Cold adaptation regulatory gene RAF3 / RAF6 and application thereof

Abstract

This invention provides a cold adaptation regulatory gene RAF3 / RAF6 This invention relates to the field of genetic engineering technology and its applications. Specifically, it provides a cold adaptation regulatory gene. RAF3 / RAF6 , wherein RAF3 The genome sequence is shown in SEQ ID No. 1. RAF6 The genome sequence is shown in SEQ ID No. 2. This invention also provides the... RAF3 / RAF6 The encoded protein, and its role in reducing RAF3 / RAF6 By reducing the expression level of a protein or decreasing its activity or content, the cold tolerance and photosynthetic efficiency of plants can be improved, meaning that under cold conditions, RAF3 and / or RAF6 It reduced photosynthesis while enhancing the expression of cold-response genes, ultimately inhibiting plant growth and promoting plant adaptation to cold stress.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a cold adaptation regulatory gene. RAF3 / RAF6 And its applications. Background Technology

[0002] Low temperatures have always been a major factor limiting agricultural production, severely inhibiting plant growth and development. Plants also have a sophisticated coping mechanism when faced with low temperatures. Chloroplasts, which are highly sensitive within plant cells and act as signal sensors, are particularly vulnerable to cold stress.

[0003] Taking rice (Oryza sativa) as an example, a key aspect of rice breeding programs is improving crop productivity by optimizing the composition of grain yield and plant structure. However, rice growth and development are frequently and severely affected by various biotic and abiotic stresses. Frost, cold waves, and low-temperature damage are major meteorological disasters that frequently occur in late spring and early autumn. Globally, the frequent occurrence of low-temperature disasters results in losses of hundreds of billions of dollars to agricultural and forestry production. Therefore, researching plant cold resistance-related genes and improving plant tolerance to low-temperature stress is of great significance.

[0004] In recent years, with the development of molecular biology, significant progress has been made in the research of rice cold resistance theory, including the cloning of several key cold resistance-related genes or QTLs. These key genes can effectively improve the plant's tolerance to low temperatures. However, current genetic engineering practices mostly utilize constitutive promoters to drive the expression of these key genes. While the resulting transgenic plants may exhibit strong cold resistance, constitutive promoters often lead to unnecessary expression, such as stunted growth, developmental delays, and wasted energy, which is detrimental to potential practical applications. Summary of the Invention

[0005] This invention provides a cold adaptation regulatory gene RAF3 / RAF6 and its applications, the gene RAF3 / RAF6 Negative regulation of cold tolerance in plants can be achieved by inhibiting genes in the target plant. RAF3 / RAF6 The expression of RAF protein can be reduced, or the content and / or activity of RAF protein in the target plant can be decreased, thereby increasing the plant's cold tolerance and photosynthetic efficiency.

[0006] This invention provides a cold adaptation regulatory gene, the cold adaptation regulatory gene comprising: RAF3 or RAF6 , wherein RAF3The genome sequence is shown in SEQ ID No. 1. RAF6 The genome sequence is shown in SEQ ID No. 2.

[0007] In a preferred embodiment of the present invention, the... RAF3 The CDS sequence is shown in SEQ ID No. 3. RAF6 The CDS sequence is shown in SEQ ID No. 4.

[0008] The present invention also provides the protein encoded by the above-mentioned cold adaptation regulatory gene.

[0009] In a preferred embodiment of the present invention, RAF3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 5. RAF6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 6.

[0010] This invention also provides the application of the above-mentioned cold adaptation regulatory genes or proteins in regulating plant cold tolerance.

[0011] In a preferred embodiment of the present invention, the regulation includes inhibiting the expression level of the cold adaptation regulatory gene or reducing the content and / or activity of the protein to improve the cold tolerance of the plant.

[0012] The present invention also provides the application of reagents that inhibit the expression of the above-mentioned cold adaptation regulatory genes or reagents that reduce the content and / or activity of the above-mentioned proteins in improving plant cold tolerance.

[0013] The present invention also provides a method for breeding cold-resistant plants, including inhibiting the expression of the above-mentioned cold adaptation regulatory genes in the target plant, or reducing the content and / or activity of the above-mentioned proteins.

[0014] In a preferred embodiment of the present invention, the target plant includes a dicotyledonous plant.

[0015] The present invention also provides a biomaterial for inhibiting the expression of the aforementioned cold adaptation-regulated genes.

[0016] Beneficial effects: This invention utilizes forward genetics to screen mutants from an Arabidopsis mutant library, and through molecular identification, clones the cold adaptation regulatory gene. RAF3 / RAF6 , wherein RAF3 The genome sequence is shown in SEQ ID No. 1. RAF6 The genome sequence is shown in SEQ ID No. 2. Upon comparison, the cold adaptation regulatory gene... RAF3 / RAF6It belongs to the MAPKKK (MAP kinase kinase kinase) kinase family. In this invention, it was demonstrated that downregulating the expression of the aforementioned cold adaptation regulatory genes in plants can improve plant cold tolerance and photosynthetic efficiency. Specifically, under cold conditions, RAF3 and / or RAF6 reduce photosynthesis while enhancing the expression of cold-response genes, ultimately inhibiting plant growth and promoting plant adaptation to cold stress. Attached Figure Description

[0017] Figure 1 for RAF3 and RAF6 Schematic diagram of gene structure;

[0018] Figure 2 for raf3 and raf6 Mutant identification diagram;

[0019] Figure 3 For 4-week-old WT (Col-0) raf3-1 , raf3-2 , raf6-1 and raf6-2 In mutants RAF3 and RAF6 Figure showing the results of RT-qPCR analysis at the transcriptional level;

[0020] Figure 4 Image showing the results of immunolocalization and quantification analysis of RAF3 and RAF6;

[0021] Figure 5 for raf3-1 , raf3-2 , raf6-1 and raf6-2 Photosynthetic phenotype of mutant plants under cold stress;

[0022] Figure 6 The photosynthetic phenotype of the RAF mutant complementary line under cold stress;

[0023] Figure 7 Figure 1 shows the growth status and quantitative analysis results of rosette leaf diameter in 3-week-old mutants under cold stress.

[0024] Figure 8 for raf3-1 , raf3-2 , raf6-1 and raf6-2 Schematic diagram of the expression pattern of cold response genes in mutants. Detailed Implementation

[0025] This invention provides a cold adaptation regulatory gene, the cold adaptation regulatory gene comprising: RAF3 or RAF6 , whereinRAF3 The genome sequence is shown in SEQ ID No. 1. RAF6 The genome sequence is shown in SEQ ID No. 2.

[0026] This invention used forward genetics to screen four mutant strains from the Arabidopsis mutant library (Arashare), two of which were RAF3 protein kinase mutants: raf3-1 (SALK_099651) and raf3-2 (SALK_046150), and the other two were RAF6 protein kinase mutants: raf6-1 (SALK_036615) and raf6-2 (SALK_029929). Through molecular identification, the cold adaptation regulatory gene was cloned. RAF3 / RAF6 The cold adaptation regulatory gene RAF3 / RAF6 It is a member of the MAPKKK (MAPkinase kinase kinase) kinase group.

[0027] The present invention RAF3 have Figure 1 The structure shown has a genome sequence as indicated in SEQ ID No. 1, which contains a 2643 bp CDS sequence (SEQ ID No. 3) encoding 880 amino acids (SEQ ID No. 5); RAF6 have Figure 1 The structure shown has a genome sequence as shown in SEQ ID No. 2, which contains a 2871 bp CDS sequence (SEQ ID No. 4) encoding 957 amino acids (SEQ ID No. 6).

[0028] The present invention also provides the protein encoded by the above-mentioned cold adaptation regulatory gene.

[0029] The present invention RAF3 The amino acid sequence of the gene-encoded protein is shown in SEQ ID No. 5. RAF6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID No. 6.

[0030] This invention also provides the application of the above-mentioned cold adaptation regulatory genes or proteins in regulating plant cold tolerance.

[0031] The regulation described in this invention includes inhibiting the expression level of the cold adaptation regulatory gene or reducing the content and / or activity of the protein, thereby improving the plant's cold tolerance and photosynthetic efficiency.

[0032] This invention demonstrates that under normal conditions, the accumulation of chloroplast-localized RAF3 and RAF6 is negligible. However, after cold treatment, the levels of chloroplast-localized RAF3-Flag and RAF6-Flag significantly increase. Furthermore, the absence of RAF3 and / or RAF6 leads to an increase in Fv / Fm during cold stress, indicating that RAF3 and / or RAF6 negatively regulate chloroplast photosynthetic activity under low-temperature stress. Specifically, under cold conditions, RAF3 and / or RAF6 reduce photosynthesis while enhancing the expression of cold-response genes, ultimately inhibiting plant growth and promoting plant adaptation to cold stress. The cold-response genes described in this invention include... DREB1 (AT1G01250) CAMTA3 (AT2G22300) CBF1 (AT4G25490) and CBF2 (AT4G25470).

[0033] The present invention also provides the application of reagents that inhibit the expression of the above-mentioned cold adaptation regulatory genes or reagents that reduce the content and / or activity of the above-mentioned proteins in improving plant cold tolerance.

[0034] This invention inhibits the expression of RAF3 / 6 by constructing T-DNA insertion mutants. For example, in one embodiment, Arabidopsis thaliana is used as an example. In Arabidopsis thaliana, the T-DNA of the raf3-1 (SALK_099651) and raf3-2 (SALK_046150) mutants is inserted into the second exon and the first exon of the AT5G11850 gene, respectively; the T-DNA of the raf6-1 (SALK_036615) and raf6-2 (SALK_029929) mutants is inserted into different sites in the first exon of the AT4G24480 gene, thereby inhibiting the expression of RAF3 / 6.

[0035] This invention reduces the content and activity of RAF3 / 6 proteins by constructing T-DNA insertion mutants. For example, in one embodiment, RAF3 mutants were constructed: raf3-1 (SALK_099651) and raf3-2 (SALK_046150) mutants were constructed, with T-DNA inserted into the second and first exons of the AT5G11850 gene, respectively; RAF6 mutants were constructed: raf6-1 (SALK_036615) and raf6-2 (SALK_029929) mutants were constructed, with T-DNA inserted into different sites in the first exon of the AT4G24480 gene. These mutants result in the loss of function of the RAF3 and RAF6 genes, thereby reducing the content and activity of the corresponding proteins, which can be used to study their effects on physiological processes such as plant photosynthesis and cold stress response.

[0036] The present invention also provides a method for breeding cold-resistant plants, including inhibiting the expression of the above-mentioned cold adaptation regulatory genes in the target plant, or reducing the content and / or activity of the above-mentioned proteins.

[0037] The target plants described in this invention include dicotyledonous plants, such as Arabidopsis thaliana in one embodiment.

[0038] The present invention also provides a biomaterial for inhibiting the expression of the aforementioned cold adaptation-regulated genes.

[0039] The biomaterials described in this invention may include the Arabidopsis mutant materials constructed to inhibit the expression of the aforementioned genes, such as the raf3 mutant and the raf6 mutant.

[0040] To further illustrate the present invention, the following examples demonstrate a cold adaptation regulatory gene provided by the present invention. RAF3 / RAF6 The invention and its applications are described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0041] In this embodiment of the invention, Arabidopsis thaliana is cultivated using a soil culture method. Specifically, the Arabidopsis thaliana seeds to be sown are placed in a 1.5 ml centrifuge tube with an appropriate amount of water added. The centrifuge tube is placed in a 4°C refrigerator in the dark for 3 days. Then, the vernalized seeds are sown on the soil surface and incubated at 22°C with 100 μmol / L water. -2 s -1 The plants were grown in a cultivation room with light intensity and a light cycle of 12h / 12h (day / night). Experiments were conducted after the plants had grown for 30 days.

[0042] Example 1

[0043] Arabidopsis mutant raf3-1 , raf3-2 , raf6-1 , raf6-2 Homozygosity identification

[0044] 1. Screening of Arabidopsis mutants

[0045] Four mutant strains were screened from the Arabidopsis mutant library (Arashare) using forward genetics, two of which were RAF3 protein kinase mutants: raf3-1 (SALK_099651) and raf3-2 (SALK_046150), the other two are RAF6 protein kinase mutants: raf6-1 (SALK_036615) and raf6-2 (SALK_029929).

[0046] 2. Extraction of total DNA from Arabidopsis thaliana

[0047] The method described in the reference (Huang, J., Ge, X., and Sun, M. (2000). Modified CTAB protocol using a silica matrix for isolation of plant genomic DNA. BioTechniques 28, 432, 434) uses the CTAB method for DNA extraction.

[0048] 3. Total RNA extraction from plants and real-time quantitative PCR (qPCR)

[0049] (1) RNA extraction

[0050] Total RNA was extracted from the plant according to the instructions of the RNA extraction kit (HiPure Total RNA Mini Kit, Magen, R4151).

[0051] (2) RNA reverse transcription

[0052] Reverse transcription was performed using the Takara RNA Reverse Transcription Kit (PrimeScript RT Reagent Kit with gDNAEraser, Takara).

[0053] ① Prepare a 15μL system: 5μg RNA, 2μL gDNA Eraser, 4μL 5x gDNA Eraser Buffer and the remainder RNase-free ddH2O; remove gDNA using a PCR instrument: 42℃ for 2min, store at 4℃.

[0054] ② Add 20 μL of reagent mixture to the reaction product from ①: PrimeScript RT Enzyme Mix I 2 μL, RT Primer Mix 2 μL, 5×PrimeScript Buffer 8 μL, and RNase-free ddH2O 8 μL. Perform reverse transcription in a PCR instrument: 37℃ for 15 min; 85℃ for 5 s; store at 4℃.

[0055] (3) Detecting relative gene expression levels

[0056] qPCR reaction premix (2×SYBR Green PCR Premix HS Taq, AG, AG11702)

[0057] qRT-PCR loading system (10 μL): 0.3 μL F end of qPCR primer, 0.3 μL R end of qPCR primer, 5 μL 2×SYBRGreen PCR Premix HS Taq (protected from light), 0.5 μL cDNA and 3.9 μL ddH2O.

[0058] The well-mixed reaction solution was added to a 384-well plate and centrifuged horizontally at 3000 rpm for 5 min at room temperature. The 384-well plate was then placed in a LightCycler 480 instrument, and the program was run. After the program finished, data analysis was performed, and the relative expression levels of the detected genes were calculated. All experiments included three technical replicates.

[0059] Program: 95℃ 30s; 95℃ 5s, 60℃ 30s, 40 cycles; 95℃ 5s, 60℃ 1min, 60-95℃ (0.11℃ / s); 50℃ 30s.

[0060] 4. Arabidopsis mutants raf3-1 , raf3-1 , raf6-1 , raf6-2 Homozygosity identification

[0061] The primers used for identification are shown in Table 1.

[0062] Table 1 Primer list for homozygosity identification

[0063]

[0064] PCR detection system (20 μL): DNA 2 μL, primer R 0.5 μL, primer F 0.5 μL, 2 Taq 10 μL and sterile water 7 μL.

[0065] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min / kb, 32 cycles; 72℃ extension for 7 min.

[0066] The results are as follows Figure 1 and Figure 2 As shown, compared to WT plants, the mutant's T-DNA is inserted in exon 2 (raf3-1, SALK_099651) and exon 1 (raf3-2, SALK_046150) of AT5G11850. In the AT5G11850 mutant... raf6-1 (SALK_036615) and raf6-2 In (SALK_029929), T-DNA is inserted at different sites in the first exon of AT4G24480.

[0067] Using the primers shown in Table 2, qPCR analysis was performed. RAF3 and RAF6 The gene expression status, the results are as follows Figure 3 As shown, mutant raf3-1 / raf3-2, raf6-1 / raf6-2 In the study, the expression of RAF3 and RAF6 genes was significantly lower than that of wild-type Col-0.

[0068] Table 2 RAF3 and RAF6 qPCR detection primer information

[0069]

[0070] Example 2

[0071] RAF3 and RAF6 Immunolocalization and quantification

[0072] 1. Experiment on the isolation and purification of chloroplasts from plant tissues

[0073] (1) Chloroplast extraction

[0074] ① Place 2g of Arabidopsis thaliana leaves in a mortar, add 5ml of Isolation Buffer, and grind the leaves into a homogenate. Then, pass the homogenate through double-layered nylon gauze and transfer the filtrate to a new 10ml centrifuge tube. Pour the residue on the gauze back into the mortar, add 5ml of Isolation Buffer, and grind again. Repeat this process 2-3 times. All the above operations should be performed on ice.

[0075] ② Centrifuge the filtrate at 1000g for 5 minutes at 4℃. The coarse chloroplasts will precipitate at the bottom of the centrifuge tube after centrifugation. Remove the supernatant and add 500μl of Isolation Buffer to resuspend the coarse chloroplasts.

[0076] (2) Chloroplast isolation and purification

[0077] ① Prepare the Percoll gradient solution (two-step): The lower layer is a mixture of 2.55 ml percoll solution and 0.45 ml gradient mixture, and the upper layer is a mixture of 2.94 ml percoll solution and 4.06 ml gradient mixture. After adding the lower layer solution to a 15 ml centrifuge tube, slowly add the upper layer solution to the surface of the lower layer solution using a syringe. Clear stratification will be visible on the liquid surface.

[0078] ② Slowly add the crude chloroplast extract to the top layer of the liquid, and then centrifuge at 1500g for 10 minutes at 4℃. The intact chloroplasts after centrifugation will be located between the upper and lower Percoll interfaces.

[0079] ③ After centrifugation, the broken chloroplasts will aggregate in the upper layer. After removing the upper layer solution, transfer the intact chloroplasts in the middle layer to a new 10 ml centrifuge tube. Then, add 5 ml of HMS Buffer to resuspend the chloroplasts, tilt the tube once to wash away Percoll, centrifuge at 1000g for 5 min at 4℃, discard the supernatant, and resuspend in 1 ml of HMS Buffer. Store at -80℃.

[0080] The formulations of each extract are shown below:

[0081] Isolation buffer: 5mM EGTA, 5mM MgCl2, 0.3M Sorbitol, 20mM HEPES-KOH (pH8.0), 5mM EDTA and 10mM NaHCO3;

[0082] HMS buffer: 50mM HEPES-NaOH, 3mM MgSO4 and 0.3M Sorbitol;

[0083] 2.55mL Percoll solution: 0.0255g Ficoll ® 400, 2.1419mL Percoll™, 0.0255gBSA, 0.0765g PEG 6000 and 0.4081mL ddH2O;

[0084] 2.94mL Percoll solution: 0.0294g Ficoll ® 400, 0.0882g PEG 6000, 2.4695mLPercoll™, 0.0294g BSA and 0.4705mL ddH2O;

[0085] Gradient Mixture: 10mM EDTA (pH 8.0), 25mM HEPES-NaOH (pH 8.0) and 5% (w / v) Sorbitol.

[0086] 2. SDS-PAGE protein electrophoresis and Western blotting

[0087] (1) Protein extraction and concentration determination

[0088] ① Place 2-3 fresh leaves from an Arabidopsis thaliana plant into a 1.5ml centrifuge tube containing steel balls and freeze in liquid nitrogen. Grind the leaves using a high-throughput tissue homogenizer and add 100μl of pre-cooled total protein extract from the leaves.

[0089] ② Centrifuge the centrifuge tube at 12,000 rpm for 5 minutes at 4°C, transfer the supernatant to a new centrifuge tube, and place it on ice for later use.

[0090] ③ Protein concentration was detected using the BCA protein quantification kit (purchased from Sangon Biotech).

[0091] (2) SDS-PAGE electrophoresis

[0092] ① Add the appropriate volume of 5×Loading Buffer to the protein sample, incubate in a 100℃ metal bath for 5 minutes, and then prepare for sample loading.

[0093] ② During the stacking stage, the instrument voltage is 70V. After the strip enters the separating gel, the voltage is adjusted to 120V.

[0094] (3) Transfer membrane

[0095] ① Prepare a PVDF membrane with a size similar to that of the gel, immerse the entire membrane in methanol for 10 seconds, and then clean the membrane using a clean transfer solution.

[0096] ② Place the transfer clamp parallel to the container containing clean transfer solution, and assemble the transfer system in the following order: anode—filter paper of appropriate thickness—PVDF membrane—gel—filter paper of appropriate thickness—cathode.

[0097] ③ Place the assembled transfer clamp vertically into the transfer tank filled with clean transfer solution, and then place the transfer tank stably into a suitable container filled with an ice-water mixture. Transfer the membrane for 90 minutes under a constant current of 350mA.

[0098] (4) Background blocking: Remove the PVDF membrane after transfer, cut the membrane to the desired target protein location according to the marker size, and rinse the membrane thoroughly with TBST. Then place the membrane in a container with 5% skim milk and place the container on a shaker overnight for blocking (4°C). The next day, wash the membrane 3-5 times with TBST, each time for 10 minutes on a shaker.

[0099] (5) Antibody incubation: Dilute the corresponding primary antibody with TBST according to the instructions. Cover the membrane with the diluted solution and incubate on a horizontal shaker at room temperature for 2 hours. After use, recover the primary antibody and wash the PVDF membrane 4-6 times with TBST, 10 minutes each time on a shaker. Dilute the corresponding secondary antibody with TBST according to the instructions. Cover the membrane with the diluted solution and incubate on a horizontal shaker at room temperature for 1 hour. Wash the membrane four times with TBST, 15 minutes each time.

[0100] (6) Development: Place the incubated membrane under the imaging analysis system, and uniformly drop a 1:1 prepared luminescent reaction solution (Thermo Scientific™ SuperSignal™ 34580) onto the membrane, and record the results.

[0101] Intact chloroplasts were isolated from Arabidopsis plants under control and cold stress conditions. Immunoblotting was performed using polyclonal antibodies targeting FLAG, PSII subunit D1, and ACTIN. Two independent biological replicates were also performed, with similar results.

[0102] The results are as follows Figure 4 As shown, immunoblotting analysis of RAF3-Flag and RAF6-Flag proteins in whole leaves and chloroplasts revealed that, under control conditions, the accumulation of chloroplast-localized RAF3 and RAF6 was negligible. However, after cold treatment, the levels of chloroplast-localized RAF3-Flag and RAF6-Flag significantly increased.

[0103] Example 3

[0104] Cold stress raf Photosynthetic activity detection of mutants

[0105] 1. Cold-treated detached blade experiment

[0106] Take plant leaves that have grown for 30 days, lay them flat in a petri dish containing water, dry them, and then place the culture medium in a constant temperature and light incubator. The culture environment temperature is 4℃, and 100μmol m -2 s -1 Light intensity, with a light cycle of 12h / 12h (day / night).

[0107] 2. Chlorophyll fluorescence parameter detection

[0108] After the Arabidopsis thaliana samples to be measured were placed in a dark environment for 20 minutes to acclimatize, chlorophyll fluorescence parameters were measured using a chlorophyll fluorescence imaging system (MAXI-type IMAGING-PAM). The measurement method involved first turning on the IMAGING-PAM instrument and software, setting the required parameters in the settings, then placing the sample to be measured in the instrument's detection area, and finally turning on the saturation pulse to measure its maximum photosynthetic efficiency (F). v / F m ).

[0109] 3. Construction of expression vectors for RAF3-GFP and RAF6-GFP fusion proteins

[0110] Table 3 Primers used for vector construction

[0111]

[0112] The PCR reaction system consisted of: 1 µL cDNA template, 0.5 µL each of forward and reverse primers, 12.5 µL 2×PCR Mix, and 10.5 µL H₂O. The amplification conditions were: 98 °C pre-denaturation for 3 min; 98 °C denaturation for 15 s, 55 °C annealing for 15 s, 72 °C extension for 120 s, for 32 cycles; and a final extension at 72 °C for 5 min. The PCR products were then separated on a 1% agarose gel.

[0113] Using wild-type Arabidopsis thaliana (Col-0) cDNA as a template, the corresponding gene fragment was amplified using specific primers. The fragment was then ligated into the expression vector PCAMIA1300-GFP by digestion with TAKERA enzymes at the BamHI and SALI restriction sites. The vector was transformed into E. coli DH5α competent cells, and single clones were picked for plasmid DNA extraction and enzyme digestion identification. Positive clones were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. Correct sequencing yielded the 35S promoter-driven fusion protein expression vector. RAF3 -GFP、 RAF6 -GFP.

[0114] Wild-type at 4℃ raf3-1 , raf3-2 , raf6-1 and raf6-2 Chlorophyll fluorescence was detected in detached leaves of the mutant. The results showed that, in the untreated state, the PSII primary light energy conversion efficiency (Fi) between the mutant and the wild type was significantly different. v / F m There was no significant difference, but under short-term treatment at 4°C, the F of the RAF-like protein kinase mutant was significantly different. v / F m It was significantly higher than the wild type. The results were as follows... Figure 5 As shown.

[0115] In addition, this invention also utilizes genetic transformation methods to achieve... raf3-1 and raf3-2 Constructing Expressions RAF3 -GFP transgenic complementary lines, in raf6-1 and raf6-2 Constructing Expressions RAF6 -GFP transgenic complementary line. Results as follows: Figure 6 As shown, the transgenic complementary line will use the F1 mutant. v / F m The level recovered to that of wild-type plants, confirming that the mutant F under cold stress... v / F m The increase is indeed due to lack of RAF3 and RAF6 .illustrate RAF3 and RAF6 It negatively regulates chloroplast photosynthetic activity under low temperature stress.

[0116] Example 4

[0117] The role of RAF in plant growth

[0118] The expression of cold-response genes in RAF mutants was detected by RT-qPCR, especially the expression of cold-response genes in the third leaf of 4-week-old Arabidopsis plants after 10 hours of low-temperature treatment. ACTIN2 (At3g18780) was used as an internal reference gene. -ΔΔCt Method for detecting cold response genes DREB1 (AT1G01250) CAMTA3 (AT2G22300) CBF1 (AT4G25490) and CBF2 The relative expression level of (AT4G25470).

[0119] Table 4 Primer information for detecting cold response gene expression levels

[0120]

[0121] The results are as follows Figure 7 and Figure 8 As shown, after prolonged low-temperature treatment, such as 15 days of cold exposure, on wild-type and RAF mutant plants, the mutants showed significantly better performance compared to the control. raf3-1 and raf6-1 The diameter of the rosette leaves is significantly smaller than that of the wild-type plant. Meanwhile, RAF The lack of photosynthesis increased but decreased the expression of cold-response genes. This indicates that under cold conditions, RAF It reduced photosynthesis while enhancing the expression of cold-response genes, ultimately inhibiting plant growth and promoting plant adaptation to cold stress.

[0122] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of cold adaptation regulatory genes in regulating plant cold tolerance, characterized in that, The regulation involves suppressing the expression of the cold adaptation regulatory gene to improve the plant's cold tolerance; the cold adaptation regulatory gene is... RAF3 or RAF6 , wherein RAF3 The genome sequence is shown in SEQ ID No.

1. RAF6 The genome sequence is shown in SEQ ID No. 2; The plant in question is Arabidopsis thaliana.

2. The application according to claim 1, characterized in that, The RAF3 The CDS sequence is shown in SEQ ID No.

3. RAF6 The CDS sequence is shown in SEQ ID No.

4.

3. The application of a reagent that inhibits the expression of cold adaptation regulatory genes in improving plant cold tolerance, characterized in that, The cold adaptation regulatory gene is RAF3 or RAF6 , wherein RAF3 The genome sequence is shown in SEQ ID No.

1. RAF6 The genome sequence is shown in SEQ ID No. 2; The plant in question is Arabidopsis thaliana.

4. A breeding method for cold-resistant plants, characterized in that, This includes suppressing the expression of cold adaptation regulatory genes in target plants, wherein the cold adaptation regulatory genes are... RAF3 or RAF6 , wherein RAF3 The genome sequence is shown in SEQ ID No.

1. RAF6 The genome sequence is shown in SEQ ID No. 2; The target plant is Arabidopsis thaliana.

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