Gene OsCOMT for regulating and controlling aluminum resistance of rice, application of gene OsCOMT and method for regulating and controlling aluminum resistance of rice

Abstract

The invention relates to the field of rice resistance genes, in particular to a gene OsCOMT for regulating and controlling the aluminum resistance of rice, application of the gene OsCOMT and a method for regulating and controlling the aluminum resistance of the rice. The CDS region sequence of the gene OsCOMT is as shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein of the gene OsCOMT is as shown in SEQ ID NO. 2. The OsCOMT gene disclosed by the invention can be applied to improving the tolerance of crops such as rice to aluminum toxicity, and rice varieties with higher aluminum resistance are cultivated, so that the yield and quality of the crops in acid soil are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of rice resistance genes, in particular to a gene OsCOMT for regulating rice aluminum tolerance and application thereof and a method for regulating rice aluminum tolerance. BACKGROUND

[0002] Rice (Oryza sativa L.) is an important food crop in the world, ranking third in the world's food crop yield. The southern region of China is mostly red soil land, which is seriously affected by aluminum toxicity, resulting in a 10% average reduction in crop yield and quality, making the southern region's overall level of rice production lower than that of the northern region.

[0003] Aluminum (Al) is the third most abundant element in the earth's crust (next to oxygen and silicon), accounting for about 8.1% of the earth's crust. The release of active aluminum in soil is closely related to the pH of the soil. Soil acidification aggravates the leaching of base cations, thereby promoting the release of active aluminum. It is generally believed that when the soil is acidified to pH ≤ 5.5, active aluminum is released, and when the soil is acidified to pH 4.3, a large amount of active aluminum is released. Therefore, it is of great production significance to mine and utilize key genes for regulating rice aluminum tolerance and breed rice varieties with aluminum tolerance.

[0004] Caffeic acid O-methyltransferase (COMT) plays a crucial role in plant stress resistance. It has been reported that it is involved in reducing the stress of cadmium metal (The mechanism of enhanced lignin regulating foliar Cd absorption and yield in rice (Oryza sativa L.), Ecotoxicology and Environmental Safety, 2022). It not only participates in the metabolic process to form the precursor of lignin, but also plays an important role by regulating the synthesis rate and yield of lignin. COMT mainly uses S-adenosylmethionine (SAM) as a source of methyl groups, and it can methylate a variety of substrates, thereby participating in the methylation in the plant body.

[0005] The present application uses rice as the research material. However, there is no report on the role of rice OsCOMT in resisting aluminum toxicity stress.

[0006] The information disclosed in this BACKGROUND section is only intended to increase an understanding of the general context in which the present application can be practiced. It is not necessarily recognized by those skilled in the art that the information constitutes prior art that is already known in all jurisdictions in the world. SUMMARY

[0007] The technical problem solved by the present application is to provide a gene OsCOMT for regulating rice aluminum tolerance and application and a method for regulating rice aluminum tolerance.

[0008] The technical solution adopted by the present application to solve the above problems is as follows:

[0009] A gene OsCOMT for regulating rice aluminum tolerance, wherein the sequence of the CDS region of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

[0010] The present application also provides an application of the above-mentioned gene OsCOMT, which includes one or more of the following:

[0011] 1) regulating the resistance of rice to aluminum stress;

[0012] 2) cultivating rice varieties resistant to aluminum stress or having aluminum tolerance;

[0013] 3) reducing the aluminum content in the root system of rice.

[0014] The present application also provides a method for regulating rice aluminum tolerance, comprising: knocking out the above-mentioned OsCOMT gene by CRISPR-Cas9 method to obtain a knockout mutant plant.

[0015] Further, the knockout method comprises: integrating the plasmid DNA fragment carrying the knockout target site of the OsCOMT gene into the rice callus by Agrobacterium to induce differentiation into seedlings; and then screening and obtaining a homozygous line by primer sequencing method.

[0016] Further, the sequence of the knockout target site is 5'-CAACGTCGTCAGGTGCGAGATGG-3', as shown in SEQ ID NO. 3. Wherein, the G at the 17th position of the target is knocked out, and the original encoding cysteine protein amino acid sequence is changed by base knockout, and the subsequent protein amino acid sequence is changed.

[0017] Further, the primer is an upstream primer and a downstream primer, the sequence of the upstream primer is 5'-ACAAGCTGCCGTCCAAGG-3', as shown in SEQ ID NO. 4, and the sequence of the downstream primer is 5'-TTGTCCTGGTTCATGAGGGC-3', as shown in SEQ ID NO. 5.

[0018] Further, the rice is Zhonghua 11 glutinous rice variety.

[0019] The present application has the following beneficial effects:

[0020] The present application adopts CRISPR-Cas9 knockout technology to knockout mutation of the rice OsCOMT gene in wild type rice ZH11, and determines the negative regulation of the OsCOMT gene in the aluminum resistance of rice, and reveals the mechanism of the OsCOMT gene in the aluminum resistance of rice, and provides a new gene target for molecular breeding of rice resistance.

[0021] After the rice OsCOMT gene is knocked out, compared with the wild type rice, the root length is significantly shortened, the water content is significantly decreased, the relative conductivity is significantly increased, and the aluminum content is significantly increased, which proves that OsCOMT participates in improving the aluminum tolerance of rice, therefore, the OsCOMT gene can be applied to improve the aluminum tolerance of crops such as rice, and cultivate rice varieties with higher aluminum tolerance, thereby improving the yield and quality of crops in acid soil.

[0022] The method for regulating the aluminum tolerance of rice is simple in operation, suitable for various rice varieties, especially for rice planted in acid soil with serious aluminum stress, and has wide applicability and popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 : Knockout vector schematic diagram;

[0024] Figure 2 : OsCOMT knockout target sequencing result;

[0025] Figure 3 : OsCOMT aluminum tolerance phenotype, WT: wild type rice material ZH11; OsCOMT is an OsCOMT gene knockout material;

[0026] Figure 4 : Root length, root water content and root relative conductivity determination of OsCOMT knockout material, WT: wild type rice material ZH11; OsCOMT is an OsCOMT gene knockout material; CK control is normal growth for 2 weeks; Al 3+ treatment is normal growth for 1 week, and treatment for 1 week; ** indicates p<0.01, which indicates that there is a highly significant difference; **** indicates p<0.0001, which indicates that there is a very high significant difference;

[0027] Figure 5 : Aluminum content determination of root cell wall, root cytoplasm and root organelle of OsCOMT knockout material, WT: wild type rice material ZH11; OsCOMT is an OsCOMT gene knockout material; CK control is normal growth for 2 weeks; Al3+ Treatment for normal growth 1 week, treatment 1 week; **** indicates p < 0.0001 indicates a very high significant difference. DETAILED DESCRIPTION

[0028] In order to make the technical problems, technical solutions and technical advantages of the present application clearer, specific examples will be described in detail below, but the protection scope of the present application is not limited to the following specific examples, the described examples are only a part of the embodiments of the present application, not all the embodiments, and not a limitation of the present application. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0029] Unless otherwise defined, all professional terms used below have the same meaning as generally understood by those skilled in the art. The professional terms used in this paper are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present application.

[0030] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0031] In this embodiment, taking ZH11 japonica rice variety as the background, transgenic seeds were obtained by knocking out the target gene fragment using conventional CRISPR-Cas9 technology. This method is the common Agrobacterium-mediated callus method on the market.

[0032] In the early stage, according to the Blast comparison of NCBI database, the rice COMT gene was obtained, and the gene registration ID was LOC_Os08g06100.

[0033] According to the gene sequence, the seed knockout gene vector from the mutation of Baige Gene was directly purchased. Figure 3 As shown in the figure, the transgenic rice seeds obtained were planted by water culture.

[0034] The specific planting method is as follows:

[0035] Rice seedling nutrition solution culture: Select uniform size, full grain rice seeds, use seed germination box for germination, first pad germination paper in the germination box, evenly spray distilled water on the paper, put the required rice seeds, cover the corresponding box cover, write the label and germination date, put into the culture room 26℃, avoid light conditions for 2-3 days, then take out the germinated seeds for culture. The germinated seeds are transferred to a plastic water culture tray, and the improved Hoagland nutrient solution is added for water culture. The nutrient solution is prepared according to the nutrient solution instruction book. According to the size of the container, the appropriate amount of nutrient solution is prepared, generally 2-3L, and the culture solution is replaced every 2-3 days. The culture conditions are temperature 26℃, light cycle 16h / 8h.

[0036] The rice grown for 2 weeks is cut to take appropriate amount of leaves, and the DNA is extracted by CTAB method as follows:

[0037] Take appropriate amount of sample (4-5cm 2 )(Take a tender leaf, too many leaves will not be ground) in a mortar, add 800μL CTAB buffer (preheat 65℃ water bath for 5min) and grind thoroughly to homogenate, then quickly transfer to a 2mL centrifuge tube; the centrifuge tube is placed in a 65℃ water bath for 20min, and is inverted evenly twice during the period; 10min once, every seven minutes; after cooling to room temperature, add an equal volume (800μL) of chloroform:isopropyl alcohol (24:1V / V) (in a fume hood), mix evenly for 15min (light shaking is to prevent DNA breakage) in the electrophoresis room, clamp two test tube racks together, pad with paper towels, place the test tubes in parallel on the shaker, then centrifuge at 12000rpm for 5min; take 400μL of supernatant to another new 1.5mL centrifuge tube, add 2 times volume (800μL) of anhydrous ethanol, mix evenly after light shaking, then vortex once and stand at-20℃ for 30min; centrifuge at 12000rpm for 10min, discard the supernatant, wash the precipitate with 1mL of 70% ethanol for 2min each time; after adding ethanol, put it for two minutes (remember, when pouring off the ethanol, do not pour off the precipitate, the precipitate is DNA, which becomes light black after oxidation); after centrifuging to remove the supernatant, centrifuge at 12000rpm for 2min, and aspirate the residual liquid; stand at room temperature for 10-20min, air dry, add 200μL ddH2O to dissolve, first add 50μL to measure the concentration, then consider whether to add water, store at-20℃ for standby.

[0038] The formula of the above CTAB is as follows:

[0039] 2% CTAB 0.5L Tris-HCl pH = 8.0 7.85g EDTA pH = 8.0 2.92g PVP 10g NaCl 40.88g CTAB 10g

[0040] The OsCOMT knockout mutant is verified, and primers are designed at the front and back of the gene target point, and the primer sequence is:

[0041] Upstream primer F: 5'-ACAAGCTGCCGTCCAAGG-3'

[0042] Downstream primer R: 5'-TTGTCCTGGTTCATGAGGGC-3'

[0043] The knockout mutant OsCOMT rice genomic DNA was used as a template for PCR amplification.

[0044] The PCR amplification system is as follows:

[0045] System 20 μL 2x Taq Master Mix 10 μL Upstream primer F 1 μL Downstream primer R 1 μL DNA 1 μL ddH2O 7 μL

[0046] The PCR amplification program is as follows: pre-denaturation at 94℃ for 3 min, denaturation at 94℃ for 15 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 20 sec, a total of 35 cycles of denaturation, annealing and extension, complete extension at 72℃ for 5 min, incubation at 20℃ for 2 min. The PCR reaction liquid with bands detected by electrophoresis was sequenced using a 1% agarose gel with a voltage of 110 V for 20 min electrophoresis. The sequencing results are shown in FIG. 2. Figure 4

[0047] The identified OsCOMT gene knockout mutant seedlings were planted in the field, and the field test was carried out in the high-standard farmland of the Baiyun campus of Zhongkai Agricultural Engineering College (23°22'22"N, 113°26'26"E). Enough homozygous seeds were harvested for subsequent experiments.

[0048] The OsCOMT gene knockout seeds and control ZH11 seeds which were already homozygous were planted in the laboratory, and the indoor test was carried out in the Guangzhou Key Laboratory for Research and Utilization of Characteristic Crop Germplasm Resources in Haizhu Campus of Zhongkai Agricultural Engineering College (23°6'36"N, 113°16'33"E).

[0049] The rice seedlings normally grown for 1 week, treated with aluminum for 2 weeks, and control normally grown for 3 weeks were subjected to phenotype identification, average root length measurement, relative water content detection, relative conductivity detection, cell wall aluminum content detection, cytoplasmic aluminum content detection, and organelle aluminum content detection.

[0050] The aluminum treatment method was as follows: 10M AlCl2 was added to the modified Hoagland nutrient solution at a concentration of 2 -1 .

[0051] The normal growth condition was as follows: the modified Hoagland nutrient solution at a concentration of 2 -1 .The culture conditions were temperature 26℃ and light cycle 16h / 8h.

[0052] The internal standard method was used to detect the aluminum content. The ratio of the mass spectrum signal of the measured element to the mass spectrum signal of the internal standard element was proportional to the concentration of the measured element. 0.3-0.5 g of rice root sample was placed in a mortar, 10 mL of extractant (250 mmol / mL sucrose + 50 mmol / L Tris-HCl buffer solution (pH = 7.5) + 1 mmol / L DTE) was added, and the homogenate was ground. The homogenate was centrifuged, and the supernatant was taken (according to whether the sample was layered, the centrifugation times were increased as needed). The remaining residue was the cell wall component, and the supernatant was placed in a high-speed centrifuge at 4°C at 16000 rpm. According to the actual situation, the centrifugation was repeated several times. The precipitate obtained by centrifugation was the organelle component, and the supernatant was the cytoplasmic component. The organelle, cell wall, and cytosol components were soaked with 5 mL of nitric acid overnight. The inner cover was covered, the stainless steel jacket was tightly screwed, and it was placed in a constant temperature drying oven at 80°C for 2 h, 120°C for 2 h, and then increased to 160°C for 4 h. The oven was cooled to room temperature naturally, and then heated to chase the acid to near dryness. The digestion solution was washed into a 25 mL volumetric flask, a small amount of nitric acid solution (1%) was used to wash the inner tank and inner cover 3 times, the washing liquid was combined and diluted to the mark with 1% nitric acid, and the mixture was mixed well for use. A reagent blank test was also performed. The test solution was measured on the instrument to determine the element content of each component.

[0053] Result calculation:

[0054]

[0055] In the formula:

[0056] c - the concentration of the element in the solution, μg / L;

[0057] c0 - the concentration of the element in the blank solution, μg / L;

[0058] V - the constant volume, mL;

[0059] D - the dilution factor;

[0060] m - the sample mass, g

[0061] The phenotype of rice was identified. After aluminum treatment, the root growth of OsCOMT mutant was inhibited compared with the wild type, the root length was shortened, the water content was significantly reduced, the electrical conductivity was significantly increased, the aluminum content in the root system was significantly increased compared with the wild type, and the loss of OsCOMT function led to the inhibition of root growth of rice under aluminum stress. Figure 4 Figure 5

[0062] ​​In summary, the OsCOMT gene can significantly improve the aluminum tolerance of rice, and provide a regulatable endogenous gene target for aluminum stress. By controlling the expression of the OsCOMT gene, the aluminum tolerance of rice can be improved, and the aluminum resistance of rice can be improved and enhanced, and a high-quality resistance resource for molecular breeding of rice resistance is provided.

[0063] It can be understood that the above specific embodiments are further illustrations of the present application and are not used to limit the protection scope of the present application, and all other decorations and modifications obtained by those skilled in the art without creative labor belong to the protection scope of the present application.

Claims

1. A gene OsCOMT that regulates aluminum tolerance in rice, characterized in that, The CDS region sequence of the gene OsCOMT is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

2. An application of the OsCOMT gene as described in claim 1, characterized in that, The application includes one or more of the following: 1) Regulating rice resistance to aluminum stress; 2) Develop rice varieties resistant to aluminum stress or with aluminum tolerance; 3) Reduce the aluminum content in rice roots.

3. A method for regulating aluminum tolerance in rice, characterized in that, include: Knockout mutant plants were obtained by targeting and knocking out the OsCOMT gene as described in claim 1 using the CRISPR-Cas9 method.

4. The method for regulating aluminum tolerance in rice according to claim 3, characterized in that, The knockout method includes: integrating a plasmid DNA fragment carrying the OsCOMT gene knockout target site into rice callus tissue using Agrobacterium to induce differentiation into seedlings; and then screening for homozygous lines using primer sequencing.

5. The method for regulating aluminum tolerance in rice according to claim 4, characterized in that, The knockout target sequence is 5'-CAACGTCGTCAGGTGCGAGATGG-3', as shown in SEQ ID NO.

3.

6. The method for regulating aluminum tolerance in rice according to claim 4, characterized in that, The primers are an upstream primer and a downstream primer. The sequence of the upstream primer is 5'-ACAAGCTGCCGTCCAAGG-3', as shown in SEQ ID NO.4, and the sequence of the downstream primer is 5'-TTGTCCTGGTTCATGAGGGC-3', as shown in SEQ ID NO.5.

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