Application of OsHRZ2 gene in regulation and control of ammonium toxicity tolerance of crops

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an OsHRZ2 gene in regulation and control of ammonium toxicity tolerance of crops, and a nucleotide sequence of the OsHRZ2 gene is shown as SEQ ID NO.1. According to the invention, a strain for overexpressing the OsHRZ2 gene is constructed, under a high ammonium condition, the root length of the OsHRZ2-OE homozygous strain is obviously greater than that of a wild type, the leaf damage is obviously reduced, and the overall growth vigor is obviously better than that of the wild type, so that the tolerance of the OsHRZ2-OE homozygous strain to high ammonium toxicity can be obviously enhanced. The ammonium toxicity tolerance function of the OsHRZ2 gene found by the invention provides an effective gene resource for cultivating ammonium toxicity-resistant crops, and meanwhile, the invention develops a channel for genetic breeding of ammonium toxicity-resistant rice, provides a new production thought for improving the ammonium toxicity resistance of rice in genetic breeding, and further ensures the food safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to application of an OsHRZ2 gene in regulating ammonium toxicity tolerance of crops. BACKGROUND

[0002] Nitrogen is an essential nutrient element in the growth process of plants, and plays a key role in maintaining plant life activities and improving crop yield. Plants mainly obtain nitrogen by absorbing ammonium nitrogen and nitrate nitrogen. As one of the main food crops in the world, rice bears the heavy responsibility of feeding more than half of the world's population, so its high and stable yield has important practical significance. However, in order to pursue high yield of rice, excessive single flying is often applied in the process of rice cultivation, and excessive ammonium nitrogen will cause toxicity to rice roots, inhibit their elongation and development, and thus have adverse effects on the growth and final yield of the whole plant. Although plants have certain physiological mechanisms to alleviate ammonium toxicity stress, under high concentration of ammonium nitrogen, these natural tolerance mechanisms often fail to play a sufficient protective role.

[0003] Therefore, mining crop ammonium toxicity tolerance related genes and enhancing the tolerance of crops to ammonium toxicity are of great significance for cultivating new crop varieties with improved nitrogen utilization efficiency by enhancing ammonium toxicity tolerance, not only helping to improve agricultural production efficiency, but also ensuring food security and meeting the growing population demand. SUMMARY

[0004] The application aims to provide application of an OsHRZ2 gene in regulating ammonium toxicity tolerance of crops, and the OsHRZ2 gene has a positive regulation effect on ammonium toxicity tolerance of crops and can be used to improve ammonium toxicity tolerance of crops.

[0005] The application provides application of an OsHRZ2 gene in regulating ammonium toxicity tolerance of crops, and the nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO. 1.

[0006] In a preferred mode of the application, the regulation comprises overexpression of the OsHRZ2 gene to improve the ammonium toxicity tolerance of crops.

[0007] The application also provides a biological material for regulating expression amount of an OsHRZ2 gene, and the nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO. 1.

[0008] The application also provides a biological material for overexpressing an OsHRZ2 gene, and the nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO. 1.

[0009] In a preferred mode of the present application, the biological material comprises at least one of the following: a recombinant expression vector containing the OsHRZ2 gene, a recombinant microorganism containing the OsHRZ2 gene, and a recombinant microorganism containing the recombinant expression vector.

[0010] In a preferred mode of the present application, when the biological material is constructed, the OsHRZ2 gene is obtained by amplification, and the primer pair for the amplification comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO. 2 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 3.

[0011] The present application also provides the use of the above-mentioned biological material in regulating the tolerance of crops to ammonium toxicity.

[0012] The present application also provides the use of the above-mentioned biological material in creating target ammonium toxicity-tolerant plant varieties.

[0013] The present application also provides a method for improving the tolerance of crops to ammonium toxicity, comprising overexpressing the OsHRZ2 gene in the genome of a target crop to obtain a crop with improved tolerance to ammonium toxicity.

[0014] The nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO. 1.

[0015] In a preferred mode of the present application, the crop comprises rice.

[0016] Beneficial effects: The present application takes wild-type japonica rice Shikarubaimo (SSBM) as the basic material, constructs OsHRZ2 overexpression transgenic homozygous rice OsHRZ2-OE, and verifies that the relative expression amount of OsHRZ2 in the OsHRZ2-OE is increased by about 13 times compared with the wild-type SSBM through qPT-PCR. Then, the agronomic traits are compared. Under normal conditions, the root length of the OsHRZ2-OE and the wild-type is not significantly different. Under high ammonium conditions, the root length of the wild-type is significantly shorter than that of the OsHRZ2-OE homozygous line, and the leaf of the wild-type shows obvious damage, while the OsHRZ2-OE homozygous line grows well. It is proved that the OsHRZ2 overexpression positively regulates the ammonium salt toxicity tolerance of crops. Overexpression of the OsHRZ2 gene can significantly increase the root length of rice under high ammonium salt toxicity conditions, and the overall growth is significantly better than that of the wild type and can also reduce leaf damage. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The plasmid map of the overexpression vector pCAMBIA1300-35S-OsHRZ2 of the present application is shown in Figure 2.

[0018] Figure 2 The relative expression amount comparison chart of OsHRZ2 in different plant materials in Example 3 is shown in Figure 3.

[0019] Figure 3 Figure 4 is a schematic diagram of the agronomic traits of root length of different plant materials in Example 4 after planting under normal and high ammonium conditions;

[0020] Figure 4 Figure 5 is a schematic diagram of the agronomic traits of leaf injury of different plant materials in Example 4 after planting under 10 mM NH4 + high ammonium conditions. DETAILED DESCRIPTION

[0021] The present application provides an application of the OsHRZ2 gene in regulating the ammonium toxicity tolerance of crops, and the nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO. 1.

[0022]

[0023] In the present application, overexpression of the OsHRZ2 gene improves the tolerance of crops to ammonium toxicity. The crops in the present application include rice, and the improvement of the tolerance of crops to ammonium toxicity mainly manifests in the improvement of the root growth of crops under ammonium stress and the reduction of leaf damage.

[0024] The present application also provides a biological material for regulating the expression amount of the OsHRZ2 gene, wherein the nucleotide sequence of the OsHRZ2 gene is shown as SEQ ID NO. 1.

[0025] The regulation in the present application includes two aspects of regulation, such as stimulating the expression of the OsHRZ2 gene, such as overexpression, and also can be inhibiting the expression of the OsHRZ2 gene, including gene silencing, siRNA or shRNA, etc., and also can be knocking out the OsHRZ2 gene, such as CRISPR-Cas9, etc.

[0026] The present application also provides a biological material for overexpressing the OsHRZ2 gene, wherein the nucleotide sequence of the OsHRZ2 gene is shown as SEQ ID NO. 1.

[0027] The biological material for overexpression in the present application includes at least one of the following: a recombinant expression vector containing the OsHRZ2 gene, a recombinant microorganism containing the OsHRZ2 gene, and a recombinant microorganism containing the recombinant expression vector. In the construction of the biological material, the OsHRZ2 gene is obtained by amplification, and the primer pair for the amplification includes a forward primer with a nucleotide sequence shown as SEQ ID NO. 2 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3.

[0028] GSP1 (SEQ ID NO. 2): 5'-gagctcggtacccggggatccATGGCGACCCCGTTGGCC-3';

[0029] GSP2 (SEQ ID NO. 3): 5'-acgacggccagtgccaagcttGAGCTGATTGTTCAACATCAGATTAA-3'.

[0030] The initial vector of the recombinant expression vector is not particularly limited in the present application, and a strong promoter capable of achieving overexpression can be used, for example, a 35S promoter of a cauliflower mosaic virus (CaMV), and in an embodiment, the initial vector is preferably pCAMBIA1300-35S, and the OsHRZ2 gene, in particular the CDS sequence of the OsHRZ2 gene, is inserted into pCAMBIA1300-35S, for example, between BamH I and Hind III, to form pCAMBIA1300-35S-OsHRZ2. The method for constructing the recombinant expression vector is not particularly limited in the present application, and a method for constructing a conventional vector in the art can be used.

[0031] The recombinant microorganism contains the OsHRZ2 gene or the recombinant expression vector, and in an embodiment, the gene is transformed and overexpressed by an agrobacterium-mediated genetic transformation method, and the agrobacterium can be EHA105, and the recombinant expression vector is transformed into the agrobacterium to obtain the recombinant microorganism.

[0032] The application further provides use of the biological material in regulating ammonium toxicity tolerance of crops.

[0033] The homozygous rice strain overexpressing the OsHRZ2 gene constructed in the embodiments of the present application has stronger ammonium toxicity tolerance than the wild type, and the overexpression strain has longer root growth and lighter leaf damage in an ammonium stress environment.

[0034] The application further provides use of the biological material in creating a target ammonium toxicity-tolerant plant variety.

[0035] The application further provides a method for improving ammonium toxicity tolerance of crops, which comprises overexpressing an OsHRZ2 gene in a target crop genome to obtain a crop with improved ammonium toxicity tolerance.

[0036] The nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO. 1.

[0037] The crop in the present application can be a monocotyledonous plant, and in an embodiment, the crop is rice. The method for introducing the OsHRZ2 gene into a target crop is not particularly limited in the present application, and a conventional method in the art can be used.

[0038] In order to further illustrate the present application, the application of the OsHRZ2 gene in regulating ammonium toxicity tolerance of crops is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of the present application.

[0039] The raw materials used in the present application are not particularly limited unless otherwise specified, and commercially available products known to those skilled in the art can be used.

[0040] Example 1

[0041] Construction of overexpression expression vector of OsHRZ2 gene

[0042] 1) RNA extraction

[0043] A rice plant sample of 20-50 mg was collected from the japonica rice variety Shiga white hair (SSBM, https: / / www.ricedata.cn / variety / varis / 608043.htm). Then, the sample was rapidly frozen with liquid nitrogen. After freezing, the sample was finely ground into powder using a special grinding device. Next, 1 ml of Invitrogen Trizol reagent (produced by Thermo Fisher Scientific) was added to the powder and mixed thoroughly. Then, 0.2 ml of chloroform was added to the mixture and shaken vigorously for 15 s to ensure that the chloroform was in contact with the mixture. After shaking, the mixture was allowed to stand at room temperature for 3 min to allow the chloroform to fully extract the impurities in the mixture. After standing, the mixture was centrifuged at 8000 rpm for 15 min. After centrifugation, the mixture was divided into three layers, with the upper layer being the aqueous phase containing RNA. The upper aqueous phase was carefully transferred to a new sterile centrifuge tube using a pipette. Then, 400 μl of isopropanol was added to the new centrifuge tube and allowed to stand at room temperature for 10 min. After standing, the mixture was again centrifuged at 8000 rpm for 15 min. After centrifugation, the supernatant was discarded and the RNA precipitate was found at the bottom of the centrifuge tube. 300 μl of RNase ethanol was added to the centrifuge tube and mixed again, and then centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. The centrifuge tube was inverted and dried. Finally, 30 μL of RNase ddH2O was added to the dried RNA precipitate and the centrifuge tube was gently shaken to dissolve the RNA. After dissolution, the RNA solution can be used for subsequent molecular biology experiments or analysis.

[0044] 2) RNA reverse transcription

[0045] The QIAGEN brand II 1stStrand cDNA Synthesis Kit II 1stStrand cDNA Synthesis Kit) was used to perform the reverse transcription process according to the kit instructions.

[0046] 3) Acquisition of target gene

[0047] In a centrifuge tube with a capacity of 200 μL, prepare the PCR reaction mixture, the specific components of which include: 5 μL of cDNA solution obtained from the previous step, 2 μL of gene-specific primer 1 (GSP1), 2 μL of gene-specific primer 2 (GSP2), 25 μL of 2x Phanta Max Master Mix, and 16 μL of ddH2O. Then, perform the PCR amplification step.

[0048] PCR reaction program: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 55°C annealing for 15 s, 72°C extension for 4 s, 35 cycles; 72°C re-extension for 1 min.

[0049] 4) Recombinant vector acquisition

[0050] The pCAMBIA1300-35S vector is subjected to enzyme cutting treatment with high-fidelity restriction endonuclease Hind III and BamH I, and then the linearized vector after treatment is recovered. Next, the linearized vector is mixed with the amplification product of step 3). This mixing process is carried out in an environment containing 5x CE II Buffer and Exnase II enzyme (ClonExpress II One-Step Cloning Kit of Vazyme Company), and the reaction lasts for 1 hour. Finally, the pCAMBIA1300-35S-OsHRZ2 recombinant vector shown in the figure is successfully constructed through the above homologous recombination reaction. Figure 1 The pCAMBIA1300-35S-OsHRZ2 recombinant vector is successfully constructed.

[0051] Example 2

[0052] Taking japonica rice Shiga white hair (SSBM) as an example, the OsHRZ2 gene is transformed into the callus of japonica rice Shiga white hair (SSBM), and the Shiga white hair (SSBM) transgenic rice is obtained.

[0053] The 0.5 μg pCAMBIA1300-35S-OsHRZ2 gene expression plasmid prepared in Example 1 is transferred into the competent cells of Agrobacterium tumefaciens strain EHA105, and then sequentially subjected to 5 min ice bath, 5 min liquid nitrogen rapid freezing, 5 min 37°C water bath and 5 min ice bath treatment. After completing these treatments, the LB medium without antibiotics is added to the system, and activated at 28°C for 1 h in a shaker, so as to obtain the Agrobacterium strain carrying the pCAMBIA1300-35S-OsHRZ2 plasmid.

[0054] The prepared EHA105 strain containing the gene expression plasmid is used to transform the callus of japonica rice Shiga white hair (SSBM), and the specific steps are as follows:

[0055] The induction and subculture of SSBM callus were performed according to the method disclosed in the reference article (Wu D X. Mechanism of rice grain filling and nitrogen utilization efficiency regulated by auxin receptor OsTIR1[D]. Nanjing Agricultural University, 2023. DOI:10.27244 / d.cnki.gnjnu.2023.000057.). The Agrobacterium carrying the plasmid was cultured in YEP liquid medium (Yeast Extract 10.0 g / L, Peptone 10.0 g / L, Sodium Chloride 5.0 g / L, pH 7.0±0.2) containing 50 mg / L kanamycin (Kan) and 50 mg / L rifampicin (Rif) at 28°C in the dark with 200 rpm shaking until its OD 600 The absorbance reached 0.8-1.0. 1 mL was taken and centrifuged at 8000 rpm for 3 min. The supernatant was taken and the bacterial pellet was resuspended with 60 μl As (acetyl-syringone) to make a suspension. The high-quality callus was taken out and placed in a sterile 50 mL centrifuge tube. The callus amount was about 10 ml. AAM (AA macroelements 100 ml / L, B5 microelements 10 ml / L, iron salt 10 ml / L, nicotinic acid 1 ml / L, pyridoxine hydrochloride 1 ml / L, thiamine hydrochloride 1 ml / L, myo-inositol 10 ml / L, MES 3.9 g / L, CH 0.5 g / L, maltose 30 g / L, pH 5.5) 30 ml was added, and the bacterial suspension was added for 5 min. The callus after infection was taken out, drained on sterile filter paper, and blown dry in a clean bench for about 30 min until dry. The callus was evenly spread on the co-culture medium with a layer of sterile filter paper on the surface, and dark culture was performed for 2.5 days

[0056] The co-cultured calli were transferred to sterile 50 mL centrifuge tubes, washed with sterile water, and finally soaked in sterile water containing Timentin for 30-60 min. The calli were then placed in the first round of selection medium, and incubated at 28°C for 2-3 weeks. The calli that grew normally in the first round of selection medium were then transferred to the second round of selection medium (the same as the first round of selection medium, but with 250 mg / L carbenicillin and 80 mg / L hygromycin added after sterilization), and incubated at 28°C for 2-3 weeks.

[0057] The calli that grew normally in the second round of selection medium were transferred to differentiation jars containing differentiation medium (N6 macroelements 50 ml / L, B5 microelements 10 ml / L, iron salt 10 ml / L, nicotinic acid 1 ml / L, pyridoxine hydrochloride 1 ml / L, thiamine hydrochloride 1 ml / L, myo-inositol 10 ml / L, L-Glu 0.5 g / L, L-pro 0.5 g / L, CH 0.3 g / L, 6-BA 3 ml / L, NAA 0.5 ml / L, sucrose 30 g / L, agar 8 g / L, pH 5.8), with 1-3 calli per jar. The jars were tightly capped, and incubated at 28°C for 3-4 weeks. When the calli differentiated into rice seedlings and grew to 3-4 cm, the seedlings were transferred to rooting medium (N6 macroelements 25 ml / L, B5 microelements 5 ml / L, iron salt 5 ml / L, nicotinic acid 0.5 ml / L, pyridoxine hydrochloride 0.5 ml / L, thiamine hydrochloride 0.5 ml / L, myo-inositol 5 ml / L, sucrose 20 g / L, agar 8.0 g / L, pH 5.5). When the seedlings grew normally in the rooting medium to a height of more than 10 cm, the seedlings were removed from the medium and incubated normally.

[0058] Example 3

[0059] Genomic DNA was extracted from the leaves of seedlings selected from Example 2. Using standard PCR techniques, the transgenic seedlings that showed positive results were successfully identified by targeting the hygromycin resistance gene. The primer sequences used for the hygromycin resistance gene identification are as follows:

[0060] hyg ID Forward Primer (SEQ ID NO. 4): 5'-ATATACGCCCGGAGTCGT-3';

[0061] hyg ID Reverse Primer (SEQ ID NO. 5): 5'-CTCTCGATGAGCTGATGCTTTG-3';

[0062] Next, total RNA was extracted from these confirmed hygromycin-resistant seedlings and converted to cDNA through a reverse transcription step. To quantify the expression level of the OsHRZ2 ammonium tolerance gene, SYBR qPCR Master Mix reagents produced by Norgen Biotek Corporation were used, and real-time quantitative PCR (qPCR) analysis was performed. In this process, the OsActin gene was used as an internal reference standard to standardize the data. The primer sequences used for qPCR are as follows:

[0063] OsHRZ2 qPCR Forward Primer (SEQ ID NO. 6): 5'-ATCACAAATGCGGCTTCTGC-3';

[0064] OsHRZ2 qPCR Reverse Primer (SEQ ID NO. 7): 5'-CCGTCTGTCTTCCATCTGGT-3';

[0065] OsActin qPCR Forward Primer (SEQ ID NO. 8): 5'-ACACCGGTGTCATGGTCGG-3'

[0066] OsActin qPCR Reverse Primer (SEQ ID NO. 9): 5'-ACACGGAGCTCGTTGTAGAA-3';

[0067] The relative expression of OsHRZ2 in wild-type japonica rice Sakumotemaki (SSBM) and the resulting homozygous OsHRZ2 overexpression transgenic rice (denoted as HRZ2-OE) is shown in Figure 2 The relative expression of OsHRZ2 in OsHRZ2-OE was increased by about 13 times compared to wild-type SSBM.

[0068] Example 4

[0069] Rice root length and leaf injury detection

[0070] Wild type japonica rice Shiga white hair (SSBM) and OsHRZ2-OE rice seeds were respectively cultivated into the soil according to the farmer's conventional cultivation method, and the agronomic management during the growth of rice was implemented according to the farmer's daily management method. Only slow-release nitrogen, phosphorus and potassium compound fertilizer was applied in the whole growth period, and the control condition was 1mM NH4 + , and the high ammonium condition was 10mM NH4 + . After the rice matured, the agronomic traits of rice aboveground root length and leaf injury were measured, as shown in Figure 3 , 4 .

[0071] As can be seen from Figure 3 , the root length of wild type japonica rice Shiga white hair (SSBM) and OsHRZ2-OE homozygous line (indicated as HRZ2-OE in the figure) under normal conditions was not much different, between 150-160mm; while under high ammonium condition, the root length of wild type japonica rice Shiga white hair (SSBM) was about 98mm, and the root length of OsHRZ2-OE homozygous line was about 171mm;

[0072] As can be seen from Figure 4 , under high ammonium salt condition, the leaves of wild type japonica rice Shiga white hair (SSBM) showed obvious injury, while the growth of OsHRZ2-OE homozygous line (indicated as HRZ2-OE in the figure) was better.

[0073] In summary, overexpression of OsHRZ2 gene can significantly increase the root length of rice under high ammonium salt toxicity, and the overall growth is significantly better than that of wild type and the leaf injury is also reduced.

[0074] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. The application of the OsHRZ2 gene in regulating crop tolerance to ammonium toxicity, characterized in that, The nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO.

1.

2. The application according to claim 2, characterized in that, The regulation includes overexpressing the OsHRZ2 gene to improve crop tolerance to ammonium toxicity.

3. A biomaterial for regulating the expression level of the OsHRZ2 gene, characterized in that, The nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO.

1.

4. A biomaterial overexpressing the OsHRZ2 gene, characterized in that, The nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO.

1.

5. The biomaterial according to claim 4, characterized in that, The biological material includes at least one of the following: a recombinant expression vector containing the OsHRZ2 gene, a recombinant microorganism containing the OsHRZ2 gene, and a recombinant microorganism containing the recombinant expression vector.

6. The biomaterial according to claim 4 or 5, characterized in that, In constructing the biomaterial, the OsHRZ2 gene is obtained by amplification, and the primer pair for amplification includes a forward primer with a nucleotide sequence as shown in SEQ ID NO.2 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.

3.

7. The application of the biomaterial according to claim 3 or any one of claims 4 to 6 in regulating crop tolerance to ammonium poisoning.

8. The use of the biomaterial according to claim 3 or any one of claims 4 to 6 in the creation of target ammonium-resistant plant varieties.

9. A method for improving crop tolerance to ammonium toxicity, characterized in that, This includes overexpressing the OsHRZ2 gene in the genome of the target crop to obtain crops with improved tolerance to ammonium toxicity; The nucleotide sequence of the OsHRZ2 gene is shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that, The crop mentioned includes rice.

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