CN112409465B - Application of protein M57 in regulating rice resistance to ammonium - Google Patents

Abstract

The invention discloses application of protein M57 in regulation and control of ammonium resistance of rice. Introduction of a recombinant vector expressing protein M57 into Nipponbare to obtain T₀Transgenic rice with OsMADS57-R gene; will T₀Continuously selfing rice with transgenic OsMADS57-R gene for three generations to obtain T₃Transgenic rice with OsMADS57-R gene. Compared with Nipponbare, T₃The generation homozygous OsMADS57-R transgenic rice has the advantages of more vigorous growth, obviously increased leaf width, obviously thickened stem, more compact plant type, obviously increased leaf angle, obviously reduced plant height in the mature period, obviously increased grain weight, grain width and yield after harvesting of grains per spike, and obviously increased grain length and grain number per spike. Therefore, the protein M57 plays an important role in regulating and controlling the ammonium resistance of rice, yield, plant height, grain weight, grain width, spike length, spike grain number, leaf width, stem diameter and leaf angle.The invention has important application value.

Description

Application of protein M57 in regulating rice resistance to ammonium

technical field

The invention belongs to the field of biotechnology, in particular to the application of protein M57 in regulating the resistance of rice to ammonium.

Background technique

Rice is one of the most important food crops in the world. Rice is grown in more than 120 countries around the world, and the cultivated area is maintained at more than 150 million hectares all year round. Rice is the staple food for 50% of the world's population. The yield of rice is very dependent on the application of nitrogen fertilizer, but the application of nitrogen fertilizer also has many serious negative effects. In the air, the nitrogen cycle causes pollution and damage to a large area of water resources, soil and ecosystems. In addition, rice is a crop that prefers ammonium nitrogen fertilizer, but excessive ammonium fertilizer is toxic to rice, affecting the growth and yield of rice. Therefore, improving the resistance of rice to ammonium is crucial for rice production.

SUMMARY OF THE INVENTION

The purpose of the present invention is to improve the resistance of plants to ammonium.

The present invention firstly protects the application of protein M57, which can be at least one of the following a1)-a10): a1) regulating plant resistance to ammonium; a2) regulating plant yield; a3) regulating plant grain weight; a4) regulating plant grain width; a5) regulation of plant ear length; a6) regulation of plant ear number of grains; a7) regulation of plant leaf width; a8) regulation of plant stem diameter; a9) regulation of plant leaf angle; a10) regulation of plant height.

In the above application, the protein M57 can be b1) or b2) or b3):

b1) The amino acid sequence is the protein shown in Sequence 3 in the sequence listing;

b2) a fusion protein obtained by linking a tag to the N-terminus or/and C-terminus of the protein shown in SEQ ID NO: 3 in the sequence listing;

b3) The resistance to ammonium and/or the yield and/or the grain weight and/or the amino acid sequence obtained by substitution and/or deletion and/or addition of one or several amino acid residues in the amino acid sequence shown in Sequence 3 in the sequence listing Proteins related to grain width and/or ear length and/or grain number per ear and/or leaf width and/or stem diameter and/or leaf angle and/or plant height.

Among them, sequence 3 in the sequence listing consists of 241 amino acid residues.

In order to facilitate purification of the protein in b1), a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 3 in the sequence listing.

Table 1. Sequence of tags

For the protein in the above b3), the substitution and/or deletion and/or addition of one or several amino acid residues is the substitution and/or deletion and/or addition of no more than 10 amino acid residues.

The protein in the above b3) can be obtained by artificial synthesis, or by first synthesizing its encoding gene and then biologically expressing it.

The coding gene of the protein in the above b3) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in Sequence 2 or Sequence 1 in the Sequence Listing, and/or performing one or several base pairs of codons. Missense mutations, and/or ligation of the coding sequences of the tags shown in Table 1 at its 5' and/or 3' ends.

The present invention also protects the application of the nucleic acid molecule encoding the protein M57, which can be at least one of the following a1)-a10): a1) regulation of plant resistance to ammonium; a2) regulation of plant yield; a3) regulation of plant grain a4) control plant grain width; a5) control plant ear length; a6) control plant ear number; a7) control plant leaf width; a8) control plant stem diameter; a9) control plant leaf angle; a10) control Plant height.

In the above application, the nucleic acid molecule encoding the protein M57 can be the DNA molecule shown in any of the following c1)-c6):

c1) a DNA molecule whose coding region is shown in sequence 2 in the sequence listing;

c2) a DNA molecule whose coding region is shown in sequence 1 in the sequence listing;

c3) The nucleotide sequence is the DNA molecule shown in sequence 2 in the sequence listing;

c4) The nucleotide sequence is the DNA molecule shown in Sequence 1 in the sequence listing;

c5) a DNA molecule having 75% or more identity with the nucleotide sequence defined in c1) or c2) or c3) or c4) and encoding the protein M57;

c6) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in c1) or c2) or c3) or c4) and encodes said protein M57.

Wherein, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

Among them, sequence 2 in the sequence listing consists of 726 nucleotides, and sequence 1 in the sequence listing consists of 726 nucleotides, and the nucleotides of sequence 2 or sequence 1 in the sequence listing encode the amino acids shown in sequence 3 in the sequence listing. sequence.

Those of ordinary skill in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the protein M57 of the present invention. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence of the protein M57 isolated by the present invention, as long as they encode the protein M57, are all derived from the nucleosides of the present invention acid sequences and are equivalent to the sequences of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or more, or 80% or more, or 85% or more of the nucleotide sequence of the protein M57 consisting of the amino acid sequence shown in SEQ ID NO: 2 of the coding sequence listing of the present invention, or 90% or more, or 95% or more identical nucleotide sequences. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In any of the above-mentioned applications, the plant can be any one of the following c1) to c5): c1) dicots; c2) monocots; c3) grasses; c4) rice; c5) Rice variety Nipponbare.

The present invention also protects a method for cultivating transgenic plants, which may include the following steps: increasing the expression and/or activity of the protein M57 in the starting plant to obtain a transgenic plant; compared with the starting plant, the resistance of the transgenic plant to ammonium Enhancement and/or increased yield and/or increased grain weight and/or increased grain width and/or increased ear length and/or increased number of grains per ear and/or increased leaf width and/or increased stem diameter and/or leaf angle Increase and/or decrease in plant height.

In the above-mentioned method, the described "improving the expression and/or activity of protein M57 in the starting plant" can achieve the improvement of the expression and/or activity of the protein M57 in the starting plant by means of multiple copies, changing promoters, regulatory factors or transgenes. /or active effect.

In the above method, the "improving the expression level and/or activity of protein M57 in the starting plant" can be realized by introducing a recombinant vector into the starting plant; the recombinant vector is to insert a nucleic acid molecule encoding protein M57 into the expression vector, and obtain recombinant plasmids.

In the above method, the nucleic acid molecule encoding protein M57 can be the DNA molecule shown in d1) or d3) or d5) or d6):

d1) a DNA molecule whose coding region is shown in sequence 2 in the sequence listing;

d3) the nucleotide sequence is the DNA molecule shown in sequence 2 in the sequence listing;

d5) a DNA molecule that has 75% or more identity with the nucleotide sequence defined in d1) or d3) and encodes the protein M57;

d6) A DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined by d1) or d3) and encodes said protein M57.

In the above method, the expression vector may be pCAMBIA1300 vector.

In the above method, the recombinant vector can be the recombinant plasmid pCAMBIA1300-MADS57-Resist. The recombinant plasmid pCAMBIA1300-MADS57-Resist can be a small DNA fragment between the restriction endonuclease BamHI recognition sequence and the restriction endonuclease SacI recognition sequence of the pCAMBIA1300 vector with the nucleotide sequence shown in Sequence 2 in the sequence table. The DNA molecule shown, the resulting recombinant plasmid.

The present invention also protects a plant breeding method, which may include the steps of increasing the expression and/or activity of the protein M57 in plants, thereby enhancing the plant's resistance to ammonium and/or increasing yield and/or increasing grain weight and /or increased grain width and/or increased ear length and/or increased number of grains per ear and/or increased leaf width and/or increased stem diameter and/or increased leaf angle and/or decreased plant height.

In the above method, the "increasing the expression and/or activity of protein M57 in plants" can increase the expression and/or the expression of protein M57 in plants by means of multiple copies, changing promoters, regulatory factors or transgenes. active effect.

Any of the above-mentioned regulation and control of plant resistance to ammonium may be to improve plant resistance to ammonium.

Any one of the plants described above may be any one of the following c1) to c5): c1) dicotyledonous plants; c2) monocotyledonous plants; c3) grasses; c4) rice; c5) rice variety Nipponbare.

The included angle of any of the above-mentioned leaves may specifically be the included angle between the flag leaf and the connected leaf sheath.

Any of the above-mentioned yields may be yield per ear.

The recombinant plasmid pCAMBIA1300-MADS57-Resist mentioned in the example was introduced into the rice variety Nipponbare to obtain T ₀ generation transgenic OsMADS57- _R gene rice _; Co-transformed rice with OsMADS57-R gene. Compared with the rice variety Nipponbare, in the vegetative growth period, the T ₃ generation homozygous transgenic rice with OsMADS57-R gene grew stronger, with significantly increased leaf width and thicker stems (shown as a significant increase in stem diameter); The plant type of the T ₃ generation homozygous transgenic rice with OsMADS57-R gene was more compact, the leaf angle was significantly increased, and the plant height was significantly decreased _; after the ear grains were harvested, the grain weight and The grain width was significantly increased, the ear length and the number of grains per ear were also significantly increased, and the yield was also significantly increased. It can be seen that protein M57 plays an important role in regulating rice resistance to ammonium, yield, plant height, grain weight, grain width, ear length, grain number per ear, leaf width, stem diameter and leaf angle. The invention has important application value.

Description of drawings

Figure 1 shows the resistance to ammonium of T ₃ generation homozygous transgenic rice with OsMADS57-R gene.

Figure 2 shows the phenotype of rice in vegetative growth and mature stages.

Figure 3 shows the leaf phenotype and ear grain phenotype of rice, as well as the statistical results of leaf width, grain weight, grain length and grain width.

Figure 4 shows the statistical results of the stem phenotype and stem internode phenotype of rice, as well as the number of grains per ear.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

Quantitative experiments in the following examples are all set up to repeat the experiments three times, and the results are averaged.

Plant cDNA synthesis kit is a product of TOYOBO company.

High-fidelity Phusion DNA polymerase is a product of Thermo.

DNA gel purification column and pGEM-T vector are products of Promega Corporation.

Leaf angle refers to the angle between the flag leaf and the attached leaf sheath.

Example 1. Acquisition of T ₃ generation homozygous transgenic rice with OsMADS57-R gene and identification of its resistance to ammonium and agronomic traits

1. Construction of recombinant plasmid pGEM-T-MADS57

1. The 10-day seedlings of the rice variety Nipponbare (hereinafter referred to as Nipponbare) were used as experimental materials, and total RNA was extracted with Trizol reagent to obtain the RNA of Nipponbare. The RNA of Nipponbare was taken, the quality of RNA was detected by DNA gel first, and then the concentration of RNA was determined by nanodrop spectrophotometer.

2. After completing step 1, take 1 μL of Nipponbare RNA, and perform reverse transcription with a plant cDNA synthesis kit to obtain 20 μL of cDNA product (the cDNA product is the cDNA of Nipponbare).

3. After completing step 2, using the cDNA product as a template, MADS57-Fp: 5'-atggggagggggaagatagt-3' and MADS57-Rp: 5'-ttaaggcagatgaagtccca-3' as primers, use high-fidelity Phusion DNA polymerase for PCR amplification , to obtain PCR amplification products.

The reaction volume was 50 μL. The concentration of each primer in the reaction system was 10 μM.

The reaction program was: 98°C for 30s; 98°C for 10s, 55°C for 15s, 72°C for 30s, 35 cycles; 72°C for 5 min.

4. After completing step 3, use a DNA gel purification column to recover a DNA fragment of about 726 bp in the PCR amplification product.

5. Connect the DNA fragment recovered in step 4 with the pGEM-T vector to obtain the recombinant plasmid pGEM-T-MADS57.

The recombinant plasmid pGEM-T-MADS57 was sequenced. The sequencing results showed that the recombinant plasmid pGEM-T-MADS57 contained the DNA molecule shown in SEQ ID NO: 1 in the sequence listing. The DNA molecule shown in sequence 1 in the sequence listing is the OsMADS57 gene.

2. Construction of recombinant plasmid pGEM-T-MADS57/Resist

1. According to the target site of miR444 (nucleotide sequence: 5′-GCAGCAAGCTTGAGGCAGCAACUGCA-3′) on the MADS57 gene, design and synthesize MADS57-Resist-Fp: 5′-gcCgcCTCcCtCCgCcaAcaGctCcaTaaTCtCcaagaaagccacaagcaactg-3′ and MADS57-Resist-Rp : 5'-agattatggagctgttggcggagggaggcggcctccctctgccaaatcttaa-3'.

2. The first round of PCR amplification reaction

(1) Using 20ng of recombinant plasmid pGEM-T-MADS57 as template and MADS57-Fp and MADS57-Rp as primers, high-fidelity Phusion DNA polymerase was used for PCR amplification to obtain PCR amplification product A.

(2) Using 20ng of recombinant plasmid pGEM-T-MADS57 as template, MADS57-Resist-Fp and MADS57-Resist-Rp as primers, high-fidelity Phusion DNA polymerase was used for PCR amplification to obtain PCR amplification product B.

In steps (1) and (2), the reaction system is 50 μL; the concentration of each primer in the reaction system is 10 μM.

In steps (1) and (2), the reaction procedures are: 98°C for 30s; 98°C for 10s, 55°C for 15s, 72°C for 30s, 35 cycles; 72°C for 5 min.

3. Recycling

(1) A DNA fragment A of about 300 bp in the PCR amplification product A was recovered by a DNA gel purification column.

(2) A DNA fragment B of about 400 bp in PCR amplification product B was recovered by a DNA gel purification column.

4. The second round of PCR amplification reaction

(1) Mix 50 ng of DNA fragment A and 50 ng of DNA fragment B thoroughly to obtain mixed DNA.

(2) Using 100ng of mixed DNA as template and MADS57-Fp and MADS57-Rp as primers, high-fidelity PhusionDNA polymerase was used for PCR amplification to obtain PCR amplification product C.

The reaction volume was 50 μL. The concentration of each primer in the reaction system was 10 μM.

The reaction procedures were: 98°C for 30s; 98°C for 10s, 55°C for 15s, 72°C for 30s, 35 cycles; 72°C for 5 min.

5. Recycling

The DNA fragment C of about 700 bp in the PCR amplification product C was recovered with a DNA gel purification column.

6. Connect the DNA fragment C recovered in step 4 with the pGEM-T vector to obtain the recombinant plasmid pGEM-T-MADS57/Resist.

The recombinant plasmid pGEM-T-MADS57/Resist was sequenced. The sequencing results show that the recombinant plasmid pGEM-T-MADS57/Resist contains the DNA molecule shown in sequence 2 in the sequence listing.

The DNA molecule shown in sequence 2 in the sequence listing is the OsMADS57-R gene.

Both the OsMADS57-R gene and the OsMADS57 gene encode the protein M57 shown in SEQ ID NO: 3 in the sequence listing.

3. Construction of recombinant plasmid pCAMBIA1300-MADS57-Resist

1. Using the recombinant plasmid pGEM-T-MADS57/Resist as the template, PCR amplification was carried out using a primer pair consisting of MADS57-recombination-Fp: 5′-gaacacgggggactctagagatggggagggggaagatagt-3′ and MADS57-recombination-Rp: 5′-cgatcggggaaattcgagctttaaggcagatgaagtccca-3′ to obtain PCR amplification products.

2. Use a DNA gel purification column to recover the DNA fragment of about 750 bp in the PCR amplification product obtained in step 1.

3. The pCAMBIA1300 vector was digested with restriction enzymes BamHI and SacI, and the vector backbone with a size of about 10 kb was recovered.

4. Use the seamless cloning system of GIBSON to carry out homologous recombination reaction, and integrate the DNA fragment recovered in step 2 into the vector backbone recovered in step 3 to obtain a recombinant plasmid pCAMBIA1300-MADS57-Resist.

The recombinant plasmid pCAMBIA1300-MADS57-Resist was sequenced. According to the sequencing results, the structure of the recombinant plasmid pCAMBIA1300-MADS57-Resist is described as follows: Replace the small DNA fragment between the restriction endonuclease BamHI recognition sequence and the restriction endonuclease SacI recognition sequence of the pCAMBIA1300 vector with the nucleotide sequence: The DNA molecule shown in the sequence 2 in the sequence listing (the DNA molecule shown in the sequence 2 in the sequence listing is the OsMADS57-R gene).

In the recombinant plasmid pCAMBIA1300-MADS57-Resist, the 35S promoter drives the expression of the OsMADS57-R gene. The recombinant plasmid pCAMBIA1300-MADS57-Resist expresses the protein M57 shown in sequence 3 in the sequence listing.

Fourth, the acquisition of recombinant Agrobacterium

The recombinant plasmid pCAMBIA1300-MADS57-Resist was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was named EHA105/pCAMBIA1300-MADS57-Resist.

The pCAMBIA1300 vector was introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant Agrobacterium, which was named EHA105/pCAMBIA1300.

5. Acquisition of OsMADS57-R gene transgenic rice in T ₀ generation

Using the method of Hiei et al. (Hiei Y, Ohta S, Komari T & Kumashiro T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 1994, 6:271-282) EHA105/pCAMBIA1300-MADS57-Resist was transformed into Nipponbare to obtain the OsMADS57-R gene transgenic rice of T ₀ generation.

6. Real-time quantitative PCR detection of OsMADS57-R gene transgenic rice in T ₀ generation

Randomly select 10 T ₀ generations to be transfected with OsMADS57-R gene rice (named respectively OsMADS57-OE1-T ₀ to OsMADS57-OE10-T ₀ ) for real-time quantitative PCR detection. The specific steps are as follows:

1. The 10-day seedling experimental materials of 10 T ₀ generations to be transfected with OsMADS57-R gene rice were used to extract total RNA with Trizol reagent, and then reverse transcribed with plant cDNA synthesis kit to obtain each T ₀ generation to be transfected with OsMADS57 - cDNA of the R gene rice.

2. Using RT-qPCR technology to detect the relative expression of OsMADS57-R gene in 10 T ₀ generation rice cDNAs to be transfected with OsMADS57-R gene (with OsACTIN gene as internal reference gene). The primers for OsMADS57-R gene detection were OsMADS57-qF: 5'-CGAAGCGTCGGAACGGGCTT-3' and OsMADS57-qR: 5'-AGGCCAGAAAGCTCCTCACCC-3'. The primers for OsACTIN gene detection were OsACTIN-qF: 5'-AGGAAGGCTGGAAGAGGACC-3' and OsACTIN-qR: 5'-CGGGAAATTGTGAGGGACAT-3'.

3. According to the above method, replace the OsMADS57-R gene rice in the T ₀ generation with Nipponbare, and other steps remain unchanged to obtain the relative expression level of the OsMADS57-R gene in Nipponbare.

Taking the relative expression level of OsMADS57-R gene in Nipponbare as 1, the relative expression level of OsMADS57-R gene in other rice plants was calculated. The results showed that, compared with Nipponbare, the relative expression of OsMADS57-R gene in the 10 T ₀ generations to be transfected with OsMADS57-R gene was significantly increased.

The above results indicated that OsMADS57-OE1-T ₀ to OsMADS57-OE10-T ₀ were all T ₀ generation transgenic OsMADS57-R rice.

7. The acquisition of OsMADS57-R gene transgenic rice with T ₃ generation homozygous and real-time quantitative PCR detection

1. The OsMADS57-OE1-T ₀ to OsMADS57-OE5-T ₀ were selfed for three consecutive generations to obtain the T ₃ generation homozygous transgenic OsMADS57-R gene rice, which were named OsMADS57-OE1-T ₃ to OsMADS57-OE5-T respectively ₃ .

2. Perform real-time quantitative PCR detection on OsMADS57-OE1-T ₃ to OsMADS57-OE5-T ₃ and Nipponbare respectively according to the method in step 6.

The detection results showed that compared with Nipponbare, the relative expression of OsMADS57-R gene in OsMADS57-OE1-T ₃ to OsMADS57-OE5-T ₃ was significantly increased.

According to the above method, EHA105/pCAMBIA1300-MADS57 _- Resist was replaced with EHA105/pCAMBIA1300.

8. Identification of ammonium resistance and agronomic traits in T ₃ generation homozygous transgenic rice with OsMADS57-R gene

The rice to be tested is OsMADS57-OE1-T ₃ , OsMADS57-OE3-T ₃ , empty vector rice or Nipponbare.

1. The resistance to ammonium in rice homozygous for T ₃ transgenic OsMADS57-R gene

The main indicator of the toxic effect of ammonium on rice is leaf yellowing, so the degree of ammonium toxicity to rice can be judged by the degree of leaf yellowing. The higher the degree of yellowing of rice leaves, the greater the toxicity of ammonium to rice, and the lower the resistance of rice to ammonium.

(1) Take 50 seeds of rice to be tested, dehull, then disinfect with 10% sodium hypochlorite for 45 minutes, and rinse with sterile water for 5 times, 5-10 minutes each time.

(2) Take 30 rice seeds to be tested that have completed step (1) and are full, and sown in 3 sterile culture bottles, with 10 seeds per bottle. The medium for each flask is 0.25mM _NH4 ⁺ , 0.5mM _NH4 ⁺ , 1 mM _NH4 ⁺ , 2.5mM _NH4 ⁺ , or 5mM NH4+ ₄₊ medium ^.

Medium containing 0.25 mM NH ₄ ⁺ : add 1 L of ultrapure water, 10 g of sucrose and 0.39 g of M531 nitrogen-free MS solid powder (PhytoTechnology Laboratories, USA), after the complete dissolution on a magnetic stirrer, adjust the pH to 5.7; then add 3 g of plant gel and 0.25 mL of 1 M NH ₄ Cl aqueous solution, and sterilize at 113° C. for 30 min.

Medium with 0.5 mM _NH4 ⁺ : Follow the preparation method for medium with 0.25 mM _NH4 ⁺ , replacing "0.25 mL of 1 M aqueous _NH4Cl " with "0.5 mL of 1 M aqueous _NH4Cl " ”, other things remain unchanged.

Medium with 1 mM _NH4 ⁺ : Following the preparation method for medium with 0.25 mM _NH4 ⁺ , replace "0.25 mL of 1 M aqueous _NH4Cl " with "1 mL of 1 M aqueous _NH4Cl ", Others remain unchanged.

Medium with 2.5 mM _NH4 ⁺ : Follow the preparation method for medium with 0.25 mM _NH4 ⁺ , replacing "0.25 mL of 1 M aqueous _NH4Cl " with "2.5 mL of 1 M aqueous _NH4Cl " ”, other things remain unchanged.

Medium with 5 mM _NH4 ⁺ : Following the preparation method for medium with 0.25 mM _NH4 ⁺ , replace "0.25 mL of 1 M aqueous _NH4Cl " with "5 mL of 1 M aqueous _NH4Cl ", Others remain unchanged.

(3) After step (2) is completed, the culture bottle is placed in a constant temperature incubator, and cultured at 25° C. and light and dark alternately for 14 days, and the degree of yellowing of the rice leaves is observed.

The light-dark culture was alternated between light and dark, and the conditions were: 12h light culture/12h dark culture; the light intensity during light culture was 90 μE/m ² /s.

Part of the results are shown in Figure 1 (A is the growth state of rice roots under different ammonium ion concentrations; B is the growth state of rice leaves when the ammonium ion concentration is 5mM). The results showed that, compared with Nipponbare, OsMADS57-OE1-T ₃ , OsMADS57-OE2-T ₃ and OsMADS57-OE3-T ₃ had significantly enhanced resistance to ammonium; Nipponbare and empty carrier rice had no significant difference in ammonium resistance. . With the increase of ammonium ion concentration, the toxicity of ammonium to Nipponbare, empty carrier rice, OsMADS57-OE1-T ₃ , OsMADS57-OE2-T ₃ or OsMADS57-OE3-T ₃ also increased to a certain extent.

2. Identification of agronomic traits

The experiment was repeated three times to obtain the average value, and the steps for each repetition were as follows:

(1) 15 seeds of the rice to be tested were sown in the field, and routine field management was performed, and then the leaves, stems, plant types, leaf angles, and plant heights of the rice to be tested were compared and counted in the vegetative growth period and the mature period respectively. , observe the phenotype of the harvested grains per ear and count the grain weight, grain length, grain width and number of grains per ear.

During the vegetative growth period, the phenotypes of some of the rice to be tested are shown in Figure 2, A (WT is Nipponbare).

At the maturity stage, the phenotypes of some of the rice to be tested are shown in B in Figure 2 (WT is Nipponbare).

The leaf phenotypes of some of the tested rice are shown in Figure 3, A (WT is Nipponbare).

The statistical results of the leaf width of some rice to be tested are shown in Figure 3, B (WT is Nipponbare).

The panicle grain phenotype of some rice to be tested is shown in Figure 3 C (WT is Nipponbare).

The statistical results of grain weight of some rice to be tested are shown in the left panel of D in Figure 3 (WT is Nipponbare), the statistical results of grain length are shown in the middle panel of D in Figure 3 (WT is Nipponbare), and the statistical results of grain width are shown in Figure 3 in D (WT is Nipponbare).

The statistics of grain number per panicle of some rice to be tested are shown in Figure 4 A (WT is Nipponbare).

The stem phenotypes of some of the tested rice are shown in Figure 4, B (WT is Nipponbare).

The stem internode length of some rice to be tested is shown in Figure 4 C (WT is Nipponbare).

The results showed that in the vegetative growth period, OsMADS57-OE1-T ₃ and OsMADS57-OE3-T ₃ grew more vigorously, with significantly increased leaf width and significantly thicker stems (shown as a significant increase in stem diameter) compared with Nipponbare. ; At the mature stage, OsMADS57-OE1-T ₃ and OsMADS57-OE3-T ₃ had more compact plant types, significantly increased leaf angle, and decreased plant height compared with Nipponbare; The grain weight and grain width of OsMADS57-OE1-T ₃ and _OsMADS57 -OE3-T ₃ were significantly increased, and the ear length and grain number per ear were also significantly increased. The yield per panicle of OE3-T ₃ was significantly increased. There was no significant difference in the above agronomic traits between Nipponbare and empty carrier rice.

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> Application of protein M57 in regulating rice resistance to ammonium

<160> 3

<170> PatentIn version 3.5

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aacatgaaaa ctgtgataga ccggtatacc aacgcaaagg aggagctact tggcgggaat 240

gcaacttcag aaattaagat ttggcagagg gaggccgcct ccctccgcca acagctccat 300

aatctccaag aaagccacaa gcaactgatg ggtgaggagc tttctggcct aggtgttaga 360

gacctacaag gtttagagaa taggcttgaa ataagtctac gtaatatcag aatgagaaag 420

gacaatcttt tgaaaagtga aatcgaggag ttacatgtga agggaagcct aattcaccag 480

gaaaacatcg aactttctag aagcctaaat gtcatgtcgc aacaaaaatt ggaactgtat 540

aacaagcttc aggcctgtga acagagaggt gccacagatg caaatgaaag ttccagcact 600

ccatacagct ttcgtatcat acaaaatgct aatatgcctc ctagtcttga attgagccaa 660

tcacagcaaa gagaagggga gtgcagcaaa acagctgctc cagaactggg acttcatctg 720

ccttaa 726

<210> 3

<211> 241

<212> PRT

<213> Oryza sativa L.

<400> 3

Met Gly Arg Gly Lys Ile Val Ile Arg Arg Ile Asp Asn Ser Thr Ser

1 5 10 15

Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Leu Leu Lys Lys Ala

20 25 30

Lys Glu Leu Ser Ile Leu Cys Asp Ala Glu Val Gly Leu Val Val Phe

35 40 45

Ser Ser Thr Gly Arg Leu Tyr Glu Phe Ser Ser Thr Asn Met Lys Thr

50 55 60

Val Ile Asp Arg Tyr Thr Asn Ala Lys Glu Glu Leu Leu Gly Gly Asn

65 70 75 80

Ala Thr Ser Glu Ile Lys Ile Trp Gln Arg Glu Ala Ala Ser Leu Arg

85 90 95

Gln Gln Leu His Asn Leu Gln Glu Ser His Lys Gln Leu Met Gly Glu

100 105 110

Glu Leu Ser Gly Leu Gly Val Arg Asp Leu Gln Gly Leu Glu Asn Arg

115 120 125

Leu Glu Ile Ser Leu Arg Asn Ile Arg Met Arg Lys Asp Asn Leu Leu

130 135 140

Lys Ser Glu Ile Glu Glu Leu His Val Lys Gly Ser Leu Ile His Gln

145 150 155 160

Glu Asn Ile Glu Leu Ser Arg Ser Leu Asn Val Met Ser Gln Gln Lys

165 170 175

Leu Glu Leu Tyr Asn Lys Leu Gln Ala Cys Glu Gln Arg Gly Ala Thr

180 185 190

Asp Ala Asn Glu Ser Ser Ser Thr Pro Tyr Ser Phe Arg Ile Ile Gln

195 200 205

Asn Ala Asn Met Pro Pro Ser Leu Glu Leu Ser Gln Ser Gln Gln Arg

210 215 220

Glu Gly Glu Cys Ser Lys Thr Ala Ala Pro Glu Leu Gly Leu His Leu

225 230 235 240

Pro

Claims (7)

1. The application of the protein M57 in enhancing the ammonium resistance of rice;

the protein M57 is b1) or b 2):

b1) the amino acid sequence is protein shown as a sequence 3 in a sequence table;

b2) and (b) fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 3 in the sequence table.

2. Use of a nucleic acid molecule encoding the protein M57 of claim 1 for enhancing the ammonium-resistant ability of rice.

3. Use according to claim 2, characterized in that: the nucleic acid molecule is a DNA molecule shown as any one of c1) -c 4):

c1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

c2) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

c3) the nucleotide sequence is a DNA molecule shown in a sequence 2 in a sequence table;

c4) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table.

4. A method for breeding transgenic rice, comprising the steps of: increasing the expression level and/or activity of the protein M57 in claim 1 in starting rice to obtain transgenic rice; compared with the starting rice, the ammonium resistance of the transgenic rice is enhanced;

the improvement of the expression level and/or activity of the protein M57 of claim 1 in the starting rice is realized by introducing a recombinant vector into the starting plant; the recombinant vector is obtained by inserting a nucleic acid molecule encoding the protein M57 into an expression vector.

5. The method of claim 4, wherein: the nucleic acid molecule for coding the protein M57 is a DNA molecule shown as d1) or d 3):

d1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

d3) the nucleotide sequence is a DNA molecule shown in a sequence 2 in a sequence table.

6. The method of claim 4, wherein: the recombinant vector is a recombinant plasmid pCAMBIA1300-MADS 57-Resist; the recombinant plasmid pCAMBIA1300-MADS57-Resist is obtained by replacing a small DNA fragment between a restriction enzyme BamHI recognition sequence and a restriction enzyme SacI recognition sequence of a pCAMBIA1300 vector with a DNA molecule of which the nucleotide sequence is shown as a sequence 2 in a sequence table.

7. A rice breeding method comprises the following steps: increasing the expression level and/or activity of the protein M57 according to claim 1 in rice, thereby increasing the ammonium resistance of rice;

the increase of the expression level and/or activity of the protein M57 according to claim 1 in rice is achieved by introducing a recombinant vector into a starting plant; the recombinant vector is obtained by inserting a nucleic acid molecule encoding the protein M57 into an expression vector.

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