Rice root length related protein OsRAC2 and coding gene and application

Abstract

This patent discloses a rice root length-related protein OsRAC2 and its encoding gene and applications, belonging to the field of biotechnology. Specifically, it discloses the protein, the encoding gene of the protein, and the application of the protein in any of the following: 1) the application of the above-mentioned protein, the above-mentioned DNA molecule, or the above-mentioned recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in regulating plant root length; 2) the application of the above-mentioned protein, the above-mentioned DNA molecule, or the above-mentioned recombinant vector, expression cassette, transgenic cell line, or recombinant bacteria in cultivating transgenic plants with increased root length; 3) a method for cultivating transgenic plants with increased root length.

Description

Technical Field

[0001] This patent belongs to the field of biotechnology and mainly relates to a rice root length-related protein OsRAC2, its encoding gene, and its applications. Background Technology

[0002] Rice is one of my country's important food crops. Compared with other crops, rice has a shallow root system, requires a large amount of water during production, and is more susceptible to drought stress. In recent years, with climate change and increasingly severe water shortages, water scarcity and drought have become one of the main stress factors affecting rice yield.

[0003] Strong and well-developed root systems can improve the efficiency of plants in absorbing water and nutrients. Improving root traits is the key to breeding excellent water-saving and drought-resistant rice varieties. Among them, root length and root thickness are considered to be the most promising traits for improvement. Long roots are more conducive to plants absorbing water and nutrients from deep soil and improving the survival ability of crops in drought environments. Therefore, cloning genes related to root development is of great significance for improving root adaptation to environmental stress and increasing yield.

[0004] Compared to the study of above-ground plant traits, the genetic study of rice roots is more difficult due to their complex underground environment. Furthermore, the number of root trait genes that can be cloned using forward genetics is relatively small, making them difficult to apply in breeding practices. Therefore, the identification and cloning of root length-related genes has broad application prospects. Genetic engineering technology can directly modify the genetic background of plants at the gene level, allowing for targeted improvement of genetic traits according to specific needs. Compared to traditional breeding methods, this can significantly shorten the breeding process, achieving breeding goals more precisely and efficiently. It plays a crucial role in gene discovery and variety development, and is one of the important approaches to selecting drought-resistant varieties. Summary of the Invention

[0005] The purpose of this invention is to provide a protein OsRAC2 that regulates rice root length, its encoding gene, and its applications.

[0006] The protein provided by this invention is as follows: 1), 2), or 3):

[0007] 1) The protein shown in sequence 2 of the sequence listing;

[0008] 2) Proteins derived from Sequence 2 with the same function, formed by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in Sequence 2 of the Sequence Listing;

[0009] The substitution and / or deletion and / or addition of one or more amino acid residues mentioned above constitutes a substitution and / or deletion and / or addition of no more than 10 amino acid residues;

[0010] 3) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of 1) or 2) above.

[0011] The DNA molecules encoding the aforementioned proteins are also within the scope of protection of this invention.

[0012] The DNA molecule mentioned above is any one of the following DNA molecules 1)-4):

[0013] 1) The coding region is the DNA molecule shown in sequence 3 of the sequence listing;

[0014] 2) The coding region is the cDNA molecule shown in sequence 1 of the sequence listing;

[0015] 3) DNA molecules that hybridize with the DNA sequence defined in 1) or 2) under strict conditions and encode proteins with the same function;

[0016] 4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in 1) or 2) and encodes a protein having the same function.

[0017] The above stringent conditions can be achieved by hybridization at 65°C in a solution of 6×SSC, 0.5% SDS, followed by washing the membrane once each with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS.

[0018] Recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria, transgenic plant tissues, or transgenic plant organs containing the aforementioned DNA molecules are also within the scope of protection of this invention.

[0019] The recombinant vector described above is the genomic cDNA of the OsRAC2 gene shown in Sequence Listing 1 that replaces the fragment between the SpeI and KpnI restriction sites on the expression vector pCM1307, resulting in the recombinant vector ubi::OsRAC2 (i.e., the OsRAC2 overexpression recombinant vector).

[0020] Primers used to amplify the full length of the aforementioned DNA molecule or any fragment thereof are also within the scope of protection of this invention.

[0021] The application of the aforementioned proteins, DNA molecules, recombinant vectors, expression cassettes, transgenic cell lines, or recombinant bacteria in regulating plant root length is also within the scope of protection of this invention.

[0022] The application of the aforementioned proteins, DNA molecules, recombinant vectors, expression cassettes, transgenic cell lines, or recombinant bacteria in the cultivation of transgenic plants with increased root length is also within the scope of protection of this invention.

[0023] In the above applications, the plant is a monocotyledonous plant or a dicotyledonous plant;

[0024] The monocotyledonous plant mentioned is specifically rice.

[0025] Another object of the present invention is to provide a method for cultivating transgenic plants with increased root length.

[0026] The method provided by the present invention includes the following steps: introducing a cDNA molecule encoding the above-mentioned protein into a target plant to obtain a transgenic plant, wherein the root length of the transgenic plant is longer than that of the target plant.

[0027] In the above method, the fact that the root length of the transgenic plant is longer than that of the target plant is reflected in the fact that the root length of the transgenic plant is longer than that of the target plant during the seedling stage;

[0028] The seedling stage refers to seedlings that are 8-10 days old.

[0029] The plant is a monocotyledonous plant or a dicotyledonous plant;

[0030] The monocotyledonous plant mentioned is specifically rice.

[0031] The experiments of this invention demonstrate that a new positively regulated rice root length gene, OsRAC2, was cloned and transferred into wild-type rice, resulting in increased rice root length and providing genetic resources for the breeding of long-rooted rice varieties. Attached Figure Description

[0032] Figure 1 Histograms showing the phenotypic distribution of root length at the seedling stage for 795 different rice varieties;

[0033] Figure 2 Discovery of the OsRAC2 gene in the QTLqRL5-2 region under the CMLM model: In the top Manhattan plot, red dots represent SNP sites with significant differences in allele frequency between the long-root and short-root material libraries; blue dots represent SNP sites used for GWAS localization other than the red dots; the bottom rectangles represent annotated genes, with light blue indicating non-candidate genes and red indicating candidate genes.

[0034] Figure 3 Expression level analysis of candidate genes in four typical materials;

[0035] Figure 4 For haplotype analysis of OsRAC2 and phenotypic statistical analysis between different haplotypes;

[0036] Figure 5 Genotyping results of OsRAC2 knockout plants;

[0037] Figure 6The results of identifying the relative expression level of the OsRAC2 gene in OsRAC2 overexpressing plants;

[0038] Figure 7 Root length phenotype and statistical results for OsRAC2 knockout and overexpression plants;

[0039] Figure 8 Phenotypic and statistical results of lateral root length and number in OsRAC2 knockout and overexpression plants. Detailed Implementation

[0040] The following are specific embodiments to further describe the present invention in detail. The embodiments given are only for illustrating the present invention and do not limit the scope of the present invention. The embodiments provided below can also serve as a guide for those skilled in the art to improve the methods and do not constitute a limitation on the present invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods and can be performed in accordance with the techniques or conditions described in the literature or product manuals in this field.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] YEP liquid culture medium: Dissolve 10g of yeast extract, 5g of NaCl and 10g of tryptone in distilled water, then bring the volume to 1L with distilled water and autoclave at 121℃ for 15min.

[0044] YEP solid medium: Dissolve 10g of yeast extract, 5g of NaCl and 10g of tryptone in distilled water, then bring the volume to 1L with distilled water, and finally add 15g of agar powder. Autoclave at 121℃ for 15min.

[0045] AAM liquid culture medium: Dissolve 50 ml of AAM (20×), 5 ml of MS (200×), 10 ml of AAM organic (100×), 10 ml of iron salt (100×), 500 mg of hydrolyzed casein, 68.5 g of sucrose, 36 g of glucose, 75 mg of glycine, 877 mg of glutamine, 266 mg of aspartic acid, and 228 mg of arginine in an appropriate amount of deionized water, then bring the volume to 1 L with deionized water, adjust the pH to 5.5, and autoclave at 115℃ for 15 min; finally, add 1 ml of 20 mg / ml acetylsuccione solution and mix well.

[0046] AAM (20×): Dissolve 6.645g CaCl2, 58.8g KCl, 3.4g KH2PO4 and 7.4g MgSO4·7H2O in an appropriate amount of distilled water, and then make up to 1L with distilled water.

[0047] MS micro-scale (200×): Dissolve 166 mg KI, 3.38 g MnSO4·H2O, 1.24 g H3BO3, 50 mg Na2MoO4·2H2O, 1.72 g ZnSO4·7H2O, 5 g CoCl2·6H2O and 5 g CuSO4·5H2O in an appropriate amount of distilled water, and then make up to 1 L with distilled water.

[0048] AAM Organic (100×): Dissolve 50mg of vitamin B1, 50mg of vitamin B5 and 10mg of vitamin B6 in an appropriate amount of distilled water, and then bring the volume up to 1L with distilled water.

[0049] Iron salt (100×): Dissolve 3.73g Na2EDTA·2H2O and 2.78g FeSO4·7H2O in an appropriate amount of distilled water, and then make up to 1L with distilled water.

[0050] NB solid culture medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, and 3 g of plant gel in 1 L of distilled water, then adjust the pH to 5.8 and autoclave for 20 min.

[0051] Co-culture medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel, and 2 mg of 2,4-D in 1 L of distilled water, adjust the pH to 5.4, sterilize at 121℃ for 15 min, and when cooled to 50-60℃, add 10 g of glucose and 20 mg of acetylsuccinone that have been filtered and sterilized with 0.22 μM.

[0052] Delayed screening medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel, and 2 mg of 2,4-D in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121 °C for 15 min, and add 500 mg of cephalosporin that has been filtered and sterilized with 0.22 μM solution when the medium has cooled to 50–60 °C.

[0053] First round of screening culture medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121℃ for 15 min, and add 500 mg of cephalosporin and 50 mg of hygromycin after filtration sterilization at 0.22 μM when the temperature has cooled to 50-60℃.

[0054] Second round of screening culture medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel and 2,4-D 2 mg in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121℃ for 15 min, and add 50 mg of hygromycin that has been filtered and sterilized with 0.22 μM when the temperature has cooled to 50-60℃.

[0055] Differentiation medium: Dissolve 20 mL of 50×N6 large volume stock solution, 10 mL of 100×B5 micro volume stock solution, 1 mL of 1000×B5 organic stock solution, 150 mg of inositol, 300 mg of hydrolyzed casein, 500 mg of glutamine, 600 mg of proline, 30.0 g of sucrose, 3 g of plant gel, 2 mg of 6-BA, 1 mg of NAA, and 1 mg of KT in 1 L of distilled water, adjust the pH to 5.8, sterilize at 121 °C for 15 min, and add 50 mg of hygromycin that has been filtered and sterilized with 0.22 μM solution when the temperature has cooled to 50–60 °C.

[0056] Seedling growth medium: Dissolve 25 mL of 20×MS large volume stock solution, 2.5 mL of 200×MS micro volume stock solution, 2.5 mL of 200×MS iron salt stock solution, 2.5 mL of 200×MS organic stock solution, 3 g of plant gel, 0.5 mg of NAA and 0.25 mg of paclobutrazol in 1 L of distilled water, adjust the pH to 5.8, and sterilize at 121℃ for 15 min.

[0057] The solutes and their concentrations in the 20×MS mother liquor were 38.00 g / L KNO3, 8.80 g / L CaCl2·2H2O, 7.40 g / L MgSO4·7H2O, 3.40 g / L KH2PO4, and 33.00 g / L NH4NO3. The solvent was water, and the pH was natural.

[0058] The solutes and their concentrations in the 200×MS trace mother liquor were 4.46 g / L MnSO4·4H2O, 0.166 g / L KI, 1.24 g / L H3BO3, 1.72 g / L ZnSO4·7H2O, 0.050 g / L Na2MoO4·2H2O, 0.005 g / L CuSO4·5H2O, and 0.005 g / L CoCl2·6H2O. The solvent was water, and the pH was natural.

[0059] The solutes and their concentrations in the 200×MS iron salt mother liquor were 5.56 g / L FeSO4·7H2O and 7.46 g / L Na2·EDTA·2H2O, with water as the solvent and natural pH.

[0060] The solutes and concentrations of the 200×MS organic mother liquor were 20 g / L inositol, 100 mg / L nicotinic acid, 100 mg / L pyridoxine hydrochloride, 100 mg / L thiamine hydrochloride, and 400 mg / L glycine, with water as the solvent and a natural pH. The solutes and concentrations of the 50×N6 bulk mother liquor were 141.50 g / L KNO3, 20 g / L KH2PO4, 23.15 g / L (NH4)2SO4, 9.25 g / L MgSO4·7H2O, and 8.30 g / L CaCl2·2H2O, with water as the solvent and a natural pH.

[0061] The solutes and their concentrations in the 100×B5 trace mother liquor were 0.3 g / L H3BO3, 1 g / L MnSO4·4H2O, 0.0025 g / L CoCl2·6H2O, 0.0025 g / L CuSO4·5H2O, 0.2 g / L ZnSO4·7H2O, 0.025 g / L Na2MoO4·2H2O, and 0.075 g / L KI. The solvent was water, and the pH was natural.

[0062] The solutes and their concentrations in the 1000×B5 organic mother liquor were 2 g / L glycine, 100 g / L inositol, 1 g / L nicotinic acid, 1 g / L pyridoxine hydrochloride, and 10 g / L thiamine hydrochloride. The solvent was water, and the pH was natural.

[0063] The rice variety Nipponbare is referred to as wild-type rice or WT in the following text.

[0064] Alternating light and dark culture, with the following conditions: 28℃; 14 hours of light culture / 10 hours of dark culture; light intensity during light culture: 90 μE / m². 2 / s.

[0065] Example 1: Obtaining the OsRAC2 gene

[0066] This invention uses the CMLM model to analyze root length traits (e.g., ...) of 795 cultivated rice germplasms. Figure 1Genome-wide association analysis was performed, revealing a novel QTL locus qRL5-2 associated with root length (Y Zhao, H Zhang, J Xu, C Jiang, Z Li, et al. (2018) Loci and natural alleles underlying robust roots and adaptive domestication of upland ecotype rice in aerobic conditions. PLoS Genet 14(8): e1007521.). Further analysis of candidate genes on qRL5-2 revealed significant signal sites located at the promoters of three genes, such as... Figure 2 As shown (the rectangles at the bottom represent annotated genes, light blue represents non-candidate genes, and red represents candidate genes). Further analysis of candidate gene expression levels excluded gene LOC_Os05g43600, such as... Figure 3 As shown in the figure. Then, haplotype analysis revealed significant differences between different haplotypes of LOC_Os05g43820, which was identified as the target gene and named OsRAC2.

[0067] The OsRAC2 gene, derived from the rice variety Nipponbare, was finally obtained through screening. The cDNA sequence of the OsRAC2 gene is shown in Sequence 1, with a full length of 645 bp, and its nucleotide sequence is shown in Sequence 3. The OsRAC2 gene encodes the OsRAC2 protein, and the amino acid sequence of the OsRAC2 protein is shown in Sequence 2.

[0068] Example 2: Functional Verification of the OsRAC2 Gene

[0069] I. Obtaining and Identifying Homozygous Knockout Plants of OsRAC2

[0070] In this embodiment, target points T1 and T2 were selected for the experiment.

[0071] The target T1 sequence is: 5'-GGCTGAGGCCACTGAGCTAC-3' (i.e., positions 209-228 from the end of sequence 1).

[0072] The target T2 sequence is: 5'-CAGATCTTACCTTGCGGACC-3' (i.e., positions 378-397 from the end of sequence 1).

[0073] The target genes corresponding to target sites T1 and T2 are the OsRAC2 gene.

[0074] 1. Obtaining the recombinant plasmid OsRAC2-CR

[0075] (1) Using the intermediate vector pCBC-MT1T2 (reference: Xing, HL, Dong, Wang, ZP, Zhang, HY, Han, CY, Liu. ACRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC PLANTBIOL, 2014) as a template,

[0076] Primer AT1-BsF was used: 5'-ATATATGGTCTCTGGCGGGCTGAGGCCACTGAGCTACGTT-3',

[0077] Primer AT1-F0: 5'-CGGGCTGAGGCCACTGAGCTACGTTTTAGAGCTAGAAATAGC-3',

[0078] Primer AT2-R0: 5'-AACCAGATCTTACCTTGCGGACCCGCTTCTTGGTGCC-3',

[0079] PCR amplification was performed using a primer combination consisting of AT2-BsR: 5'-ATTATTGGTCTCTAAACCAGATCTTACCTTGCGGACCC-3', and approximately 964bp of PCR amplification product was recovered.

[0080] In the PCR amplification system, the concentrations of primers AT1-BsF and AT2-BsR were 10 μM, and the concentrations of primers AT1-F0 and AT2-R0 were 0.5 μM.

[0081] (2) Construction of the enzyme digestion-ligation system. The enzyme digestion-ligation system consisted of 15 μL, which was composed of 2 μL of PCR amplification product recovered in step (1), 2 μL of vector pBUE411 (reference: Xing, HL, Dong, Wang, ZP, Zhang, HY, Han, CY, Liu. Acrispr / cas9 toolkit for multiplex genome editing in plants. BMC PLANT BIOL, 2014), 1.5 μL of 10×NEB T4 Buffer (a component of T4 Ligase), 1.5 μL of T4 Ligase (NEB), 1 μL of restriction endonuclease BsaI (NEB), 1 μL of 10×Cutsmart buffer (a component of restriction endonuclease BsaI), and water.

[0082] (3) Take the enzyme digestion-ligation system constructed in step (2) and react to obtain the recombinant plasmid OsRAC2-CR. Reaction conditions: 37℃ for 5 hours; 50℃ for 5 minutes; 80℃ for 10 minutes.

[0083] (4) The recombinant plasmid OsRAC2-CR was sequenced. The sequencing results showed that the recombinant plasmid OsRAC2-CR was obtained by inserting the cDNA double-stranded molecule shown in sequence 1 into the recognition site of the restriction endonuclease BsaI of the vector pBUE411.

[0084] 2. Obtaining OsRAC2 homozygous mutant plants

[0085] Rice is a diploid plant. When Cas9 begins to cleave specific genes, both alleles on two homologous chromosomes within the same cell can be edited, producing the same or different types of mutations. Therefore, two alleles in a plant are considered two gene editing events. A homozygous mutant is one where the OsRAC2 gene on both homologous chromosomes of the plant has the same mutation. A biallelic mutant is one where both OsRAC2 genes on both homologous chromosomes of the plant have mutations, but in different forms. A heterozygous mutant is one where the OsRAC2 gene on one of the plant's two homologous chromosomes has a mutation, while the OsRAC2 gene on the other homologous chromosome has no mutation. A wild-type is one where neither of the two homologous chromosomes on the plant has a mutation in the OsRAC2 gene.

[0086] (1) The recombinant plasmid OsRAC2-CR was transferred to Agrobacterium tumefaciens EHA105 by liquid nitrogen freezing to obtain recombinant Agrobacterium, which was named EHA105 / OsRAC2-CR.

[0087] (2) The method in the reference (Mei-Rong, XU, Xia, ZH, Zhai, WX, Jian-Long, XU, Zhou, YL, & Zhi-Kang, LI (2008). Construction of double right-border binary vector carrying non-host gene rxo1 resistant to bacterial leafstreakofrice. Rice Science, 15(3), 243-246.) was used to transform EHA105 / OsRAC2-CR into Nipponbare rice. After screening, differentiation and rooting, T0 generation OsRAC2 gene knockout rice was obtained.

[0088] (3) Using genomic DNA from T0 generation rice leaves as templates.

[0089] Primers T1F: 5'-TTGTGTGCATTTCTTGGTAGCG-3' and

[0090] PCR amplification was performed using a primer pair consisting of primer T1R: 5'-TGCATCCGGTACAAACCTAACT-3', yielding the corresponding PCR amplification products. The PCR amplification products were then subjected to Sanger sequencing. The sequencing results were compared with the Cas9 target sequence of the OsRAC2 gene (shown in sequence 3) to identify mutation types. Several heterozygous mutant plants were obtained.

[0091] (4) Self-pollinate the heterozygous mutant obtained in step (3) to obtain the seeds, which are the T1 generation seeds. The plants grown from the T1 generation seeds are the T1 generation plants. Self-pollinate the T1 generation plants to obtain the seeds, which are the T2 generation seeds. The plants grown from the T2 generation seeds are the T2 generation plants.

[0092] (5) Using genomic DNA from leaves of T2 generation plants as templates, PCR amplification was performed using primer pairs consisting of primers T1F: 5'-TTGTGTGCATTTCTTGGTAGCG-3' and primer T1R: 5'-TGCATCCGGTACAAACCTAACT-3', yielding the corresponding PCR amplification products. The PCR amplification products were then subjected to Sanger sequencing. The sequencing results were compared with the Cas9 target sequence of the OsRAC2 gene (shown in sequence 3) to identify mutation types.

[0093] Test results as follows Figure 5 As shown, two homozygous mutant strains of OsRAC2 were obtained, named OsRAC2-KO1 (KO-1) and OsRAC2-KO2 (KO-2), respectively.

[0094] The OsRAC2 gene on both homologous chromosomes of KO-1 underwent the same mutation. Specifically, an insertion of one "A" nucleotide occurred at target site T1 (i.e., insertion after position 734 from the 5' end of sequence 3) on both homologous chromosomes, and a deletion of 16 nucleotides occurred at target site T2 (i.e., deletion of nucleotides 1231 to 1246 from the 5' end of sequence 3), which caused a frameshift and resulted in the loss of function of the protein OsRAC2.

[0095] The OsRAC2 gene on both homologous chromosomes of KO-2 underwent the same mutation. Specifically, a "G" nucleotide was inserted at target site T1 (i.e., the insertion of sequence 3 from position 734 on the 5' end) and a 3 nucleotide was deleted at target site T2 (i.e., the deletion of bases 1243 to 1245 on the 5' end of sequence 3), which caused a frameshift and resulted in the loss of function of the protein OsRAC2.

[0096] II. Obtaining and Identifying OsRAC2 Genetically Modified Rice

[0097] 1. Construction of recombinant vector ubi::OsRAC2 and obtaining recombinant Agrobacterium EHA105 / ubi::OsRAC2

[0098] (1) Using cDNA from the rice variety Nipponbare as a template, the following formula was used: A-HA-F: 5'-ATCGATACCGTCGACGAGCTCTCTAGA ACTAGT ATGAGCGGCGCCACCAAGTTCA-3' (underlined is the recognition site of the restriction endonuclease SpeI) and A-HA-R:5'-AGACTGGTGATTTTTGCGGAGTACCCG GGTACC PCR amplification was performed using a primer pair consisting of CTAAACAACACATGCACTGCC-3' (the underlined part is the recognition site of the restriction endonuclease KpnI), yielding a PCR amplification product of approximately 711 bp.

[0099] (2) The PCR amplification product obtained in step (1) was sequenced. The results showed that the PCR amplification product contained the cDNA fragment shown in sequence 1.

[0100] (3) The PCR amplification product obtained in step (1) and the vector pCM1307-3FLAG (described in the following literature: Rice SPL10 positively regulates trichome development through expression of HL6 and auxin-related genes[J]. Journal of Integrative Plant Biology, 2021., named pCM1307-3FLAG vector in the literature) were homologously recombinated to obtain the recombinant vector ubi::OsRAC2.

[0101] The recombinant vector ubi::OsRAC2 was sequenced. Sequencing results showed that the recombinant vector ubi::OsRAC2 was obtained by replacing a small DNA fragment between the restriction endonucleases SpeI and KpnI in the vector pCM1307-3FLAG with the cDNA molecule shown in sequence 1.

[0102] In the recombinant vector ubi::OsRAC2, the OsRAC2 gene is inserted downstream of the maize ubiquitin promoter. The recombinant vector ubi::OsRAC2 expresses the OsRAC2 protein as shown in sequence 2.

[0103] (4) The recombinant vector ubi::OsRAC2 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was named EHA105 / ubi::OsRAC2.

[0104] 2. Obtaining T3 generation homozygous transgenic OsRAC2 rice

[0105] The method described in the reference (Mei-Rong, XU, Xia, ZH, Zhai, WX, Jian-Long, XU, Zhou, YL, & Zhi-Kang, LI (2008). Construction of double right-border binary vector carrying non-host gene rxo1 resistant to bacterial leafstreakofrice. Rice Science, 15(3), 243-246.) was used to transform EHA105 / ubi::OsRAC2 into Nipponbare rice, and then T3 generation homozygous transgenic OsRAC2 rice was obtained through self-pollination. The specific steps are as follows:

[0106] (1) Acquisition of callus

[0107] Mature Nipponbare seeds were shelled, sterilized with alcohol, then with sodium hypochlorite, rinsed once with sterile water, and air-dried for 3 hours to obtain sterile Nipponbare seeds. These sterile Nipponbare seeds were inoculated onto NB solid medium and cultured in the dark at 28°C for 2 weeks to obtain embryogenic callus. The embryogenic callus was subcultured twice to obtain callus tissue. Each culture method involved inoculating the embryogenic callus onto fresh NB solid medium and incubating in the dark at 28°C for 2 weeks.

[0108] (2) Preparation of the infiltration solution

[0109] EHA105 / ubi::OsRAC2 was spread on YEP solid medium containing kanamycin and rifampin and incubated in the dark at 28°C for 2 days; then Agrobacterium was scraped onto AAM liquid medium to obtain an infection solution with an OD of approximately 0.3 at 600 nm.

[0110] (3) Co-cultivation

[0111] Select naturally dispersed, bright yellow granular callus tissue with a diameter of about 3-5 mm from the callus tissue obtained in step (1) and place it in an Erlenmeyer flask. Then add the infection solution prepared in step (2) and let it stand for 10 min for infection. After that, use sterile filter paper to absorb the excess infection solution and place the callus tissue on a co-culture medium covered with a layer of filter paper. Co-culture at 20℃ for 2-3 days.

[0112] (4) Screening of resistant callus

[0113] ① Take the callus tissue after completing step (3), first wash it with sterile water by shaking 5-6 times, then wash it with sterile water containing cephalosporin and carbenicillin for 20 minutes, then place it on sterile filter paper to drain for 3 hours, and finally transfer it to delayed selection medium and incubate it in the dark at 28°C for 1 week.

[0114] ②After completing step ①, transfer the callus tissue to the first round of screening medium and incubate in the dark at 28°C for 2 weeks.

[0115] ③ After completing step ②, the callus tissue was transferred to the second round of screening medium and cultured in the dark at 28°C for 2 weeks to obtain resistant callus tissue.

[0116] (5) Differentiation

[0117] ① Transfer the resistant callus obtained from step (4) to a predifferentiation medium and incubate in the dark at 28°C for 2 weeks.

[0118] ②After completing step ①, the resistant callus tissue is transferred to the differentiation medium and cultured under alternating light for 2-3 weeks to obtain transgenic seedlings.

[0119] (6) Strong seedlings

[0120] The transgenic seedlings obtained in step (5) were transferred to a seedling culture medium. After the seedlings took root, they were removed from the culture bottle, the culture medium on the roots was washed off, and the seedlings were hardened off for 1-2 weeks before being transplanted to the field for planting until maturity, thus obtaining T0 generation rice transgenic with the OsRAC2 gene.

[0121] (7) Select positive seedlings of the T0 generation of rice to be transgenic into the OsRAC2 gene obtained in step (6), and then perform self-pollination to obtain the T1 generation of rice transgenic into the OsRAC2 gene.

[0122] (8) Select positive seedlings of the T1 generation OsRAC2 gene transgenic rice obtained in step (7), and then perform self-pollination to obtain the T2 generation OsRAC2 gene transgenic rice.

[0123] (9) Select positive seedlings of T2 generation transgenic OsRAC2 rice obtained in step (8), and then perform self-pollination to obtain T3 generation homozygous transgenic OsRAC2 rice.

[0124] The above method for screening positive seedlings is as follows: Genomic DNA is extracted from rice seedlings and used as a template. PCR amplification is performed using primer pairs consisting of OEP-AF: 5'-TACCCATACGATGTTCCAGAT-3' and OEP-AR: 5'-TAATCCTCTTGTCCAGCAGTG-3' to obtain PCR amplification products. Then, the following judgment is made: If a PCR amplification product contains a DNA fragment of approximately 288 bp, the rice seedling corresponding to that PCR amplification product is a positive seedling.

[0125] 3. Real-time quantitative PCR detection of the relative expression level of the OsRAC2 gene in T3 generation homozygous transgenic OsRAC2 rice.

[0126] (1) The seedlings of each T3 generation homozygous transgenic OsRAC2 rice that had grown to 10 days were placed in liquid nitrogen for preservation to obtain the corresponding test samples. Nipponbare seeds were taken and cultured at 28℃ with alternating light and dark conditions for 10 days to obtain the rice seedlings to be tested; the rice seedlings to be tested were placed in liquid nitrogen for preservation to obtain the corresponding test samples.

[0127] (2) Total RNA was extracted from the sample to be tested using the Trizo1 method, and then the first-strand cDNA was reverse transcribed using a reverse transcription kit (TransGen). The cDNA was diluted 50 times with sterile water as a template, and the relative expression level of the OsRAC2 gene was detected by real-time quantitative PCR (with Actin gene Os03g0718100 as an internal reference gene).

[0128] The primers for detecting the OsRAC2 gene were F1: 5'-CCCACCGTGTTCGACAACT-3' and R1: 5'-TCCAGCAGTGTCCCATAATCC-3'.

[0129] The primers for detecting the Actin gene are ActinF: 5'-CACAGGTATTGTGTTGGACTCTG-3' and ActinR: 5'-AGTAACCACGCTCCGTCAGG-3'.

[0130] Some test results can be found Figure 6The results showed that, compared with Nipponbare, the relative expression level of the OsRAC2 gene was significantly increased in all T3 generation homozygous transgenic OsRAC2 rice lines. Three of these T3 generation homozygous transgenic OsRAC2 rice lines were named OsRAC2-OE1 (abbreviated as OE1) and OsRAC2-OE2 (abbreviated as OE2), respectively.

[0131] III. Statistics on taproot length, lateral root density, lateral root length, and crown root number of OsRAC2 homozygous mutant plants and OsRAC2 transgenic rice

[0132] The experiment was repeated three times and the average value was taken. The steps for each repetition were as follows:

[0133] 1. Remove the husks from 15 rice seeds (Nipponbare, KO-1, KO-2, OE-1 or OE-2), disinfect them with alcohol, then disinfect them with sodium hypochlorite, rinse them once with sterile water, and air dry them for 3 hours to obtain sterile rice seeds for testing.

[0134] 2. Inoculate sterile rice seeds to be tested onto NB solid medium and incubate vertically at 28°C for 5 days.

[0135] 3. During the cultivation process in step 2, measure the root length daily.

[0136] Root systems of some rice seeds tested after 6 days of vertical culture are shown below. Figure 7 The statistical results for root lengths of A and D in the figure are shown in the figure. Figure 7 The statistical results of the daily root length of some rice seeds tested during the cultivation period (B and E) are shown in [the original text]. Figure 7 C and F. The results showed that, compared with Nipponbare, the root length of OsRAC2 transgenic rice (such as OE-1 and OE-2) was significantly increased, and the root elongation rate was also significantly increased; compared with Nipponbare, the root length of OsRAC2 homozygous mutants (such as KO-1 and KO-2) was significantly shorter, and the root elongation rate was also significantly reduced.

[0137] 4. After completing step 2, check the lateral root density (i.e., the number of lateral roots 2-3 cm below the base of the main root), lateral root length (the average of the three longest lateral roots 2-3 cm below the base of the main root), and crown root number.

[0138] The lateral root phenotypes of some rice seeds tested after 6 days of vertical culture are shown in the figure. Figure 5 The statistical results for lateral root density and lateral root length are shown in Figure A. Figure 5 In sections B and C. The results showed that, compared with Nipponbare, transgenic OsRAC2 rice (such as OE-1 and OE-2) had no statistically significant difference in lateral root density but significantly increased lateral root length, while homozygous OsRAC2 mutants (such as KO-1 and KO-2) had no significant difference in lateral root density but significantly decreased lateral root length.

[0139] Therefore, overexpression of the OsRAC2 gene in Nipponbare significantly increased both the primary root length and lateral root length; while suppression of OsRAC2 gene expression in Nipponbare significantly decreased both the primary root length and lateral root length. The OsRAC2 protein positively regulates rice root elongation.

[0140] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for increasing the root length of rice, characterized in that, This includes increasing rice root length by upregulating or enhancing or increasing the expression of protein-coding genes in rice, and / or the activity and / or content of said proteins; The protein is any of the following proteins: 1) The protein shown in sequence 2 of the sequence listing; 2) Proteins derived from Sequence 2 with the same function, formed by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in Sequence 2 of the Sequence Listing; The substitution and / or deletion and / or addition of one or more amino acid residues mentioned above constitutes a substitution and / or deletion and / or addition of no more than 10 amino acid residues; 3) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of 1) or 2) above.

2. A method for cultivating long-rooted rice, characterized in that, The transgenic rice plant obtained by upregulating or enhancing or increasing the expression level of the gene encoding the protein of claim 1 in the target rice plant, and / or the activity and / or content of the protein, wherein the root length of the transgenic rice plant is longer than that of the target rice plant.

3. The application of the protein and the gene encoding the protein in increasing rice root length; The protein described herein can be any of the following proteins: 1) The protein shown in sequence 2 of the sequence listing; 2) Proteins derived from Sequence 2 with the same function, formed by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence shown in Sequence 2 of the Sequence Listing; The substitution and / or deletion and / or addition of one or more amino acid residues mentioned above constitutes a substitution and / or deletion and / or addition of no more than 10 amino acid residues; 3) Fusion proteins obtained by attaching protein tags to the N-terminus and / or C-terminus of 1) or 2) above: The gene encoding the protein that regulates it is any one of the following: 1) The coding region is the DNA molecule shown in sequence 3 of the sequence listing; 2) The coding region is the cDNA molecule shown in sequence 1 of the sequence listing; 3) DNA molecules that hybridize with the DNA sequence defined in 1) or 2) under strict conditions and encode proteins with the same function; 4) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in 1) or 2) and encodes a protein having the same function.

4. Primers for amplifying the full length of the above-mentioned DNA molecule or any fragment thereof are also within the scope of protection of this invention.

5. Recombinant vectors, expression cassettes, transgenic cell lines, recombinant bacteria, transgenic plant tissues, or transgenic plant organs containing the above-mentioned DNA molecules are also within the scope of protection of this invention.

6. The method according to claim 1 or 2, characterized in that, The upregulation, enhancement, or increase of the expression of the gene encoding the protein of claim 1 or 2 in the plant includes introducing the gene encoding the protein of claim 3 or the recombinant vector or expression cassette of claim 5 into the target plant; In the above applications, the plant is a monocotyledonous plant or a dicotyledonous plant; The monocotyledonous plant mentioned is specifically rice.

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