Rice coding gene OsPRR1 and application thereof in direct seeding of rice

Abstract

The invention belongs to the technical field of biology, and discloses a rice coding gene OsPRR1 and application thereof in rice direct seeding, the gene OsPRR1 is a gene for coding any one of the following proteins: (1) a protein composed of an amino acid sequence as shown in SEQ ID NO.1; (2) a protein which is derived from the amino acid sequence as shown in SEQ ID NO.1 through substitution and / or deletion and / or addition of one or more amino acid residues and can regulate and control plant mesocotyl elongation and direct seeding emergence rate; and (3) a protein which is composed of an amino acid sequence having at least 75%-99% homology with the amino acid sequence as shown in SEQ ID NO.1 and is related to plant mesocotyl elongation and direct seeding emergence rate. The application refers to the application of the protein in regulation and control of rice mesocotyl elongation and direct seeding emergence rate. The regulatory protein and the coding gene thereof provided by the invention are of great significance to increase the length of mesocotyl of rice and increase the direct seeding emergence rate of rice.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and relates to the application of plant coding genes, in particular to the rice coding gene OsPRR1 and its application in direct seeding of rice. Background Art

[0002] With the shift of rural labor and the transformation of agricultural planting patterns, traditional intensive rice seedling cultivation methods, such as transplanting and raising rice seedlings, are no longer able to meet the demands of modern agricultural production. Direct-seeding rice cultivation, characterized by its labor-saving, time-saving, and water-saving advantages, has gradually become a highly efficient planting method. However, in practice, direct-seeding rice still faces challenges such as difficulty in seedling emergence, uneven emergence, and poor growth potential during the seedling stage, severely hindering its widespread adoption and the achievement of high and stable yields.

[0003] Faced with the current difficulties in direct-seeded rice seedling emergence and slow seedling growth, utilizing modern molecular biology and gene editing technologies to clone key genes regulating rice seedling emergence rate and cultivate high-yielding, tolerant rice varieties through gene aggregation and molecular breeding is an important approach to addressing the current problem of poor direct-seeded rice seedling emergence. Studies have shown that mesocotyl and coleoptile elongation are important traits affecting seedling emergence and early seedling vigor in direct-seeded rice, and both are quantitatively inherited traits controlled by multiple genes. To date, researchers have identified several key genes regulating rice mesocotyl elongation through forward cloning, including CYC U2, OsGY1, OsMHZ7, OsSMAX1, and OsGSK2. For example, Sun et al. discovered that OsGSK2 phosphorylates the cell cycle protein CYC U2, significantly reducing its protein stability. In this process, strigolactones inhibit rice mesocotyl elongation by mediating the proteasomal degradation of phosphorylated CYC U2 via the D3 ubiquitin ligase. Antagonistic regulation is achieved by brassinosteroids, which inhibit the activity of OsGSK2 kinase, blocking the phosphorylation of CYC U2, ultimately relieving growth inhibition and promoting mesocotyl elongation. Although some studies have reported the genetic regulatory mechanisms of rice mesocotyl elongation, the relevant genes identified so far are still insufficient to meet the needs of direct-seeding rice variety breeding.

[0004] This study, based on the Nipponbare variety, a rice variety characterized by non-elongated mesocotyls, knocked out and overexpressed the OsPRR1 gene. The researchers found that knocking out OsPRR1 significantly promoted mesocotyl elongation and improved seed emergence compared to the wild-type Nipponbare. This study provides a gene encoding a circadian rhythm-related protein, OsPRR1, that can be used to regulate mesocotyl length and seedling emergence rate in rice under deep burial conditions. Summary of the Invention

[0005] The present invention provides the rice encoding gene OsPRR1 and its application in direct seeding of rice to solve the technical problems of time-consuming and labor-intensive rice transplanting and low emergence rate of direct seeding rice seedlings, providing important theoretical support for the cultivation of rice varieties suitable for direct seeding.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides an encoding gene OsPRR1, wherein the gene OsPRR1 is a gene encoding any one of the following proteins:

[0008] (1) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1;

[0009] (2) A protein capable of regulating plant mesocotyl elongation and direct seeding emergence rate derived from the amino acid sequence shown in SEQ ID NO. 1 by substitution and / or deletion and / or addition of one or more amino acid residues;

[0010] (3) A protein consisting of an amino acid sequence that is at least 75% to 99% homologous to the amino acid sequence shown in SEQ ID NO. 1 and is associated with plant mesocotyl elongation and direct seeding emergence rate.

[0011] It should be noted that the encoding gene OsPRR1 is a protein derived from the japonica rice variety Nipponbare (Oryza sativa L. japonica. cv. Nipponbare, NIP); SEQ ID NO. 1 consists of 518 amino acid residues.

[0012] Preferably, the substitution and / or deletion and / or addition of the amino acid residues is caused by natural variation or artificial mutagenesis.

[0013] Preferably, the above protein can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0014] Preferably, the gene OsPRR1 is any one of the following:

[0015] (1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO. 2;

[0016] (2) A DNA molecule having a nucleotide sequence such as that shown in SEQ ID NO. 2 with one or more nucleotides substituted, deleted, and / or added;

[0017] (3) a DNA sequence that hybridizes with the DNA sequence defined in (1) and encodes a protein related to plant mesocotyl elongation and direct seeding emergence rate;

[0018] (4) A DNA molecule that has 70% to 99% homology with the nucleotide sequence defined in (1) or (2) and can encode a protein related to plant mesocotyl elongation and direct seeding emergence rate.

[0019] It is necessary to explain that SEQ ID NO. 2 consists of 1,557 nucleotides, all of which are the coding sequence of OsPRR1 protein.

[0020] The present invention also provides a recombinant vector, a knockout vector, an expression cassette, a transgenic cell line or a recombinant bacterium of the above-mentioned gene OsPRR1.

[0021] The present invention also provides primers for amplifying the full length or any fragment of the above-mentioned gene OsPRR1, wherein the primers are: a primer pair consisting of OsPRR1-F1 with a nucleotide sequence as shown in SEQ ID NO.3 and OsPRR1-R1 with a nucleotide sequence as shown in SEQ ID NO: 4, or a primer pair consisting of OsPRR1-F2 with a nucleotide sequence as shown in SEQ ID NO.5 and OsPRR1-R2 with a nucleotide sequence as shown in SEQ ID NO: 6.

[0022] The present invention also provides the use of the above-mentioned gene OsPRR1 in regulating rice mesocotyl elongation and direct seeding emergence rate, wherein the regulation refers to increasing the mesocotyl length and direct seeding emergence rate of the plant by knocking out the gene OsPRR1 in the plant species or reducing or inhibiting the expression of the gene OsPRR1, or reducing the mesocotyl length and direct seeding emergence rate of the plant by overexpressing the gene OsPRR1.

[0023] The present invention also provides the use of a knockout vector, an expression cassette, a transgenic cell line or a recombinant bacterium of the above-mentioned gene OsPRR1 in increasing the length of the mesocotyl and the direct seeding emergence rate of rice.

[0024] The present invention also provides a method for increasing the length of the mesocotyl and the direct seeding emergence rate of a plant, comprising the step of knocking out the above-mentioned gene OsPRR1 in the plant.

[0025] Preferably, the method comprises the following steps: constructing a knockout vector encoding the OsPRR1 gene as claimed in any one of claims 1 to 3 using the CRISPR / Cas9 genome editing system through two specific sgRNA target sites; then transferring the knockout vector into rice cells by infecting rice callus with Agrobacterium and integrating it into the chromosome, and screening cells, tissues or organs in which the OsPRR1 gene is successfully knocked out to regenerate into plants.

[0026] Preferably, the nucleotide sequences of the two specific sgRNA targets are shown as SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0027] It should be noted that in the embodiment of the present invention, the sgRNA sequence designed for the gene OsPRR1 is inserted into the CRISPR / Cas9 knockout vector using the CRISPR / Cas9 knockout vector to obtain the knockout recombinant vector CRISPR / Cas9-OsPRR1.

[0028] To facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors can be appropriately processed. For example, the following types of genes can be introduced: (1) genes encoding enzymes that can cause color change or fluorescence production, such as the GUS gene and the luciferase gene; (2) antibiotic resistance marker genes, such as the nptII gene that confers kanamycin resistance, the corresponding marker gene that confers gentamicin resistance, and the hph gene that confers hygromycin resistance; (3) marker genes for herbicide or chemical resistance, such as the EPSPS gene that confers glyphosate resistance, the bar gene that confers phosphinothricin resistance, and the dhfr gene that confers methotrexate resistance; (4) functional metabolic genes, such as the mannose-6-phosphate isomerase gene that confers the ability to metabolize mannose.

[0029] The present invention provides a method for cultivating transgenic plants, which introduces a gene editing vector into a target plant to obtain a transgenic plant having a mesocotyl length and a direct seeding emergence rate higher than that of the target plant. The transgenic plant refers not only to the first-generation transgenic plant obtained by transforming the target plant with a gene editing vector, but also includes its offspring. At the same time, the gene can be transmitted within the plant species through reproduction, and can also be transferred to other varieties of the same plant, especially commercial varieties, through conventional breeding techniques. By introducing the gene editing vector into the target plant, the synthesis of a specific target protein in the target plant can be inhibited, thereby increasing the mesocotyl length and direct seeding emergence rate of the target plant.

[0030] The protein coding gene may be modified as follows before introduction into the host to improve its expression efficiency:

[0031] 1) Optimize according to actual needs to ensure that the knockout vector works efficiently and accurately; select Cas proteins from different bacterial species based on the experimental design, and design sgRNAs based on their corresponding PAM sequence types to improve the accuracy of targeted recognition; the knockout site is located in the coding region (CDS), preferably at the front end of the protein or in the key functional domain area; select targets with high editing efficiency and no off-target effects; and increasing the number of sgRNAs to achieve effective knockout of single or multiple genes is also a common practice in experimental design.

[0032] 2) In the promoter region of the Cas gene region and the CRISPR unit, select an appropriate high-efficiency expression promoter based on the target crop to achieve more effective gene knockout effects; such promoters may include constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter should be based on the temporal and spatial requirements of expression and depend on the target species; for example, a promoter specific for expression in a specific tissue or organ can be selected based on the requirements of the receptor at a specific developmental stage; although many promoters derived from monocots can function in dicots and vice versa, it is generally recommended to prioritize monocot promoters for use in monocots and dicot promoters for use in dicots;

[0033] 3) Linking to a suitable transcriptional terminator can also improve the knockout efficiency of the gene of the present invention. For example, the 35S terminator derived from CaMV virus or the NOS terminator derived from plants; the gene of the present invention can be linked to any terminator known to be functional in plants and available.

[0034] 4) Introducing enhancer sequences, such as intron sequences (e.g., from Adhl and bronze1) and viral leader sequences (e.g., from TMV, MCMV, and AMV).

[0035] In practical applications, the functional localization of the gene of the present invention can be achieved through cell targeting technology. Specifically, the target gene sequence targeting an organelle can be fused with the gene sequence of the present invention and then transferred into plant cells to achieve localization.

[0036] Specifically, the gene can be introduced into the target plant via a recombinant vector. The recombinant vector carrying the gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, or Agrobacterium-mediated transformation, and the transformed plant tissue can be cultivated into plants. The target plants include monocotyledonous plants such as rice, corn, sorghum, and millet.

[0037] Compared with the existing technology, this solution has the following beneficial effects:

[0038] The present invention uses the CRISPR / Cas9 knockout system to achieve the knockout of the gene OsPRR1 in rice, obtains a transgenic rice line with the gene knocked out, and performs phenotypic statistics and comparisons on the wild-type Nipponbare and the transgenic line. The results showed that after culturing for 8 days under conditions of 6 cm burial depth, the knockout gene OsPRR1 can significantly increase the length of the mesocotyl and the rate of emergence of rice. The average mesocotyl length reached 6.4 mm, while the mesocotyl length of the wild-type Nipponbare was 1.8 mm; the mutant emergence rate reached 50.3%, while that of the wild type was 35.1%. In actual production, this gene can optimize the key agronomic trait of mesocotyl elongation, and is expected to significantly increase the emergence rate under direct seeding conditions of rice. The protein involved in the present invention and the gene encoding it have broad application prospects in the breeding of direct seeding-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A and Figure 1 B shows the detection results of the amplification of the nucleic acid sequence encoding the gene OsPRR1 and the overexpression vector OsPRR1-OE (lane 1 shows the amplified fragment of OsPRR1, lane 2 shows the double-enzyme digested fragment of the pBI121 empty vector, and lane 3 shows the double-enzyme digested fragment of the overexpression vector OsPRR1-OE);

[0040] Figure 2 This is a schematic diagram of the plant overexpression vector pBI121-OsPRR1.

[0041] Figure 3 is the molecular identification of rice overexpression lines OsPRR1-OE1 / 2 (lane 1 shows the amplified fragment using the overexpression vector OsPRR1-OE as a template (positive control), lane 1 shows the amplified fragment using water as a template (negative control), and lanes 3 and 4 show the amplified fragment using gDNA of the overexpression transgenic lines as a template);

[0042] Figure 4 The expression of OsPRR1 gene was detected by qRT-PCR;

[0043] Figure 5 Molecular identification of the rice knockout line osprr1.

[0044] Figure 6 The mesocotyl length and seedling emergence rate of the control line WT, the knockout line osprr1 and the overexpression line OsPRR1-OE1 / 2 under the condition of 6 cm burial depth. DETAILED DESCRIPTION

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All primers used were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The rice transgenic process was performed by Shanghai Boyi Biotechnology Co., Ltd. The T2 generation in the following examples refers to seeds produced by selfing the T1 generation and the resulting plants.

[0048] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0049] Example 1 Cloning of the rice encoding gene OsPRR1

[0050] 1. Synthesis of cDNA sequences

[0051] Total RNA was extracted from the leaves of Nipponbare rice using the TriZol method and reverse transcribed into cDNA sequences according to the instructions of the TaKaRa reverse transcription kit. The full-length gene (including the entire coding sequence of the gene) was amplified by PCR using cDNA as a template. Figure 1 A), the primer sequences for PCR amplification are as follows:

[0052] OsPRR1-F1 (SEQ ID NO. 3):

[0053] 5'-ATGGTGGGCGCCCGGCGAGGGGGATC-3';

[0054] OsPRR1-R1 (SEQ ID NO.4):

[0055] 5'-CTCTGGAGAAGAAAACCATCTCTACCTC-3'.

[0056] 2. Cloning of the OsPRR1 gene

[0057] 2.1 Purify and recover the PCR product according to the kit (QIAGEN). The purified PCR product was ligated into the pMD18-T vector (purchased from TAKARA) and transformed into Escherichia coli DH5α competent cells (purchased from TAKARA). Single clones were selected for PCR verification, and the amplified sequence was sent to Nanjing GenScript Biotechnology Co., Ltd. for Sanger sequencing to verify that the sequence was correctly cloned.

[0058] 2.2 Sequence sequencing results showed that the fragment obtained by PCR reaction had the nucleotide sequence shown in the sequence listing SEQ ID NO.2 and encoded the amino acid sequence of the protein shown in the sequence listing SEQ ID NO.1; the protein shown in SEQ ID NO.1 was named protein OsPRR1, and the gene encoding the protein shown in SEQ ID NO.1 was named gene OsPRR1.

[0059] Protein OsPRR1 (SEQ ID NO. 1):

[0060] MVGAGEGDRVGGGAAVGGGQQFVDRSKVRILLCDSDPSSSREVLRLLCNCSYQVTCAKSPRQVINVLNCEAGEIDIILAEVDLPVSKCFKMLKYIARNKELRHIPIIMMSNRDEVSVVVKCLRLGAAEY LVKPLRMNELLNLWTHVWRRRRMLGLSEKNFFNDNFELALSEPSDANTNSTTLLSDDTDDKPKENINQETSTSNQHEYESNPSDAEPKQKGTPEGLLVSTEGGDQASSPGVMFSRPIKTNLRVAESSAFL AYVKSSTPTTSSFDSELQKGGNRLDSSDHRGNFSSTTDRSDTGTDVNIRDKEAFEMPVQYPVVCFSSSNLHLERSNEGQNDASGTPPVYHFPFYYPGMMDHGMTHPPVQNFQGNINNAQVHTPQTLLPQY NVYPQCHGVSMMPPFQYNPAGMSIQSNQLPTQNMWPQASSTPMPEETCSRSERRAAALAKFRLKRKERCFDKKVRYVNRKKLAETRPRVRGQFVRQANYTDITSTGDDISEDEDDDPSSREVEMVSSPE;

[0061] Gene OsPRR1 (SEQ ID NO. 2):

[0062]

[0063] 3. Construction of OsPRR1 overexpression vector

[0064] The above sequenced monoclonal bacterial suspension was used as a template and a specific primer pair with XbaI and SmaI adapter sequences was added for PCR amplification. At the same time, the plant binary expression vector pBI121 plasmid was double-digested with XbaI and SmaI endonucleases, and the PCR product and linearized pBI121 plasmid were purified and recovered. The purified PCR product was inserted into the pBI121 expression vector plasmid according to the homologous recombination method provided by the One Step Cloning Kit to obtain the overexpression vector OsPRR1-OE (see Figure 2 ), double enzyme digestion with XbaI and SmaI was performed to verify the successful construction of the vector ( Figure 1 B).

[0065] The primer sequences are as follows:

[0066] OsPRR1-F2 (SEQ ID NO. 5, the underlined sequence is the vector linker sequence):

[0067] 5'- GAGAACACGGGGGACTCTAGA ATGGTGGGCGCCGGCGAGGG

[0068] GGATC-3';

[0069] OsPRR1-R2 (SEQ ID NO. 6, the underlined sequence is the vector linker sequence):

[0070] 5'- ATAAGGGACTGACCACCCGGG CTCTGGAGAAGAAACCATCT

[0071] CTACCTC-3'.

[0072] Example 2: Acquisition and detection of transgenic plants

[0073] 1. Identification of Transformed Rice and Transgenic Plants

[0074] 1. Use a plasmid extraction kit (QIAGEN) to extract the constructed OsPRR1-OE expression vector plasmid, and transform the plasmid into Agrobacterium EHA105 by electroporation to obtain recombinant Agrobacterium.

[0075] 2. Infect callus tissue with Agrobacterium and transfer the recombinant Agrobacterium to Nipponbare. After culturing in the dark at 25°C for 3 days, screen for resistant calli and transgenic plants on a selective medium containing 150 mg / L G418. Identify the resulting T0 generation resistant plants, harvest positive T0 generation plants, and propagate them to the T3 generation.

[0076] 2. Molecular identification of OsPRR1 transgenic plants

[0077] 1. Use the CTAB method to extract rice genomic DNA and use the genomic DNA as a template to detect transgenic rice using the designed specific primers (see Figure 3 ), the primer sequences are as follows:

[0078] OsPRR1-F3(SEQ ID NO.7):5'-TCTCAGAAGACCAAAGGGC-3'

[0079] OsPRR1-R3 (SEQ ID NO. 8): 5'-TCTCCGACAAACCAAGCAT-3'.

[0080] 2. Detection of OsPRR1 gene expression by qRT-PCR

[0081] Total RNA from T3 transgenic plants and control plants was extracted using the TriZol method and reverse transcribed into cDNA. qRT-PCR was performed using the OsPRR1-F4 / R4 primer pair using cDNA as a template. The results are shown in Figure 4 The primer sequences are as follows:

[0082] OsPRR1-F4 (SEQ ID NO.9):5'-GCCAATACCAACAGCACCA-3';

[0083] OsPRR1-R4 (SEQ ID NO. 10): 5'-AAAAGCAGAAGACTCAGCAACC-3'.

[0084] Depend on Figure 4 It can be seen that the relative expression level of OsPRR1 in the overexpression plants was significantly higher than that in the wild type, and a transgenic line overexpressing OsPRR1 was successfully obtained.

[0085] Example 3: Obtaining and detecting OsPRR1 mutant plants

[0086] 1. Construction of gene knockout vector

[0087] An 18-bp sequence containing the NGG trinucleotide specifically recognized by Cas9 was screened from the full-length CDS sequence of OsPRR1. The screened 18-bp sequence was ligated into the VK005-01 vector using a plant Cas9 / gRNA plasmid vector construction kit to generate the OsPRR1-sgRNA gene knockout vector. The specific steps are as follows:

[0088] 1. Design and synthesize specific sgRNA target site primers based on the OsPRR1 gene sequence. The primer sequences are as follows:

[0089] OsPRR1-sgRNAF1 (SEQ ID NO.11):

[0090] 5'-CAGGCAGTTCGTGGACCGGAGCA-3';

[0091] OsPRR1-sgRNAR1 (SEQ ID NO.12):

[0092] 5'-AACTGCTCCGGTCCACGAACTGC-3'.

[0093] 2. Reaction to form dimers: Pipette 5 μL of OsPRR1-sgRNAF (10 μM), 5 μL of OsPRR1-sgRNAR (10 μM) and 15 μL of MilliQ water, mix well and incubate the mixture at 95°C for 3 minutes, then slowly cool to 25°C (-1°C / 20 seconds), and finally incubate at 16°C for 5 minutes;

[0094] 3. Use the method provided in the Plant Cas9 / gRNA Plasmid Construction Kit to ligate the primer dimer into the vector. The ligation system is: 1 μL of Cas9 / gRNA Vecter, 1 μL of primer dimer formed in the above reaction, 1 μL of Solution, 21 μL of Solution, and finally, add MilliQ water to 10 μL. Mix thoroughly by pipetting with a pipette tip. Incubate the mixture at 16°C for 2 hours and place on ice. Transfer 10 μL of this ligation reaction to competent E. coli DH5α cells and select clones for sequencing and verification.

[0095] 2. Acquisition and identification of OsPRR1 mutants

[0096] 1. The process of obtaining OsPRR1 mutants is as follows:

[0097] 1) The OsPRR1-sgRNA plasmid was extracted using a plasmid extraction kit (QIAGEN), and the plasmid was transformed into Agrobacterium EHA105 by electroporation to obtain a recombinant Agrobacterium strain containing the knockout vector OsPRR1-sgRNA.

[0098] 2) Nipponbare callus tissue was infected with a recombinant Agrobacterium strain containing the OsPRR1-sgRNA plasmid. After culturing in the dark at 25°C for 3 days, resistant calli and transgenic plants were screened on a selection medium containing 50 mg / L HygB.

[0099] 3) The genomic DNA of the T0 generation resistant plants was extracted by CTAB method. The genomic DNA was used as template and a specific primer pair (SEQ ID NO.3 and SEQ ID NO.4) was used for PCR amplification and sequencing identification. The homozygous mutant OsPRR1 (see Figure 5 ).

[0100] 2. Phenotypic analysis and identification of homozygous mutant OsPRR1 transgenic rice

[0101] Analysis of phenotypic data from transgenic rice materials and wild-type control lines revealed that, when buried at a depth of 6 cm, the mesocotyl length of the transgenic lines with the OsPRR1 knockout gene reached an average of 6.4 mm, with a seedling emergence rate of 50.3%. Compared with the wild-type control, the mesocotyl length increased significantly by approximately 4.6 mm, and the seedling emergence rate increased by approximately 15.2% (see Figure 6 However, the mesocotyl length of the transgenic lines overexpressing OsPRR1 (OsPRR1-OE1 / 2) was not significantly different from that of the wild-type control, but the seedling emergence rate was significantly lower than that of the wild-type.

[0102] In summary, the OsPRR1 gene plays an important role in regulating rice mesocotyl elongation and direct seeding emergence rate.

[0103] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A coding gene OsPRR1, characterized in that The gene OsPRR1 is a gene encoding any one of the following proteins: (1) A protein consisting of the amino acid sequence shown in SEQ ID NO. 1; (2) A protein capable of regulating plant mesocotyl elongation and direct seeding emergence rate derived from the amino acid sequence shown in SEQ ID NO. 1 by substitution and / or deletion and / or addition of one or more amino acid residues; (3) A protein consisting of an amino acid sequence that is at least 75% to 99% homologous to the amino acid sequence shown in SEQ ID NO. 1 and is associated with plant mesocotyl elongation and direct seeding emergence rate.

2. The gene OsPRR1 according to claim 1, characterized in that The substitution and / or deletion and / or addition of the amino acid residues are caused by natural variation or artificial mutagenesis.

3. The gene OsPRR1 according to claim 1, characterized in that The gene OsPRR1 is any one of the following: (1) a DNA molecule whose nucleotide sequence is shown in SEQ ID NO. 2; (2) A DNA molecule having a nucleotide sequence such as that shown in SEQ ID NO. 2 with one or more nucleotides substituted, deleted, and / or added; (3) a DNA sequence that hybridizes with the DNA sequence defined in (1) and encodes a protein related to plant mesocotyl elongation and direct seeding emergence rate; (4) A DNA molecule that has at least 70% to 99% homology with the nucleotide sequence defined in (1) or (2) and can encode a protein related to plant mesocotyl elongation and direct seeding emergence rate. 4 . The knockout vector, expression cassette, transgenic cell line or recombinant bacterium of the OsPRR1 gene according to claim 1 .

5. Primers for amplifying the full length or any fragment of the gene OsPRR1 according to any one of claims 1 to 3, characterized in that: The primers are: a primer pair consisting of OsPRR1-F1 with a nucleotide sequence as shown in SEQ ID NO.3 and OsPRR1-R1 with a nucleotide sequence as shown in SEQ ID NO: 4, or a primer pair consisting of OsPRR1-F2 with a nucleotide sequence as shown in SEQ ID NO.5 and OsPRR1-R2 with a nucleotide sequence as shown in SEQ ID NO:

6.

6. Use of the gene OsPRR1 according to any one of claims 1 to 3 in regulating mesocotyl elongation and direct seeding emergence rate in rice, characterized in that: The regulation refers to increasing the mesocotyl length and direct seeding emergence rate of the plant by knocking out the gene OsPRR1 or reducing or inhibiting the expression of the gene OsPRR1 in the plant species, or reducing the mesocotyl length and direct seeding emergence rate of the plant by overexpressing the gene OsPRR1.

7. Use of the knockout vector, expression cassette, transgenic cell line or recombinant bacteria of the OsPRR1 gene according to claim 4 for increasing the mesocotyl length and direct seeding emergence rate of rice.

8. A method for increasing the length of the mesocotyl and the direct seeding emergence rate of plants, characterized in that: The method comprises the step of knocking out the gene OsPRR1 according to any one of claims 1 to 3 in a plant.

9. The method according to claim 8, characterized in that The following steps are involved: The CRISPR / Cas9 genome editing system is used to construct a knockout vector for the OsPRR1 gene according to any one of claims 1 to 3 through two specific sgRNA target sites; the knockout vector is then transferred into rice cells by infecting rice callus with Agrobacterium and integrated into the chromosome, and cells, tissues or organs in which the OsPRR1 gene is successfully knocked out are screened to regenerate into plants.

10. The method according to claim 9, characterized in that The nucleotide sequences of the two specific sgRNA targets are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

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