Application of OsMAPK6 gene in improving cold tolerance of rice

Abstract

The application of OsMAPK6 gene in improving the cold tolerance of rice at the seedling stage relates to the technical field of plant genetic engineering, and particularly relates to the application of OsMAPK6 gene in improving the cold tolerance of rice at the seedling stage. The application aims to solve the problem of low survival rate of rice at the seedling stage after suffering from cold damage in the existing rice growth process. The application of OsMAPK6 gene in improving the cold tolerance of rice at the seedling stage. After suffering from low-temperature stress, the survival rate of OsMAPK6 mutant dsg1 significantly decreases, the ion permeability significantly increases, the aniline blue staining significantly strengthens, and the expression level of low-temperature response genes significantly decreases, while the OsMAPK6 constitutive overexpression transgenic plant CAMAPK6-OE is completely opposite. The application provides important theoretical support for rice breeding, and has important reference value for cultivating high-quality and stress-resistant rice germplasm.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of the OsMAPK6 gene in improving the cold tolerance of rice. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops, with more than half of the world's population relying on it as their staple food. However, rice is a cold-sensitive crop, and with global climate change, low temperatures have become one of the main threats to rice yield and quality. Identifying and analyzing the functions of genes that regulate cold stress in rice is of significant theoretical and applied value for stabilizing national economic development and ensuring food security.

[0003] Chilling injury in rice includes delayed chilling injury and chilling injury during the booting stage. The seedling stage is a critical period for delayed chilling injury in rice. Chilling injury during this stage manifests as yellowing leaves, reduced tiller number, decreased root activity, or even death. The booting stage is the most sensitive to low-temperature stress. Chilling injury during this stage leads to pollen abortion, poor differentiation of branches and spikelets, reduced grains per panicle, and decreased seed setting rate, all of which severely impact rice yield and quality.

[0004] The MAPK cascade signaling pathway plays a crucial role in plant growth and development, as well as in responses to various biotic and abiotic stresses. In rice, it comprises 75 MAPKKK genes, 8 MAPKK genes, and 17 MAPK family genes.

[0005] OsMAPK6 is a member of the MAPK family. It can positively regulate rice grain shape and rice blast response, and negatively regulate the number of grains per panicle. However, its function in seedling cold tolerance has not been reported. Summary of the Invention

[0006] The present invention aims to address the problem of low survival rate of rice seedlings after encountering cold damage during the existing rice growth process, and to provide the application of the OsMAPK6 gene in improving the cold tolerance of rice.

[0007] This invention provides the application of the OsMAPK6 gene in improving the cold tolerance of rice. The nucleotide sequence of the OsMAPK6 gene is shown in SEQ ID NO: 1 in the sequence listing.

[0008] Furthermore, under cold stress conditions, the OsMAPK6 gene can improve the survival rate of rice seedlings.

[0009] Furthermore, under cold stress conditions, the OsMAPK6 gene can reduce ion permeability in rice seedlings.

[0010] Furthermore, under cold stress conditions, the OsMAPK6 gene can reduce aniline blue staining in rice seedlings.

[0011] Furthermore, under cold stress conditions, the OsMAPK6 gene can enhance the expression level of low-temperature response genes in rice.

[0012] Furthermore, the rice low-temperature response genes are OsCBF1, OsCBF2, OsCBF3 and OsTPP1.

[0013] The beneficial effects of this invention are:

[0014] This invention is the first to discover that OsMAPK6 positively regulates cold tolerance in rice seedlings. This provides important theoretical support for rice breeding and has significant reference value for cultivating high-quality, stress-resistant rice germplasm.

[0015] This invention demonstrates through low-temperature stress treatment experiments that the OsMAPK6 mutant dsg1 is hypersensitive to low-temperature stress and its survival rate is significantly reduced, while the survival rate of the OsMAPK6 overexpressing transgenic plant CAMAPK6-OE is significantly increased.

[0016] This invention demonstrates through ion permeability measurement experiments that after low-temperature stress treatment, the ion permeability of dsg1 increases significantly, while the ion permeability of CAMAPK6-OE decreases significantly, indicating that low-temperature stress causes more severe damage to dsg1.

[0017] This invention demonstrates through aniline blue staining experiments that dsg1 staining is stronger after low-temperature stress treatment, while CAMAPK6-OE staining is weaker, indicating that low-temperature stress causes more severe cell damage to dsg1.

[0018] This invention demonstrates, through the detection of low-temperature response gene expression levels, that the expression levels of OsTPP1, OsCBF1, OsCBF2, and OsCBF3 in dsg1 were significantly downregulated, while CAMAPK6-OE was significantly upregulated, regardless of whether the temperature was normal or after low-temperature stress treatment. This further illustrates that OsMAPK6 positively regulates the cold tolerance of rice seedlings. Attached Figure Description

[0019] Figure 1 The overall morphology of Zhonghua 11 (ZH11), dsg1, and CAMAPK6-OE before low-temperature treatment is shown.

[0020] Figure 2 The overall morphology of Zhonghua 11 (ZH11), dsg1, and CAMAPK6-OE after 3 days of low-temperature treatment is shown.

[0021] Figure 3 The overall morphology of Zhonghua 11 (ZH11), dsg1, and CAMAPK6-OE after 5 days of recovery at normal temperature is shown.

[0022] Figure 4 The survival rates of Zhonghua 11 (ZH11), dsg1, and CAMAPK6-OE under low-temperature stress treatment;

[0023] Figure 5 The results of ion permeability analysis for Zhonghua 11 (ZH11), dsg1 and CAMAPK6-OE under low temperature stress treatment;

[0024] Figure 6 The results of aniline blue staining under low-temperature stress treatment of Zhonghua 11 (ZH11), dsg1 and CAMAPK6-OE;

[0025] Figure 7 The expression characteristics of OsCBF1 in ZH11, dsg1 and CAMAPK6-OE under ambient and low temperature stress conditions;

[0026] Figure 8 The expression characteristics of OsCBF2 in ZH11, dsg1 and CAMAPK6-OE under ambient and low temperature stress conditions;

[0027] Figure 9 The expression characteristics of OsCBF3 in ZH11, dsg1 and CAMAPK6-OE under ambient and low temperature stress conditions;

[0028] Figure 10 The expression characteristics of OsTPP1 in ZH11, dsg1 and CAMAPK6-OE under ambient and low temperature stress conditions. Detailed Implementation

[0029] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0030] This invention experimentally demonstrates that the OsMAPK6 gene can positively regulate cold tolerance in rice seedlings. Specifically, after exposure to low-temperature stress, the survival rate of the OsMAPK6 mutant dsg1 significantly decreased, ion permeability significantly increased, aniline blue staining significantly intensified, and the expression level of low-temperature response genes significantly decreased. In contrast, the constitutive overexpression transgenic plant CAMAPK6-OE of OsMAPK6 showed the complete opposite. This indicates that OsMAPK6 positively regulates cold tolerance in rice seedlings. This discovery of a novel function of the OsMAPK6 gene provides genetic resources for breeding high-quality, stress-resistant new rice germplasm.

[0031] Example 1: Analysis of Low Temperature Stress Treatment

[0032] 1. Wild-type ZH11 (Zhonghua 11), dsg1 (OsMAPK6 mutant), and CAMAPK6-OE (OsMAPK6 constitutive overexpression transgenic plant) were used as experimental materials. Seeds were soaked in distilled water for approximately 24 hours, then germinated at 37℃ for approximately 48 hours. Once the seeds showed signs of sprouting, they were inoculated into 96-well hydroponic containers. The seeds of dsg1 and CAMAPK6-OE used in this example were donated to the research group of Professor Li Yunhai at the State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The experimental materials dsg1 and CAMAPK6-OE used have been published in the following articles: (1) Xu, R., Duan, P., Yu, H., Zhou, Z., Zhang, B., Wang, R., Li, J., Zhang, G., Zhuang, S., Lyu, J., Li, N., Chai, T., Tian, ​​Z., Yao, S., and Li, Y. (2018b). Control of Grain Size and Weight by the OsMKKK10-OsMKK4-OsMAPK6 Signaling Pathway in Rice. Mol Plant 11, 860-873. (2) Liu, SY, Hua, L., Dong, SJ, Chen, HQ, Zhu, XD, Jiang, JE, Zhang, F., Li, YH, Fang, XH, and Chen, F. (2015). OsMAPK6, a mitogen-activated protein kinase,influences ricegrain size and biomass production.Plant Journal 84,672-681.

[0033] 2. Under 28℃ conditions, light for 14 hours and dark culture for 10 hours until the product reaches the size of a 2-week-old plant. During this period, change the nutrient solution daily.

[0034] Nutrient solution formula: Add 1ml of Coolaber's 800×Yoshida rice nutrient solution component one (containing nitrogen + phosphorus + potassium), 1ml of 800×Yoshida rice nutrient solution component two (containing CaCl2), and 1ml of 800×Yoshida rice nutrient solution component three (containing trace elements such as Mg) to 797ml of water, and adjust the pH to 5.5-5.8.

[0035] 3. Adjust the low-temperature incubator to 4℃ in advance, transfer the ZH11, dsg1, and CAMAPK6-OE seedlings from step 2 to the low-temperature incubator, and incubate under light for 14 hours, then in the dark for 10 hours, and then treat with low temperature for 3 days.

[0036] 4. Transfer the seedlings from step 3 to a normal temperature incubator and continue culturing for 5 days.

[0037] 5. Calculate the survival rate.

[0038] Figure 1-3 The images show the overall morphology of ZH11, dsg1, and CAMAPK6-OE before, 3 days after, and 5 days after recovery at normal temperature, respectively. It can be seen that after low-temperature treatment, dsg1 exhibited wilting and curling leaves compared to the wild-type ZH11, while CAMAPK6-OE remained in good condition compared to the control. Survival rate statistics revealed that CAMAPK6-OE achieved a survival rate of approximately 90%, while dsg1 only had a survival rate of approximately 37% (e.g., ...). Figure 4 The results indicate that OsMAPK6 dysfunction significantly reduces cold tolerance in rice seedlings, while OsMAPK6 function recovery significantly improves cold tolerance in rice seedlings. This demonstrates that OsMAPK6 can positively regulate cold tolerance in rice seedlings.

[0039] Example 2: Ion Permeability Analysis

[0040] 1. Using ZH11, dsg1, and CAMAPK6-OE as experimental materials, soak the seeds in distilled water for about 24 hours, then germinate them at 37℃ for 48 hours. When the seeds show signs of sprouting, inoculate them into a 96-well hydroponic box.

[0041] 2. Under 28℃ conditions, light for 14 hours and dark culture for 10 hours until the product reaches the size of a 3-week-old plant. During this period, change the nutrient solution daily.

[0042] 3. Take 0.5g of leaf parts from ZH11, dsg1, and CAMAPK6-OE seedlings at 28℃, cut them into small pieces, place them in 15ml of deionized water, and then shake them at 200rpm / min for 2 hours at 28℃.

[0043] 4. Use a conductivity pen to measure the conductivity of the sample from step 3 at room temperature, which is C1.

[0044] 5. Then boil the sample from step 4 for 20 minutes, and after cooling to room temperature, measure the conductivity of the sample as C2.

[0045] 6. The conductivity of deionized water is C0.

[0046] 7. Use the formula: Relative ion permeability = (C1-C0) / (C2-C0)×100% to calculate the relative ion permeability of ZH11, dsg1, and CAMAPK6-OE at room temperature.

[0047] 8. Adjust the low-temperature incubator to 4℃ in advance, and transfer the remaining seedlings of ZH11, dsg1, and CAMAPK6-OE from step 3 to the low-temperature incubator. Illuminate for 14 hours, dark culture for 10 hours, and treat with low temperature for 24 hours and 48 hours.

[0048] 9. After 24h and 48h of low-temperature treatment, take 0.5g of leaf parts from ZH11, dsg1 and CAMAPK6-OE seedlings respectively, cut them into small pieces and place them in 15ml of deionized water, and then shake them at 200rpm / min for 2h at 28℃.

[0049] 10. After processing the samples in step 9 for 24 hours and 48 hours, place them at room temperature and boil them, respectively. Then, measure the conductivity of the samples after room temperature and after boiling.

[0050] 11. Using the formula in step 7, calculate the relative ion permeability of ZH11, dsg1, and CAMAPK6-OE after 24h and 48h of cold treatment.

[0051] 12. Statistical analysis of data, and whether the differences in ion permeability of ZH11, dsg1, and CAMAPK6-OE after cold treatment are significant.

[0052] The ion permeability analysis results of ZH11, dsg1, and CAMAPK6-OE under low-temperature stress treatment are as follows: Figure 5 As shown, Indicates ZH11, Indicates CAMAPK6-OE, The figure represents dsg1. After 24 hours of low-temperature stress treatment, the ion permeability of CAMAPK6-OE reached approximately 30%, significantly lower than that of the wild type; while the ion permeability of dsg1 reached approximately 70%, significantly higher than that of the wild type. Similarly, after 48 hours of low-temperature stress treatment, the ion permeability of CAMAPK6-OE reached approximately 55%, significantly lower than that of the wild type; while the ion permeability of dsg1 reached approximately 90%, significantly higher than that of the wild type. All of these findings indicate that OsMAPK6 can positively regulate the cold tolerance of rice seedlings.

[0053] Example 3: Aniline Blue Staining

[0054] 1. Using ZH11, dsg1, and CAMAPK6-OE as experimental materials, the seeds were soaked in distilled water for 24 hours, then germinated at 37℃ for 48 hours. When the seeds showed signs of sprouting, they were inoculated into 96-well hydroponic boxes.

[0055] 2. Under 28℃ conditions, light for 14 hours and dark culture for 10 hours until the product reaches the size of a 2-week-old plant. During this period, change the nutrient solution daily.

[0056] 3. Transfer the rice seedlings from step 2 to a low-temperature light incubator and subject them to low-temperature stress treatment for 48 hours.

[0057] 4. Immerse the leaf parts of ZH11, dsg1, and CAMAPK6-OE from step 3 into an aniline blue solution at 70°C, heat in boiling water for 2 minutes, and observe the staining process at any time.

[0058] Aniline blue solution formulation: 2.5 mg / ml aniline blue + 25% lactic acid (w / v) + 23% water-saturated phenol + 25% glycerol

[0059] 5. Use hydrated trichloroacetaldehyde solution to decolorize the sample from step 3. If the decolorization effect is not good, you can use 70% ethanol + 30% glacial acetic acid for decolorization.

[0060] Formula for hydrated trichloroacetaldehyde solution: Dissolve 25g of hydrated trichloroacetaldehyde in 10ml of water.

[0061] 6. Transfer the sample from step 4 to 70% glycerol and take a picture to record the results.

[0062] The results of aniline blue staining under low-temperature stress conditions of ZH11, dsg1, and CAMAPK6-OE are as follows: Figure 6 As shown, aniline blue staining is one of the indicators of cell integrity. Cells with good integrity cannot be stained by aniline blue, while cells with poor integrity are easily stained by aniline blue. From Figure 6 As can be seen, after low temperature treatment, the coloring degree of dsg1 was significantly enhanced, while the coloring degree of CAMAPK6-OE was significantly weakened compared with the control. This indicates that low temperature stress caused more severe damage to dsg1, while the damage to CAMAPK6-OE was relatively less, further demonstrating that the OsMAPK6 gene can positively regulate the cold tolerance of rice seedlings.

[0063] Example 4: Detection of low-temperature response gene expression levels

[0064] 1. Low temperature treatment of rice seedlings: ZH11, dsg1, and CAMAPK6-OE seedlings that were 2 weeks old were treated in a 4℃ low temperature light incubator for 6 hours.

[0065] 2. RNA extraction:

[0066] 1) Take the leaf tissues of ZH11, dsg1, and CAMAPK6-OE from step 1 before and after cold treatment for 6 hours, wrap them in tin foil and freeze them in liquid nitrogen.

[0067] 2) Preheat the mortar with alcohol to remove RNase, and then use it after it has cooled to room temperature.

[0068] 3) Quickly transfer the cryogenically frozen RNA extract sample to a mortar pre-cooled with liquid nitrogen, and grind the tissue thoroughly with a pestle until it becomes a bright green powder. Liquid nitrogen can be added during the process.

[0069] 4) Transfer the sample from step 3) to a 1.5 ml centrifuge tube to remove RNase, add 1 ml TransZol Up, mix thoroughly with a vortex mixer, and let stand at room temperature for 5 min.

[0070] 5) Add 0.2 ml of chloroform to step 4), shake vigorously for 30 seconds, and incubate at room temperature for 3 minutes.

[0071] 6) Centrifuge at 10000×g, 4℃ for 15 min. At this point, the sample separates into three layers: a colorless aqueous phase (upper layer), a middle layer, and a pink organic phase (lower layer). RNA is mainly in the aqueous phase.

[0072] 7) Transfer 0.5 ml of colorless aqueous phase to a new RNase-free 1.5 ml centrifuge tube, add 0.5 ml of isopropanol, invert to mix, and incubate at room temperature for 10 min.

[0073] 8) Centrifuge at 10000×g, 4℃ for 10min. Discard the supernatant, as a gel-like precipitate forms on the sides and bottom of the tube.

[0074] 9) Add 1 ml of 75% ethanol (prepared with DEPC water) and vortex vigorously.

[0075] 10) Centrifuge at 7500×g, 4℃ for 5 min. Discard the supernatant, and centrifuge again for 1 min to thoroughly remove excess liquid.

[0076] 11) Let the sediment settle at room temperature for about 5 minutes.

[0077] 12) Add 30 μl of RNA lysis solution to step 11).

[0078] 13) Incubate in a metal bath at 65℃ for 10 min, measure the sample concentration, and store the sample in a -20℃ refrigerator.

[0079] 3. RNA is reverse transcribed into cDNA

[0080] 1) Adjust the concentration of the RNA samples in step 2 to approximately 500 ng.

[0081] 2) Add 1 μl of 5×gDNA Eraser Buffer, 0.5 μl of gDNA Eraser, 2.5 μl of RNase-free H2O, and 1 μl of RNA to a dedicated PCR tube.

[0082] 3) Run the sample from step 2) in a PCR instrument at 42°C for 2 minutes.

[0083] 4) Add 0.5 μl primer Script RT Emzyme Mix 2, 0.5 μl RTprimer Mix, 2 μl 5×primer Script Buffer 2, and 2 μl RNase free H2O to the sample from step 3).

[0084] 5) Run the sample from step 4) in a PCR instrument at 37°C for 15 min, then at 85°C for 5 s, and store at 4°C.

[0085] 6) The PCR system is 10 μl.

[0086] 7) Reverse transcribe RNA into cDNA.

[0087] 4. RT-qPCR detection

[0088] 1) Add 40 μl of deionized water to the sample from step 3 for reverse transcription to dilute.

[0089] 2) Reaction system: 5 μl 2×T5 Fast qPCR Mix + 0.4 μl forward primer + 0.4 μl reverse primer + 4.2 μl DNA, OsCBF1 primer (F1+R1), OsCBF2 primer (F2+R2), OsCBF3 primer (F3+R3), OsTPP1 primer (F4+R4), ubiquitin primer (F5+R5).

[0090] Forward primer F1: 5'-GAGACCTTCGCCAACGATG-3'

[0091] Reverse primer R1: 5'-CACCGGCAACACGTCCTT-3'

[0092] Forward primer F2: 5'-TACGGCAACATGGACTTCGA-3'

[0093] Reverse primer R2: 5'-CCCATCCCGTCGTAGTAGTAG-3'

[0094] Forward primer F3: 5'-AGCGACCTGGCGTTCG-3'

[0095] Reverse primer R3: 5'-TCGCGTAGTACAGGTCCCA-3'

[0096] Forward primer F4: 5'-TGTCTCCCGTGATGAGAGCTG-3'

[0097] Reverse primer R4: 5'-AAACACCTTATTGCGGGACCTT-3'

[0098] Forward primer F5: 5'-TGTGAAGATTGTGATGCCCTAC-3'

[0099] Reverse primer R5: 5'-CCACGCCAACGGATAAAA-3'

[0100] 3) Establish a qPCR reaction system in a 96-well plate specifically designed for ABI real-time PCR instrument.

[0101] 4) The reaction procedure is a two-step method: pre-denaturation at 95℃ for 2 min; denaturation at 95℃ for 10 s; annealing and extension at 60℃ for 15 s (35 cycles, signal acquisition).

[0102] 5) RT-qPCR statistics: Data were obtained from the Bio-Rad chromo 4 real-time PCR detector; fold change was analyzed using the 2-ΔΔCT method.

[0103] like Figure 7-10 The figures show the expression characteristics of the low-temperature response genes OsCBF1, OsCBF2, OsCBF3, and OsTPP1 before and after low-temperature treatment in ZH11, dsg1, and CAMAPK6-OE, respectively. The results showed that the expression levels of these four low-temperature response genes were significantly decreased in dsg1 and significantly increased in CAMAPK6-OE, indicating that OsMAPK6 can activate the expression of OsCBF1, OsCBF2, OsCBF3, and OsTPP1. Similarly, after low-temperature stress treatment, low temperature can induce the expression of OsCBF1, OsCBF2, OsCBF3, and OsTPP1 genes, with a more significant upregulation in CAMAPK6-OE and a more significant downregulation in dsg1. This further demonstrates that OsMAPK6 positively regulates the cold tolerance of rice seedlings.

Claims

1. Application of OsMAPK6 gene in positive regulation of cold tolerance in rice seedlings.

2. The application according to claim 1, characterized in that: Under cold stress conditions, the OsMAPK6 gene can improve the survival rate of rice seedlings.

3. The application according to claim 1, characterized in that: Under cold stress conditions, the OsMAPK6 gene can reduce ion permeability in rice seedlings.

4. The application according to claim 1, characterized in that: Under cold stress conditions, the OsMAPK6 gene can reduce aniline blue staining in rice seedlings.

5. The application according to claim 1, characterized in that: Under cold stress conditions, the OsMAPK6 gene can enhance the expression level of low-temperature response genes in rice.

6. The application according to claim 5, characterized in that: The rice low-temperature response gene is OsCBF1, OsCBF2 , OsCBF3 and OsTPP1 .

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