Application of rice OsMAPK7 gene in improving rice submergence tolerance

Abstract

The application discloses application of a rice OsMAPK7 gene in improving waterlogging resistance of rice and belongs to the technical field of gene functions. The nucleotide sequence of the OsMAPK7 gene is shown in SEQ ID NO:1. It is found in the application that by knocking out the OsMAPK7 gene of rice, common non-waterlogging-resistant cultivated rice can be changed into waterlogging-resistant cultivated rice, the germination rate of rice seeds can be improved under waterlogging conditions, the growth of rice seedling roots can be promoted, the survival rate of rice seedlings can be improved, the bottleneck problem faced by direct seeding production of rice can be solved, and thus the production area and yield of rice can be ensured. The application also provides a method for improving waterlogging resistance of rice, and the method has important utilization value in rice production.

Description

Technical Field

[0001] This invention relates to the field of gene function technology, specifically to the application of the rice OsMAPK7 gene in improving the waterlogging tolerance of rice. Background Technology

[0002] Rice is an important food crop. Natural disasters have a significant impact on rice production. For example, floods are common in tropical and subtropical regions. Continuous heavy rainfall during the rice planting season can severely affect rice germination rates, leading to reduced yields. With the migration of young rural laborers to cities, rice production faces a labor shortage. To address this issue, many rural areas are abandoning traditional seedling raising and transplanting methods in favor of direct seeding. Large-scale direct seeding saves a significant amount of labor, alleviates seasonal labor shortages, and is crucial for achieving lighter, more specialized, and larger-scale rice production. It also ensures sufficient rice planting area and yield, showing broad prospects for widespread application. However, during direct seeding, if the paddy field is uneven, some areas become waterlogged, or rain during rice germination causes waterlogging, rice seeds falling into waterlogged areas will not germinate or will have a reduced germination rate, affecting overall seedling emergence. Furthermore, direct-seeded rice suffers from severe weed infestation, necessitating chemical weed control, which increases agricultural input and labor costs, impacts environmental safety, and makes it prone to lodging due to its shallow root system. If the direct-seeded rice variety is flood-tolerant, seed germination can be unaffected even in flooded areas, and the paddy field can even remain submerged for a period after direct seeding. This not only preserves seed germination but also suppresses weed growth, reducing weed damage and eliminating the need for chemical weed control, thus achieving environmental friendliness. In flooded conditions, the more developed root systems of flood-tolerant varieties can further mitigate the lodging problem of direct-seeded rice. Therefore, research on the flood tolerance of rice is a crucial step in ensuring rice production area and yield. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide the application of the rice OsMAPK7 gene in improving the waterlogging tolerance of rice. By knocking out the rice OsMAPK7 gene, the germination rate of rice seeds under waterlogging conditions can be improved, and the root growth of rice seedlings under waterlogging conditions can be promoted.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides the application of the rice OsMAPK7 gene in improving the waterlogging resistance of rice, and the nucleotide sequence of the OsMAPK7 gene is shown in SEQ ID NO:1.

[0006] Preferably, the rice varieties include japonica rice and indica rice.

[0007] Preferably, the application includes improving the germination rate of rice seeds under flooded conditions.

[0008] Preferably, the application includes promoting root growth in rice seedlings under flooded conditions.

[0009] The present invention also provides a method for improving the waterlogging resistance of rice, the method comprising: knocking out the rice OsMAPK7 gene.

[0010] Preferably, the gene knockout method employs CRISPR / Cas9 technology.

[0011] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0012] OsMAPK7 is a gene commonly found in cultivated rice. This invention is the first to discover that the OsMAPK7 gene in rice is related to rice's tolerance to flooding. By knocking out the OsMAPK7 gene in rice, ordinary cultivated rice that is not tolerant to flooding can be transformed into cultivated rice that is tolerant to flooding. Under flooded conditions, this can improve the germination rate of rice seeds, promote the growth of rice seedling roots, and increase the survival rate of rice seedlings, thus solving the bottleneck problem faced by direct-seeded rice production and ensuring the rice production area and yield. Therefore, it has important utilization value in rice production. Attached Figure Description

[0013] Figure 1 Results of sequencing of some CRISPR / Cas9-OsMAPK7 knockout lines;

[0014] Figure 2 Germination and seedling growth of seeds of Zhonghua 11 (ZH11), OsMAPK7-Cas9 and OsMAPK7-OE after 14 days of water immersion in test tubes;

[0015] Figure 3 The growth of seeds of Yasi 881 and Huang Huazhan and their OsMAPK7-Cas knockout plants after 14 days under field flooding conditions. Detailed Implementation

[0016] This invention provides the application of the rice OsMAPK7 gene in improving the waterlogging resistance of rice. The OsMAPK7 gene is a gene that is commonly found in cultivated rice, and its nucleotide sequence is shown in SEQ ID NO:1.

[0017] In this invention, the rice varieties include japonica rice and indica rice, wherein the japonica rice is preferably japonica rice ZH11, and the indica rice is preferably indica rice Yasi 881 and Huanghuazhan.

[0018] The applications described in this invention include improving the germination rate of rice seeds under flooded conditions and promoting the root growth of rice seedlings under flooded conditions.

[0019] The present invention also provides a method for improving the waterlogging resistance of rice, the method comprising: knocking out the rice OsMAPK7 gene, wherein the gene knockout method preferably employs CRISPR / Cas9 technology.

[0020] As an optional implementation method, the method of knocking out the rice OsMAPK7 gene using CRISPR / Cas9 technology includes: designing a target site corresponding to the OsMAPK7 gene, constructing an sgRNA expression cassette by ligating the target primers and the sgRNA expression cassette using the repeat stacking method, cloning the sgRNA expression cassette into a plant gene editing vector, and then constructing transgenic rice by transforming the ligation product with Agrobacterium tumefaciens to obtain rice with the OsMAPK7 gene knocked out.

[0021] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0022] In this embodiment, the plant gene editing vector used was pYLCRISPR / Cas9Pubi-H, the promoters selected were U6a and U6b, and the method used was overlapping PCR and Kinmen cloning.

[0023] Example 1

[0024] The cDNA sequence of the OsMAPK7 gene was obtained from the NCBI website https: / / www.ncbi.nlm.nih.gov. CRISPR-GE primers were used to design target primers for the gene editing vector. Based on the principles of high-efficiency target site design, such as GC content of 45%–70% and off-target < 0.6, the target sites were all promoters U6a and U6b. The multiple cloning site for insertion into the rice OsMAPK7 gene was BsaI.

[0025] The ligation of the target primers to the sgRNA expression cassette was performed using overlapping PCR to construct the sgRNA expression cassette, which involved two rounds of PCR reactions:

[0026] First round of PCR reaction: The target site is introduced downstream of the U6 promoter and upstream of the sgRNA sequence.

[0027] The first step of the PCR reaction is as follows:

[0028] Table 1. PCR reaction system for the first step

[0029]

[0030]

[0031] Table 2. First step PCR reaction procedure

[0032]

[0033] Second round of PCR: Construct the promoter, target site, and sgRNA into a complete expression cassette. Dilute the product from the first round of PCR tenfold for use in the second round of PCR.

[0034] Table 3. PCR reaction system for the second step

[0035]

[0036] Table 4. Second step PCR reaction procedure

[0037]

[0038] Product purification: The PCR product from the previous step was purified to remove proteins, oligonucleotides, etc., to obtain a relatively pure sgRNA expression cassette for subsequent experiments. In this experiment, the Novizan FastPure Gel DNA Extraction Mini Kit was used for PCR product purification. The specific steps are as follows:

[0039] (1) Add ddH2O to the product from the previous step to make up to 100 μL, then add 500 μL of Buffer GDP, mix by inverting, transfer the mixture to the adsorption column, put the adsorption column into the collection tube, put it into the centrifuge, and centrifuge at 13400×g for 60s.

[0040] (2) Remove the centrifuge tube, discard the filtrate, add 700 μL of Buffer GW with anhydrous ethanol to the adsorption column, put the collection tube into the centrifuge, and centrifuge at 13400×g for 60s.

[0041] (3) Repeat step (2);

[0042] (4) Remove the centrifuge tube, discard the filtrate, put the adsorption column back into the collection tube, put the collection tube into the centrifuge, and centrifuge at 13400×g for 2min.

[0043] (5) Take out the centrifuge tube, put the adsorption column into a new 1.5mL centrifuge tube, add 30μL of ddH2O preheated at 55℃ to the center of the adsorption column membrane, let stand at room temperature for 2min, put the collection tube into the centrifuge, centrifuge at 13400×g for 1min, and the filtrate is the purified product.

[0044] sgRNA expression cassette cloned into pYLCRISPR / Cas9Pubi-H vector: The purified product from the previous step and the pYLCRISPR / Cas9Pubi-H vector were digested with Bsa I and simultaneously ligated with T4 DNA ligase. The sgRNA expression cassette was inserted into the pYLCRISPR / Cas9Pubi-H vector using a "golden gate" cloning method, digesting and ligating simultaneously. This reaction was performed on a PCR instrument with the heat shield closed. The specific reaction system and procedure are as follows:

[0045] Table 5. Kinmen Cloning System

[0046]

[0047] Table 6. Kinmen Cloning Procedure

[0048]

[0049] The primers used are as follows:

[0050] OsMAPK7-OsU6a-F: GCCGTACGGCATTGCTCCGTTCAA (SEQ ID NO: 2);

[0051] OsMAPK7-OsU6a-R: AAACTTGAACGGAGCAATGCCGTA (SEQ ID NO: 3);

[0052] OsMAPK7-OsU6b-F: GTTGACTGCTGCGATGCCGTTGAA (SEQ ID NO: 4);

[0053] OsMAPK7-OsU6b-R: AAACTTCAACGGCATCGCAGCAGT (SEQ ID NO: 5).

[0054] The ligation products from the above steps were transferred into DH5α competent *E. coli* using a heat shock method and plated on LB selection plates containing 25 μg / mL kanamycin. The plates were incubated at 37°C for 12–16 h until positive plaques appeared. The plasmids of the positive clones were then transferred into *Agrobacterium*, and *Agrobacterium* was used to transform rice callus tissue.

[0055] Construction of transgenic rice: Callus tissue was induced from mature seeds of Zhonghua 11 (japonica rice), Huanghuazhan (indica rice), and Yasi 881 (indica rice), respectively. After 21 days, the callus was subcultured. Subculture was repeated every 10 days, and after two subcultures, well-formed embryogenic callus tissue was selected for infection with Agrobacterium tumefaciens EHA105. After three days of co-culture, resistant callus tissue was selected for screening. Transgenic plant regeneration was achieved by screening using 50 mg / L hygromycin medium, with screening every two weeks. Well-formed resistant callus tissue was selected for two weeks of pre-differentiation culture, followed by two weeks of differentiation culture. Differentiated seedlings were then transferred to rooting medium to obtain transgenic plants. PCR detection of the hygromycin gene (Hpt) and sequencing of the target gene after PCR reaction yielded CRISPR-Cas9-OsMAPK7 knockout transgenic positive plants. Figure 1 The sequencing results of partial CRISPR / Cas9-OsMAPK7 knockout lines indicate that OsMAPK7 has been edited.

[0056] Seeds of CRISPR-Cas9 transgenic plants with OsMAPK7 knocked out were subjected to germination period flood tolerance experiments under both in vitro and field conditions. The results are shown in [Figure number missing]. Figures 2-3 .

[0057] Figure 2 This figure shows the germination and growth of seeds from wild-type Zhonghua 11 (ZH11), OsMAPK7 knockout (OsMAPK7-Cas9), and OsMAPK7 overexpression (OsMAPK7-OE) plants after 14 days of waterlogging in test tubes. As can be seen from the figure, seeds from OsMAPK7-Cas9 plants germinate normally, seedlings elongate above the water surface, and their root systems are longer than those of wild-type ZH11. This indicates that knocking out OsMAPK7 in japonica rice ZH11 can transform the water-intolerant ZH11 into a water-tolerant ZH11.

[0058] Figure 3The germination and growth of seeds of wild-type Yasi 881, wild-type Huanghuazhan, and their respective OsMAPK7 knockout (OsMAPK7-Cas9) plants after 14 days of flooding in the field are shown in the figure. In the figure, OsMAPK7-Cas-Y1, OsMAPK7 Cas-Y2, and OsMAPK7 Cas-Y3 represent the OsMAPK7 knockout plants of Yasi 881 in three replicate experiments, respectively; OsMAPK7-Cas-H1, OsMAPK7 Cas-H2, and OsMAPK7 Cas-H3 represent the OsMAPK7 knockout plants of Huanghuazhan in three replicate experiments, respectively. When sown under flooded conditions in the field, seeds of the wild-type Yasi 881 failed to germinate, and the germination rate of Huang Huazhan seeds was very low (3%). However, transgenic seeds with the OsMAPK7 gene knocked out exhibited a high germination rate (75% average germination rate in Yasi 881 with OsMAPK7 knocked out) or complete germination under flooded conditions (90% average germination rate in Huang Huazhan with OsMAPK7 knocked out). This indicates that OsMAPK7 knockout (OsMAPK7-Cas9) plants possess flood tolerance.

[0059] The above experimental results show that, compared with wild-type seeds, transgenic plants with the OsMAPK7 gene knocked out in indica and japonica rice varieties have higher germination rates, longer root systems, and seedlings that can extend beyond the water surface under long-term flooding conditions.

[0060] The above description is only a preferred embodiment 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. The application of knocking out the rice OsMAPK7 gene in improving the waterlogging tolerance of rice, characterized in that, The nucleotide sequence of the OsMAPK7 gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The rice varieties mentioned include japonica rice and indica rice.

3. The application according to claim 1, characterized in that, The application includes improving the germination rate of rice seeds under flooded conditions.

4. The application according to claim 1, characterized in that, The application includes promoting root growth in rice seedlings under flooded conditions.

5. A method for improving the waterlogging resistance of rice, characterized in that, The method includes knocking out the rice OsMAPK7 gene, the nucleotide sequence of which is shown in SEQ ID NO:

1.

6. The method according to claim 5, characterized in that, The gene knockout method uses CRISPR / Cas9 technology.

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