Application of rice gene OsROHAN in improving plant tolerance to ammonium toxicity and nitrogen use efficiency

Abstract

The application belongs to the field of genetic engineering, and relates to application of rice gene OsROHAN in improving plant tolerance to ammonium toxicity and nitrogen utilization efficiency, a nucleotide sequence of the gene is shown as SEQ ID NO:1, and an amino acid sequence coded by the gene is shown as SEQ ID NO:2. The gene sequence is obtained by cloning from rice through a PCR technique. Experiments prove that a transgenic plant obtained by using the plant overexpression vector constructed by the gene OsROHAN and performing plant transgenic operation is significantly improved in tolerance to ammonium toxicity, and is significantly improved in nitrogen utilization efficiency and yield of rice, indicating that implementation of the gene OsROHAN can create a new type of plant tolerant to ammonium toxicity and high in nitrogen, can provide a candidate gene for subsequent crop variety improvement, and has great significance for agricultural production in China.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of rice gene OsROHAN in improving plant tolerance to ammonium toxicity and nitrogen utilization efficiency, and belongs to the technical field of genetic engineering. BACKGROUND

[0002] With the base of the world population still increasing, the demand for food will be greater and greater. As one of the main food crops, how to improve the yield of rice and reduce the application of chemical fertilizers (excessive application of fertilizers causes environmental pollution and changes in soil environment) has been a technical problem that scientists have been trying to solve.

[0003] Nitrogen, as one of the essential and most deficient macronutrients for plants, is an important component of amino acids necessary for protein synthesis, nucleic acids necessary for DNA / RNA synthesis, chlorophyll, and other natural compounds necessary for plant growth. Therefore, the efficient sensing, absorption and utilization of nitrogen by crops play a decisive role in their development, growth and yield (Xu et al., 2012). In the past few decades, in order to overcome the low utilization rate of nitrogen fertilizer and improve crop yield, inorganic nitrogen fertilizer has been applied in large quantities in farmland, causing soil acidification, serious eutrophication of water bodies, excessive emission of greenhouse gases and a series of ecological and environmental problems; at the same time, excessive application of nitrogen fertilizer also has negative effects on crop growth, such as late maturity, plant lodging, quality decline, high ammonium toxicity and inhibition of root growth. Therefore, reducing the application of nitrogen fertilizer in farmland has social, economic and ecological significance. Revealing the response mechanism of crops to soil nitrogen supply, excavating key genes that regulate nitrogen absorption and utilization, and improving the nitrogen absorption and utilization efficiency of crops are one of the important biological approaches to ensure high yield, stability and quality of crops under the condition of limited resources such as fertilizer and water, and are the inevitable demand for the green and sustainable development of modern agriculture.

[0004] Nowadays, there are few studies on the ammonium tolerance genes of plants and their application in rice, an important food crop. Studying the ammonium tolerance of rice plays an important role in improving the yield and quality of rice in high-ammonium stress soil. The excavation of nitrogen-responsive genes in rice has also become a hot spot in the research of rice genetic resources and variety improvement. Therefore, cloning and identifying plant genes with ammonium tolerance function from rice and elucidating the mechanism of these genes to play tolerance will help to deepen the understanding of the molecular mechanism of plant response to nitrogen from a scientific perspective, and enrich the resources of plant response to nitrogen genes; from an application perspective, these new ammonium tolerance genes will be used in the cultivation of new varieties of crops such as rice, which will help to improve rice through genetic means and improve the stress resistance of rice. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to solve the problem of providing a rice ammonium toxicity tolerance related gene OsROHAN and its application in improving plant ammonium toxicity tolerance, using genetic engineering means to tap the plant's own ammonium toxicity resistance, providing an effective strategy for solving ammonium toxicity in acid soil and sustainable development of agricultural production.

[0006] In one aspect, the present application provides an ammonium toxicity tolerance gene, the nucleic acid sequence of which is shown in SEQ ID NO: 1.

[0007]

[0008] The application also provides a protein encoded by the gene, and the amino acid sequence of the protein is shown as SEQ ID NO: 2.

[0009] MAAATSQSFLSPAPNPLLRPRILPFPAGGSVSLRGRRPAFPSVAAASTSMASSESEERKETKLWGGRFEEGVTDAVEGFTESISYDWQLYKYDIMGSKAHASMLAAQGLITAGDKDIILEGLDQIEKLIQDGKFEWRTDREDVHMNIEAALIEKVGEPAKKLHTARSRNDQIVTDLRLWCRDAIDKILFRIKQFQVSLVLLASKYVDLIVPGYTHLQRAQPVLLPHLLLSYVEQLERDAGRLVNCRERLNFCPLGACALAGTGLPIDRFKTAKDLKFTAPMKNSIDAVSDRDFVLEFLAANSIAAVHLSRIGEEWVLWASEEFGFLTPSDSVSTGSSIMPQKKNPDPMELVRGKSARVFGDLMTVLTLCKGLPQAYNRDLQEDKEPLFDSVKAVLGMLEVCTEFAQNISFNSKRIQSSLPAGYLDATTLADYLVKKGVPFRTSHEIVGRSVALCVSKNCQLAELGLDDLKSVHPVFEGDVYEYLGVENAVNKFISYGSTGSEQVKKQLEDWRTQLGISS.

[0010] The application provides application of a rice gene OsROHAN in improving plant ammonium toxicity tolerance and nitrogen utilization efficiency, a nucleotide sequence of the rice gene OsROHAN is shown as SEQ ID NO: 1, and an amino acid sequence encoded by the rice gene OsROHAN is shown as SEQ ID NO: 2.

[0011] Further, the plant is a plant in the family Poaceae.

[0012] The application also provides a recombinant expression vector containing the nucleotide sequence shown as SEQ ID NO: 1.

[0013] The application provides, in another aspect, a mutant sensitive to ammonium nitrogen, and the mutant has a gene with a nucleotide sequence shown as SEQ ID NO: 1.

[0014] The application also provides a preparation method of the mutant, comprising the following steps.

[0015] Step 1, preparing a mutant library;

[0016] Step 2, taking each single clone in the mutant library to germinate;

[0017] Step 3, treating in an ammonium nitrogen solution, observing root growth inhibition, and selecting candidate plants for breeding;

[0018] Step 4, after harvesting seeds of the candidate mutant, selecting an ammonium nitrogen sensitive mutant by comparing relative root elongation before and after treatment in an ammonium nitrogen solution.

[0019] Preferably, in the above step 1, the specific method for preparing the mutant library is as follows: wild type seeds of Wu Yunj 7 are soaked in water for 8 hours, then soaked in 1% ethyl methanesulfonate for 16 hours, and stirred during the soaking; after washing, the seeds are sowed, and M2 generation seeds are harvested.

[0020] Preferably, in the above step 3, the treatment is performed in a 2.5 mM ammonium sulfate solution for 6 days.

[0021] In still another aspect, the present application provides a method for breeding ammonium tolerant plants, which comprises introducing the nucleotide sequence OsROHAN as shown in SEQ ID NO: 1 into a target plant.

[0022] Preferably, the method for overexpressing OsROHAN comprises the following steps:

[0023] Step 1, designing a primer sequence on the OsROHAN gene;

[0024] Step 2, introducing into an expression vector by using a Gateway connection method;

[0025] Step 3, transforming the plasmid with correct sequencing into Agrobacterium;

[0026] Step 4, preparing a bacterial floating liquid from the cultured Agrobacterium with a bacterial liquid;

[0027] Step 5, infecting callus tissue of the target plant with the bacterial floating liquid, and placing on a co-culture medium for dark culture;

[0028] Step 6, screening the callus tissue on a selection medium, and culturing on a differentiation medium until differentiation into seedlings.

[0029] Preferably, the primer sequence is as shown in SEQ ID NO: 3-6.

[0030] More preferably, in step 4, the bacterial floating liquid is prepared with an AAM bacterial liquid containing 200 μM acetosyringone.

[0031] More preferably, in step 5, the infection time is 5-10 min.

[0032] More preferably, in step 5, the dark culture is carried out at 25°C for 2.5 days.

[0033] More preferably, in step 6, the selection medium contains cephradine and hygromycin, and 3 rounds of screening are carried out.

[0034] Preferably, in the above method, the plant of interest is a plant of the family Poaceae.

[0035] More preferably, the plant of interest is rice.

[0036] More preferably, the plant of interest is japonica rice and / or indica rice.

[0037] The application also provides use of the above ammonium toxicity-tolerant gene in improving the ammonium toxicity tolerance of a plant.

[0038] Preferably, the plant is a plant of the family Poaceae.

[0039] More preferably, the plant of the family Poaceae is rice, corn, wheat, sorghum or oat. In addition, the plants also include soybean and quinoa.

[0040] Beneficial effects

[0041] The application clones and identifies a gene sequence related to ammonium toxicity tolerance from rice, which is named rice ammonium toxicity-tolerant gene OsROHAN. It is proved that the gene sequence can enhance the tolerance of plants to ammonium toxicity, and improve the nitrogen utilization efficiency and yield of plants. After searching, there is no report on the application of the rice ammonium toxicity-tolerant gene OsROHAN in improving the ammonium toxicity tolerance and nitrogen utilization efficiency of plants. The ammonium toxicity-tolerant rice cultivated by the application has a relative root length increased by 23% under the treatment of 10 mM ammonium sulfate relative to the main cultivar Wuyunjing 7 in Jiangsu Province, and the yield is increased by 16% in a field plot with 350 kg / ha of nitrogen application. The application uses genetic engineering means to tap the ammonium toxicity resistance of plants, which provides an effective strategy for solving the ammonium toxicity in acid soil and making agricultural production sustainable. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1a The change in the root length phenotype of wild type and mutant (rohan) under the conditions of no ammonium salt and with ammonium salt is shown in the more preferred embodiment of the application; Figure 1b The statistical results of the seed root length phenotype in Figure 1a are shown; Figure 1c The reaction of the root system of wild type and mutant (rohan) to ammonium toxicity under different ammonium nitrogen concentrations is shown;

[0043] Figure 2aThe overexpression vector related information in the preferred embodiments of the present application is shown. Figure 2b The complementation vector related information in the preferred embodiments of the present application is shown.

[0044] Figure 3a The root phenotype figures of wild type, mutant (rohan) and two independent complementation lines (C1, C2) under ammonium supply in the preferred embodiments of the present application are shown, Figure 3b The relative length statistics of root growth are shown. Figure 3a The relative length statistics of root growth are shown.

[0045] Figure 4a The relative expression amount of OsROHAN gene is induced under ammonium treatment in the preferred embodiments of the present application. Figure 4b The subcellular localization of OsROHAN in the plastid of plants is shown.

[0046] Figure 5a The root phenotype figures of wild type and two independent overexpression lines (OE-1, OE-2) under nitrogen deficiency and high ammonium in the preferred embodiments of the present application are shown, Figure 5b The relative length statistics of root growth of wild type and two independent overexpression lines (OE-1, OE-2) under different ammonium concentrations are shown, Figure 5c The relative expression amount results of OsROHAN of wild type and two independent overexpression lines (OE-1, OE-2) are shown.

[0047] Figure 6a The field agronomic traits under three different fertilization concentrations, i.e. the field phenotypes of wild type and overexpression line (OE-1) under 75 kg / ha urea, 150 kg / ha urea and 350 kg / ha urea, and the phenotypes of single plant seed yield in the preferred embodiments of the present application are shown. Figure 6b The statistics of single plant tiller number of wild type and overexpression line (OE-1) under different fertilization concentrations are shown, Figure 6c The single plant yield of wild type and overexpression line (OE-1) under different fertilization concentrations is shown, Figure 6d The nitrogen utilization efficiency of wild type and overexpression line (OE-1) under different fertilization concentrations is shown. Figure 6e The protein content in the harvested seeds of wild type and overexpression line (OE-1) under different fertilization concentrations is shown. DETAILED DESCRIPTION

[0048] Before the present application is described, it is to be understood that this application is not limited to the particular methodology and experimental conditions described, as such can of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.

[0049] Example 1

[0050] Preparation of ammonium-sensitive mutants

[0051] In this example, the model crop rice is used as the research object, and the mutants sensitive to ammonium are prepared according to the following method.

[0052] About 2.5 kg of wild-type seeds of Wuyunj 7 were soaked in water for 8 h, then soaked in 1% ethyl methanesulfonate solution for 16 h, and stirred several times during the period. After washing, the seeds were sown, and the M1 generation seeds were harvested, thereby obtaining a mutant library.

[0053] 100 seeds from each sub-library in the mutant library were germinated and treated in 2.5 mM ammonium sulfate solution for 6 days, and the root growth inhibition was observed. The selected plants were multiplied; after the candidate mutant seeds were harvested, the relative elongation before and after ammonium sulfate solution treatment was measured and compared, and the ammonium-sensitive mutant rohan was selected. After multiplying another generation, the relative root elongation before and after ammonium salt treatment was measured and compared to confirm the ammonium sensitivity of the mutant.

[0054] Table 1 Composition of rice nutrient solution (Kimura's culture solution)

[0055] Rice nutrient solution formula

[0056]

[0057]

[0058] Each component was dissolved in water to prepare the rice nutrient solution mother liquor, and water was used to dilute it to the final concentration when used.

[0059] Identification of ammonium-sensitive mutants

[0060] In order to locate the mutant gene of the rohan mutant, a backcross population with wild-type Wuyunj 7 was constructed. Using the high-throughput sequencing results of the BSA library of the F2 generation of the backcross population, the mutant gene ROHAN was accurately located, and the gene location result was confirmed by transgenic back complementation. We constructed a reverse vector containing the entire coding region driven by the self-promoter of the candidate gene, and the primers for amplifying it were as follows.

[0061] Gene promoter upstream primer:

[0062] GGGGACAACTTTGTATAGAAAAGTTGCGCCAGCCTACGACGCCACGAA (SEQ ID NO: 3)

[0063] Gene promoter downstream primer:

[0064] GGGGACAAGTTTGTACAAAAAAGCAGGCTATATGGCCGCCGCCACCTCCCA (SEQ ID NO: 3)

[0065] Gene upstream primer:

[0066] GGGGACAAGTTTGTACAAAAAAGCAGGCTATATGGCCGCCGCCACCTCCCA (SEQ ID NO: 3)

[0067] Gene downstream primer:

[0068] GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATGAACTAATGCCAAGCT (SEQ ID NO: 6)

[0069] The mutant rohan induced callus was transformed with the reply vector. The transformation method and transformation strain identification were the same as in Example 2. Figure 3a and Figure 3b The relative root elongation of wild type (WT), mutant (rohan), and two independent complementation lines (C1, C2) was shown, and the results showed that the relative root elongation of the two independent complementation lines (C1, C2) was restored to the wild type level, and did not show stronger sensitivity to ammonium nitrogen, confirming the results of gene mapping.

[0070] Example 2

[0071] OsROHAN overexpression vector construction and rohan mutant complementation vector construction and introduction into rice

[0072] The amplification primers of the gene were designed on the OsROHAN genomic DNA sequence as follows:

[0073] Upstream primer:

[0074] GGGGACAAGTTTGTACAAAAAAGCAGGCTATATGGCCGCCGCCACCTCCCA (SEQ ID NO: 3)

[0075] Downstream primer:

[0076] GGGGACCACTTTGTACAAGAAAGCTGGGTCTTATGAACTAATGCCAAGCT (SEQ ID NO: 6)

[0077] The amplification primers of the promoter were designed on the promoter sequence of about 2000bp of the OsROHAN genomic DNA as follows:

[0078] Upstream primer: GGGGACAACTTTGTATAGAAAAGTTGCGCCAGCCTACGACGCCACGAA (SEQ ID NO: 5)

[0079] Downstream primer: GGGGACTGCTTTTTTGTACAAACTTGCAGTATCGTCGCCGAGGGTG (SEQ ID NO: 6)

[0080] Gateway ligation method as shown was used to introduce into overexpression vector and complementation expression vector Figure 2a and Figure 2b ).

[0081] OsROHAN overexpression vector and complementation vector were introduced into rice plants and positive transformant was identified

[0082] Transformation was performed using Agrobacterium (EHA105) mediated rice genetic transformation method, and the process was as follows:

[0083] 1. The medium and solution used in the process of rice transgene were as shown in the following table.

[0084] Table 2. Formula of rice tissue culture medium mother liquor

[0085]

[0086]

[0087] Table 3. Induction medium of mature embryo callus of japonica rice

[0088] Induction medium of mature embryo callus of japonica rice (1L)

[0089]

[0090] Table 4. Subculture medium of mature embryo callus of japonica rice

[0091] Subculture medium of mature embryo callus of japonica rice (1L)

[0092]

[0093] Table 5. Co-culture medium of japonica rice

[0094] Co-culture medium of japonica rice (1L)

[0095]

[0096] Table 6. Callus selection medium

[0097] Callus selection medium (1L)

[0098]

[0099]

[0100] Table 7 Induction medium for japonica rice

[0101] Induction medium for japonica rice (1 L)

[0102]

[0103] Table 8 Rooting medium for japonica rice

[0104] Rooting medium for japonica rice (1 L)

[0105]

[0106] Table 9 Medium for Agrobacterium suspension infection of callus

[0107] Medium for Agrobacterium suspension infection of callus (1 L)

[0108]

[0109] 2. Preparation of mature callus of rice:

[0110] After shelling, the mature seeds of wild-type Wu Yun japonica 7 rice were selected and placed in a beaker. The seeds were sterilized with 70% alcohol for 2 min. The alcohol was poured out, and the seeds were sterilized with 30% (v / v) sodium hypochlorite solution for 30 min. The sodium hypochlorite solution was poured out, and the seeds were washed with sterile water for 5 times. The last time, the seeds were soaked in sterile water for 30 min. The sterile water was poured out, and the seeds were dried on sterile filter paper. The pre-processed rice seeds were placed flat on the induction medium for mature embryos of japonica rice, and cultured in the dark at 28°C for about 10 days. The Petri dishes were opened on the clean bench, and the buds and endosperm were removed with sterile tweezers, leaving the embryonic callus (light yellow, dense and irregular). The embryonic callus was transferred to the subculture medium for japonica rice, and cultured in the dark at 28°C for 5-10 days. The mature embryo callus of rice was obtained.

[0111] 3. Culture of Agrobacterium:

[0112] The OsROHAN gene's back vector or overexpression vector is transformed into Agrobacterium EHA105 competent cells using the freeze-thaw method. Specifically, 2 μL of the constructed plasmid is added to 100 μL of Agrobacterium competent cells. After mixing, place on ice for 15 min, place in liquid nitrogen for 90 s, 37°C for 5 min, and then add 800 μL of antibiotic-free YEP liquid medium and shake at 28°C for 2 h. Take 200 μL of bacterial solution and spread on YEP solid medium containing 50 mg / L spectinomycin (Spec) and 50 mg / L streptomycin (Str). After 2 days of growth, pick EHA105 single colony bacteria on 5 ml of YEP liquid medium containing 50 mg / L spectinomycin (Spec) and 50 mg / L streptomycin (Str) resistance, 28°C, 250 rpm shaking culture overnight, until the bacterial solution OD600 is about 2.0; take 500 μL from the above bacterial solution and add to 30 ml of YEP liquid medium containing 50 mg / L spectinomycin (Spec) and 50 mg / L streptomycin (Str) resistance, 28°C, 250 rpm shaking culture on a shaker for 14 h until the bacterial solution OD600 = 1.0, to obtain Agrobacterium bacterial solution. The YEP liquid medium is composed of yeast extract 10 g / L, tryptone powder 10 g / L, NaCl 5 g / L, solvent water, pH 7.0. The YEP solid medium is added with 15 g / L agar in the YEP liquid medium.

[0113] 4. Co-culture and screening of resistant callus:

[0114] Take 15 ml of the well-cultured Agrobacterium bacterial solution in step 3 and place it in a 50 ml centrifuge tube, 4°C, 4000 rpm, centrifuge for 10 min, collect the bacterial cells; use 30 ml of AAM bacterial solution containing 200 μmol / L of acetosyringone (As) to make an Agrobacterium suspension, so that the final concentration of bacterial solution OD600 is 0.6.

[0115] The step 2 mature rice embryo callus is picked out, cut into granular, put into the agrobacterium suspension, 28℃ oscillation culture 30min; the callus is carefully taken out, is placed on the sterile filter paper and is drained for 30min; then the callus is placed on the japonica (indica) rice co-culture medium containing 200 μmol / L of acetyl-syringone (As) with a layer of sterilized filter paper; after 2.5 days of dark culture at 25℃, the callus is taken out, washed with sterile water for 5-6 times, and needs to be shaken constantly during the period; then washed with sterile water containing 250mg / L carbenicillin sodium for 1-2 times; finally placed on the sterile filter paper and drained for 2h; the dried callus is transferred to the selection medium containing 250mg / L carbenicillin sodium and 50mg / L hygromycin for the first round of resistance screening, and dark culture at 28℃ for 14 days, and the initial callus is transferred to the selection medium containing 250mg / L carbenicillin sodium and 50mg / L hygromycin for the second round of selection, and dark culture at 28℃ for 14 days, at this time the callus grown is the resistant callus with hygromycin resistance gene.

[0116] 5. Differentiation of resistant callus and seedling:

[0117] The differentiation medium is added to 500ml differentiation tank (1 / 3 volume), and 2-3 resistant calluses of the same callus from step 4 are placed on the differentiation medium, covered with a lid, and cultured at 25℃ under light (16h / 8h light cycle, light intensity 2000LUX) for about 30 days until the callus differentiates into seedlings, and when the seedlings grow to about 3-5cm, they are transferred to the rooting medium test tube, sealed with sealing film, and cultured at 25℃ under light (16h / 8h light cycle, light intensity 2000LUX).

[0118] 6. Exercise and transplanting of transgenic seedlings:

[0119] After 14 days of rooting in step 5, the test tubes of seedlings with well-differentiated roots and stems and leaves are picked out, the sealing film is opened, 30ml sterile water is added, and the seedlings are hardened in the culture room at 30℃ for 2-3 days; the agar is washed away, and the seedlings are cultured in the rice nutrient solution (same as in example 1), and seeds are collected.

[0120] 7. Screening and identification of positive transgenic plants of T0 generation

[0121] After the T0 generation transgenic seedlings are cultured in normal (temperature: 30℃ in the daytime, 22℃ at night; humidity 60%; light intensity 30000LUX, daytime and nighttime time respectively 12h) rice nutrient solution for one week, 1cm long leaves are taken, genomic DNA is extracted using CTAB method buffer, and the transgenic seedlings are identified using the resistance screening marker gene, positive transformed lines are screened, and two lines of overexpression materials OE-1 and OE-2 are obtained.

[0122] Extraction of genomic DNA by CTAB method:

[0123] Take 1 cm leaf blade in 2 ml centrifuge tube, add a 3 mm diameter steel ball, cover the centrifuge tube cover, the centrifuge tube containing the leaf blade is pretreated with liquid nitrogen, then oscillate in the sample instrument (Beijing Dinghao Yuan) for 2 min, the frequency is 25 times / sec, to obtain the crushed plant sample. Add 400 μΐ CTAB solution buffer (500 ml mother liquor, 10 g CTAB, 100 mM Tris-HCl pH 7.5, 0.7 mol NaCl, 20 mM EDTA). Place in 60°C water bath for 30 min, add 250 μΐ chloroform, centrifuge at 8000 g for 10 min, take the supernatant to another new 2 ml centrifuge tube, add 300 μΐ isopropanol, mix well and put into -20°C refrigerator for at least one hour or overnight. Centrifuge at 10000 g for 20 min, discard the liquid, add 500 μΐ 70% ethanol, then centrifuge at 10000 g for 5 min, then discard the ethanol and blow dry the centrifuge tube in the clean bench. Add 100 μΐ deionized sterile water to the final blow-dried centrifuge tube. Take 1 μΐ as a PCR template.

[0124] Hygromycin resistance gene detection:

[0125] Hygromycin resistance gene primer sequence is:

[0126] Upstream primer: CGAGTACTTCTACACAGCCATC (SEQ ID NO: 7)

[0127] Downstream primer: TAGCGAGAGCCTGACCTATT (SEQ ID NO: 8)

[0128] The PCR condition is denaturation at 95°C for 5 min, then enter the cycle reaction: 95°C, 30 s, 59°C, 30 s, 72°C, 1 min, the cycle number is 38, and the last extension is 5 min. Take 10 μΐ of PCR product for electrophoresis detection on 1% agarose gel, then record the experimental results by GelDoc™ XR+ (BIO-RAD, USA) imaging system. The transgenic positive plants have amplification bands.

[0129] Example 3

[0130] OsROHAN is a protein induced by nitrogen and located in plant plastid

[0131] Through gene cloning and construction of complementation material, the complementation material of OsROHAN can complement the sensitivity of mutant rohan to ammonium nitrogen Figure 3a and 3bAs shown in the figure; RT-PCR at specific time points and under treatment with 2.5 mM ammonium sulfate (ammonium nitrogen) and 2.5 mM potassium nitrate (nitrate nitrogen) showed that the OsROHAN gene was significantly induced by nitrogen. Figure 4a As shown in the figure; further, by examining the roots of the regenerated material under a laser confocal microscope at an excitation wavelength of 514 nm, it was found that the OsROHAN protein is located in the plastids of plant cells. Figure 4b As shown in the figure.

[0132] Example 4

[0133] Evaluation of OsROHAN overexpression resistance to ammonium poisoning

[0134] The tolerance to ammonium toxicity of OsROHAN overexpression plants obtained above was evaluated using relative root elongation as an indicator. Figure 5a and 5b The responses of wild-type and two independent overexpression lines (OE-1 and OE-2) to ammonium toxicity were shown at different ammonium sulfate concentrations (0 mM, 2.5 mM, 5 mM, 10 mM). The results showed that the seed roots of the overexpression lines treated with high concentrations of ammonium sulfate were longer than those of the wild type, indicating that the overexpression lines exhibited stronger tolerance to ammonium toxicity under high concentrations of ammonium sulfate.

[0135] Example 5

[0136] Evaluation of field yield and nitrogen use efficiency of OsROHAN overexpression

[0137] The agronomical value of OsROHAN overexpressing plants obtained above was evaluated using three fields with different fertilization concentrations (75 kg / ha, 150 kg / ha, and 350 kg / ha) as indicators of tillering at maturity, yield per plant, nitrogen use efficiency, and protein content in seeds. Figure 6a The plant phenotypes in the field and the seed phenotypes of individual plants harvested are shown for wild-type (WT) and an independent overexpression line (OE-1) in three fields with different fertilization concentrations. The results indicate that the independent overexpression line (OE-1) exhibited increased tiller number, increased yield per plant, improved nitrogen use efficiency, and increased seed protein content compared to the wild-type (WT) in the three fertilization concentrations (75 kg / ha, 150 kg / ha, 350 kg / ha urea). Figure 6b (As shown in 6c, 6d, and 6e). This demonstrates that overexpression of OsROHAN can improve nitrogen use efficiency and yield in rice, and has extremely high application value for rice germplasm innovation.

[0138] The preferred embodiments of the present application have been described above in detail. It should be understood that modifications and variations can be made by those of ordinary skill in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all such modifications and variations be included within the scope of the claims determined by the following claims.

Claims

1. Overexpression of a rice gene OsROHAN In use for improving rice tolerance to ammonium toxicity and nitrogen use efficiency, characterized in that, The nucleotide sequence of the rice gene OsROHAN is shown as SEQ ID NO: 1; the rice gene OsROHAN encoded amino acid sequence is shown as SEQ ID NO:

2.

2. A method for breeding ammonium-tolerant water rice, characterized by, The nucleotide sequence as shown in SEQ ID NO: 1 OsROHAN The overexpression is introduced into the plant of interest.

3. The method for breeding an ammonium-toxicity-tolerant plant according to claim 2, characterized by, overexpressing OsROHAN the method comprises the following steps: Step 1, in OsROHAN Genetically designing primer sequences; Step 2, import into the expression vector by using Gateway connection method; Step 3, transform the plasmid with correct sequencing into Agrobacterium; Step 4, prepare the bacterial floating liquid by using the cultured Agrobacterium with bacterial liquid; Step 5, infect the callus of the target plant with the bacterial floating liquid and place on the co-culture medium for dark culture; Step 6, screen the callus on the selection medium and culture on the differentiation medium until differentiation into seedlings.

4. The method for breeding an ammonium-toxicity-tolerant plant according to claim 3, characterized by, The amplification primer sequence is shown in SEQ ID NO: 3~6.

5. The method of claim 3, wherein, In step 4, the bacterial floating liquid is prepared by using the AAM bacterial liquid containing 200 μM acetosyringone.

6. The method of claim 3, wherein, The selection medium contains cefradine and hygromycin, and the screening times are 3 times.

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