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

Abstract

The invention relates to the technical field of plant molecular biology, in particular to a ZmHPE3 gene based on multi-omics intelligent design and application of the ZmHPE3 gene. The CDS sequence of the ZmHPE3 gene is as shown in SEQ ID No.2, and the amino acid sequence of the protein coded by the ZmHPE3 gene is as shown in SEQ ID No.1. After overexpression of the gene ZmHPE3 in crop rice, the photosynthetic efficiency and the grain number per ear of a plant can be increased, the plant height can be properly reduced, the yield is remarkably increased, and gene resources and material resources are provided for creation of high-photosynthetic-efficiency and high-yield crops.

Description

Technical Field

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

[0002] Photosynthesis is the basis for crop yield formation. More than 90% of the dry matter comes from photosynthesis. Currently, the light energy utilization efficiency of major crops is only about 1%, and it is even lower under adverse conditions such as drought, high temperature / low temperature. For crops, photosynthesis is not only the main way for them to obtain energy, but also one of the key factors determining yield. Improving photosynthetic efficiency is one of the effective ways to increase crop yield. C4 plants, such as corn, sorghum and sugarcane, etc., have a photosynthetic efficiency 50% higher than that of C3 plants. Introducing the high-light efficiency system of the C4 pathway into C3 crops such as rice and wheat, improving the inherent genetic basis of C3 crop photosynthesis, and achieving an increase in biological yield by significantly improving photosynthetic efficiency is a frontier topic in plant biological science research. Through the joint analysis of multi-omics data, cloning genes that regulate crop photosynthetic efficiency and yield, and exploring the practical application value of high-light efficiency genes in high yield, are of great significance for cultivating high-light efficiency and high-yield crops. Summary of the Invention

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

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

[0005] Provide the application of the maize gene ZmHPE3 or the protein encoded by it or the substance that regulates the expression of the encoding gene of the protein or the substance that regulates the activity and / or content of the protein in any one of the following:

[0006] D1) Regulating the yield of plants;

[0007] D2) Preparing a product for regulating the yield of plants;

[0008] D3) Cultivating plants with increased yield;

[0009] D4) Preparing a product for cultivating plants with increased yield;

[0010] D5) Regulating the photosynthetic efficiency of plants;

[0011] D6) Preparing a product for regulating the photosynthetic efficiency of plants;

[0012] D7) Cultivating plants with increased photosynthetic efficiency;

[0013] D8) Products for preparing plants with increased photosynthetic efficiency;

[0014] D9) Regulating the plant height;

[0015] D10) Products for preparing products for regulating the plant height;

[0016] D11) Cultivating plants with reduced plant height;

[0017] D12) Products for preparing products for cultivating plants with reduced plant height;

[0018] D13) Regulating the number of grains per ear of plants;

[0019] D14) Products for preparing products for regulating the number of grains per ear of plants;

[0020] D15) Cultivating plants with increased number of grains per ear;

[0021] D16) Products for preparing products for cultivating plants with increased number of grains per ear.

[0022] The protein is any one of the following:

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

[0024] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in A1), having more than 80% identity with the protein shown therein and having the same function;

[0025] A3) A fusion protein with 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).

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

[0027] Those of ordinary skill 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 methods. Those nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of the protein isolated from the present invention, as long as they encode the aforementioned protein and have the function of the aforementioned protein, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0028] In the above application, 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 downregulates the expression of a protein-encoding gene, or an RNA molecule that inhibits or reduces or downregulates 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] A transgenic plant organ containing the nucleic acid molecule of B9), or a transgenic plant organ containing the expression cassette of B10).

[0044] Furthermore, the recombinant vector is obtained by replacing the SmaI and XbaI sites of the pCam23A vector with the nucleotide shown in SEQ ID No. 2.

[0045] In the above application, the expression cassette containing the nucleic acid molecule encoding the aforementioned protein (the gene expression cassette of the encoding gene of the aforementioned protein) refers to the DNA capable of expressing the encoding gene of the aforementioned protein in a host cell. This DNA may not only include the promoter that initiates the transcription of the encoding gene of the aforementioned protein, but also include the terminator that terminates the transcription of the encoding gene of the aforementioned protein. Further, the expression cassette may also include an enhancer sequence. The 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 tumefaciens nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine synthase terminator.

[0046] A recombinant vector containing the coding gene expression cassette of the aforementioned protein can be constructed using existing expression vectors. Plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant particle bombardment, etc., such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (from CAMBIA), etc. The plant expression vector may also contain the 3' untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylate to the 3' end of the mRNA precursor. For example, the nopaline synthase gene Nos of the Agrobacterium tumefaciens Ti plasmid gene and the 3' untranslated regions transcribed from plant genes such as rice or maize storage protein genes have similar functions. When constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translational enhancers or transcriptional enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as 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 initiation region can be from the transcription initiation region or the structural gene. For the convenience of identifying and screening transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change in plants (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, the hph gene conferring resistance to the antibiotic hygromycin, and the dhfr gene conferring resistance to methotrexate, the EPSPS gene conferring resistance to glyphosate) or a marker gene for anti-chemical reagents (such as an anti-herbicide gene), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress conditions.

[0047] In the above application, B1) the RNA molecule targets the mRNA transcribed from the gene 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, cosmid, phage or viral vector.

[0050] In the above applications, the microorganism can be yeast, bacteria, algae or fungi, such as Agrobacterium.

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

[0052] In the above applications, the substance that regulates the expression of the coding gene of 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 coding gene of the aforementioned protein in the cell.

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

[0054] G1) Monocotyledonous plants;

[0055] G2) Gramineous plants;

[0056] G3) Oryza plants;

[0057] G4) Rice species plants;

[0058] G5) Rice.

[0059] The beneficial effects of the present invention are as follows:

[0060] The maize gene ZmHPE3 provided by the present invention encodes a transcriptional repressor protein. The CDS length of this gene is 1149 bp. There are no research reports on this gene and its homologous genes in terms of plant photosynthetic efficiency, grain number per ear, plant height and yield, etc., nor is there any application regarding its use in increasing crop yield. After overexpression of the gene ZmHPE3 in rice, the photosynthetic efficiency and grain number per ear of the plants are significantly increased, the plant height is reduced, and at the same time the yield is significantly increased. By optimizing the source-sink-flow of plant photosynthetic products, the purpose of increasing crop yield is achieved, providing gene resources and material resources for cultivating new crop varieties with high photosynthetic efficiency and high yield. Brief Description of the Drawings

[0061] Figure 1 It is a schematic diagram of the relative expression levels of the maize 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 overexpressing the maize gene ZmHPE3 in Example 3;

[0063] Figure 3 It is an ear type diagram of the control CK and the transgenic lines obtained by overexpressing the maize gene ZmHPE3 in Example 3;

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

[0065] Figure 5 Statistical analysis results of the agronomic traits of the control CK and the overexpressed maize gene ZmHPE3 lines in Example 3: A is the statistical analysis result of plant height; B is the statistical analysis result of yield per plant; C is the statistical analysis result of grain length; D is the statistical analysis result of grain width; E is the statistical analysis result of 1000-grain weight; F is the statistical analysis result of the number of grains per ear. Specific implementation manners

[0066] The specific implementation manners of the present invention will be described below to facilitate those skilled in the art of the present technology to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation manners. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.

[0067] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0068] Measurement of plant height: At the mature stage, the height from the ground surface to the top of the highest ear of a single plant (excluding awns). Take the average value of 15 plant heights within the line.

[0069] Measurement of photosynthetic efficiency parameters: One week after the initial ear emergence, on a sunny day with a temperature above 30 degrees, start measuring the photosynthetic rate of the flag leaves of the selected rice plants at 9:00 am every day. The measurement site is the front 1 / 3 of the flag leaf blade; each measurement data takes 1 - 5 minutes; each group of materials is limited to be measured within 1 hour; repeat the morning measurement at 13:00 on the same day; repeat the measurement 2 more times within the next week; the photosynthetic measurement system is a single Li-cor6800 portable photosynthetic rate meter produced in the United States, which can automatically measure the net photosynthetic rate, photorespiration rate, and intercellular CO2 concentration.

[0070] pCam23A vector: described 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. This biological material is only used for repeating the relevant experiments of the present invention and is not used for other purposes.

[0071] Grain type statistical analysis: For the determination of grain length, grain width and 1000-grain weight, a Wanshen automatic seed measurement analyzer and 1000-grain weight instrument model SC-G were used, and the specific method was referred to the instruction manual.

[0072] 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 is preserved by the Crop High Photosynthetic 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.

[0073] All primers used in the following examples were synthesized by Beijing Tsingke Biotechnology Co., Ltd., and sequencing was completed by Beijing Tsingke Biotechnology Co., Ltd. Restriction enzymes SmaI and XbaI, Infusion recombinase, and T4 ligase were all purchased from Dalian Lihexingtong Co., Ltd. (Takara); pTEAY-T1 Cloning Kit, Taq enzyme, Trans5α competent cells and related kits were all purchased from Beijing TransGen Biotech Co., Ltd.; RNA extraction and reverse transcription kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Agrobacterium AGL1 was purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., product number ZK296; antibiotics were purchased from SIGMA Company, USA; the rest of the reagents were all domestic analytical pure.

[0074] The reagent formulations used are as follows:

[0075] (I) MS vitamins and organics (1000×): 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 (1000×): nicotinic acid 1 g / L, Thiamine-HCl 10 g / L, Pyridoxine-HCl

[0077] 1 g / L.

[0078] (III) N6 Vitamins and Organic (1000×): Nicotinic acid 0.5 g / L, Thiamine-HCl 1.0 g / L, Pyridoxine-HCl 0.5 g / L, Glycine 2.0 g / L.

[0079] (IV) MS Micronutrients (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. (V) N6 Micronutrients (1000×): MnSO4·4H2O 5.6 g / L, ZnSO4·7H2O 1.7 g / L, CuSO4·5H2O

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

[0081] 1.53 g / L.

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

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

[0084] 2.9 g / L.

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

[0086] (VIII) N6 Macronutrients (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.

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

[0088] (X) AA Trace (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

[0089] 0.65 g / L.

[0090] (XI) MS Macro (20×): 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) 2,4-D (2 mg / mL): First, dissolve 2,4-D in a microwave oven with 5 ml - 10 ml of 1N KOH,

[0092] add ultrapure water to make up the volume. Store at room temperature.

[0093] (XIII) 6-BA (3 mg / mL): Weigh 150 mg of BA, first dissolve it with 5 ml of 1N KOH, and then make up the volume to 50 ml with sterile water. Filter sterilize.

[0094] (XIV) AS (40 mg / mL): Dissolve and make up the volume of AS with dimethyl sulfoxide (DMSO).

[0095] (XV) Timentin TIM (200 mg / mL): Dissolve with sterile water, make up the volume and filter sterilize.

[0096] (XVI) Hygromycin HYG (50 mg / mL): Dissolve with sterile water, make up the volume and filter sterilize.

[0097] (XVII) G418 (150 mg / mL): Dissolve with sterile water, make up the volume and filter sterilize.

[0098] (XVIII) ABA (5 mg / mL): Dissolve and make up the volume of ABA with 95% analytical pure ethanol.

[0099] (XIX) Rifampicin RIF (25 mg / mL): First dissolve rifampicin (rif) with 1N NaOH, and then make up the volume with methanol. (Or directly make up the volume with DMSO and filter sterilize).

[0100] (XX) NAA (2 mg / mL): Dissolve with 1N KOH, make up the volume with distilled water and filter sterilize.

[0101] The following examples used GraphPad Prism 8 statistical software to process the data. The experimental results were expressed as mean ± standard deviation and analyzed by Student’s t test. P < 0.05 (*) indicated significant difference, and P < 0.01 (**) indicated extremely significant difference.

[0102] Example 1: Intelligent prediction of key genes in the transcriptional regulatory network by multi-omics joint analysis

[0103] The transcriptional regulatory network within plants is determined by transcription factors. Currently, there are few studies on transcription factor binding in plants, making it impossible to construct a comprehensive picture of complex regulatory networks. Based on the multi-omics data obtained by our team using microdissection technology in the early stage (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 the joint analysis of a large amount of multi-omics data such as ChIP-seq, the regulatory network of transcription factors was reconstructed in plants, revealing the complexity and redundancy of the plant transcriptional regulatory network and providing a data basis for mining high-yield genes.

[0104] Through the intelligent prediction of the transcriptional regulatory network and gene mining, combined with the collected genetic data, multiple genes at the key nodes of the transcriptional regulatory network were predicted. Among them, the gene ZmHPE3 encodes a transcriptional repressor protein, and the CDS length of this gene is 1149 bp. Retrieval found that there were no research reports on this gene and its homologous genes in aspects such as plant yield, nor any applications in improving crop yield. Therefore, an overexpression vector of this gene was constructed and transferred into the crop rice for functional verification.

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

[0106] I. Extraction of maize RNA

[0107] The total plant RNA of maize B73 was extracted using the RNA Easy Fast Plant Tissue RNA Rapid Extraction Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. For each tissue sample used for detection, after sampling in the field, it was placed in tin foil, quickly frozen in liquid nitrogen, and then taken back to the Beijing laboratory and stored in an ultra-low temperature refrigerator. The specific steps for RNA extraction are as follows:

[0108] 1. Sample pretreatment: Grind the plant leaves or fruit pulp into powder rapidly in liquid nitrogen. Take 30 - 150 mg of the sample and add 600 μl of lysis buffer SG and 10 μl of Proteinase K. Immediately vortex vigorously to mix evenly, and then let it stand at room temperature for 5 min.

[0109] 2. Centrifuge at 12,000 rpm (~13,400×g) for 2 min, and take about 500 μl of the supernatant for the following operations.

[0110] 3. Add the obtained supernatant to the genomic DNA removal column, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, and retain the filtrate.

[0111] 4. Slowly add 0.5 times the volume of absolute ethanol (about 250 μl) of the supernatant to the above filtrate, mix well (precipitation may occur at this time), and transfer the obtained solution and precipitate together into the RNase-Free adsorption column CR4 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0112] 5. Add 700 μl of protein removal solution RW3 to the RNase-Free adsorption column CR4, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, discard the waste liquid, and put the adsorption column back into the collection tube.

[0113] 6. Add 500 μl of rinsing solution RW (please check whether ethanol has been added before use) to the RNase-Free adsorption column CR4, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm (~13,400×g) for 30 - 60 sec, discard the waste liquid, and put the adsorption column back into the collection tube.

[0114] 7. Repeat step 6.

[0115] 8. Centrifuge at 12,000 rpm (~13,400×g) for 2 min, pour out the waste liquid. Place the RNase-Free adsorption column CR4 at room temperature for 2 min to thoroughly dry the residual rinsing solution in the adsorption material.

[0116] 9. Transfer the RNase-Free adsorption column CR4 into a new RNase-Free centrifuge tube. Pipette 30 - 100 μl of RNase-Free ddH2O onto the middle part of the adsorption membrane in a suspended manner. Incubate at room temperature for 2 min, then centrifuge at 12,000 rpm (~13,400×g) for 2 min to obtain the RNA solution.

[0117] II. RNA Reverse Transcription

[0118] Use the FastKing cDNA First Strand Synthesis Kit (Genomic DNA Removal) from Tiangen Biochemical Technology (Beijing) Co., Ltd. to perform the reverse transcription of cDNA. The specific steps are as follows:

[0119] 1. Prepare the mixture according to the genomic DNA removal system in the following table, mix thoroughly. Centrifuge briefly and incubate at 42 °C for 3 min. Then place on ice.

[0120] Component Dosage 5×gDNA Buffer 2 μl Total RNA <![CDATA[RNase-Free ddH2O]]> Make up to 10 μl

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

[0122] Reagent Dosage 10×King RT Buffer 2 μl FastKing RT Enzyme Mix 1 μl FQ-RT Primer Mix 2 μl <![CDATA[RNase-Free ddH2O]]> Make up to 10 μl

[0123] 3. Add the Mix in the reverse transcription reaction to the reaction solution in the gDNA removal step and mix well.

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

[0125] 5. Incubate at 95 °C for 3 min and then place on ice. The obtained cDNA can be used for subsequent experiments or stored at low temperature.

[0126] III. Construction of the Overexpression Vector of Gene ZmHPE3 CDS

[0127] Use the primer design software DNAMAN to design the primers ZmHPE3-CDS-F and ZmHPE3-CDS-R:

[0128] ZmHPE3-CDS-F:

[0129] 5'-GTAGAAGAGGTACCCGGGATGGAGTACCCGGCGACGG-3';

[0130] ZmHPE3-CDS-R:

[0131] 5'-GCAGGTCGACTCTAGATCAATCTTCATCTGGTGTTTCCT-3'.

[0132] Using the obtained cDNA as a template, PCR amplification was performed with primers ZmHPE3-CDS-F and ZmHPE3-CDS-R (using high-fidelity KOD enzyme in the amplification system) to obtain a PCR product.

[0133] The obtained PCR product was subjected to gel electrophoresis and gel recovery. The recovered product was recombined with the pCam23A vector digested with SmaI and XbaI using Infusion recombinase. After sequencing, the recombinant vector pCam23A-ZmHPE3 was a vector obtained by replacing the SmaI and XbaI sites of the pCam23A vector with the nucleotides shown in SEQ ID No.2.

[0134] Example 3. Application of Maize Gene ZmHPE3 in Improving Photosynthetic Efficiency and Yield of Rice

[0135] I. Preparation of Overexpressing Plants of Maize Gene ZmHPE3 Transformed into Rice Nipponbare

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

[0137] (1) The prepared recombinant vector pCam23A-ZmHPE3 was introduced into Agrobacterium tumefaciens AGL1 to obtain the recombinant bacterium AGL1 / pCam23A-ZmHPE3. After digestion verification, positive recombinant bacteria were obtained.

[0138] (2) The recombinant bacterium AGL1 / pCam23A-ZmHPE3 was transformed into rice Nipponbare by rice genetic transformation to obtain the T0 generation overexpressing rice lines of maize 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 plump and intact dehulled rice seeds were placed in a 50 mL sterilized centrifuge tube. The seeds were washed three times with sterilized ultrapure water. Then, they were surface sterilized with 40 mL of 75% ethanol for 5 min, sterilized with 40 mL of 50% sodium hypochlorite for 5 min, and the sterilization was repeated once. Finally, they were rinsed 10 times with sterile water until the water was clear, and the sterilized seeds were blotted dry with sterile filter paper and placed on the MS+ medium, 20 seeds per dish, and cultured for 28 days under dark conditions at 28 °C until calli the size of shed millet grains grew.

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

[0142] The well-developed embryogenic calli were transferred to the MS medium. About 100 calli could be placed in each dish. They were cultured for 7 days under dark conditions at 28 °C.

[0143] 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.

[0144] S3. Preparation of Agrobacterium:

[0145] 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.

[0146] 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)

[0147] S4. OD value adjustment:

[0148] 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.

[0149] S5. Infection and co-cultivation:

[0150] 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.

[0151] S6. Resistance screening of transformed callus:

[0152] Collect the callus that has completed the co-culture stage into a 50 mL sterilized centrifuge tube, and rinse the callus 10 times with sterile water until the washing liquid is clear. Then pour 1 mL of the washing liquid of NBL + Tim200 mg / mL, shake it on a shaker at 100 g for 1 h. After shaking, pour out the filtrate and dry the moisture with filter paper. Transfer the callus to the selection medium (the concentration of G418 is 50 mg / L), and use forceps to place the callus particles evenly to prevent contact inhibition and large-area contamination. Screening with 2 - 3 dishes for one vector is sufficient. Incubate in the dark at 28 °C for 2 weeks, and this process is the first screening. Pay attention to observing whether there is any contamination during this period. After two weeks, subculture on the same medium once again, doubling the number of screening culture dishes, and 4 - 6 dishes for each vector are enough. The total selection takes about 4 weeks. This process is the second screening. Wait until the callus has obvious yellow round solid particles the size of millet grains falling off before proceeding to the next stage.

[0153] S7. Pre-differentiation stage:

[0154] Transfer the fresh creamy yellow round and dense embryogenic callus grown from the transformed old callus to the pre-differentiation medium. When picking, pick the newly fallen callus from the top, bottom, left, and right of one piece of callus without repeated picking. About 50 pieces can be inoculated in each dish. Pick for pre-differentiation as appropriate, and 3 - 4 dishes for each vector are sufficient. Incubate in the dark at 28 °C for 7 - 10 d. The pre-differentiation stage can be appropriately extended according to the growth of the callus. If the callus is too small, it is not easy to differentiate. When it grows to the size of a soybean, hard in texture, and slightly yellow, it can be differentiated. (The pre-differentiation medium also needs to distinguish between hygromycin HYG and G418 resistance. Dry the water vapor before use, pay attention to numbering, and do not mix them up).

[0155] S8. Differentiation and rooting:

[0156] Pick the white and dense callus and transfer it to the differentiation medium. Be sure to dry the water vapor before using the differentiation medium. About 20 pieces can be inoculated in each dish. Pay attention not to place them on the edge of the culture dish as much as possible, as it is easy to come into contact with water. Incubate under light at 28 °C for 3 - 4 weeks. Pay attention to placing the materials layer by layer when placing. Prevent high temperature generated by the light heat under the materials from burning the callus and affecting the differentiation ability. It is best to place it on the bottom layer of the tissue culture room shelf to prevent the generation of water vapor and affect the differentiation of the callus. Then subculture on the same medium once again, pay attention to handling gently to prevent the water droplets on the lid from dripping onto the callus. The callus that comes into contact with water will no longer differentiate. The callus just subcultured onto the differentiation medium needs to be placed for two days or covered with a black plastic bag for shading treatment to prevent the callus from overheating and browning.

[0157] S9. Seedling strengthening:

[0158] If relatively strong seedlings appear, transfer them to the 1 / 2MS seedling strengthening medium. Incubate under light at 28 °C for 2 - 3 weeks. The seedlings just transferred to the seedling strengthening medium need to be placed for two days before light treatment.

[0159] S10. Transplantation of tissue culture seedlings:

[0160] Wash the residual culture medium on the roots, transfer the seedlings with good roots to the greenhouse, and keep the soil moist in the first few days. The regenerated seedlings obtained are the overexpression rice lines of transgenic ZmHPE3 in Nipponbare rice. The T0 plants are self-crossed to obtain T1 generation plants overexpressing the ZmHPE3 gene, which are used for subsequent analysis.

[0161] 2. Identification of plants overexpressing the ZmHPE3 gene

[0162] Extract the young leaf RNA of three random lines 5-155-1, 5-155-2, and 5-155-3 of the transgenic ZmHPE3 overexpressing rice in the T1 generation (the method is the same as one in Example 1), and reverse transcribe to obtain cDNA as a template (the method is the same as two in Example 1). Select a pair of primers ZmHPE3-QRT1 and ZmHPE3-QRT2 in the CDS region for fluorescence quantitative analysis of the transformed seedlings, and perform fluorescence quantitative PCR amplification with the ZmHPE3-QRT1 primer and the ZmHPE3-QRT2 primer.

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

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

[0165] Use the iQ5 Muticolor Real-Time PCR Detection System (Bio-Rad) instrument for fluorescence quantitative PCR. The reagent used is SYBR Green Mix, and all experiments are independently repeated 3 times.

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

[0167]

[0168]

[0169] Reaction program: 95°C for 5 min, 95°C for 10 s, 60°C for 20 s, 72°C for 30 s, collect fluorescence at 80°C for 10 s, 72°C for 5 min, rise from 55°C to 95°C at 0.5°C / s, make a melting curve, and run steps 2-5 for 40 cycles.

[0170] The internal reference primers used are 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, compared with the control CK, the expression levels of the gene ZmHPE3 in 5-155-1, 5-155-2, and 5-155-3 were all significantly increased, indicating successful overexpression.

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

[0175] Recombinant Agrobacterium tumefaciens AGL1 / pCam23A was used to replace recombinant Agrobacterium tumefaciens AGL1 / pCam23A-ZmHPE3 and operated according to Step 1 to obtain rice plants transformed with empty vectors, which were subsequently called control CK.

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

[0177] The plants transformed with empty vectors (i.e., control CK) and the transgenic ZmHPE3 overexpressed rice materials were planted in the field, and their photosynthetic efficiency was measured, phenotypes were observed, and agronomic traits were counted. 15 plants were counted for each plant line.

[0178] Measure the parameters related to photosynthesis: 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 ). The results are shown in the following table. The photosynthetic efficiency of the transgenic ZmHPE3 overexpressed rice materials increased significantly, indicating that overexpression of the gene ZmHPE3 in the crop rice can significantly improve the photosynthetic efficiency of rice.

[0179] Measurement of photosynthetic efficiency of control CK and ZmHPE3 overexpressed lines

[0180]

[0181] The statistical results of agronomic traits are as Figure 5 shown. CK is the control of the empty vector transformation, and 5-155-1, 5-155-2, and 5-155-3 are three rice lines with overexpression of transgenic ZmHPE3 in rice Nipponbare. It can be seen that, compared with the control CK, the transgenic ZmHPE3 overexpressed plants showed a significant decrease in plant height (Figure 5 in A), the yield per plant increased significantly ( Figure 5 in B), the number of grains per panicle increased significantly ( Figure 5 in F), while the grain length ( Figure 4 and Figure 5 in C), the grain width ( Figure 4 and Figure 5 in D) and the 1000-grain weight ( Figure 5 in E) did not change significantly, indicating that overexpression of the gene ZmHPE3 in the crop rice can significantly improve the photosynthesis efficiency, the number of grains per panicle and the yield of rice, while reducing the plant height, and it is a newly discovered high photosynthetic efficiency and high-yield gene.

[0182] In summary, in the early stage of the present invention, through multi-omics combined analysis, key genes in the transcriptional regulatory network were mined, an overexpression vector of the ZmHPE3 gene was constructed and transferred into the crop rice for functional verification. The obtained transgenic plants had significantly improved photosynthetic efficiency, the number of grains per panicle and the yield compared with the control group, and the plant height was reduced. At present, there are no research reports on this gene and its homologous genes in terms of plant photosynthetic efficiency, the number of grains per panicle and yield, nor is there any application in increasing crop yield. This indicates that the gene ZmHPE3 (SEQ ID No.2) provided by the present invention and the protein sequence (SEQ ID No.1) expressed by it have important theoretical and practical significance for cultivating new high-yield crop materials with increased photosynthetic efficiency.

[0183] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any aspect, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present invention.

Claims

1. The ZmHPE3 gene based on multi-omics intelligent design, characterized in that, Its CDS sequence is shown in SEQ ID No.

2.

2. The protein encoded by the ZmHPE3 gene is characterized in that, Its amino acid sequence is shown in SEQ ID No.

1.

3. Recombinant vector, host bacterium and / or expression cassette of the coding region of the ZmHPE3 gene.

4. Application of the ZmHPE3 gene based on multi-omics intelligent design, characterized in that, Overexpression of the gene ZmHPE3 can improve the photosynthetic efficiency of rice.

5. Application of the ZmHPE3 gene based on multi-omics intelligent design, characterized in that, Overexpression of the gene ZmHPE3 can increase the number of grains per panicle of rice.

6. Application of the ZmHPE3 gene based on multi-omics intelligent design, characterized in that, Overexpression of the gene ZmHPE3 can reduce the plant height of rice.

7. Application of the ZmHPE3 gene based on multi-omics intelligent design, characterized in that, Overexpression of the gene ZmHPE3 can increase the yield of rice.

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