Application of osdrs1 gene in improving drought resistance of rice

Abstract

The application provides application of an OsDRS1 gene in improving drought resistance of rice, a nucleotide sequence of the OsDRS1 gene is shown as SEQ ID NO:1, and an amino acid sequence of a coded protein of the OsDRS1 gene is shown as SEQ ID NO:2. Compared with a wild type (WT), a survival rate of osdrs1 mutant plant under PEG-induced and soil dehydration drought stress is significantly higher than that of the WT, and a leaf relative water loss rate after dehydration is reduced; a survival rate of an OsDRS1 overexpression plant OsDRS1-OE under drought stress is significantly lower than that of the WT, and a leaf relative water loss rate after dehydration is higher. Compared with the WT, more stomata of the osdrs1 mutant plant are closed after PEG-induced drought stress, and accumulation of hydrogen peroxide and superoxide anion is reduced; less stomata of the OsDRS1 overexpression plant OsDRS1-OE are closed after PEG-induced drought stress, and accumulation of hydrogen peroxide and superoxide anion is increased. Compared with the WT, more abiotic stress response factors OsABF2 and OsABI5 proteins are accumulated in the osdrs1 mutant plant after PEG-induced drought stress.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological genes and drought resistance of rice, and particularly relates to application of an OsDRS1 gene in improving drought resistance of rice. BACKGROUND

[0002] Rice is one of the most important food crops in the world. In recent years, with the change of climate, the available fresh water resources are reduced. Because rice has a high requirement for water, drought will seriously affect the yield of rice. Therefore, it is of great significance to explore the genes related to drought response of rice and elucidate the regulation mechanism for breeding drought-resistant rice with high efficiency of water use.

[0003] Abscisic acid (ABA) is a very important hormone in plants, which is involved in the response of plants to abiotic stress (drought, high temperature, high salt, etc.), and is therefore called "stress hormone". When plants are subjected to stress, the ABA in the body will increase sharply, and then transported to the aboveground part through the xylem, inhibits the opening of stomata, and then closes the stomata, thereby reducing water transpiration and regulating (inducing or inhibiting) the expression of stress-related genes. For example, there are 9 AREB / ABFs transcription factors in Arabidopsis, and ABF2 and ABF4 can be induced to express by drought and high salt stress, and positively regulate drought resistance and salt tolerance in the ABA-dependent signal pathway; AtAREB3 can bind to the promoter region of stomatal regulation gene AtADF5 to activate and start transcription, and enhance the drought resistance of plants by regulating the opening of leaf stomata. It is found that ABFs can not only induce the expression of downstream resistance genes, but also can play a function by interacting with some proteins, such as AtABF2 improves the drought resistance of Arabidopsis by interacting with AtDELLA. The above shows the importance of ABA signal transduction factor ABFs in regulating the drought resistance of plants.

[0004] Autophagy is considered as a basic defense and stress regulation mechanism, which promotes cell repair, reconstruction and regeneration. Autophagy can be induced by nutritional stress, drought stress, osmotic stress, salt stress and other stresses. Plants resist abiotic stress by activating autophagy. It is found that overexpression of MdATG10 in apple has more biomass accumulation under drought conditions than wild type, less accumulation of reactive oxygen species under long-term drought stress, and lower damage to biological membrane system, which significantly enhances the drought resistance of plants. Arabidopsis atg5 and atg7 mutants are not tolerant to drought stress. MtCAS31 forms MtATG8a-MtCAS31-MtPIP2;7 protein complex as an autophagy receptor, enhances autophagic degradation of water channel protein MtPIP2;7, and thus reduces water loss under drought stress and improves plant drought tolerance. When screening drought resistance of rice genetic materials, it is found that overexpression of autophagy-related protein DRS1 (Drought sensitivity 1) is sensitive to drought stress. However, there are few reports on the regulation of autophagy to drought resistance in rice, and the role of autophagy-mediated ABFs regulation mechanism in the response of rice to drought stress is not clear. SUMMARY

[0005] To overcome the problems in the related art, the purpose of the present application is to provide the application of OsDRS1 gene in improving the drought resistance of rice, which has important significance for improving the drought resistance of rice and can be used as a new target for improving the drought resistance of rice.

[0006] The application of OsDRS1 gene in improving the drought resistance of rice, the nucleotide sequence of the OsDRS1 gene is shown as SEQ ID NO: 1, the drought resistance of mutant rice osdrs1 obtained by mutation of the OsDRS1 gene is enhanced, and the drought resistance of overexpression strain OsDRS1-OE obtained by overexpression of the OsDRS1 gene is weakened.

[0007] In the preferred technical solution of the present application, the amino acid sequence of the encoded protein of the OsDRS1 gene is shown as SEQ ID NO: 2.

[0008] In the preferred technical solution of the present application, the improvement of the drought resistance of mutant rice osdrs1 is to improve the survival rate of plants in response to soil dehydration of rice and drought stress caused by PEG.

[0009] The application of the mutant rice osdrs1 of the OsDRS1 gene to improve the survival rate of rice under PEG simulated drought stress conditions.

[0010] The application of the mutant rice osdrs1 of the OsDRS1 gene to improve the survival rate of rice under soil drought stress conditions.

[0011] In the preferred technical solution of the present application, the improved drought resistance of the mutant rice osdrs1 is that the leaf of the mutant rice osdrs1 plant osdrs1 closes more stomata and reduces the leaf water loss rate when responding to the drought stress caused by PEG.

[0012] In the preferred technical solution of the present application, the improved drought resistance of the mutant rice osdrs1 is that the leaf of the mutant rice osdrs1 plant osdrs1 reduces the accumulation of hydrogen peroxide and superoxide anion when responding to the drought stress caused by PEG.

[0013] In the preferred technical solution of the present application, the mutant rice osdrs1 of the rice OsDRS1 gene accumulates more ABA abiotic stress response proteins OsABF2 and OsABI5 under the condition of PEG simulated drought stress.

[0014] In the preferred technical solution of the present application, the mutant rice osdrs1 is obtained by knocking out the OsDRS1 gene of the rice through gene editing technology, or producing one or more than one deoxynucleotide mutation at the coding region of the OsDRS1 gene using gene editing technology.

[0015] In the preferred technical solution of the present application, the rice is japonica rice.

[0016] In the preferred technical solution of the present application, the japonica rice is Zhonghua 11.

[0017] The present application has the following beneficial effects:

[0018] (1) Compared with the wild type, the survival rate of the mutant rice osdrs1 of the OsDRS1 gene after soil drought stress and PEG simulated drought stress is significantly higher than that of the wild type plant, and the survival rate of the OsDRS1 gene overexpression plant OsDRS1-OE is significantly lower than that of the wild type plant. It is inferred that OsDRS1 negatively regulates the drought resistance of rice.

[0019] (2) The mutant rice osdrs1 of the OsDRS1 gene can improve the drought resistance of rice to the drought stress caused by PEG, and the leaf closes more stomata and reduces the leaf water loss rate.

[0020] (3) The mutant rice osdrs1 of the OsDRS1 gene can reduce the accumulation of hydrogen peroxide and superoxide anion when responding to the drought stress caused by PEG.

[0021] (4) The mutant rice osdrs1 of the OsDRS1 gene can improve the drought resistance of rice to the drought stress caused by PEG, and increase the accumulation of ABA abiotic stress response proteins OsABF2 and OsABI5.

[0022] OsDRS1 gene is a potential new target for the study of rice drought resistance mechanism. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1 is the performance of OsDRS1 mutant and overexpression plants under PEG simulated drought treatment. Wherein, Figure a is the drought treatment phenotype of wild type plants and mutant osdrs1; Figure b is the drought treatment phenotype of wild type plants and overexpression line OsDRS1-OE; Figure c is the survival rate statistical result of wild type plants and mutant osdrs1 after drought treatment; Figure d is the survival rate statistical result of wild type plants and overexpression line OsDRS1-OE after drought treatment.

[0024] Figure 2 Figure 2 is the performance of OsDRS1 mutant and overexpression plants under soil drought treatment. Wherein, Figure a is the drought treatment phenotype of wild type plants and mutant osdrs1; Figure b is the drought treatment phenotype of wild type plants and overexpression line OsDRS1-OE; Figure c is the survival rate statistical result of wild type plants and mutant osdrs1 after drought treatment; Figure d is the survival rate statistical result of wild type plants and overexpression line OsDRS1-OE after drought treatment.

[0025] Figure 3 Figure 3 is the stomatal conductance and relative water loss rate analysis of wild type, mutant osdrs1 and OsDRS1-OE overexpression plants after PEG simulated drought treatment. Wherein, Figure a is a representative photo of completely closed, partially open and completely open stomata of rice leaves. Figure b is the proportion of different stomata types. ** indicates extremely significant, n=3. Figure c is the relative water loss rate of leaves after PEG treatment.

[0026] Figure 4 Figure 4 is the antioxidant activity detection of wild type, mutant osdrs1 and OsDRS1-OE overexpression plants after PEG simulated drought treatment. Wherein, Figure a is DAB staining, DAB staining method is also called diaminobenzidine method, which is used to detect the active site of peroxidase in cells. Figure b is NBT staining, NBT staining method is also called nitro blue tetrazolium staining method, which is used to detect the active site of peroxidase and superoxide anion in cells.

[0027] Figure 5Western blot analysis of wild type, mutant osdrs1 after PEG simulated drought treatment. Among them, Anti-ABF2: rice OsABF2 specific antibody, Anti-ABI5: rice OsABI5 specific antibody, Anti-Actin: plant internal reference antibody. CK: normal growth conditions, PEG: 20% PEG treatment. The numbers in figures a and b are the ratio of the gray value of the protein of WT CK treatment as a reference. DETAILED DESCRIPTION

[0028] Preferred embodiments of the present application will be described in more detail with reference to the accompanying drawings. Although preferred embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present application is more thorough and complete, and the scope of the present application is fully conveyed to those skilled in the art.

[0029] Plant material: The test material of this study is japonica rice (Oryza sativa L.). The wild type (WT) is Zhonghua 11 (ZH11) (publicly available rice variety, commercially available). The genetic transformation of OsDRS1 genetic material is constructed by Baige Gene Technology (Jiangsu) Co., Ltd.

[0030] Chemical reagents: Polyethylene glycol 6000 (PEG6000) was purchased from Shengong Bioengineering (Shanghai) Co., Ltd., NBT, DAB staining solution was purchased from Regen Biochemical Co., Ltd.; Anti-Actin was purchased from Yixing Biological Co., Ltd., Shengong Bioengineering (Shanghai) Co., Ltd., OsABF2 and OsABI5 antibodies were prepared in the laboratory.

[0031] Example 1: PEG drought stress phenotype analysis

[0032] The present application sowed japonica rice varieties wild type (WT), OsDRS1 mutant plant (osdrs1), OsDRS1 overexpression plant (OsDRS1-OE) in Guangzhou, Guangdong Province, South China Agricultural University farm after harvest.

[0033] The mutant rice osdrs1 knocks out the OsDRS1 gene of rice by gene editing technology, or produces one or more deoxy nucleotide mutations at the coding region of the OsDRS1 gene using gene editing technology.

[0034] Tested rice: wild type, osdrs1, OsDRS1-OE

[0035] The specific steps are as follows: just after germination, wild type, osdrs1, OsDRS1-OE seeds are placed in a 96-well plate with holes at the bottom, one seed in each well, 5 columns for each of the two strains, and then placed in a culture box containing rice nutrient solution, cultured at 28°C with light (16 hours light / 8 hours dark), and after the rice seedlings grow to 2 weeks old, treated with 20% polyethylene glycol 6000 (PEG6000) for 5 days, and then added with rice nutrient solution for 7 days. The survival and death of seedlings of each strain are counted, and the survival rate is calculated. Each rice variety is repeated three times.

[0036] The results are shown in Figure 1 Figure 1 The scale is 5 cm. Each rice variety is repeated three times, ** indicates t test compared with wild type (WT), * indicates significant, ** indicates extremely significant, n=3. Phenotypic analysis shows that the growth state of wild type, osdrs1, OsDRS1-OE before treatment is consistent, and wild type, osdrs1, OsDRS1-OE all appear different degrees of damage after treatment and recovery. Compared with WT, but the damage degree of osdrs1 plant is lighter ( Figure 1 a), and the damage degree of OsDRS1-OE plant is heavier ( Figure 1 b). The survival rate is counted, the survival rate of wild type is 25%-30%, the survival rate of osdrs1 mutant plant is 60%-70% ( Figure 1 c), which is significantly higher than that of wild type, and the survival rate of OsDRS1-OE overexpression plant is about 10% ( Figure 1 d), which is significantly lower than that of wild type. It shows that the mutation of osdrs1 improves the tolerance of rice under drought stress, and the overexpression of OsDRS1-OE reduces the tolerance of rice under drought stress. It is inferred that OsDRS1 negatively regulates the drought resistance of rice.

[0037] Example 2: Soil drought stress phenotype analysis

[0038] The present application sows wild type (WT), OsDRS1 mutant plant (osdrs1), and OsDRS1 overexpression plant (OsDRS1-OE) of japonica rice variety in Guangzhou, Guangdong Province, and plants are sowed in pots for soil drought stress.

[0039] Tested rice: wild type, osdrs1, OsDRS1-OE

[0040] ​Specific steps are as follows: just after the wild type, osdrs1, OsDRS1-OE seedlings are planted in the wet soil, each strain of various 30 seedlings, 28°C light culture (16 hours light / 8 hours dark), when the rice seedlings grow 4 weeks old, stop watering, drought treatment for 7 days and then add rice nutrient solution for 7 days. Statistics of each strain of survival and death of seedlings, calculate the survival rate. Each rice variety is repeated three times.

[0041] Results are shown in Figure 2 Phenotype analysis found that, after drought treatment and rehydration, wild type, osdrs1, OsDRS1-OE all appear different degrees of damage. Compared with WT, but osdrs1 plants are less damaged ( Figure 2 a), OsDRS1-OE plants are more damaged ( Figure 2 b). Statistics of survival rate, the survival rate of wild type is 10%-20%, the survival rate of osdrs1 mutant plants is about 50% ( Figure 2 c), significantly higher than that of wild type, the survival rate of OsDRS1-OE overexpression plants is about 5% ( Figure 2 d), significantly lower than that of wild type. It shows that the mutation of osdrs1 improves the tolerance of rice under drought stress, and the overexpression of OsDRS1-OE reduces the tolerance of rice under drought stress. It is inferred that OsDRS1 negatively regulates drought resistance of rice.

[0042] Example 3: Analysis of stomatal conductance and leaf water loss rate of plant leaves after PEG treatment

[0043] Tested rice: wild type, osdrs1, OsDRS1-OE

[0044] Specific steps are as follows:

[0045] (1) Analysis of stomatal conductance of rice leaves: wild type, osdrs1, OsDRS1-OE rice seedlings are cultured for 5 weeks, and then treated with PEG for 3 days to simulate drought. The same part of the flag leaf of rice is cut and placed in glutaraldehyde for preservation. The specific method is referred to the sample preparation method of scanning electron microscope. Then the sample is prepared by alcohol grading dehydration, gold spraying, and mounting. The opening and closing of stomata of rice leaves are observed by scanning electron microscope, photographed and counted.

[0046] Results are shown in Figure 3As shown, the proportion of completely open stomata in OsDRS1-OE leaves was significantly higher than that in wild-type rice leaves under normal conditions, and the proportions of partially open and completely closed stomata were significantly lower than those in wild-type rice. Compared with normal culture conditions, the proportion of completely open stomata in wild-type rice leaves decreased after PEG treatment, and the proportion of completely closed stomata increased. After PEG treatment, the proportion of completely closed stomata in OsDRS1-OE leaves was significantly lower than that in wild-type rice, and the proportions of partially open and completely open stomata were significantly higher than those in wild-type rice. Figure 3 b) The results show that osdrs1 mutant leaves can close more stomata after PEG stress treatment, and the number of completely open and partially open stomata is reduced. The osdrs1 mutant enhances drought resistance by controlling the water consumption of stomata.

[0047] (2) Water loss rate analysis of rice leaves: Wild-type, osdrs1, and OsDRS1-OE rice seedlings were cultured for 5 weeks, and then the flag leaves of the same part were cut and placed in a culture dish in a light incubator for natural drying and dehydration. The leaf weight was measured and counted according to the time.

[0048] The results are shown in Figure 3 As shown, the relative water loss rate of OsDRS1-OE leaves under drought conditions was significantly higher than that of wild-type rice, and the relative water loss rate of osdrs1 was significantly lower than that of wild-type rice Figure 3 c), and the survival rate of osdrs1 after rehydration was about 50%.

[0049] The above results show that the osdrs1 mutant has stronger water retention ability, and the osdrs1 mutant regulates drought resistance by reducing the opening degree of stomata and / or closing more stomata to reduce water consumption.

[0050] Example 4: DAB and NBT staining analysis of plant leaves after PEG treatment

[0051] Tested rice: wild-type, osdrs1, and OsDRS1-OE

[0052] The specific steps are as follows: Newly sprouted wild-type, osdrs1, and OsDRS1-OE seeds were placed in 96-well plates with holes at the bottom, one seed per well, with 5 rows for each of the two lines. The plates were then placed in culture boxes containing rice nutrient solution and cultured at 28°C under light (16 hours light / 8 hours dark). After the rice seedlings had grown for 2 weeks, they were treated with 20% polyethylene glycol 6000 (PEG6000) to simulate drought for 24 hours, and then samples were taken. The samples were stained according to the instructions of the NBT staining kit (catalog number DP0035) and the DAB staining kit (catalog number DP0041). Each rice variety was repeated three times.

[0053] (1) The results are as follows Figure 4 As shown, under normal culture conditions, the leaves of wild-type, osdrs1, and OsDRS1-OE plants showed no significant difference after DAB staining, all turning white after decolorization. After PEG-simulated drought treatment, compared to WT, OsDRS1-OE plants showed more dark brown spots on their leaves, while osdrs1 plants showed significantly fewer dark brown spots. These results indicate that osdrs1 plants accumulated significantly less catalase in their leaves after PEG treatment, resulting in less reactive oxygen species (ROS) production. Conversely, OsDRS1-OE plants accumulated significantly more catalase in their leaves after PEG treatment, resulting in more ROS production.

[0054] (2) Under normal culture conditions, the leaves of wild-type, osdrs1, and OsDRS1-OE plants showed no significant difference after NBT staining, all turning white after decolorization. After PEG-simulated drought treatment, compared with WT, OsDRS1-OE plant leaves accumulated more blue spots, while osdrs1 plant leaves had significantly fewer blue spots than WT. The results indicate that osdrs1 plant leaves accumulated significantly fewer superoxide anions after PEG treatment than wild-type, and the leaves produced less reactive oxygen species; OsDRS1-OE plant leaves accumulated significantly more superoxide anions after PEG treatment than wild-type, and the leaves produced more reactive oxygen species.

[0055] The fewer staining spots in the two staining experiments above, the stronger the plant's ability to scavenge reactive oxygen species, and the lower the damage the plant suffers. This shows that the osdrs1 mutant plants suffer less damage under drought conditions than the WT mutant, while the OsDRS1-OE plants suffer more damage than the WT mutant. This indicates that the osdrs1 mutant plants improve rice drought resistance by enhancing their antioxidant capacity.

[0056] Example 5: Immunoblot analysis of protein in leaves of plants treated with PEG

[0057] Rice varieties tested: wild type, osdrs1

[0058] The specific steps are as follows: wild type, osdrs1 rice seedlings are normally cultured to 2 weeks old, and then sampled after 24 h of simulated drought treatment with 20% polyethylene glycol 6000 (PEG6000). Then the proteins are extracted, and the samples are subjected to Western blot analysis using specific antibodies.

[0059] The results are shown in Figure 5 Compared with normal growth conditions, PEG treatment promotes the accumulation of OsABFs protein in the leaves of wild type rice and autophagy mutant osdrs1. Compared with WT, autophagy mutant osdrs1 accumulates more OsABFs protein. According to the above results, we preliminarily speculate that autophagy mutant osdrs1 cannot normally induce autophagy activity under drought conditions, and enriches more OsABFs protein, so osdrs1 has higher drought resistance.

[0060] The relative arrangement of parts and steps, numerical expressions, and numerical values set forth in these examples are not meant to limit the scope of the present application, unless otherwise specifically stated. In all of the examples shown and discussed herein, any particular value is to be interpreted as merely an example, and not a limitation. Thus, other examples of example embodiments can have different values. It is noted that like numbers and letters refer to like items throughout the several views, and once an item is defined in one view, it is not necessary to discuss it further in subsequent views.

[0061] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

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Claims

1. Mutations OsDRS1 The application of genes in improving drought resistance of rice is characterized in that, the mutation OsDRS1 genes are mutated OsDRS1 genes are mutated osdrs1 The mutant rice osdrs1 By knocking out the genes of rice through gene editing technology OsDRS1 Genes; The OsDRS1 The nucleotide sequence of the gene is shown as SEQ ID NO: 1; The OsDRS1 The amino acid sequence of the encoded protein of the gene is set forth in SEQ ID NO:

2.

2. The mutation of claim 1 OsDRS1 The application of the gene in improving drought resistance of rice is characterized in that, the mutant rice osdrs1 The improvement of drought tolerance is to improve the survival rate of plants when responding to drought stress caused by rice soil dehydration and PEG.

3. The mutation of claim 1 OsDRS1 The application of the gene in improving drought resistance of rice is characterized in that, the mutant rice osdrs1 The improvement of drought resistance is that the plant closes more stomata and reduces the water loss rate of leaves when the rice is subjected to drought stress caused by PEG.

4. The mutation of claim 1 OsDRS1 The application relates to the use of a gene for improving drought resistance of rice, characterized in that, the mutant rice osdrs1 The increased drought tolerance is the ability of the rice to reduce the accumulation of hydrogen peroxide and superoxide anion in the leaves of the plant under PEG-induced drought stress.

5. The mutation of claim 1 OsDRS1 The application of the gene in improving drought resistance of rice is characterized in that, The mutant rice osdrs1 The improvement of drought resistance is to accumulate more ABA non-biological stress response proteins OsABF2 and OsABI5 proteins when the rice is under drought stress caused by PEG.

6. The mutation according to any one of claims 1 to 5 The rice is japonica. The application relates to the use of a gene for improving drought resistance of rice, characterized in that, OsDRS1 7. The mutation of claim 6 The japonica is Zhonghua No.

11. The application of the gene in improving drought resistance of rice is characterized in that, ​

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