Rice osrdr5 gene and coding protein, site-directed editing system, application and application method

Abstract

The application discloses a rice OsRDR5 gene and a coding protein, a site-directed editing system, application and an application method, a nucleotide sequence of the OsRDR5 gene is shown as SEQ ID NO. 1; and an amino acid sequence of the coding protein of the rice OsRDR5 gene is shown as SEQ ID NO. 2. The gene has a Coa3_cc domain and belongs to a cytochrome c oxidase assembly factor 3 gene family. Knockout mutants of the OsRDR5 gene show a higher deep root ratio, and simultaneously change the gravitropism of rice root systems. The rice OsRDR5 gene of the application regulates the drought avoidance of rice, the knockout mutants have a higher deep root ratio, and can be applied in breeding of new water-saving and drought-resistant rice varieties, so that the drought resistance of rice is improved.

Description

Technical Field

[0001] This invention relates to the field of rice genetic engineering technology, and in particular to the rice OsRDR5 gene and its encoded protein, a site-directed editing system, its applications, and application methods. Background Technology

[0002] Rice is one of the most important food crops in my country and even globally. It has a high water consumption rate, accounting for nearly 70% of total agricultural water use. Drought stress has a particularly severe impact on rice production, occurring at any stage of rice growth and representing the largest abiotic stress factor affecting high and stable rice yields. Utilizing modern biotechnology to study the physiological, biochemical, and genetic mechanisms of drought resistance in rice, isolating and cloning important drought-resistant genes, and transferring them into widely promoted superior varieties to improve their drought resistance, and cultivating new water-saving and drought-resistant rice varieties, is an effective way to ensure rice production and alleviate food security pressures against the backdrop of increasingly scarce global freshwater resources.

[0003] Plant drought resistance is extremely complex, with root-based drought avoidance being a crucial component. When drought strikes, plants reduce water loss by regulating stomatal closure and maintain high water potential by promoting root water absorption, effectively mitigating damage from drought stress. Enhancing root water absorption capacity is key to improving drought resistance. Rice roots can be categorized into deep and shallow roots based on their soil depth (angles between the downward-growing roots and the horizontal are 50-90° and 0-50°). When the soil is dry, only deep roots can absorb water from deeper soil layers. Therefore, the number and proportion of deep roots largely determine rice's water absorption capacity under drought stress. Kato and Uga et al. used the deep-root ratio to assess this characteristic, which is the ratio of the number of deep roots to the total number of roots in rice. Generally, deep-root characteristics are influenced by apical gravimetric distribution; a higher deep-root ratio indicates a stronger ability to absorb deeper water and greater drought resistance.

[0004] Previous studies have shown that root system is closely related to drought resistance, but these studies mainly focus on increasing root density, root depth, root length, and root weight to promote nutrient and water absorption and enhance rice's ability to resist drought stress. Deep roots are particularly important for rice drought resistance, but research on this topic is limited.

[0005] Current research has conducted genetic mapping and gene resource mining on the root depth ratio in rice, but only a few genes controlling the root depth ratio have been cloned. Among them, DRO1, the first major QTL for deep rooting in rice located on chromosome 9, is the most typical. Studies have reported that DRO1 alters root morphology by affecting root tip cell elongation, leading to asymmetrical root growth and downward bending in response to gravity. Overexpression of this gene can increase the root's growth angle relative to the horizontal plane, causing the roots to grow more vertically, thereby improving drought resistance. Furthermore, introducing DRO1 into the shallow-rooted variety IR64 significantly improves its root depth ratio and drought resistance. Researchers subsequently mapped the major QTL for deep rooting, DRO2, on chromosome 4 using three F2 mapping populations. Using the extremely deep-rooted variety Kinandang Patong and the shallow-rooted variety IR64 as materials, the major QTL for root angle, DRO3, was mapped on chromosome 7. Using IRAT109 as the parent material, the OsSAUR11 gene was cloned, and overexpression of this gene significantly improved the deep root ratio and drought resistance of rice.

[0006] Currently, only a few genes controlling the root-to-root ratio have been isolated and cloned in rice, and more drought-resistant genes need to be discovered to facilitate the breeding of new water-saving and drought-resistant rice varieties. Summary of the Invention

[0007] In view of this, the present invention provides the rice OsRDR5 gene and its encoded protein, a site-directed editing system, its application, and its application method.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] The rice OsRDR5 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] The protein encoded by the rice OsRDR5 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0011] Application of rice OsRDR5 gene and its encoded protein in regulating rice drought avoidance.

[0012] The application of the rice OsRDR5 gene and its encoded protein in regulating drought avoidance in rice includes the following steps:

[0013] 1) Based on the DNA sequence shown in SEQ ID NO.1, an OsRDR5 gene knockout vector was constructed using CRISPR-Cas9 gene editing technology;

[0014] 2) The constructed rice OsRDR5 gene knockout vector was transformed into rice;

[0015] 3) Knockout mutants were obtained through breeding and screening, and named rdr5-1 and rdr5-2:

[0016] Preferably, in step 3), mutant rdr5-1 has a deletion of one base in the 22-24 bp interval after the transcription initiator of the rice OsRDR5 gene; mutant rdr5-2 has a deletion of two bases in the 21-24 bp interval after the transcription initiator of the rice OsRDR5 gene.

[0017] A site-directed editing system for the rice OsRDR5 gene, comprising: an sgRNA target sequence, target sequence primers, a cleavage-ligation reaction system, and a recombinant vector.

[0018] Preferably, the sgRNA target sequence is SEQ ID NO:5; the target sequence primers are SEQ ID NO:6-7.

[0019] Preferably, the recombinant vector is a Ti plasmid or a plant virus vector.

[0020] Preferably, the recombinant vector is the pYLCRI / Cas9Pubi-H vector.

[0021] This invention, through the isolation and cloning of the rice OsRDR5 gene, discovered that the protein encoded by this gene contains a Coa3_cc domain and belongs to the cytochrome c oxidase assembly factor 3 gene family. The OsRDR5 loss-of-function mutant created using CRISPR-Cas9 gene editing technology exhibits reduced root gravitropism but a higher deep root ratio, potentially enhancing the drought resistance of rice and showing great promise for application in drought-resistant rice breeding. Attached Figure Description

[0022] Figure 1 This is a diagram showing the results of the OsRDR5 gene domain analysis of the present invention;

[0023] Figure 2 This is a diagram showing the results of comparing the protein sequence of the OsRDR5 gene with its homologous protein sequences using DNAMAN software in this invention.

[0024] Figure 3 This diagram shows the changes in gene sequence (A) and protein sequence (B) of the knockout mutant of the OsRDR5 gene created using CRISPR-Cas9 gene editing technology according to the present invention.

[0025] Figure 4 This is a diagram showing the subcellular localization results of OsRDR5 in this invention;

[0026] Figure 5 This is a comparison diagram of root tip gravity in OsRDR5 gene knockout mutant and wild-type rice.

[0027] WT: Wild-type rice (Nipponbare), rdr5-1, rdr5-2: OsRDR5 gene knockout mutant;

[0028] Figure 6 This is a comparison diagram of the root depth ratio of the OsRDR5 gene knockout mutant of this invention and wild-type rice;

[0029] WT: Wild-type rice (Nipponbare), rdr5-1, rdr5-2: OsRDR5 gene knockout mutant. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1: Cloning of the rice OsRDR5 gene

[0032] 1. Seedling cultivation

[0033] The rice variety IRAT109 was disinfected with a 5% sodium hypochlorite solution for 25 minutes, then rinsed clean and placed in a 30℃ incubator for 48 hours to germinate. After the seeds showed signs of sprouting, they were sown in a germination box for hydroponic growth. When the rice plants had grown to 3-5 leaves, DNA or RNA was extracted.

[0034] 2. RNA isolation

[0035] RNA extraction: The sample was frozen in liquid nitrogen in a mortar and then ground into powder. The powder was added to a 2 mL EP tube containing 1 mL of LTRNzol-A+ reagent (Tiangen Biotech Co., Ltd.). After thorough shaking, the tube was incubated at room temperature for 5 min. Then, 0.2 mL of chloroform was added, and the tube was shaken vigorously for 15 s, followed by incubation at room temperature for 3 min. The tube was then centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was transferred to a new 2 mL EP tube, and an equal volume of isopropanol was added to precipitate the RNA. The RNA was then dissolved in 100 μL of RNase-free ddH2O. The total RNA mass was identified by electrophoresis, and the RNA content was determined by spectrophotometry.

[0036] 3. Reverse transcription to synthesize first-strand cDNA

[0037] The first-strand cDNA was synthesized using materials from Shanghai TransGen Biotech Co., Ltd. The One-step gDNARemoval and cDNA Synthesis SuperMix kit was used for reverse transcription of total RNA. The reaction volume was 20 μL, and the specific steps are as follows:

[0038]

[0039] Incubate the above reaction system at 42°C for 15 min; then heat at 85°C for 5 s, and place at 4°C or on ice for 5 min. The prepared cDNA can be used immediately or stored at -20°C for later use.

[0040] 4. Amplification of the coding region (CDS) of the rice OsRDR5 gene

[0041] The CDS sequence of the coding region of the rice OsRDR5 gene (gene number: LOC_Os05g01330 / Os05g0103800) was obtained by searching the rice genome and full-length gene database. PCR amplification primers were designed based on the predicted information. The primer sequences are: OsRDR5-F:ATGGACGACGACGACCATG, SEQ ID NO:3, OsRDR5-R:TTAGGATCCCGCGGTGGA, SEQ ID NO:4.

[0042] The CDS sequence of the OsRDR5 gene was directly cloned from the cDNA of IRAT109, recovered by gel extraction, ligated into the pEASY-Blunt vector, identified, and sequenced. The sequencing results were confirmed by BLAST alignment. The results showed that the full-length CDS sequence of the rice OsRDR5 gene in this invention is 558 bp. The detailed sequence is shown in SEQ ID NO:1.

[0043] SEQ ID NO:1

[0044] ATGGACGACGACCATGACCACCACGGCAACGGCAACACCCCCTTCTCCTTGGCCTTGGCGCGCTCCTGCGGTGAAGTTCATCTCATCGGGCCGGCTTGCAGTTTAGCCCAGCCCAGCAAGGCCCATTGGCCCACTGGGAGTGGGAGGAACAGGAAACCGACCTCCTC ATCCTCGAAGACGAAGACCTCTCTCTCGCTCGCCTGGCGCCGCCTTCCTCCACCCCACTCTTCCTCCTCCGCCGCCGCATCCTCACCAACCCCCAACAGCCCTCCCTCCCTCCTCGCCAAAGGCTATGTTCTGGTATGGTTCAATCCCCTCCCCACTGCTAATTTCTG CAACAACCGGTGCAAACTAAAGATGGCGGGATTCGGCAGCCTGGCACCCAAGACCAAGAACTTTGTGGTGGCTGGAGGACTGTCTGCTTTCGTCCTCGGCGTGTATTACTACACCATGAGAGCGGTGGGAGGCACAGATGAGCTGCAGGTCGCCATTGATAAGTTTGAAGACATGAAGAAAAATGATGCTGGGAACTCATCCACCGCGGGATCCTAA

[0045] SEQ ID NO:3

[0046] ATGGACGACGACGACCATG

[0047] SEQ ID NO:4

[0048] TTAGGATCCCGCGGTGGA

[0049] Example 2: Protein sequence information and homology analysis of rice OsRDR5

[0050] The amino acid sequence of rice OsRDR5 was deduced from the ORF of the new rice OsRDR5 gene (gene number: LOC_Os05g01330 / Os05g0103800) according to the present invention. It consists of 185 amino acids and has a molecular weight of 19380 Daltons. The detailed sequence is shown in SEQ ID NO:2.

[0051] SEQ ID NO:2

[0052] MDDDDHDHHGNGNTPFSLALARSCGEVHLIGPACSLAQPSKAHWTGSGRNRKPTSSSSKTKTSLSRSPGAAFLHPTLSSSSAAASSPTPNSPPSLLAKGYVLVWFNPLPTANFCNNRCKLKMAGFGSLAPKTKNFVVAGGLSAFVLGVYYYYTMRAVGGTDELQVAIDKFEDMKKNDAGNSSTAGS*

[0053] Predicting the conserved amino acid domains encoded by this gene using the BLASTP online database in NCBI revealed that the OsRDR5 protein possesses a Coa3_cc domain, belonging to the cytochrome c oxidase assembly factor 3 gene family. Figure 1 As shown.

[0054] Multiple sequence alignment of some OsRDR5-encoded proteins from different species of Poaceae revealed that this protein exhibits high conservation with its homologs, all containing a conserved Coa3_cc domain, such as... Figure 2 As shown.

[0055] Example 3: Creation of OsRDR5 gene knockout mutant in rice

[0056] 1. Construct an OsRDR5 gene knockout vector using the CRISPR-Cas9 gene editing system.

[0057] (1) Guide RNA target sequence selection and primer design

[0058] Based on the genomic sequence of OsRDR5, sgRNA target sequence information was designed. Among a series of sgRNAs, the 20nt nucleotide sgRNA target sequence was designed according to the 5'-GN19NGG-3' (SEQ ID NO:5) sequence to cover the exon region as much as possible and reduce off-target effects.

[0059] SEQ ID NO:5

[0060] GACGACCATGACCACCACGG

[0061] Simultaneously, target sequence primers OsRDR5-gRT+ and OsRDR5-OsU6aT- were designed, with 16-17 nt at their 3' ends to pair with the sgRNA and U6a promoters, respectively. The designed sgRNA target sequences were compared with the rice genome database to exclude non-specific target cleavage sites. The specific target nucleotide sequences are as follows.

[0062] OsRDR5-gRT+:5'-ACGACGACCATGACCACCAgttttagagctagaaat-3', SEQ ID NO:6

[0063] OsRDR5-OsU6aT-:5'-TGGTGGTCATGGTCGTCGTcaacacaagcggcagc-3', SEQ ID NO:7

[0064] (2) Denature and anneal the adapter primers, and dilute the primers to a working concentration of 10 μmol for later use.

[0065] Reaction system: 1 μL OsRDR5-gRT primer + 1 μL OsRDR5-OsU6aT primer + 8 μL ddH2O.

[0066] Reaction conditions: 90℃ for 30 seconds, then naturally cooled.

[0067] (3) The reaction system with simultaneous cutting and bonding is 10 μL.

[0068]

[0069] The above reaction system was subjected to five cycles at 37℃ for 5 min and 20℃ for 5 min before being put into use.

[0070] (4) The product of the cleavage and ligation reaction was used as a template for the first round of PCR. The reaction system was 20 μL.

[0071]

[0072] The reaction conditions were 98℃ for 3 min, 98℃ for 15 s, 58℃ for 20 s, 68℃ for 20 s, 68℃ for 2 min, and 12℃ for 10 min, for 30 cycles before use. The gel was recovered, and the products were named product1 and product2, respectively.

[0073] (5) The U6 promoter was fused with sgRNA by overlap PCR in a 20 μL reaction volume.

[0074] Take 2 μL product1 + 2 μL product2 + 16 μL ddH2O, dilute the product 10 times, mix well, and name it product1+2.

[0075]

[0076] The reaction conditions were 98℃ for 3 min, 98℃ for 15 s, 58℃ for 20 s, 68℃ for 20 s, 68℃ for 2 min, and 12℃ for 10 min, for 30 cycles before use. The gel was recovered, and the product was named product RDR5.

[0077] (6) The enzyme vector pYLCRISPR / Cas9Pubi-H was digested in a 50 μL reaction system.

[0078]

[0079]

[0080] The reaction system was digested with enzymes at 37℃ for 30 min, and the target fragment was recovered after gel electrophoresis.

[0081] (7) Recombination reaction, the reaction system is 10 μL.

[0082]

[0083] The reaction conditions were 37℃ for 30 min.

[0084] After the reaction, the cells were transformed into E. coli, plated on LB agar plates containing kanamycin to screen for positive clones, selected single clones for sequencing verification, and plasmids containing target site sgRNA that were correctly sequenced were extracted and stored at -20℃ for later use.

[0085] 2. Genetic transformation of rice

[0086] (1) Seed disinfection

[0087] Select plump and uniform Nipponbare seeds, soak them in 75% ethanol for 1 minute, rinse them twice with sterile water, then sterilize them with 1.5% sodium hypochlorite solution by shaking for 30 minutes, and finally rinse them five times with sterile water. After washing, dry the seeds with absorbent paper and inoculate them on callus induction medium. Incubate them in the dark at 25°C for 2 weeks.

[0088] Callus induction medium: The induction medium in Table 1 was used. 0.3g proline, 0.6g hydrolyzed casein, 30g sucrose and 2.5mL 2,4-D (concentration 1mg / mL) were added to prepare a 1L solution. The pH was adjusted to 5.9, 7g agar powder was added, and the solution was sterilized by high temperature and high pressure.

[0089] (2) Subculture

[0090] Embryogenic callus was excised and inoculated into subculture medium, and cultured in the dark at 25°C for 2 weeks.

[0091] Subculture medium: The subculture medium in Table 1 was used. 0.5 g proline, 0.6 g hydrolyzed casein, 30 g sucrose and 2 mL 2,4-D (concentration 1 mg / mL) were added to prepare a 1 L solution. The pH was adjusted to 5.9, 7 g agar powder was added, and the solution was autoclaved at high temperature.

[0092] (3) Preparation of Agrobacterium tumefaciens (EHA105) competent cells

[0093] Agrobacterium tumefaciens culture was cultured at 28℃ until OD600 = 0.5, and the cells were collected by centrifugation at 4℃. The cells were resuspended in 500 μL of 0.1 mol / L ice-bath CaCl2, and centrifuged again after 30 min on ice. The supernatant was discarded, and the cells were resuspended in 100 μL of 0.1 mol / L ice-bath CaCl2 and stored at 4℃.

[0094] (4) Agrobacterium-mediated transformation

[0095] Add 5 μL of plant expression vector plasmid DNA to 100 μL of Agrobacterium competent cells, mix gently, incubate in an ice-water bath for 30 min, and then freeze-shock in liquid nitrogen for 2 min. Add 400-800 μL of LYEP culture medium (containing kanamycin, Kan). Incubate at 28℃ with shaking at 200 rpm for 3-5 h. Centrifuge at room temperature (5000 rpm, 5 min), retain 100 μL of supernatant to resuspend the cells, spread on LB solid medium (containing Kan), and incubate upside down at 28℃ for 2 days until colonies of suitable size grow. Pick single clones for PCR detection to obtain positive strains.

[0096] (5) Co-cultivation

[0097] Rice callus tissue was placed in a suspension culture medium containing Agrobacterium tumefaciens and cultured with shaking for about 20 minutes. The infected callus tissue was then dried on sterile filter paper and transferred to a co-culture medium, and cultured in the dark at 25°C for 5 days.

[0098] Suspension culture medium: Using the suspension culture medium in Table 1, add 0.08 g hydrolyzed casein, 2 g sucrose, and 0.2 mL 2,4-D (concentration 1 mg / mL) to prepare a 100 mL solution. Adjust the pH to 5.4, divide into two bottles (50 mL each), and autoclave. Before use, add 1 mL of 50% glucose and 100 μL LAS (100 mM).

[0099] Co-culture medium: Use the co-culture medium in Table 1, add 0.8 g hydrolyzed casein, 20 g sucrose and 3.0 mL 2,4-D (concentration 1 mg / mL) to prepare a 1 L solution, adjust the pH to 5.6, add 7 g agar powder, and autoclave. Before use, add 20 mL 50% glucose and 1 mL AS (100 mM).

[0100] (6) Selection of culture

[0101] Selected co-cultured callus tissues were inoculated into selective culture medium and cultured in the dark at 25°C for 2 weeks, followed by two selection cultures.

[0102] Selection medium: Use the selection medium in Table 2, add 0.6 g hydrolyzed casein, 30 g sucrose and 2.5 mL 2,4-D (concentration 1 mg / mL) to make a 1 L solution, adjust the pH to 6.0, add 7 g agar powder, and autoclave. Add 1 mL Hn and 1 mL Cn (100 ppm) before use.

[0103] (7) Differentiation culture

[0104] Embryogenic callus tissue was picked and inoculated into differentiation medium, and cultured at 24℃ for 16h / 8h light and dark to induce differentiated shoots (4-6 weeks).

[0105] Differentiation medium: The differentiation medium in Table 2 was used. 2.0 mg / L 6-BA, 2.0 mg / L KT, 0.2 mg / L NAA, 0.2 mg / L IAA, 1.0 g hydrolyzed casein and 30 g sucrose were added to prepare a 1 L solution. The pH was adjusted to 6.0, 7 g agar powder was added, and the solution was autoclaved at high temperature.

[0106] (8) Rooting culture

[0107] When the buds grow to about 2cm, cut them off and insert them into the rooting medium. Incubate at about 25℃ for 16h / 8h in light and dark to induce rooting.

[0108] Rooting medium: Use the rooting medium in Table 2, add 30g of sucrose to make 1L solution, adjust the pH to 5.8, add 7g of agar powder, and autoclave.

[0109] (9) Transformation plant culture

[0110] After the root system has developed, open the test tube, add sterile water to harden the seedlings for 2-3 days, then take out the plants, wash off the attached solid culture medium with sterile water, and transplant them into the soil. Initially, provide shade and shelter from the wind. Once the plants are healthy, carry out conventional field or greenhouse management and cultivation.

[0111] Table 1: Components of Basic Culture Medium

[0112]

[0113]

[0114] Table 2: Components of Basic Culture Medium

[0115]

[0116]

[0117] (10) Identification of T0 generation transgenic positive lines

[0118] Genomic DNA extraction: Take the top tender leaves of a single rice plant, put them into a 2 mL centrifuge tube, add 2 steel balls, add 500 μL of CTAB buffer, grind for 1 min to form a paste, place in a 65℃ oven to dry for 1 h, add 500 μL of chloroform, gently shake for about 15 min, centrifuge at 1000 r / min for 10 min; transfer 300 μL of supernatant to a 1.5 mL centrifuge tube, add 600 μL of ice-cold ethanol, gently shake by hand for 1 min, freeze at -20℃ for at least 30 min, centrifuge at 1000 r / min for 10 min, discard the supernatant, add 500 mL of 75% ethanol to precipitate for 5 min, centrifuge at 1000 r / min for 5 min, discard the ethanol waste liquid, invert the centrifuge tube on absorbent paper to dry, add 100 μL of sterile water to dissolve the DNA.

[0119] PCR amplification and cloning sequencing of the knockout mutant OsRDR5 gene: Same as in Examples 1-4.

[0120] The results showed that sequence analysis of the OsRDR5 gene in the two knockout mutants revealed a one-base deletion in the 22–24 bp region in the rdr5-1 mutant and a two-base deletion in the 21–24 bp region in the rdr5-2 mutant. Figure 3 As shown in A, both types of deletions lead to premature termination of translation, resulting in changes in the amino acid sequence encoding the protein, such as... Figure 3 As shown in B, the OsRDR5 gene in the mutant was successfully knocked out.

[0121] Example 4: Subcellular localization of OsRDR5 in rice

[0122] 1. Rice RNA extraction and cDNA reverse transcription are the same as in Example 1.

[0123] 2. Primers OsRDR5-F and OsRDR5-R were designed targeting the OsRDR5 gene sequence.

[0124] OsRDR5-F: ATGGACGACGACGACCATGACC, SEQ ID NO:8

[0125] OsRDR5-R: AGATCTCCTCCAGATCCTCCTC, SEQ ID NO:9

[0126] SEQ ID NO:8

[0127] ATGGACGACGACGACCATGACC

[0128] SEQ ID NO:9

[0129] AGATCCTCCTCCAGATCCTCCTC

[0130] 3. Using the cDNA from step 1 as a template, amplify the CDS sequence of the OsRDR5 gene. Ligate the amplified fragment to the pAN580 vector, fusing OsRDR5 to the N-terminus of the fluorescent protein eGFP, constructing the RDR5-eGFP expression vector. Simultaneously, fuse the endoplasmic reticulum localization signal protein SPER with the fluorescent protein mKATE, constructing the SPER-mKATE fusion expression vector. Take 200 μL of rice protoplast suspension, add 10 μL of the RDR5-eGFP vector plasmid and 10 μL of the SPER-mKATE plasmid for co-transformation, and incubate in the dark at 28℃ for 18-24 h. Observe the fluorescence position using a laser confocal microscope (Nikon C2-ER).

[0131] The results showed that the OsRDR5 protein is located in the endoplasmic reticulum, such as... Figure 4 As shown.

[0132] Example 5: Evaluation of the effect of OsRDR5 knockout mutant on drought avoidance

[0133] 1. Evaluation of root tip gravitropism in transgenic rice

[0134] Select plump, uniformly sized seeds from WT and rdr5 mutants. Disinfect the seeds by soaking them in a 1:1 mixture of 5% sodium hypochlorite solution and water for 15 minutes. Rinse thoroughly with tap water and soak in petri dishes lined with filter paper for 1 day. Then, germinate the seeds at 30℃. Sow the newly sprouted seeds in vertical plates containing 0.8% agar gel and place them in a 30℃ incubator for dark cultivation or use aluminum foil to block light, ensuring sufficient moisture in the agar gel. Allow the seeds to grow for approximately 2 days. When the radicles of the same material reach 1-2 cm in length, draw a tangent along the growth direction of the radicle tip, record the time, mark the root tip growth position, and immediately place the vertical plate in the incubator after rotating it 90°. Measure the root tip bending angle after 2 hours to analyze gravitropism. Root tip gravitropism measurements were performed in at least 12 biological replicates.

[0135] 2. Detection of root depth ratio in genetically modified rice

[0136] The deep root ratio was evaluated using the field "basket method." The baskets used had a top diameter of 18cm, a bottom diameter of 10cm, and a height of 7cm. Fields with good soil homogeneity were selected, and the baskets were buried in the soil at a density of 30cm x 30cm (the distance between the centers of two baskets), ensuring the top of the basket was level with the soil surface. WT and rdr5 mutant seeds with plump, uniform size were selected and sown in 96-well PCR plates for germination. When seedlings reached the three-leaf stage, they were transplanted to the center of the baskets. After approximately 50 days, the number of shallow and deep roots was counted, and the deep root ratio was calculated. Roots penetrating the mesh at the bottom of the basket were defined as deep roots, and roots penetrating the mesh on the four sides of the basket were defined as shallow roots. The ratio of deep roots to the total number of roots was the deep root ratio. At least 12 baskets were planted for each line, with one seedling planted in each basket.

[0137] The results showed that, compared with the wild type, the root tip gravity of the OsRDR5 transgenic plants was weakened, such as... Figure 5 As shown, however, the ratio of deep roots increases, as... Figure 6 The results show that OsRDR5 gene knockout affects the root depth ratio and gravimetric response in rice, playing an important role in rice drought avoidance.

[0138] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. Knock out rice plants OsRDR5 The application of genes in enhancing drought resistance in rice, the OsRDR5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Knock out rice plants OsRDR5 The method of using genes to increase the drought resistance of rice is characterized by, The OsRDR5 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and includes the following steps: 1) Based on the DNA sequence shown in SEQ ID NO.1, construct using CRISPR-Cas9 gene editing technology. OsRDR5 Gene knockout vector; 2) Construct rice OsRDR5 The gene knockout vector was transformed into rice; 3) Cultivate and screen to obtain knockout mutants.

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