Method for visually screening rice mutants and application thereof

Abstract

The invention provides a method for visually screening rice mutants and application thereof, and belongs to the technical field of biology. The method for visually screening the rice mutants comprises the following steps: transferring a recombinant expression vector based on an inducible promoter into rice to obtain a transgenic plant, carrying out seed reproduction on the transgenic plant, and then screening to obtain the mutants, the recombinant expression vector based on the inducible promoter comprises a promoter of a candidate gene and a reporter gene, and the screening comprises a step of low-temperature treatment. According to the screening method disclosed by the invention, the time required for screening the rice mutant phenotype is shortened, and meanwhile, the phenotype screening result is more accurate.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for visually screening rice mutants and its application. Background Technology

[0002] Rice evolved in tropical and subtropical regions and is a warm-season crop, highly sensitive to low temperatures. Chill damage to rice refers to natural disasters that occur when rice plants experience temperatures below the critical range during their growth period, leading to stunted growth and reduced yields. It is one of the main obstacles to rice production in cold regions. Therefore, research on rice cold tolerance is of great significance for steadily increasing rice yields, helping to improve rice's resistance to low temperatures, expand its global geographical distribution, and ultimately increase total rice production.

[0003] Research on cold tolerance in rice generally involves using molecular genetic techniques to identify cold-tolerant genes, explore their molecular mechanisms of cold resistance, and then utilize them. Currently, identifying cold-tolerant genes in rice mainly relies on traditional mutant screening methods.

[0004] Chinese patent application CN116515893A discloses the application of the OsbHLH6 gene and its protein in improving crop cold tolerance. The method involves a series of steps: inducing cold stress on the OsbHLH6 gene; constructing an OsbHLH6 gene overexpression vector; constructing an Osbhlh6 mutant vector (knockout vector); transforming the constructed Osbhlh6 mutant vector with Agrobacterium to obtain transgenic plants; and then performing cold treatment phenotypic analysis on the plants to determine the effect of the Osbhlh6 gene on rice cold tolerance. However, the above technical solution requires a long plant growth period for phenotypic assessment and cannot distinguish between subtle mutations or loss-of-function phenotypes with similar appearances. Summary of the Invention

[0005] In view of this, the present invention provides a method for visually screening rice mutants and its application, so as to shorten the time required to screen rice mutant phenotypes and make the screening results more accurate.

[0006] To achieve the above objectives, the present invention provides a method for visually screening rice mutants, comprising the following steps: transferring a recombinant expression vector based on an inducible promoter into rice to obtain transgenic plants, propagating the transgenic plants, and then screening to obtain mutants. The recombinant expression vector based on an inducible promoter includes a promoter of a candidate gene and a reporter gene, and the screening includes a low-temperature treatment step.

[0007] Optionally, the propagation strain is obtained as a homozygous propagation strain.

[0008] Optionally, the reporter gene is the RUBY reporter gene.

[0009] Optionally, the candidate gene is one of OsDREB1B, OsABA8ox1, OsCYP19-4, OsTPP1, and OsLEA9.

[0010] Optionally, the candidate gene is OsDREB1B, and the promoter sequence of the OsDREB1B gene is shown in SEQ ID NO.1.

[0011] Optionally, the amplification primers for the promoter of the OsDREB1B gene are as follows: F: 5'-AGTTGTCCTTCTAGTAACAA-3'; R: 5'-TGAACCAGAGAGAGTCATCC-3'.

[0012] Optionally, the candidate gene is OsABA8ox1, and the promoter sequence of the OsABA8ox1 gene is shown in SEQ ID NO.2.

[0013] Optionally, the amplification primers for the promoter of the OsABA8ox1 gene are as follows: F: 5'-TCATATTCCTCATAGATAAACT-3'; R: 5'-TAAGTGAAGTGAGCAGGCGCCA-3'.

[0014] Optionally, the candidate gene is OsCYP19-4, and the promoter sequence of the OsCYP19-4 gene is shown in SEQ ID NO. 3.

[0015] Optionally, the amplification primers for the promoter of the OsCYP19-4 gene are as follows: F: 5'-TTTATTTTTTTTCAGGTATGCC-3'; R: 5'-GCTTGCTCATTCTGTTTATATC-3'.

[0016] Optionally, the candidate gene is OsTPP1, and the promoter sequence of the OsTPP1 gene is shown in SEQ ID NO.4.

[0017] Optionally, the amplification primers for the promoter of the OsTPP1 gene are as follows: F: 5'-CACAATGTATATGAAACTTTTAG-3'; R: 5'-GTAGTAGGATTTAAAAGGGTT-3'.

[0018] Optionally, the candidate gene is OsLEA9, and the promoter sequence of the OsLEA9 gene is shown in SEQ ID NO. 5.

[0019] Optional primers for amplifying the OsLEA9 gene promoter are as follows: F: 5'-TTTGGAGACCGTGTCACTGT-3'; R: 5'-AGAAAGAAAGTTGATCATCC-3'.

[0020] Optionally, the recombinant expression vector based on an inducible promoter may further include a marker gene.

[0021] Optionally, the marker gene includes a hygromycin resistance gene.

[0022] Optionally, the low-temperature treatment involves culturing the propagated seeds at a temperature not exceeding 26°C for up to 10 days. Optionally, the temperature of the low-temperature treatment is not higher than 16°C. Optionally, the duration of the low-temperature treatment is up to 5 days. More preferably, the temperature of the low-temperature treatment is not higher than 14°C. The duration of the low-temperature treatment is up to 2 days.

[0023] Optionally, the mutant does not exhibit a red phenotype after low-temperature treatment.

[0024] Optionally, the method for transforming the recombinant expression vector based on the inducible promoter into rice to obtain transgenic plants includes one of Agrobacterium-mediated transformation and gene gun transformation.

[0025] Optionally, the method for constructing the recombinant expression vector based on the inducible promoter includes the following steps: selecting a sequence 1.7~2.5kb upstream of the transcription start site of the candidate gene as the promoter sequence; The empty vector was subjected to double enzyme digestion to obtain the enzyme digested vector. Fragment 1 was amplified using the promoter of the candidate gene as a template, and fragment 2 was amplified using the RUBY reporter gene as a template. Fragment 1, fragment 2 and the enzyme digested vector were ligated to obtain the recombinant expression vector.

[0026] Optionally, the carrier is the pCAMBIA1300 carrier.

[0027] This invention also provides an application of a method for visually screening rice mutants in rice breeding.

[0028] The above-described technical solution of the present invention has at least the following beneficial effects: 1. Compared with traditional screening methods, the visual screening method of this invention for rice mutants has several advantages. First, it allows for phenotypic differentiation from the seed stage through low-temperature treatment, eliminating the need for subsequent low-temperature treatment after seedling development. Second, this method uses the presence or absence of red germination as a distinguishing phenotypic feature, which, compared to existing techniques where plants exhibit similar phenotypes after low-temperature treatment, reduces the time required to differentiate phenotypes and yields more accurate results. Third, this method eliminates the need for complex equipment, simplifying the experimental process and reducing additional costs and operational steps.

[0029] 2. The method for visually screening rice mutants in this invention uses a recombinant expression vector based on an inducible promoter. By constructing a vector that connects a candidate gene promoter to the RUBY reporter gene, and then subjecting the gene to low-temperature treatment, the RUBY reporter gene enables intuitive mutant identification through color change. Seeds that do not show red after mutagenesis are identified as mutants. The combination of these steps screens out mutants of cold-resistant genes and provides a convenient and efficient tool for discovering cold-resistant genes in rice and studying cold resistance mechanisms. Attached Figure Description

[0030] Figure 1 This is a comparison diagram of the expression of each candidate gene under different temperature conditions according to the present invention; Figure 2 This is a comparison diagram of the expression of each candidate gene after treatment at different time points according to the present invention; Figure 3 This is a map of the recombinant expression vector constructed in Example 1 of the present invention; Figure 4 The images show the phenotypic diagrams of the wild-type material and the double-copy material in Example 2 of this invention under different temperatures and times. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figure 1-4 The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.

[0032] The rice variety used in this invention is Zhongkefa No. 5.

[0033] The candidate genes used in this invention are rice cold-resistant genes: OsDREB1B, OsABA8ox1, OsCYP19-4, OsTPP1 and OsLEA9.

[0034] After two weeks of hydroponics, “Zhongkefa No. 5” rice seedlings were treated at 4℃, 6℃, 8℃, 10℃ and 26℃ for 24 hours. Then, rice genomic RNA was extracted from rice leaves and roots using the Trizol method for subsequent gene expression analysis. The experiment was conducted in an RNase-free environment as much as possible to reduce RNA degradation.

[0035] The specific steps for extracting rice genomic RNA using the Trizol method are as follows: ① Quickly freeze the sample material (rice leaves or roots) in liquid nitrogen and grind it under low temperature conditions to prevent RNA degradation; ② Add 1 mL of TRIzol reagent to the sample, shake to mix, and let stand at room temperature for 5-10 min to allow the cells to fully lyse and release nucleic acids; ③ Add 750 μL of chloroform, shake thoroughly to mix, place on ice and let stand for 10 min, then centrifuge at 12000 rpm for 15 min at 4℃. ④ Transfer 500 μL of supernatant to an RNase-free 1.5 mL centrifuge tube, add isopropanol at a 1:1 volume ratio, gently invert to mix to induce RNA precipitation, and then incubate at -20℃ for 30 min. Centrifuge at 12000 rpm for 10 min at 4℃, and then discard the waste liquid; ⑤ Wash the white precipitate with 75% ethanol, centrifuge at 7500 rpm for 5 min at 4℃, discard the supernatant, and repeat this step twice; ⑥ After the RNA precipitate has been properly dried, add 20-50 μL of RNase-free water to dissolve it completely. It can be stored for a long time at -80℃.

[0036] The obtained RNA was subjected to reverse transcription PCR using a cDNA synthesis kit from Beijing TransGen Biotech Co., Ltd. The cDNA synthesis kit model was AT311. Detailed steps were described in the product manual. The reverse transcription PCR reaction system is shown in Table 1 below. Table 1 Reverse transcription PCR reaction system

[0037] After the reaction mixture was thoroughly mixed, it was incubated at 42°C for 15 min, then heated to 85°C and held for 5 s. Finally, it was stored at -20°C for subsequent gene expression analysis.

[0038] A quantitative PCR reaction system was prepared on ice, and quantitative PCR amplification was performed using a three-step procedure.

[0039] The specific steps for PCR amplification are as follows: (1) Search for the target gene information through the National Rice Data Center (www.ricedata.cn) and download the corresponding gene sequence (FASTA format) for subsequent primer design; (2) Use the FASTA sequence of the input gene in Primer-BLAST from the NCBI website (www.ncbi.nlm.nih.gov), adjust the PCR product length to 80-200bp, select Primer must span an exon-exonjunction, select Refseq mRNA database, select Oryza sativa Japonica Group (taxid:39947) for the organism species, click Get Primers, and the appropriate primers will be automatically generated. Select primers with good specificity and close to the 3'UTR as much as possible for subsequent gene expression analysis; verify the specificity of the primers; (3) Measure the cDNA concentration using a nucleic acid quantification system and dilute it to an appropriate concentration; (4) Mix the reaction system and use the real-time PCR system to perform quantitative analysis of the transcription level of the target gene (three biological replicates are set up for each treatment sample).

[0040] The qRT-PCR reaction system is shown in Table 2: Table 2 qRT-PCR reaction system

[0041] Reaction program: 95℃ 5min; 95℃ 10s, 60℃ 10s, 72℃ 10s, 40 cycles; 95℃ 5s, 65℃ 1min, 97℃ 5s; 40℃ 30s.

[0042] Results were interpreted by analyzing melting and amplification curves, and differential expression was performed using Ct values. The expression analysis results of the OsDREB1B, OsABA8ox1, OsCYP19-4, OsTPP1, and OsLEA9 genes are shown below. Figure 1 As shown. Figure 1 The expression of each gene under different temperature conditions.

[0043] Specifically, by Figure 1It can be seen that the OsDREB1B gene used in this invention has the highest expression level in both rice leaves and roots at 4℃, the OsABA8ox1 gene has high expression levels in both rice leaves and roots at 4℃, 8℃ and 10℃, the OsCYP19-4 gene has high expression levels in both rice leaves and roots at 8℃ and 10℃, the OsTPP1 gene has the highest expression level in both rice leaves and roots at 4℃, the OsLEA9 gene has high expression levels in rice leaves at 4℃ and 10℃, and the OsLEA9 gene has the highest expression level in rice roots at 4℃. However, the expression levels of OsDREB1B, OsABA8ox1, OsCYP19-4, OsTPP1 and OsLEA9 genes are all the lowest at 26℃.

[0044] After two weeks of hydroponic cultivation, 'Zhongkefa 5' rice seedlings were treated at 4℃ for 0h, 1h, 2h, 4h, 8h, 1d, 2d, 3d, 4d, 5d, 6d, 7d, and 8d. Then, genomic RNA was extracted from rice leaves and roots using the Trizol method for subsequent gene expression analysis. The experiment was conducted in an RNase-free environment to minimize RNA degradation. The specific steps for extracting rice genomic RNA using the Trizol method are as follows: ① Quickly freeze the sample material (rice leaves or roots) in liquid nitrogen and grind it under low temperature conditions to prevent RNA degradation; ② Add 1 mL of TRIzol reagent to the sample, shake to mix, and let stand at room temperature for 5-10 min to allow the cells to fully lyse and release nucleic acids; ③ Add 750 μL of chloroform, shake thoroughly to mix, place on ice and let stand for 10 min, then centrifuge at 12000 rpm for 15 min at 4℃. ④ Transfer 500 μL of supernatant to an RNase-free 1.5 mL centrifuge tube, add isopropanol at a 1:1 volume ratio, gently invert to mix to induce RNA precipitation, and then incubate at -20℃ for 30 min. Centrifuge at 12000 rpm for 10 min at 4℃, and then discard the waste liquid; ⑤ Wash the white precipitate with 75% ethanol, centrifuge at 7500 rpm for 5 min at 4℃, discard the supernatant, and repeat this step twice; ⑥ After the RNA precipitate has been properly dried, add 20-50 μL of RNase-free water to dissolve it completely. It can be stored for a long time at -80℃.

[0045] The obtained RNA was subjected to reverse transcription PCR using the cDNA synthesis kit from Beijing TransGen Biotech Co., Ltd. (model AT311-02). For detailed steps, please refer to the product manual. The reverse transcription PCR reaction system and procedure are as described in Table 1 and the aforementioned methods.

[0046] Then, a quantitative PCR reaction system was prepared on ice, and quantitative PCR amplification was performed using a three-step procedure.

[0047] The specific steps for PCR amplification are as follows: (1) Search for the target gene information through the National Rice Data Center (www.ricedata.cn) and download the corresponding gene sequence (FASTA format) for subsequent primer design; (2) Use the FASTA sequence of the input gene in Primer-BLAST from the NCBI website (www.ncbi.nlm.nih.gov), adjust the PCR product length to 80-200bp, select "Primer must span an exon-exonjunction" for exon junction span, select Refseq mRNA database, select Oryza sativa Japonica Group (taxid:39947) for biological species, click Get Primers, and appropriate primers will be automatically generated. Select primers with good specificity and close to the 3'UTR for subsequent gene expression analysis; verify the specificity of the primers; the primers here are consistent with the primers designed when testing each candidate gene at different temperatures.

[0048] (3) Measure the cDNA concentration using a nucleic acid quantification system and dilute it to an appropriate concentration; (4) Mix the reaction system and use the real-time PCR system to perform quantitative analysis of the transcription level of the target gene (three biological replicates are set up for each treatment sample).

[0049] The qRT-PCR reaction system is shown in Table 2 and the methods described above.

[0050] Results were interpreted by analyzing melting and amplification curves, and differential expression was performed using Ct values. The expression analysis results of the OsDREB1B, OsABA8ox1, OsCYP19-4, OsTPP1, and OsLEA9 genes are shown below. Figure 2 As shown. Figure 2 The expression of each gene after treatment at 4℃ for different time periods is shown.

[0051] Specifically, by Figure 2 It can be seen that the peak expression levels of the OsDREB1B, OsABA8ox1, OsCYP19-4, OsTPP1 and OsLEA9 genes used in this invention in rice roots and leaves generally occur before 2 days. Except for OsABA8ox1, whose peak expression level in roots occurs on the 6th day, the peak expression levels of the other genes in roots and leaves all occur before 5 days.

[0052] Example 1 Carrier construction The sequences of rice cold-resistance genes OsDREB1B, OsABA8ox1, OsCYP19-4, OsTPP1, and OsLEA9 were obtained from the National Rice Data Center. The sequences 1.7–2.5 kb upstream of the transcription start site of these rice cold-resistance genes were selected as the promoter regions for subsequent vector construction. The promoter sequence of OsDREB1B is shown in SEQ ID NO. 1; the promoter sequence of OsABA8ox1 is shown in SEQ ID NO. 2; the promoter sequence of OsCYP19-4 is shown in SEQ ID NO. 3; the promoter sequence of OsTPP1 is shown in SEQ ID NO. 4; and the promoter sequence of OsLEA9 is shown in SEQ ID NO. 5.

[0053] Primers for each promoter were designed using SnapGene 6.0. The primer design parameters were: primer length: 18-24 bp, annealing temperature (Tm): 52-62℃, GC content: 40%-60%, and good specificity.

[0054] The amplification primers for the promoter of the OsDREB1B gene are as follows: F: 5'-AGTTGTCCTTCTAGTAACAA-3'; R: 5'-TGAACCAGAGAGAGTCATCC-3'.

[0055] The amplification primers for the promoter of the OsABA8ox1 gene are as follows: F: 5'-TCATATTCCTCATAGATAAACT-3'; R: 5'-TAAGTGAAGTGAGCAGGCGCCA-3'.

[0056] The primers for amplifying the promoter of the OsCYP19-4 gene are as follows: F: 5'-TTTATTTTTTTTCAGGTATGCC-3'; R: 5'-GCTTGCTCATTCTGTTTATATC-3'.

[0057] The primers for amplifying the promoter of the OsTPP1 gene are as follows: F: 5'-CACAATGTATATGAAACTTTTAG-3'; R: 5'-GTAGTAGGATTTAAAAGGGTT-3'.

[0058] The primers for amplifying the promoter of the OsLEA9 gene are as follows: F: 5'-TTTGGAGACCGTGTCACTGT-3'; R: 5'-AGAAAGAAAGTTGATCATCC-3'.

[0059] The promoter sequence was amplified using KOD FX reagent from Toyobo Biotechnology Co., Ltd. The pCAMBIA1300 empty vector was digested with KPN1 and BamH1 enzymes to obtain a digested vector. Fragment 1 was amplified using the candidate gene promoter as a template, and fragment 2 was amplified using the RUBY reporter gene as a template. Fragments 1, 2, and the digested vector were then ligated to obtain the constructed recombinant expression vector based on the inducible promoter. The fragments were then assembled using the pEASY-Basic Seamless Cloning and Assembly Kit from Beijing TransGen Biotech Co., Ltd. The RUBY sequence is shown in SEQ ID NO. 6.

[0060] The pCAMBIA1300 used in this embodiment was purchased from Shanghai Newp Biotechnology Co., Ltd., plasmid number V008765, vector size 8958bp, vector containing kanamycin resistance gene and linked to hygromycin B (Hyg) resistance gene. The linkage map of the hygromycin B resistance gene and RUBY gene in the constructed recombinant expression vector based on an inducible promoter is shown below. Figure 3 As shown. The kanamycin resistance gene is used as a bacterial selection marker, and the hygromycin B resistance gene is used as a plant cell selection marker.

[0061] Escherichia coli DH5α competent cells were transformed using the heat shock method. The DH5α competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., product specification DL1001. After overnight culture, identification was performed to confirm whether the recombinant expression vector was inserted. The specific transformation steps are as follows: (1) Melt Escherichia coli DH5α competent cells on ice; (2) Add 10 μL of recombinant expression vector to 100 μL of thawed competent cells, mix well, and let stand on ice for 30 min to obtain bacterial culture; (3) Heat shock the bacterial solution in a 42℃ water bath for 30 seconds, then immediately transfer it to ice to cool for 3 minutes; (4) Add the cooled bacterial culture to 500 μL of LB liquid medium and incubate at 37°C and 250 rpm for 1 h in a shaker. (5) Pour the LB solid medium containing kanamycin (Kan) into a sterile petri dish and let it stand and cool until solidified; (6) After centrifugation, collect the bacterial precipitate from the transformed competent cells in step (4), and resuspend the bacterial precipitate in 100 μL of supernatant. Inoculate the resuspended bacterial solution evenly onto LB solid medium plates containing kanamycin selection pressure, and invert the plates in a 37°C incubator for about 12 h. Since the recombinant vector contains the kanamycin resistance gene, the strains that can grow normally are confirmed to have inserted the recombinant expression vector. The recombinant DNA molecules in the transformed competent cells are sequenced. If the sequence is consistent with the inserted recombinant expression vector sequence, it is confirmed that the recombinant expression vector has no mutation.

[0062] The promoter sequence (SEQ ID NO.1) of OsDREB1B is as follows: The promoter sequence of OsABA8ox1 (SEQ ID NO.2) is as follows: The promoter sequence (SEQ ID NO.3) of OsCYP19-4 is as follows: The promoter sequence of OsTPP1 (SEQ ID NO.4) is as follows: The promoter sequence of OsLEA9 (SEQ ID NO.5) is as follows: The RUBY sequence (SEQ ID NO. 6) is as follows: Example 2 The mutation-free recombinant expression vector based on the inducible promoter from Example 1 was sent to Weimi Biotechnology Co., Ltd., where the inducible promoter was the promoter of the OsABA8ox1 gene. Using Zhongkefa 5 rice as the background material, Agrobacterium transformation was performed. First, Agrobacterium competent cells were transformed. The Agrobacterium competent cells were EHA105 Agrobacterium tumefaciens purchased from Shanghai Weidi Biotechnology Co., Ltd., product specification AC1010, containing a rifampicin resistance gene. Then, the transformed Agrobacterium competent cells were infected into rice cells, obtaining T0 positive plants.

[0063] The steps for transforming Agrobacterium competent cells are as follows: Referring to the EHA105 Chemically Competent Cell product manual from Weidi Biotechnology Co., Ltd., the recombinant plasmid was transformed using the Freeze-Thaw Method. The specific steps are as follows: ① Take 500 μL of EHA105 competent cells and thaw them in an ice bath; ② Add 0.4 μg of non-mutated recombinant expression vector based on inducible promoter to every 100 μL of thawed competent cells, gently pipette to mix, and let stand on ice for 10 min; ③Then, the cells were rapidly frozen in liquid nitrogen for 5 minutes to form micropores in the cell membrane, ensuring that the recombinant expression vector could enter competent cells. Immediately afterwards, the cells were transferred to a 37°C water bath for 5 minutes for heat shock, followed by a 5-minute ice bath to stabilize the cells. ④ Add 800 μL of YEB medium and incubate at 28℃ and 180 rpm for 3 hours to revive Agrobacterium; ⑤ Centrifuge the Agrobacterium cultured in step ④ at 5000 rpm for 3 min to obtain supernatant and bacterial precipitate. Take 100 μL of supernatant to resuspend the bacterial precipitate, and then gently pipette to mix to obtain resuspended bacterial solution. Spread the resuspended bacterial solution evenly on YEB solid plates containing the corresponding antibiotics. Invert the plates in a 28℃ constant temperature incubator and incubate for 2-3 days until clear single colonies grow. ⑥ Pick a single colony and place it on YEB liquid medium, shake and culture, OD600=0.7, centrifuge at 4000rpm for 10min, resuspend in infection solution (YEB+10mM MgCl2+100μM AS), adjust OD600=0.1-0.2 to obtain Agrobacterium resuspension, for later use.

[0064] The specific steps for the transformed Agrobacterium competent cells to infect rice cells are as follows: (1) Infecting plant materials ① Take mature and plump Zhongkefa 5 rice seeds, remove the husk, sterilize with 75% C2H5OH for 2 minutes, soak in 1% NaClO solution for 15 minutes, and rinse with sterile water 4-7 times. ② The sterilized seeds were cultured on an induction medium and induced at 27°C in the dark for 2 weeks to obtain embryogenic callus tissue; ③ Select vigorous, golden-yellow, and firm embryogenic callus tissues for subculture, and use them for infection after 3-5 days of growth; ④ Place the callus tissue in Agrobacterium resuspension, gently shake, and soak for 15 minutes; ⑤ Remove excess bacterial solution from the surface, transfer the embryogenic callus to a co-culture medium, and co-culture at 28°C in the dark for 2-3 days; ⑥ Wash the callus tissue with sterile water 5-7 times, transfer it into a selection medium containing Hyg resistance and Cef resistance to remove the transformed Agrobacterium and prevent continuous infection of plant cells by Agrobacterium. Culture at 28°C under light. ⑦ Subculture every two weeks. After 2-3 rounds of selection to obtain resistant callus, transfer it to differentiation medium. Once the resistant callus forms shoots, transfer it to rooting medium to cultivate seedlings.

[0065] The culture medium formulation for the infection of plant materials is as follows: Induction medium: N6 medium is the basal medium, supplemented with 2 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.8 g / L Proline and 80 mg / L Cysteine.

[0066] Co-culture medium: N6 medium is the basal medium, supplemented with 2 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 100 μM AS (acetylsyleugenol) and 8 g / L Agar (agar).

[0067] Screening medium: N6 medium was used as the basal medium, supplemented with 2 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 40 mg / L Hygromycin B, 300 mg / L Cefotaxime sodium and 8 g / L Agar.

[0068] Differentiation medium: MS medium was used as the basal medium, supplemented with 2.0 mg / L 6-BA (6-benzylaminopurine), 0.5 mg / L NAA (α-naphthaleneacetic acid), 40 mg / L Hygromycin B, 300 mg / L Cefotaxime sodium, and 8 g / L Agar. Rooting medium: based on 2.2 g / L MS medium, with the addition of 10 g / L Sucrose and 4 g / L Phytagel.

[0069] (2) Positive identification After the transformed material emerges, T0 positive seedlings are obtained. Rice genomic DNA is then extracted using the AB solution extraction method, suitable for large-scale screening of positive plants. The specific steps are as follows: ① Prepare solutions A and B. Solution A (lysis buffer): 10 mL 20% Tween-20, 2 mL 5M NaOH, 88 mL H2O; Solution B (equilibration buffer): 10 mL 1M Tris-HCl, 0.4 mL 0.5M EDTA-Na2, 89.6 mL H2O. ② Cut the tender leaves of rice into small pieces and add them to a 96-well plate in sequence to obtain multiple samples; ③ Add 80 μL of solution A to each sample and heat in a PCR instrument at 95℃ for 10 min to obtain a mixture; ④ In step ③, when the temperature of the mixture is reduced to room temperature, add 80 μL of solution B, and then shake to mix thoroughly to obtain the total mixture; ⑤ Centrifuge the total mixture at 12000 rpm for 5 min, collect the supernatant, detect the DNA concentration, dilute the DNA concentration to <1µg / μL, and perform PCR amplification.

[0070] Then, referring to the product instructions for the 2× M5 HiPer plus Taq HiFi PCR mix from Polymer Biotechnology Co., Ltd., PCR amplification was used to confirm that the exogenous DNA fragment had been transferred into the rice genome. The specific reaction system is shown in Table 3: Table 3 Positive Identification Reaction System

[0071] Reaction procedure: 95℃ for 3 min; 94℃ for 25 s, 57℃ for 25 s, 72℃ for 15 s, 35 cycles; 72℃ for 5 min. Positive results were identified by electrophoresis.

[0072] T0 positive seedlings were propagated by planting and growing them normally to obtain seeds. After propagation, high-quality DNA was extracted and a system was prepared using the AceQ qPCR Probe Master Mix from Nanjing Novizan Biotechnology Co., Ltd. Copy number was then determined using qPCR. Single-copy materials were selected for propagation, and copy number was determined again. Double-copy materials were then selected for further experiments.

[0073] The qPCR reaction system is shown in Table 2. The reaction program is as follows: 95℃ for 5 min; 95℃ for 10 s, 60℃ for 10 s, 72℃ for 10 s, 40 cycles; 95℃ for 5 s, 65℃ for 1 min, 97℃ for 5 s; 40℃ for 30 s.

[0074] The phenotypes of the wild-type material and the double-copy material of "Zhongkefa 5" were obtained after treatment at room temperature (26℃) and low temperature (14℃) for 0 days, 5 days, and 10 days, as shown below. Figure 4 As shown. WT represents the phenotype of the wild-type material, and RUBY represents the phenotype of the double-copy material.

[0075] Figure 4 After being treated at low temperatures for two days, the wild-type material showed a normal-colored green embryo, while Figure 4 The double-copy material prepared in this invention exhibits a red embryo phenotype after low-temperature treatment, indicating that the promoter expression of the cold-resistance gene activates the RUBY gene under low temperature, thus producing a red signal. Therefore, the RUBY gene can be used to identify rice plants with normal expression of the cold-resistance gene.

[0076] Example 3 The double-copy materials from the same batch as those showing red signals in Example 2 were mutagenized and then treated at 14°C for 5-10 days. The materials showing red signals were rice plants with normal expression of the cold-resistant gene, while the materials without red signals were rice mutants of the cold-resistant gene.

[0077] In summary, the screening method provided by this invention is used for screening mutants of rice. It achieves intuitive mutant identification through color change, and phenotypic differentiation can be achieved in a maximum of 10 days of plant culture. It is suitable for screening mutants, and the phenotypic screening results are more explicit.

[0078] The above are preferred embodiments of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method of visualizing screening of rice mutants, characterized in that, The method comprises the following steps: transferring a recombinant expression vector based on an inducible promoter into rice to obtain a transgenic plant, breeding the transgenic plant, and then screening the transgenic plant to obtain a mutant, wherein the recombinant expression vector based on the inducible promoter comprises a promoter of a candidate gene and a reporter gene, and the screening comprises a step of low-temperature treatment; the candidate gene is OsDREB1B, and a promoter sequence of the OsDREB1B gene is shown as SEQ ID NO. 1; or the candidate gene is OsABA8ox1, and a promoter sequence of the OsABA8ox1 gene is shown as SEQ ID NO. 2; or the candidate gene is OsCYP19-4, and a promoter sequence of the OsCYP19-4 gene is shown as SEQ ID NO. 3; or the candidate gene is OsTPP1, and a promoter sequence of the OsTPP1 gene is shown as SEQ ID NO. 4; or the candidate gene is OsLEA9, and a promoter sequence of the OsLEA9 gene is shown as SEQ ID NO.

5.

2. The method of visualizing and screening rice mutants according to claim 1, wherein, the reporter gene is a RUBY reporter gene.

3. The method of visualizing and screening rice mutants according to claim 1, wherein, amplification primers of the promoter of the OsDREB1B gene are as follows: F: 5'-AGTTGTCCTTCTAGTAACAA-3'; R: 5'-TGAACCAGAGAGAGTCATCC-3'.

4. The method of visualizing and screening rice mutants according to claim 1, wherein, amplification primers of the promoter of the OsABA8ox1 gene are as follows: F: 5'-TCATATTCCTCATAGATAAACT-3'; R: 5'-TAAGTGAAGTGAGCAGGCGCCA-3'.

5. The method of visualizing and screening rice mutants according to claim 1, wherein, amplification primers of the promoter of the OsCYP19-4 gene are as follows: F: 5'-TTTATTTTTTTTCAGGTATGCC-3'; R: 5'-GCTTGCTCATTCTGTTTATATC-3'.

6. The method of visualizing and screening rice mutants according to claim 1, wherein, amplification primers of the promoter of the OsTPP1 gene are as follows: F: 5'-CACAATGTATATGAAACTTTTAG-3'; R: 5'-GTAGTAGGATTTAAAAGGGTT-3'.

7. The method of visualizing and screening rice mutants according to claim 1, wherein, amplification primers of the promoter of the OsLEA9 gene are as follows: F: 5'-TTTGGAGACCGTGTCACTGT-3'; R: 5'-AGAAAGAAAGTTGATCATCC-3'.

8. The method of visualizing and screening rice mutants according to claim 1, wherein, the low-temperature treatment is to culture the seeds after breeding at not higher than 26 ℃ for at most 10 days.

9. The method of visualizing and screening rice mutants according to claim 2, wherein, the construction method of the recombinant expression vector based on the inducible promoter comprises the following steps: selecting a sequence in 1.7-2.5 kb upstream of a transcription start site of a candidate gene as a promoter sequence; subjecting an empty vector to double enzyme digestion to obtain an enzyme-digested vector, amplifying a fragment 1 by taking the promoter of the candidate gene as a template, amplifying a fragment 2 by taking a RUBY reporter gene as a template, and subjecting the fragment 1, the fragment 2 and the enzyme-digested vector to multi-fragment ligation to obtain the recombinant expression vector.

10. Application of the method for visualizing and screening rice mutants according to any one of claims 1-9 in rice breeding.

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