Rice OsRDR5 gene, encoding protein, fixed-point editing system, application and application method

Abstract

The invention discloses a rice OsRDR5 gene, an encoding protein, a fixed-point editing system, application and an application method. The nucleotide sequence of the OsRDR5 gene is shown as SEQ ID NO.1; the amino acid sequence of the encoding protein of the rice OsRDR5 gene is as shown in SEQ ID NO. 2. The gene has a Coa3cc structural domain and belongs to a cytochrome c oxidase assembly factor 3 gene family. A knockout mutant of the OsRDR5 gene shows a relatively high deep root ratio, and meanwhile, the gravitational property of a rice root system is changed. The rice OsRDR5 gene disclosed by the invention is used for regulating and controlling the drought resistance of rice, and a knockout mutant of the rice OsRDR5 gene has a relatively high deep root ratio and can be applied to breeding of a new variety of water-saving and drought-resistant rice, so that the drought resistance of the rice is improved.

Description

Technical Field

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

[0002] Rice is one of the most important food crops in my country and globally. It consumes a significant amount of water, with rice production accounting for nearly 70% of total agricultural water use. Drought stress has a particularly severe impact on rice production, occurring at any stage of its growth. Drought stress is the single most significant abiotic stressor affecting high and stable rice yields. Using modern biotechnology to study the physiological, biochemical, and genetic mechanisms of rice drought resistance, isolate and clone key drought-resistant genes, and transfer them into widely distributed, improved varieties to improve their drought resistance and cultivate new, water-saving, and drought-resistant rice varieties is an effective way to ensure rice production and alleviate the pressure of food security amidst the increasing global shortage of freshwater resources.

[0003] Plants' drought tolerance 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 uptake, effectively avoiding damage caused by drought stress. Enhancing root water uptake is crucial for improving drought tolerance. Rice roots can be divided into deep roots and shallow roots based on their depth in the soil (the angles of downward-growing roots with respect to the horizontal are 50-90° and 0-50°, respectively). When the soil is dry, only deep roots can absorb water from deeper layers of the soil. Therefore, the number and proportion of deep roots largely determine rice's water uptake capacity under drought stress. Kato and Uga et al. used the deep root ratio—the ratio of deep roots to total roots—to assess this characteristic. Deep rooting is generally influenced by root tip gravitropism. A larger deep root ratio indicates a greater ability of the root system to absorb water from deeper layers and, consequently, greater drought tolerance.

[0004] Previous research has shown that the root system is closely linked to drought tolerance, but this has primarily focused on enhancing rice's ability to withstand drought stress by increasing root density, depth, length, and weight to promote nutrient and water absorption. Deep rooting is particularly important for rice drought resistance, but this has been less studied.

[0005] Studies have been conducted on genetic mapping and gene resource mining of the deep root ratio in rice, but only a few genes controlling this ratio have been cloned. The first major QTL for the deep rooting trait in rice, DRO1, located on chromosome 9, is the most prominent. Studies have reported that DRO1 alters root morphology by affecting root tip cell elongation, leading to asymmetric root growth and downward bending in response to gravity. Overexpression of this gene can also increase the root growth angle with the horizontal plane, causing roots to grow in a more vertical direction, thereby improving drought resistance. Furthermore, introducing DRO1 into the shallow-rooting variety IR64 significantly improved its deep root ratio and drought resistance. Subsequently, researchers used three F2 mapping populations to locate the major QTL for the deep rooting trait, DRO2, on chromosome 4. Using the extremely deep-rooting variety Kinandang Patong and the shallow-rooting variety IR64 as materials, they mapped the major QTL for root angle, DRO3, on chromosome 7. Using IRAT109 as the parent material, the OsSAUR11 gene was cloned, and overexpression of this gene significantly improved the deep rooting ratio and drought resistance of rice.

[0006] At present, only a few genes that control the deep root ratio in rice have been isolated and cloned. 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 a rice OsRDR5 gene and its encoded protein, a site-directed editing system, and applications and application methods.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

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

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

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

[0012] The method for using the rice OsRDR5 gene and its encoded protein in regulating rice drought avoidance comprises 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) Transforming the constructed rice OsRDR5 gene knockout vector into rice;

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

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

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

[0018] Preferably, the sgRNA target sequence is: SEQ ID NO: 5; the target sequence primer is: 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 a pYLCRI / Cas9Pubi-H vector.

[0021] This study isolated and cloned the rice OsRDR5 gene and discovered that the protein it encodes contains a Coa3_cc domain and belongs to the cytochrome c oxidase assembly factor 3 gene family. OsRDR5 loss-of-function mutants, created using CRISPR-Cas9 gene editing technology, exhibit reduced root gravitropism but a higher root depth ratio, potentially enhancing rice's drought tolerance. This approach holds great promise for application in rice drought-tolerance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[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 the result of comparing the protein sequence of the OsRDR5 gene with its homologous protein sequence using DNAMAN software in the present invention;

[0024] Figure 3 Graph showing the changes in the gene sequence (A) and protein sequence (B) of the knockout mutant of the OsRDR5 gene created based on CRISPR-Cas9 gene editing technology of the present invention;

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

[0026] Figure 5 This is a comparison diagram of the root tip gravitropism of the OsRDR5 gene knockout mutant of the present invention and the wild-type rice;

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

[0028] Figure 6 This is a comparison diagram of the deep rooting ratio between the OsRDR5 gene knockout mutant of the present invention and the wild-type rice;

[0029] WT: wild-type rice (Nipponbare), rdr5-1, rdr5-2: OsRDR5 gene knockout mutants. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts 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 5% sodium hypochlorite solution for 25 minutes, then cleaned and placed in a 30°C incubator for germination for 48 hours. After the seeds turned white, they were sown in a germination box for hydroponic growth. When the rice grew to 3-5 leaves, DNA or RNA was prepared for extraction.

[0034] 2. RNA Isolation

[0035] RNA extraction: Samples were frozen in liquid nitrogen in a mortar and ground into a powder. The powder was then added to a 2 mL EP tube containing 1 mL of TRNzol-A+ reagent (Tiangen Biochemical Technology Co., Ltd.). After vigorous shaking, the mixture was incubated at room temperature for 5 minutes. 0.2 mL of chloroform was then added, and the mixture was shaken vigorously for 15 seconds before being incubated at room temperature for 3 minutes. The mixture was then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new 2 mL EP tube, and an equal volume of isopropanol was added to precipitate RNA. The mixture was then dissolved in 100 μL of RNase-free ddH2O. Total RNA quality was assessed by electrophoresis, and RNA content was determined spectrophotometrically.

[0036] 3. Reverse transcription synthesis of first-strand cDNA

[0037] First-strand cDNA was synthesized using the Reverse transcription of total RNA was performed using the One-Step gDNA Removal and cDNA Synthesis SuperMix Kit. The reaction volume was 20 μL and the specific steps were as follows:

[0038]

[0039] Incubate the reaction at 42°C for 15 minutes, then heat at 85°C for 5 seconds and place at 4°C or on ice for 5 minutes. The prepared cDNA can be used immediately or stored at -20°C until needed.

[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 (LOC_Os05g01330 / Os05g0103800) was obtained by searching the rice genome and full-length gene databases. PCR amplification primers were designed based on the predicted information: OsRDR5-F: ATGGACGACGACGACCATG, SEQ ID NO: 3, and 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, and ligated into the pEASY-Blunt vector. After identification, the sequence was determined. The sequencing results were confirmed by BLAST comparison. The results showed that the full length of the CDS sequence of the rice OsRDR5 gene in the present invention was 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 based on the ORF of the novel rice OsRDR5 gene (gene number: LOC_Os05g01330 / Os05g0103800) of the present invention, which contains 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] The amino acid conserved domain encoded by the gene was predicted by BLASTP online website in NCBI database, and it was found that OsRDR5 protein has Coa3_cc domain and belongs to cytochrome c oxidase assembly factor 3 gene family. Figure 1 shown.

[0054] By performing multiple sequence alignment on some OsRDR5-encoded proteins from different species of Poaceae, it was found that the protein is highly conserved with its homologous proteins and contains a conserved Coa3_cc domain, such as Figure 2 shown.

[0055] Example 3: Creation of a Rice OsRDR5 Knockout Mutant

[0056] 1. Use the CRISPR-Cas9 gene editing system to construct a gene knockout vector for OsRDR5.

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

[0058] The sgRNA target sequence information was designed based on the OsRDR5 genomic sequence. Across a series of sgRNAs, the 20-nt nucleotide sgRNA target sequence was designed according to the 5'-GN19NGG-3' (SEQ ID NO: 5) sequence, maximizing sgRNA coverage within the exon region to minimize off-target effects.

[0059] SEQ ID NO:5

[0060] GACGACCATGACCACCACGG

[0061] Target sequence primers OsRDR5-gRT+ and OsRDR5-OsU6aT- were also designed, with 16-17 nt at their 3' ends pairing with the sgRNA and U6a promoter, respectively. The designed sgRNA target sequences were compared against the rice genome database to exclude nonspecific target cleavage sites. The specific target nucleotide sequences are shown below.

[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 linker primers, dilute the primers to a working concentration of 10 μmol, and set aside.

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

[0066] Reaction conditions: 90°C for 30 seconds and then naturally cool.

[0067] (3) The cutting and ligating reaction system is 10 μL.

[0068]

[0069] The above reaction system was subjected to 5 cycles of 5 min at 37°C and 5 min at 20°C.

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

[0071]

[0072] The reaction conditions were 98°C for 3 min, 98°C for 15 s, 58°C for 20 s, 68°C for 20 s, 68°C for 2 min, and 12°C for 10 min, after 30 cycles. The gel was recovered and the products were named product 1 and product 2.

[0073] (5) The U6 promoter was fused to the sgRNA by overlapping PCR, and the reaction system was 20 μL.

[0074] Take 2 μL product 1 + 2 μL product 2 + 16 μL ddH2O, dilute the product 10 times, mix well, and name it product 1 + 2.

[0075]

[0076] The reaction conditions were 30 cycles of 98°C for 3 min, 98°C for 15 s, 58°C for 20 s, 68°C for 20 s, 68°C for 2 min, and 12°C for 10 min. The gel was recovered and designated product RDR5.

[0077] (6) Enzyme digestion of the vector pYLCRISPR / Cas9Pubi-H, the reaction system is 50 μL.

[0078]

[0079]

[0080] The reaction system was digested at 37°C 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°C for 30 min.

[0084] After the reaction, E. coli was transformed and LB plates containing kanamycin were spread to screen positive clones. Single clones were selected for sequencing verification, and plasmids with correct sequencing and containing target site sgRNA were extracted and stored at -20°C until use.

[0085] 2. Rice genetic transformation

[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 oscillate and disinfect them with 1.5% sodium hypochlorite solution for 30 minutes, and finally rinse them with sterile water for 5 times. Dry the washed seeds with absorbent paper and inoculate them on callus induction medium, and culture them in the dark at 25℃ for 2 weeks.

[0088] Callus induction medium: Use the induction medium in Table 1, add 0.3 g proline, 0.6 g tyrosinase, 30 g sucrose and 2.5 mL 2,4-D (concentration 1 mg / mL) to make 1 L solution, adjust the pH to 5.9, add 7 g agar powder, and sterilize at high temperature and high pressure.

[0089] (2) Subculture

[0090] The embryonic callus was cut off, inoculated into the subculture medium, and cultured in the dark at 25°C for 2 weeks.

[0091] Subculture medium: Use the subculture medium in Table 1, add 0.5 g proline, 0.6 g tyrosinase, 30 g sucrose, and 2 mL 2,4-D (concentration 1 mg / mL) to make 1 L of solution, adjust the pH to 5.9, add 7 g agar powder, and sterilize at high temperature and high pressure.

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

[0093] Agrobacterium tumefaciens culture liquid was cultured at 28°C until OD600 = 0.5, and then the cells were collected by centrifugation at 4°C, resuspended with 500 μL of 0.1 mol / L ice-bathed CaCl2, and centrifuged after ice-bath for 30 minutes. The supernatant was removed, and the cell was resuspended with 100 μL of 0.1 mol / L ice-bathed CaCl2 and stored at 4°C.

[0094] (4) Agrobacterium transformation

[0095] Add 5 μL of plant expression vector plasmid DNA to Agrobacterium competent cells (100 μL), mix gently, place in an ice-water bath for 30 minutes, and then quickly freeze in liquid nitrogen for 2 minutes; add 400-800 μL YEP culture medium (containing kanamycin, Kan); culture at 28°C, 200 rpm, and shake for 3-5 hours; centrifuge at room temperature (5000 rpm, 5 minutes), retain 100 μL of supernatant, resuspend the bacteria, spread on LB solid medium (containing Kan), and culture inverted at 28°C for 2 days until colonies of appropriate size grow. Pick a single clone for PCR detection to obtain a positive strain.

[0096] (5) Co-culture

[0097] Place the rice callus in a suspension culture medium containing Agrobacterium and shake culture for about 20 minutes. Dry the infected callus on sterile filter paper and transfer it to co-cultivation medium and culture it in the dark at 25°C for 5 days.

[0098] Suspension medium: Use the suspension medium in Table 1, add 0.08 g tyrosinase hydrolyzate, 2 g sucrose, and 0.2 mL 2,4-D (1 mg / mL) to make 100 mL of solution. Adjust the pH to 5.4, divide the solution into two bottles (50 mL each), and sterilize by autoclaving. Add 1 mL of 50% glucose and 100 μL of 100 mM AS immediately before use.

[0099] Co-culture medium: Use the co-culture medium in Table 1, add 0.8 g tyrosinase hydrolyzate, 20 g sucrose, and 3.0 mL 2,4-D (1 mg / mL) to make a 1 L solution, adjust the pH to 5.6, add 7 g agar powder, and sterilize by high temperature and high pressure. Before use, add 20 mL 50% glucose and 1 mL AS (100 mM).

[0100] (6) Selective training

[0101] The co-cultured callus tissue was selected and inoculated into the selection medium, cultured in the dark at 25°C for 2 weeks, and then selectively cultured twice.

[0102] Selective medium: Use the screening medium in Table 2, add 0.6 g of tyrosinase hydrolyzate, 30 g of sucrose, and 2.5 mL of 2,4-D (1 mg / mL) to a 1 L solution. Adjust the pH to 6.0, add 7 g of agar powder, and sterilize by autoclaving. Add 1 mL of Hn and 1 mL of Cn (100 ppm) immediately before use.

[0103] (7) Differentiation culture

[0104] Embryogenic callus was selected and inoculated into differentiation medium, cultured at 24°C with 16h / 8h light / dark cycle to induce bud differentiation (4-6 weeks).

[0105] Differentiation medium: Use the differentiation medium in Table 2, add 2.0 mg / L 6-BA, 2.0 mg / L KT, 0.2 mg / L NAA, 0.2 mg / L IAA, 1.0 g tyrosinase hydrolyzate, and 30 g sucrose to make 1 L of solution, adjust the pH to 6.0, add 7 g agar powder, and sterilize at high temperature and high pressure.

[0106] (8) Rooting culture

[0107] When the buds grow to about 2 cm, cut them off and insert them into the rooting medium. Culture them at about 25°C with 16h / 8h light and dark conditions to induce rooting.

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

[0109] (9) Transformed plant culture

[0110] After the root system is well developed, open the test tube, add sterile water to harden the seedlings for 2-3 days, take out the plants, wash the attached solid culture medium with sterile water, and move them into the soil. Provide shade and shelter from wind at the beginning, and carry out conventional field or greenhouse management and cultivation after the plants are strong.

[0111] Table 1: Basic medium components 1

[0112]

[0113]

[0114] Table 2: Minimal medium components 2

[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, place them in a 2mL centrifuge tube, add 2 steel balls, add 500μL CTAB buffer, grind for 1min into a slurry, place it in a 65℃ oven for 1h, add 500μL chloroform, shake gently for about 15min, and centrifuge at 1000r / min for 10min; aspirate 300μL supernatant and transfer it to a 1.5mL centrifuge tube, add 600μL ice-cold ethanol, shake gently by hand for 1min, freeze at -20℃ for at least 30min, centrifuge at 1000r / min for 10min, discard the supernatant, add 500mL75% ethanol to precipitate for 5min, centrifuge at 1000r / min for 5min, discard the ethanol waste liquid, invert the centrifuge tube on absorbent paper to dry, and add 100μL sterile water to dissolve the DNA.

[0119] PCR amplification and cloning sequencing of knockout mutant OsRDR5 gene: same as Example 1-4.

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

[0121] Example 4: Subcellular localization of rice OsRDR5

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

[0123] 2. Design primers OsRDR5-F and OsRDR5-R based on 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 into the pAN580 vector, fusion OsRDR5 to the N-terminus of the fluorescent protein eGFP to construct the RDR5-eGFP expression vector. Simultaneously, fuse the endoplasmic reticulum localization signal protein SPER to the fluorescent protein mKATE to construct the SPER-mKATE fusion expression vector. Add 10 μL of the RDR5-eGFP vector plasmid and 10 μL of the SPER-mKATE plasmid to 200 μL of rice protoplast suspension. Incubate in the dark at 28°C for 18-24 hours, and observe the fluorescence position using a laser confocal microscope (Nikon C2-ER).

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

[0132] Example 5: Evaluation of the Effect of OsRDR5 Knockout Mutants on Drought Avoidance

[0133] 1. Evaluation of gravitropism of transgenic rice root tips

[0134] WT and rdr5 mutant seeds with plump, uniform grains were selected and disinfected by soaking them in a 1:1 mixture of 5% sodium hypochlorite solution and water for 15 minutes. The seeds were then rinsed repeatedly with tap water and placed in a Petri dish lined with filter paper for 1 day. Germination was then accelerated in a 30°C incubator. Seeds that had just turned white were sown on a vertical plate filled with 0.8% agar gel and incubated in a 30°C incubator in the dark or shaded with tin foil, ensuring sufficient moisture in the agar gel. The seeds were allowed to grow for approximately 2 days. When the radicles of the same seed material reached 1-2 cm in length, a line was drawn tangentially along the root tip, the time was recorded, and the position of root tip growth was marked. The vertical plate was then rotated 90° and immediately placed in the incubator. After 2 hours, the root tip curvature angle was measured and analyzed for gravitropism. Root tip gravitropism was measured in at least 12 biological replicates.

[0135] 2. Detection of deep root ratio of transgenic rice

[0136] The deep root ratio was evaluated using the field "basket method". The specifications of the baskets used were: top diameter 18 cm, bottom diameter 10 cm, and height 7 cm. A field with good soil uniformity was selected, and baskets were buried in the soil at a density of 30 cm × 30 cm (the distance between the centers of the two baskets), so that the top of the basket was at the same level as the soil surface. WT and rdr5 mutant seeds with full grains and uniform size were selected and sown in a 96-well PCR plate for germination. When the seedlings grew to the three-leaf and one-heart stage, they were transplanted and planted in the center of the basket. After about 50 days, the number of shallow roots and deep roots were counted to calculate the deep root ratio. The roots that passed through the mesh at the bottom of the basket were defined as deep roots, and the roots that penetrated the mesh on the four walls of the basket were defined as shallow roots. The ratio of the number of deep roots to the total number of roots was the deep root ratio. At least 12 baskets were planted for each strain, and one seedling was planted in each basket.

[0137] The results showed that compared with the wild type, the root tip gravitropism of OsRDR5 transgenic plants was weakened. Figure 5 As shown, but the deep root ratio increases, as Figure 6 The results showed that knockout of OsRDR5 affected the deep rooting ratio and gravitropism of rice, and played an important role in rice drought avoidance.

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

Claims

1. Rice OsRDR5 gene, characterized in that The nucleotide sequence of the OsRDR5 gene is shown in SEQ ID NO.

1.

2. The protein encoded by the rice OsRDR5 gene is characterized in that: The amino acid sequence of the protein encoded by the rice OsRDR5 gene is shown in SEQ ID NO.

2.

3. Use of the rice OsRDR5 gene and the encoded protein according to claims 1 to 2 in regulating drought tolerance in rice.

4. The method for using the rice OsRDR5 gene and the encoded protein in regulating drought tolerance of rice according to claim 3, characterized in that: The following steps are involved: 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; 2) Transforming the constructed rice OsRDR5 gene knockout vector into rice; 3) Knockout mutants were obtained by breeding and screening and named rdr5-1 and rdr5-2.

5. The method for using the rice OsRDR5 gene and the encoded protein in regulating drought tolerance of rice according to claim 4, characterized in that: In the step 3), the mutant rdr5-1 has a base deletion in the 22-24 bp region after the transcription initiator of the rice OsRDR5 gene; the mutant rdr5-2 has a base deletion in the 21-24 bp region after the transcription initiator of the rice OsRDR5 gene.

6. A site-directed editing system for the rice OsRDR5 gene, characterized in that: The system includes: an sgRNA target sequence, a target sequence primer, a cleavage-and-ligation reaction system, and a recombinant vector.

7. The site-directed editing system of rice OsRDR5 gene according to claim 6, characterized in that: The sgRNA target sequence is: SEQ ID NO: 5; the target sequence primer is: SEQ ID NO: 6-7.

8. The site-directed editing system of rice OsRDR5 gene according to claim 6, characterized in that: The recombinant vector is a Ti plasmid or a plant virus vector.

9. The site-directed editing system of rice OsRDR5 gene according to claim 6, characterized in that: The recombinant vector is a pYLCRI / Cas9Pubi-H vector.

Images