:

[0075] (I) MS vitamins and organics (1000x): nicotinic acid 0.5 g / L, Thiamine-HCl 0.5 g / L, Pyridoxine-HCl 0.5 g / L, Glycine 2.0 g / L.

[0076] (II) B5 vitamins and organics (1000x): nicotinic acid 1 g / L, Thiamine-HCl 10 g / L, Pyridoxine-HCl

[0077] 1g / L.

[0078] (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.

[0079] (IV) MS trace (1000×): MnSO4.4H2O 22.7g / L, ZnSO4.7H2O 8.97g / L, CuSO4.5H2O0.055g / L, Na2MoO4.2H2O 0.55g / L, KI 0.76g / L, H3BO3 7.3g / L. (V) N6 trace (1000×): MnSO4.4H2O 5.6g / L, ZnSO4.7H2O 1.7g / L, CuSO4.5H2O

[0080] 0.033g / L, CoCl2.6H2O 0.031g / L, Na2MoO4.2H2O 0.23g / L, KI 0.78g / L, H3BO3

[0081] 1.53g / L.

[0082] (VI) B5 trace (1000×): MnSO4.H2O 7.58g / L, ZnSO4.7H2O 3.11g / L, CuSO4.5H2O

[0083] 0.032g / L, CoCl2.6H2O 0.023g / L, Na2MoO4.2H2O 0.24g / L, KI 0.82g / L, H3BO3

[0084] 2.9g / L.

[0085] (VII) Fe-EDTA: FeSO4·7H2O 4.66g / L, Na2-EDTA 6.87g / L.

[0086] (VIII) N6 large amount (20×): KNO3 57.77g / L, NH4NO3 8.78g / L, MgSO4.7H2O 4.32g / L, KH2PO4 7g / L CaCl2.2H2O 3.32g / L.

[0087] (IX) AA large amount (1000×): KCl 59g / L, MgSO4.7H2O 5g / L, Ca(H2PO4)2 3.4g / L, CaCl2.2H2O 3.0g / L.

[0088] (X) AA trace (1000x): 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

[0089] 0.65 g / L.

[0090] (XI) MS macro (20x): KNO3 38 g / L, KH2PO4 4.01 g / L, MgSO4.7H2O 8.0 g / L, NH4NO3 31.9 g / L, CaCl2.2H2O 9.1 g / L.

[0091] (XII) 2,4D (2 mg / mL): 2,4D was dissolved by 5-10 ml of 1 N KOH, heated by microwave oven,

[0092] Add ultrapure water to constant volume. Store at room temperature.

[0093] (XIII) 6-BA (3 mg / mL): 150 mg of BA was dissolved by 5 ml of 1 N KOH, and then dissolved in sterile water to 50 ml. Filter sterilization.

[0094] (XIV) AS (40 mg / mL): AS was dissolved in dimethyl sulfoxide (DMSO) and dissolved to constant volume.

[0095] (XV) Timentin TIM (200 mg / mL): dissolved in sterile water, dissolved to constant volume, and filter sterilization.

[0096] (XVI) Hygromycin HYG (50 mg / mL): dissolved in sterile water, dissolved to constant volume, and filter sterilization.

[0097] (XVII) G418 (150 mg / mL): dissolved in sterile water, dissolved to constant volume, and filter sterilization.

[0098] (XVIII) ABA (5 mg / mL): ABA was dissolved in 95% analytical pure ethanol and dissolved to constant volume.

[0099] (XIX) Rifampicin RIF (25 mg / mL): Rifampicin (rif) was dissolved in 1 N NaOH, and then dissolved in methanol. (Or directly dissolved in DMSO to constant volume, and filter sterilization).

[0100] (XX) NAA (2 mg / mL): dissolved in 1 N KOH, distilled water to constant volume, and filter sterilization.

[0101] The following examples use GraphPad Prism 8 statistical software to process data, and the experimental results are expressed as mean ± standard deviation, Student's t test is used for inspection, P<0.05 (*) indicates significant difference, P<0.01 (**) indicates extremely significant difference.

[0102] Example 1, Multi-omics joint analysis intelligent prediction of transcription regulation network key genes

[0103] The transcription regulation network inside the plant is determined by transcription factors, and the number of transcription factor binding studies in plants is currently small, which cannot construct the overall picture of the complex regulation network. Based on the multi-omics data obtained by our team's previous microdissection technology (Sun, J., C. Deng, X. R. Dai, H. S. Li, L. Y. Zhang, J. K. Wang, H. Zhao, Y. R. Yang, N. Phung, Z. G. Zhang, P. H. Li, X. H. Sun, and T. G. Lu (2024). Gene expression profiles in early leaf of rice (Oryza sativa) and foxtail millet (Setaria italica). Crop Journal 12(3): 776-787), and through a large number of ChIP-seq and other multi-omics data joint analysis, the transcription factor regulation network in plants was reconstructed, revealing the complexity and redundancy of plant transcription regulation network, providing data basis for mining high yield genes.

[0104] Through intelligent prediction of transcription factor regulation network and gene mining, combined with collected genetic data, multiple genes of the key nodes of the transcription regulation network were predicted, among which the gene ZmHPE3 encodes a transcriptional repressor protein, and the CDS length of the gene is 1149 bp. It was found that there was no research report on the gene and its homologous genes in plant yield and other aspects, and there was no application in improving crop yield, so the overexpression vector of the gene was constructed and introduced into crops rice for function verification.

[0105] Example 2, Cloning and vector construction of maize gene ZmHPE3

[0106] I. Extraction of maize RNA

[0107] The total RNA of corn B73 was extracted using RNA Easy Fast Plant Tissue RNA Fast Extraction Kit of Tiangen Biochemical Biological Company. Each tissue sample for detection was placed in a tin foil paper after sampling in the field, frozen in liquid nitrogen and taken back to the Beijing laboratory, and stored in an ultra-low temperature refrigerator. The specific steps for extracting RNA are as follows:

[0108] 1. Sample pretreatment: The plant leaves or fruit pulp were quickly ground into powder in liquid nitrogen, 30-150 mg of sample was added into 600 μl of lysis solution SG and 10 μl of Proteinase K, and immediately vortexed to mix, and then placed at room temperature for 5 min.

[0109] 2. Centrifuged at 12,000 rpm (~ 13,400 x g) for 2 min, and about 500 μl of supernatant was taken for the following operation.

[0110] 3. The obtained supernatant was added into a genomic DNA removal column, centrifuged at 12,000 rpm (~ 13,400 x g) for 30 sec, and the filtrate was reserved.

[0111] 4. Anhydrous ethanol (about 250 μl in volume) was slowly added into the above filtrate at 0.5 times the volume of the supernatant, mixed (at this time, a precipitate may appear), and the obtained solution and precipitate were transferred into an RNase-Free adsorption column CR4 (the adsorption column was placed in a collection tube), centrifuged at 12,000 rpm (~ 13,400 x g) for 30 sec, and the waste liquid in the collection tube was discarded, and the adsorption column was placed back into the collection tube.

[0112] 5. 700 μl of deproteinization liquid RW3 was added into the RNase-Free adsorption column CR4, centrifuged at 12,000 rpm (~ 13,400 x g) for 30 sec, the waste liquid was discarded, and the adsorption column was placed back into the collection tube.

[0113] 6. 500 μl of rinse liquid RW (before use, please check whether ethanol has been added) was added into the RNase-Free adsorption column CR4, and placed at room temperature for 2 min, centrifuged at 12,000 rpm (~ 13,400 x g) for 30-60 sec, the waste liquid was discarded, and the adsorption column was placed back into the collection tube.

[0114] 7. Step 6 was repeated.

[0115] 8. Centrifuged at 12,000 rpm (~ 13,400 x g) for 2 min, and the waste liquid was discarded. The RNase-Free adsorption column CR4 was placed at room temperature for 2 min to completely dry the residual rinse liquid in the adsorption material.

[0116] 9. RNase-Free Absorbent Column CR4 was transferred into a new RNase-Free centrifuge tube, 30-100 μl RNase-Free ddH2O was added to the middle of the absorbent membrane, and the mixture was placed at room temperature for 2 min, then centrifuged at 12,000 rpm (~ 13,400 x g) for 2 min to obtain the RNA solution.

[0117] II. Reverse transcription of RNA

[0118] The reverse transcription of cDNA was performed using FastKing cDNA First-Strand Synthesis Kit (Degenerate) from Tiangen Biochemical Biological Co., Ltd., and the specific steps were as follows:

[0119] 1. The mixture was prepared according to the genome DNA removal system in the following table, thoroughly mixed, briefly centrifuged, and incubated at 42°C for 3 min, then placed on ice.

[0120] Ingredient Amount 5x gDNA Buffer 2 μl Total RNA RNase-Free ddH2O Make up to 10 μl

[0121] 2. The mixture was prepared according to the reverse transcription reaction system in the following table.

[0122] Reagent Amount 10x King RT Buffer 2 μl FastKing RT Enzyme Mix 1 μl FQ-RT Primer Mix 2 μl RNase-Free ddH2O Make up to 10 μl

[0123] 3. The Mix in the reverse transcription reaction was added to the reaction solution in the gDNA removal step, and thoroughly mixed.

[0124] 4. Incubated at 42°C for 15 min.

[0125] 5. Incubated at 95°C for 3 min, then placed on ice, and the obtained cDNA could be used for subsequent experiments or stored at low temperature.

[0126] III. Construction of gene ZmHPE3 CDS overexpression vector

[0127] Primers ZmHPE3-CDS-F and ZmHPE3-CDS-R were designed using primer design software DNAMAN:

[0128] ZmHPE3-CDS-F:

[0129] 5'-GTAGAAGAGGTACCCGGGATGGAGTACCCGGCGACGG-3';

[0130] ZmHPE3-CDS-R:

[0131] 5'-GCAGGTCGACTCTAGATCAATCTTCATCTGGTGTTTCCT-3'.

[0132] The above obtained cDNA was used as a template, and primers ZmHPE3-CDS-F and ZmHPE3-CDS-R were used for PCR amplification (high-fidelity KOD enzyme was used in the amplification system), to obtain a PCR product.

[0133] The above obtained PCR product was subjected to gel running and gel recovery, and the recovered product was recombined with the pCam23A vector subjected to Smal and Xbal enzyme digestion using an Infusion recombinase. Through sequencing, the recombinant vector pCam23A-ZmHPE3 was a vector obtained by replacing the nucleotides shown in SEQ ID No. 2 at the Smal and Xbal sites of the pCam23A vector.

[0134] Example 3, Application of the Corn Gene ZmHPE3 in Improving Photosynthetic Efficiency and Yield of Rice

[0135] I. Preparation of the Transgenic Rice Nipponbare Overexpressing the Corn Gene ZmHPE3

[0136] 1. Genetic Transformation of the pCam23A-ZmHPE3 Overexpression Vector

[0137] (1) The above prepared recombinant vector pCam23A-ZmHPE3 was introduced into Agrobacterium AGL1 to obtain a recombinant bacterium AGL1 / pCam23A-ZmHPE3, which was verified by enzyme digestion to obtain a positive recombinant bacterium.

[0138] (2) The recombinant bacterium AGL1 / pCam23A-ZmHPE3 was introduced into rice Nipponbare using rice genetic transformation to obtain a T0 generation of transgenic rice overexpressing the corn gene ZmHPE3 in rice Nipponbare. The specific operation steps are as follows:

[0139] S1, Seed Sterilization and Callus Induction Stage:

[0140] The mature seeds were dehulled, and 300 full and complete dehulled rice seeds were selected and placed in a 50 mL sterilized centrifuge tube. The seeds were washed with sterilized ultrapure water three times. Then the surface was sterilized with 40 mL of 75% ethanol for 5 min, and then sterilized with 40 mL of 50% sodium hypochlorite for 5 min. The sterilization was repeated once, and finally washed with sterile water for 10 times until the water was clear. The sterilized seeds were dried with sterile filter paper, placed on the MS+ medium, 20 seeds per dish, and cultured at 28°C in the dark for 28 days until small millet-sized callus was formed.

[0141] S2, Subculture and Pre-culture Stage of Callus:

[0142] The good embryogenic callus was transferred to the MS medium. About 100 seeds per dish can be placed. Cultured at 28°C in the dark for 7 days.

[0143] The selected callus can be returned to continue culture, and a batch of induction can be selected 3-4 times, and the large pieces of unshed callus can be placed on new MS+ medium to make it shed again and be selected again.

[0144] S3, preparation of Agrobacterium:

[0145] The Agrobacterium was plated on YEP (or LB medium) with the corresponding screening resistance 1 day in advance, and kanamycin + rifampicin or spectinomycin + rifampicin was added; the Agrobacterium colony on the original plate was scraped with a coating rod and evenly coated on a new culture medium, and if it was a bacterial solution, the bacterial solution was poured into the culture medium in a small amount, and then coated with a coating rod. Label the carrier, place it upside down, and incubate it in a 28°C incubator overnight.

[0146] The transformed colonies can be directly cultured for 4-5 days without activation treatment. Before shaking the bacteria, use a sterilized spoon to evenly spread all the colonies, then scrape an appropriate amount of bacteria and shake them (those stored at 4°C must be activated).

[0147] S4, OD value adjustment:

[0148] The Agrobacterium was scraped with a key, and the bacteria were placed in AAM liquid medium and cultured at 28°C for 2h using a 200rpm shaker. Then the OD value of the bacterial solution was adjusted to 0.12-0.15 with AAM.

[0149] S5, infection and co-culture:

[0150] About 100 embryogenic calli were collected in a 100mL conical flask; the adjusted concentration of Agrobacterium was poured into the conical flask for infection, and the shaker was shaken at 100g for 20min. After the shaking was completed, the immersion liquid was poured out, the callus was dried with filter paper, and the infected callus was moved to the co-culture medium, covered with sterile filter paper, and all the callus was in contact with the filter paper surface. 22°C dark culture for 4d.

[0151] S6, resistance screening of transformed callus:

[0152] The callus that has completed the co-cultivation stage is collected in a 50 mL sterile centrifuge tube, and the callus is rinsed 10 times with sterile water until the wash is clear. The callus is then poured into NBL + Tim 200 mg / mL 1 ml wash, shaken at 100 g for 1 h on a shaker, and after shaking is complete, the filtrate is discarded and the callus is blotted dry with filter paper. The callus is transferred to selection medium (G418 concentration is 50 mg / L), and the callus particles are placed evenly with forceps to prevent contact inhibition and large-area contamination. One carrier 2-3 dishes are screened. Cultured at 28°C in the dark for 2 weeks, and this process is a screening. During the period, attention should be paid to observe whether there is contamination. After two weeks, subculture again on the same medium, and the number of screening medium dishes is doubled, and each carrier can be 4-6 dishes. The total selection is about 4 weeks. This process is a second screening. When the callus has obvious millet-sized yellow round solid particles, the next stage can be carried out.

[0153] S7, pre-differentiation stage:

[0154] The fresh, creamy yellow, round, solid embryogenic callus grown from the transformed old callus is transferred to the pre-differentiation medium. When picking, the new callus that has fallen off from all sides of the callus is picked, and repeated picking is avoided. About 50 particles per dish can be inoculated. Pre-differentiation is picked according to the situation, and 3-4 dishes per carrier can be used. Cultured at 28°C for 7-10 days in the dark. The pre-differentiation stage can be appropriately extended depending on the growth of the callus. The callus is too small to differentiate, and it grows to the size of a soybean, and the quality is hard and yellow. Differentiation can be carried out. (The pre-differentiation medium also needs to be differentiated between hygromycin HYG and G418 resistance. Dry the water vapor before use, and pay attention to numbering to avoid confusion.)

[0155] S8, differentiation and rooting:

[0156] The white, solid callus is transferred to the differentiation medium, and the water vapor is dried before use. Each dish can inoculate 20 particles, and attention should be paid to avoid placing them on the edge of the dish, which is easy to contact water. Cultured at 28°C under light for 3-4 weeks. When placing the material, it must be placed in layers to prevent the lower layer of material from being heated by the light and causing high temperature to burn the callus and affect the differentiation ability. It is best to place it on the lowest layer of the shelf in the tissue culture room to prevent water vapor from affecting the differentiation of the callus. Then subculture again on the same medium, and pay attention to handle it gently to prevent water droplets on the cover from falling on the callus, which will not differentiate when it comes into contact with water. The callus just subcultured to the differentiation medium needs to be placed for two days or treated with a black plastic bag to prevent the callus from browning due to overheating.

[0157] S9, strong seedlings:

[0158] If there are relatively strong seedlings, they are inoculated into 1 / 2MS strong seedling medium. Cultured at 28°C under light for 2-3 weeks. The seedlings just inoculated into the strong seedling medium are placed for two days before light treatment.

[0159] S10, transplanting the tissue culture seedlings:

[0160] The residual culture medium on the roots was washed off, and the seedlings with good root systems were transferred to a greenhouse while keeping the soil moist for the first few days. The regenerated seedlings were T0 generation transgenic ZmHPE3 overexpression rice strains in rice Nipponbare, and the T0 generation plants were selfed to obtain T1 generation ZmHPE3 overexpression plants, which were used for subsequent analysis.

[0161] 2. Identification of ZmHPE3 overexpression plants

[0162] RNA was extracted from the young leaves of three random T1 generation transgenic ZmHPE3 overexpression rice strains 5-155-1, 5-155-2, and 5-155-3 (the method was the same as in Example 1), and cDNA was obtained by reverse transcription (the method was the same as in Example 1), a pair of primers ZmHPE3-QRT1 and ZmHPE3-QRT2 were selected for the fluorescence quantitative analysis of the transformed seedlings, and fluorescence quantitative PCR amplification was performed using ZmHPE3-QRT1 and ZmHPE3-QRT2 primers.

[0163] ZmHPE3-QRT1: 5'-CGAGAGCAATGCCTTCTGAC-3';

[0164] ZmHPE3-QRT2: 5'-TTGCCTCCATCTTCTTCCGT-3'.

[0165] Fluorescence quantitative PCR was performed using an iQ5 Muticolor Real-Time PCR Detection System (Bio-Rad) instrument, and the reagent used was SYBR Green Mix. All experiments were independently repeated 3 times.

[0166] The reaction system is shown in the following table:

[0167]

[0168]

[0169] Reaction program: 95℃ 5min, 95℃ 10s, 60℃ 20s, 72℃ 30s, 80℃ 10s to collect fluorescence, 72℃ 5min, 55℃ to 95℃ 0.5℃ / s, and a melting curve was made. The second to fifth steps were run for 40 cycles.

[0170] The reference primers used were RICE-actin-F and RICE-actin-R:

[0171] RICE-actin-F: 5'-TGCTATGTACGTCGCCATCCAG-3';

[0172] RICE-actin-R: 5'-AATGAGTAACCACGCTCCGTCA-3'

[0173] From Figure 1 It can be seen that the expression of gene ZmHPE3 in 5-155-1, 5-155-2 and 5-155-3 is significantly increased compared with the control CK, indicating that overexpression is successful.

[0174] II. Preparation of rice plants transformed with empty vector

[0175] Recombinant Agrobacterium AGL1 / pCam23A was used instead of recombinant Agrobacterium AGL1 / pCam23A-ZmHPE3, and step one was performed to obtain rice plants transformed with empty vector, which is hereinafter referred to as control CK.

[0176] III. Agronomic trait analysis of gene ZmHPE3 in rice Nipponbare overexpression plants

[0177] The empty vector transformed plants (i.e. control CK) and the transgenic ZmHPE3 overexpression rice materials were planted in the field and the photosynthetic efficiency was measured, the phenotype was observed, and the agronomic traits were counted, with 15 plants per plant system.

[0178] The photosynthesis related parameters: photosynthetic efficiency (μmol m -2 s -1 ), transpiration efficiency (mmol m -2 s -1 ), intercellular CO2 concentration (μmol mol -1 ), and CO2 stomatal conductance (mol m -2 s -1 ) were measured, and the results are shown in the following table. The photosynthetic efficiency of the transgenic ZmHPE3 overexpression rice material was significantly increased, indicating that overexpression of gene ZmHPE3 in crops rice can significantly improve the photosynthetic efficiency of rice.

[0179] Photosynthetic efficiency determination of control CK and ZmHPE3 overexpression lines

[0180]

[0181] The agronomic trait statistical results are shown in Figure 5 The CK is the empty vector control, and 5-155-1, 5-155-2 and 5-155-3 are three rice lines overexpressing transgenic ZmHPE3 in rice Nipponbare. It can be seen that compared with the control CK, the transgenic ZmHPE3 overexpression plants show a significant decrease in plant height (Figure 5 Significant increase in single plant yield Figure 5 Significant increase in spike grain number Figure 5 Significant increase in single plant yield Figure 4 Significant increase in single plant yield Figure 5 Significant increase in single plant yield Figure 4 Significant increase in single plant yield Figure 5 Significant increase in single plant yield Figure 5 Significant increase in single plant yield

[0182] In summary, in the early stage of the present application, the key genes of the transcriptional regulatory network were mined by multi-omics joint analysis, the ZmHPE3 gene overexpression vector was constructed and transferred into the crop rice for function verification. The transgenic plants obtained have significantly improved photosynthetic efficiency, spike grain number and yield, and reduced plant height compared with the control group. At present, there is no research report on the gene and its homologous genes in the aspects of plant photosynthetic efficiency, spike grain number and yield, and there is no application of the gene in improving crop yield. It is indicated that the gene ZmHPE3 (SEQ ID No. 2) and the expressed protein sequence (SEQ ID No. 1) provided in the present application have important theoretical and practical significance for cultivating new high-yield crop materials with increased photosynthetic efficiency.

[0183] It is apparent to those skilled in the art that the present application is not limited to the details of the foregoing exemplary embodiments, but can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, and the scope of the present application should be defined by the appended claims rather than the above description, and it is intended to include all changes falling within the meaning and scope of the equivalent elements of the claims.

Claims

1. ZmHPE3 The application of genes in improving photosynthetic efficiency of rice, characterized in that, By overexpressing a gene ZmHPE3 improving photosynthetic efficiency of rice, ZmHPE3 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID No.

1.

2. ZmHPE3 The use of the gene in increasing the number of grains per panicle of rice is characterized in that, By overexpressing a gene ZmHPE3 increasing the number of grains per panicle of rice, ZmHPE3 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID No.

1.

3. ZmHPE3 The use of a gene in reducing plant height in rice, characterized in that, By overexpressing a gene ZmHPE3 reducing the plant height of rice, ZmHPE3 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID No.

1.

4. ZmHPE3 The application of the gene in improving the yield of rice is characterized in that, By overexpressing a gene ZmHPE3 Increasing the yield of rice, ZmHPE3 ZmHPE3 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID No. 1.

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