Application of Rice OsMYBS2 Gene in Regulating Plant Phosphorus Nutrient Accumulation

Abstract

This application relates to the field of biotechnology, specifically to the application of the rice OsMYBS2 gene in regulating the accumulation of plant phosphorus nutrients. After overexpressing this gene in the recipient plant, it can promote the increase in the expression levels of phosphate transporter genes and proteins, so as to promote the absorption, distribution and utilization of phosphorus by plants, significantly increase the inorganic phosphorus content in the plant body, and thus improve the yield and quality of plants.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically relates to the application of the rice OsMYBS2 gene in regulating the accumulation of plant phosphorus nutrients. Background Art

[0002] Phosphorus is an essential macronutrient for plant growth and development. It is not only an important component of many biomolecules in plants, including nucleic acids, proteins, phospholipids, phosphosugars, and adenylates, but also participates in many important life processes in plants, including photosynthesis, respiration, energy transfer, and signal transduction. Since the inorganic phosphorus that can be absorbed by plants in the soil is easily fixed, the available phosphorus content in most soils is low, making phosphorus one of the main factors limiting crop yield and quality. In production, a large amount of phosphate fertilizer is often applied to ensure stable and high yields of crops, which accelerates the depletion of phosphate rock resources. On the other hand, the large application of phosphate fertilizer causes eutrophication of surface runoff water bodies, resulting in agricultural non-point source pollution.

[0003] To cope with low phosphorus stress, plants have evolved a complex regulatory mechanism to promote the absorption, distribution, and utilization of phosphorus. Regulatory proteins play a key role in the phosphorus starvation response mechanism. PHRs and SPXs proteins are core regulatory factors in the plant phosphorus signal and phosphorus balance regulation system (Paz-Ares J, Puga M I, Rojas-Triana M, Martinez-Hevia I, Diaz S, Poza-Carrion C, Minambres M, Leyva A. Plant adaptation to low phosphorus availability: Core signaling, crosstalks, and applied implications. Molecular Plant, 2022, 15, 104-124). Under phosphorus starvation stress, PHRs directly activate the expression of phosphorus starvation-induced genes by binding to the P1BS (PHR1 Binding Site, GNATATNC) cis-acting element in the promoter region of downstream genes, including phosphate transporter PTs (Phosphate Transporters) genes, to promote the absorption and utilization of phosphorus by plants. SPXs proteins are negative regulators of phosphorus signal and phosphorus balance. When phosphorus supply is sufficient, SPXs inhibit the transcriptional activation activity of PHRs by interacting with PHRs proteins (Paz-Ares J, Puga M I, Rojas-Triana M, Martinez-Hevia I, Diaz S, Poza-Carrion C, Minambres M, Leyva A. Plant adaptation to low phosphorus availability: Core signaling, crosstalks, and applied implications. Molecular Plant, 2022, 15, 104-124). The discovery of phosphorus regulatory proteins is of great significance for the cultivation of phosphorus-efficient crop varieties. However, the currently discovered phosphorus regulatory proteins are still very limited. Summary of the Invention

[0004] In view of this, the present invention provides the application of rice OsMYBS2 gene in regulating the accumulation of plant phosphorus nutrients. The discovery of this new application of the gene and protein is of great significance for the cultivation of phosphorus-efficient crop varieties.

[0005] To achieve the above invention object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides an application of a gene in regulating phosphorus nutrient accumulation in plants, and the gene comprises at least one of the following sequences:

[0007] (1) The nucleotide sequence shown in SEQ ID NO:1;

[0008] (2) The complementary sequence, degenerate sequence or homologous sequence of the nucleotide sequence shown in SEQ ID NO:1, wherein the homologous sequence is a nucleotide sequence having 95% or more identity with the nucleotide sequence shown in SEQ ID NO:1.

[0009] In the present invention, genes having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the nucleotide sequence shown in SEQ ID NO:1 and having the same function are all within the protection scope of the present invention.

[0010] Among them, the gene can be a CDS sequence (coding sequence), genomic DNA or recombinant DNA.

[0011] In the embodiments of the present invention, the plants include monocotyledonous plants and / or dicotyledonous plants.

[0012] In the specific embodiments of the present invention, the monocotyledonous plants include rice, wheat, sorghum or corn.

[0013] Preferably, the monocotyledonous plant is rice.

[0014] In the specific embodiments of the present invention, the dicotyledonous plants include soybean, rapeseed, cotton or Arabidopsis thaliana.

[0015] In a second aspect, the present invention provides an application of a protein in regulating phosphorus nutrient accumulation in plants, and the protein comprises at least one of the following sequences:

[0016] (1) The protein sequence shown in SEQ ID NO:2;

[0017] (2) The fusion protein sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the protein sequence shown in SEQ ID NO:2.

[0018] In a third aspect, the present invention provides an application of a biological material in regulating phosphorus nutrient accumulation in plants, and the biological material is one of the following (1) or (2):

[0019] (1) A recombinant vector containing the gene of claim 1;

[0020] (2) A recombinant microorganism containing the gene of claim 1.

[0021] In an embodiment of the present invention, the vector is a plasmid vector. The plasmid vector is, for example, pCAMBIA1300, pCAMBIA1302, pCAMBIA2300, pCAMBIA1300-GFP, pCAMBIA2301, pCAMBIA1301, pGWB411, pGWB412, pGWB405, pBin438, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb.

[0022] In an embodiment of the present invention, the microorganism is Agrobacterium. For example, Agrobacterium EHA105 and the like.

[0023] Fourthly, the present invention provides a method for cultivating a plant with efficient phosphorus absorption, the method comprising: overexpressing the above-mentioned gene and / or protein in a recipient plant to obtain a target plant; the phosphorus content of the target plant is increased compared with the recipient plant.

[0024] In a specific embodiment of the present invention, the method for overexpressing the gene and / or protein of the present invention in a recipient plant is: inserting the gene into a vector to obtain a recombinant vector; transferring the recombinant vector into the recipient plant. However, the overexpression method is not limited thereto, and it can also be promoter editing technology, codon optimization, using a strong promoter, inserting introns, using a viral vector, fusion protein technology, etc.

[0025] In the present invention, over-expression (OE) of a gene refers to constructing a plasmid vector by fusing the full-length sequence of a target gene with a highly active constitutive promoter, and obtaining an organism in which the gene product accumulates in large amounts through transformation.

[0026] In an embodiment of the present invention, when the gene of the present invention is overexpressed in a recipient plant, the relative expression level of the gene in the obtained target plant is increased by at least 50%, at least 100%, at least 500%, at least 1000%, at least 2000%, at least 3000%, at least 4000%, at least 5000%, at least 6000% or at least 7000%.

[0027] Preferably, the phosphorus content of the target plant is increased by at least 20%, at least 25%, at least 30%, at least 35% or at least 40% compared with the recipient plant.

[0028] In a specific embodiment of the present invention, the phosphorus content of the above-ground part of the target plant is increased by at least 40% compared with the recipient plant.

[0029] In a specific embodiment of the present invention, the phosphorus content of the root of the target plant is increased by at least 20% compared with the recipient plant.

[0030] In the embodiments of the present invention, the method for cultivating plants with efficient phosphorus absorption further includes: compared with the recipient plant, the expression level of the phosphate transporter gene in the target plant is increased.

[0031] In the embodiments of the present invention, the phosphate transporter gene includes at least one of OsPT2, OsPT4, OsPT8, and OsPT10.

[0032] Preferably, compared with the recipient plant, the expression level of the phosphate transporter gene in the target plant is increased by at least 100%, at least 110%, at least 130%, at least 150%, at least 170%, at least 190%, at least 200%, or at least 210%.

[0033] In the specific embodiments of the present invention, compared with the recipient plant, the expression level of the phosphate transporter gene OsPT2 in the target plant is increased by at least 170%.

[0034] In the specific embodiments of the present invention, compared with the recipient plant, the expression level of the phosphate transporter gene OsPT4 in the target plant is increased by at least 170%.

[0035] In the specific embodiments of the present invention, compared with the recipient plant, the expression level of the phosphate transporter gene OsPT8 in the target plant is increased by at least 110%.

[0036] In the specific embodiments of the present invention, compared with the recipient plant, the expression level of the phosphate transporter gene OsPT10 in the target plant is increased by at least 210%.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] The present invention provides the application of the rice OsMYBS2 gene in regulating the accumulation of plant phosphorus nutrients. After overexpressing this gene in the recipient plant, it can promote the increase in the expression levels of the phosphate transporter gene and protein, so as to promote the absorption, distribution, and utilization of phosphorus by the plant, significantly increase the content of inorganic phosphorus in the plant body, and thus improve the plant yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Showing the relative expression level of the OsMYBS2 gene.

[0040] Figure 2 Showing the relative expression levels of the OsPT2, OsPT4, OsPT8, and OsPT10 genes.

[0041] Figure 3 Showing the results of the phenotypic observation of rice leaves.

[0042] Figure 4 Showing the results of the detection of the inorganic phosphorus content.

[0043] In the figure, * indicates P-Value < 0.05, and ** indicates P-Value < 0.01. Detailed implementation manners

[0044] The present invention discloses the application of rice OsMYBS2 gene in regulating the accumulation of plant phosphorus nutrients. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0045] Term explanation:

[0046] N-terminus: Refers to the amino terminus of a peptide chain or protein;

[0047] C-terminus: Refers to the carboxyl terminus of a peptide chain or protein;

[0048] Deoxyribonucleic acid (DNA): It is one of the four biological macromolecules contained in biological cells and is a type of nucleic acid. DNA carries the genetic information necessary for the synthesis of RNA and proteins and is an essential biological macromolecule for the development and normal operation of organisms. DNA is composed of deoxynucleotides, and deoxynucleotides are composed of bases, deoxyribose, and phosphoric acid. Among them, there are 4 types of bases: adenine (A), guanine (G), thymine (T), and cytosine (C).

[0049] CDS sequence: CDS is the abbreviation of Coding sequence. DNA is transcribed into mRNA, and after mRNA is processed such as splicing, it is translated into a protein. The so-called CDS is the DNA sequence that corresponds one-to-one with the protein sequence, and there is no other sequence in the middle that does not correspond to this protein, regardless of the sequence changes during mRNA processing and other processes.

[0050] Ribonucleic acid (RNA): The genetic information carrier existing in biological cells and some viruses and viroids. RNA is a long-chain molecule formed by the condensation of ribonucleotides through phosphodiester bonds. A ribonucleotide molecule is composed of phosphoric acid, ribose, and a base. The bases of RNA are mainly 4 types, namely A (adenine), G (guanine), C (cytosine), and U (uracil). Among them, U (uracil) replaces T (thymine) in DNA. The main function of ribonucleic acid in vivo is to guide the synthesis of proteins.

[0051] Phosphorus is the main phosphorus source for plants, and its occurrence form and content greatly affect the yield and quality of crops. In the soil environment, phosphorus is mainly divided into two forms: inorganic phosphorus and organic phosphorus. Among them, the inorganic phosphorus that can be absorbed by plant roots mainly includes primary mineral phosphorus, secondary phosphate, hydrolyzed phosphorus, and adsorbed phosphorus. Plant roots mainly obtain the essential phosphorus for growth by absorbing soluble phosphate in the soil, and most organic phosphorus needs to be converted into inorganic phosphorus before it can be absorbed and utilized by plants.

[0052] The reagents, instruments, biological materials, etc. used in the present invention can all be obtained through commercial channels.

[0053] The experimental materials used in the following examples:

[0054] Rice material: Ishikari Shiro (SSBM);

[0055] Strain: Agrobacterium strain EHA105;

[0056] Overexpression vector: pCAMBIA1300.

[0057] The present invention will be further described below in conjunction with examples:

[0058] Example 1: Preparation of transgenic rice plants overexpressing OsMYBS2

[0059] The nucleotide sequences and protein sequences used in this example are as follows:

[0060] OsMYBS2 DNA sequence:

[0061] ATGGAGCAGCATGAGGAGGCAGCGGAGAGGAAGCCTTCGCCGCCGGTGA

[0062] TATTCCGGCTGTTCGGCGTCGAGGTCCGCGGCGGCGGCGGCGGAGTTGAC

[0063] GAGGAGGAGTACGAGGAGGAGGAGGTGGAGGGTGGATTGTTCATCAAGA

[0064] AGAGCTCCAGTATGCCCAACCTCACCTCCATCGACCCGCTGCCGGTGCCG

[0065] GCCGACGGCGGCAAACGGCGCGCCTCCGACGACTCCGAGCTCGCCTCCGG

[0066] CCAGCAGAAGCGCCGCCGCCGCAAGGTGCAGGAGAGGAAGAAAGGGGTA

[0067] CCATGGACTGAGGAGGAGCACAAGAAATTCCTGGAAGGGCTGAGGCAGC

[0068] TGGGGAAAGGGGACTGGAGAGGCATCTCCAAGAACTTTGTGACCAGCAG

[0069] GACGGCGACTCAGGTGGCCAGCCACGCCCAGAAGTACTTCCTCCGGCAGA

[0070] CCAACCCTGGCAAAAAGAAGCGCCGGGCCAGCCTCTTTGATGTTGTTGCT

[0071] GAGTGCAGTGATGATCAGCTTCCAAGTCCTCAGAGTGTTGGAACTAAGCC

[0072] TCCTACCCAGGATATAATTCATACAGATCGCGGCGATGTCCCGATACTAA

[0073] GCTATCCAGTTGCTAGAGGCTTTAGAGGCGATAGCGTGCAGGTTGATGAA

[0074] CTAACTGAATATGTGAAGAGATTAAAGGCCGCCGAGGACATGTCGCTCTC

[0075] CATGATCTCTGGACTGGAAATGGCATCATCATCCATCAGCAGTCTAGAGC

[0076] TCAGTATCGCGCCTCCTCATTGCGCGATCGAGGCGGCCATCAAGGTGCTG

[0077] TGA

[0078] OsMYBS2 protein sequence:

[0079] MEQHEEAAERKPSPPVIFRLFGVEVRGGGGGVDEEEYEEEEVEGGLFIKKSSS

[0080] MPNLTSIDPLPVPADGGKRRASDDSELASGQQKRRRRKVQERKKGVPWTEEE

[0081] HKKFLEGLRQLGKGDWRGISKNFVTSRTATQVASHAQKYFLRQTNPGKKKR

[0082] RASLFDVVAECSDDQLPSPQSVGTKPPTQDIIHTDRGDVPILSYPVARGFRGDS

[0083] VQVDELTEYVKRLKAAEDMSLSMISGLEMASSSISSLELSIAPPHCAIEAAIKVL*

[0084] The specific preparation method of the overexpressing transgenic plants of rice OsMYBS2 includes the following steps:

[0085] (1) Construction of the pCAMBIA1300-OsMYBS2 overexpression vector:

[0086] Using the cDNA of the above-ground part of rice Shishou Baimao (SSBM) under hydroponic conditions as a template, PCR amplification was carried out using the upstream primer 5’-CGCGGATCCATGGAGCAGCATGAGGAGGC-3’ and the downstream primer 5’-ACGCGTCGACCAGCACCTTGATGGCCGC-3’. The DNA fragment of the OsMYBS2 gene about 800bp was purified and recovered. The DNA fragment of the OsMYBS2 gene was double digested with BamHI endonuclease and SalI endonuclease (NEB), and it was ligated into the vector of pCAMBIA1300 that was also double digested with BamHI endonuclease and SalI endonuclease to obtain the recombinant vector pCAMBIA1300-OsMYBS2.

[0087] (2) Preparation of the overexpressing transgenic plants of OsMYBS2:

[0088] Using the wild-type rice plant SSBM as the background material, rice was infected with the Agrobacterium tumefaciens EHA105 strain containing the pCAMBIA1300-OsMYBS2 vector to obtain the T0 generation transgenic positive plants; the T0 generation transgenic plants were self-crossed for 2 generations to obtain the homozygous OsMYBS2 overexpressing transgenic rice material. Two overexpressing homozygous lines #1 and #2 were selected and denoted as OsMYBS2-OE-1 and OsMYBS2-OE-2 respectively. Real-time fluorescence quantitative PCR detection was carried out using the primer pair composed of the upstream 5'-CAGACCAACCC TGGCAAAAA-3' and the downstream 5'-GAGGACTTGGAAGCTGATCATCA-3'.

[0089] The results are as Figure 1 shown. Compared with wild-type plants, the relative expression levels of the OsMYBS2 gene in the OsMYBS2-OE-1 and OsMYBS2-OE-2 plant lines were significantly increased. Example 2: Increased expression levels of phosphate transporter genes OsPT2, OsPT4, OsPT8, and OsPT10 in rice OsMYBS2 overexpressing transgenic plants

[0090] Experimental material treatment: Wild-type rice plants SSBM, overexpressing transgenic plants OsMYBS2-OE-1 and OsMYBS2-OE-2 plant lines were hydroponically treated. The hydroponic full nutrient solution formula was: 1.4 mM NH4NO3, 0.2 mM NaH2PO4·2H2O, 0.19 mM K2SO4, 0.55 mM MgSO4·7H2O, 0.36 mM CaCl2, 0.1 mM EDTA-FeNa, 0.005 mM 24 MnCl2·4H2O, 0.03 mM H3BO3, 0.001 mM (NH4)6Mo7O

[0091] ·4H2O, 0.004 mM ZnSO4·7H2O, 0.002 mM CuSO4·5H2O, and the pH of the nutrient solution was 5.5 - 6.0. The culture conditions for the plants were 12 h light / 12 h dark, 30°C / 22°C. After culturing in the full nutrient solution for 7 days, the roots of the rice plants were sampled.

[0092] PT2-F: 5’-GAAACCCCACAAATCCACAAC-3’

[0093] PT2-R: 5’-CACAAACTTCCTCGGTATGCT-3’

[0094] PT4-F: 5’-ACCAGAATAATCATCCATTGCACAT-3’

[0095] PT4-R: 5’-CGTCATCGGGTTCTTCTTCAC-3’

[0096] PT8-F: 5’-TGAATTGTTGGACAGCGTATTT-3’

[0097] PT8-R: 5'-TTCCCTGCCTGGCTATTTATC-3'

[0098] PT10-F: 5'-GAGTTCACTCACACGGAGACC-3'

[0099] PT10-R: 5'-GAGCTCGCACCTCAGCAT-3'

[0100] Actin-F: 5'-CAACACCCCTGCTATGTACG-3'

[0101] Actin-R: 5'-CATCACCAGAGTCCAACACAA-3'

[0102] The results are as Figure 2 shown. Compared with the wild-type plants, the relative expression levels of OsPT2, OsPT4, OsPT8, and OsPT10 genes in the plants of OsMYBS2-OE-1 and OsMYBS2-OE-2 lines were significantly increased.

[0103] Example 3: Observation of leaf phenotypes and detection of inorganic phosphorus content in rice OsMYBS2 overexpression transgenic plants under normal culture conditions

[0104] I. Observation of rice leaf phenotypes

[0105] Treatment of experimental materials and phenotypic observation: After culturing the wild-type rice plants SSBM and the OsMYBS2 overexpression transgenic plants OsMYBS2-OE-1 and OsMYBS2-OE-2 in a complete nutrient solution for 14 days, the phenotypes of the first old leaves were observed.

[0106] The results are as Figure 3 shown. The leaves of the wild-type plants did not show the withered and chlorotic phenotypes of phosphorus accumulation, while the leaves of the OsMYBS2-OE-1 and OsMYBS2-OE-2 plants showed the withered and chlorotic phenotypes of phosphorus accumulation.

[0107] II. Detection of inorganic phosphorus content in rice plants

[0108] Treatment of experimental materials: After culturing the wild-type rice plants SSBM and the OsMYBS2 overexpression transgenic plants OsMYBS2-OE-1 and OsMYBS2-OE-2 in a complete nutrient solution for 14 days, samples of the above-ground parts and roots of the rice were taken.

[0109] The inorganic phosphorus contents in the above-ground parts and roots of the rice were measured. The specific measurement method is as follows:

[0110] (I) Solution preparation

[0111] (1) 2,6-Dinitrophenol or 2,4-dinitrophenol indicator: Weigh 0.25 g of dinitrophenol powder and dissolve it in deionized water. Stir carefully and, after cooling, make up to 100 mL with deionized water.

[0112] (2) 4 M sodium hydroxide (NaOH) solution: Weigh 16 g of sodium hydroxide pellets and dissolve them in deionized water. Finally, make up to 100 mL with deionized water.

[0113] (3) 5 M sulfuric acid (H₂SO₄) solution: Measure 227.8 mL of concentrated sulfuric acid and slowly pour it into 500 mL of deionized water while stirring. After cooling, make up to 1000 mL with deionized water.

[0114] (4) 2 M H₂SO₄ solution: Dilute the 5 M sulfuric acid solution 2.5 times.

[0115] (5) 0.5% potassium antimonyl tartrate solution: Weigh 0.5 g of potassium antimonyl tartrate and dissolve it in deionized water. Make up to 100 mL with deionized water.

[0116] (6) Molybdenum-antimony mixture: Weigh 10 g of ammonium molybdate and dissolve it in 450 mL of deionized water. Then slowly add 153 mL of concentrated sulfuric acid while stirring. Next, pour the previously prepared 100 mL of 0.5% potassium antimonyl tartrate solution into it. Finally, make up to 1000 mL with deionized water, shake well and store in a brown bottle.

[0117] (7) Molybdenum-antimony-ascorbic acid solution: This solution is prepared immediately before use. Weigh 1.5 g of L-ascorbic acid and dissolve it in 100 mL of the molybdenum-antimony mixture.

[0118] (II) Preparation of phosphorus standard curve

[0119] (1) Weigh an appropriate amount of potassium dihydrogen phosphate (KH₂PO₄) (analytical grade) and dry it to constant weight in an oven at 105 °C.

[0120] (2) Accurately weigh 0.2195 g of the dried KH₂PO₄ above, dissolve it in deionized water and make up to 1000 mL to obtain a 50 μg / mL phosphorus standard solution.

[0121] (3) Accurately pipette 25 mL of the 50 μg / mL phosphorus standard solution prepared in the previous step and make up to 250 mL with deionized water to obtain a 5 μg / mL phosphorus standard solution (this concentration of phosphorus standard solution needs to be prepared immediately before use).

[0122] (4) Accurately pipette 0, 2, 4, 6, 8, 10 mL of the 5 μg / mL phosphorus standard solution into a 50 mL volumetric flask, and then add deionized water to about 30 mL.

[0123] (5) Add 200 μL of dinitrophenol indicator to the above solution, dropwise add 4 M NaOH solution to the solution, and observe the color of the solution until it just turns yellow, then add 2 M H2SO4 solution dropwise until the solution just turns colorless.

[0124] (6) Add 5 mL of molybdenum antimony anti-solution to the above solution, make up the volume to 50 mL with deionized water, after standing for 30 min, measure the OD value (zero with a blank sample) using a spectrophotometer, and then prepare a phosphorus standard curve. 700nm Value (zero with a blank sample), and then prepare a phosphorus standard curve.

[0125] (7) The standard curve equation is Y = 1.948X + 0.0016; X is the OD value, Y is the phosphorus concentration (unit: μg / mL); R 700nm Value, Y is the phosphorus concentration (unit: μg / mL); R 2 = 1.

[0126] (III) Extraction of inorganic phosphorus

[0127] Grind the plant sample with liquid nitrogen, weigh about 0.1 g of the sample powder into a 2 mL centrifuge tube, add 0.5 mL of 5 M H2SO4 solution, mix well and let stand for 30 min, then add 1 mL of deionized water, mix well, then centrifuge at 13000 rpm for 10 min, and take the supernatant for testing.

[0128] (IV) Detection of inorganic phosphorus content

[0129] Prepare a 10 mL centrifuge tube, add 5 mL of deionized water to the centrifuge tube, add 20 μL of dinitrophenol indicator, and then add 100 μL of the supernatant from step (III) (adjust the volume of the added supernatant according to the content of Pi (inorganic phosphorus) in the sample). Add 4 M NaOH solution dropwise to the solution and observe the color of the solution until it just turns yellow, then add 2 M H2SO4 solution dropwise until the solution just turns colorless. Make up the above reaction solution to 9 mL with deionized water, then add 1 mL of molybdenum antimony anti, and finally mix well. After standing for 30 min, measure the OD 700nm Value.

[0130] Substitute the OD 700nm Value into the standard curve equation in step (II) to obtain the phosphorus content.

[0131] Phosphorus content in rice plants = phosphorus content (μg) obtained in step (IV) ÷ fresh weight of plant sample (g) obtained in step (III)

[0132] The results are as Figure 4 shown. Compared with SSBM, the inorganic phosphorus content of OsMYBS2 overexpressing transgenic plants OsMYBS2 - OE - 1 and OsMYBS2 - OE - 2 is significantly increased.

[0133] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Use of a gene in regulating inorganic phosphorus nutrient accumulation in rice, characterized in that, The gene is the nucleotide sequence shown in SEQ ID NO:

1.

2. Use of a protein in regulating inorganic phosphorus nutrient accumulation in rice, characterized in that, The protein is at least one of the following sequences: (1) The protein sequence shown in SEQ ID NO:2; (2) A fusion protein sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the protein sequence shown in SEQ ID NO:

2.

3. Application of a biological material in regulating inorganic phosphorus nutrient accumulation in rice, characterized in that, The biological material is one of the following (1) or (2): (1) A recombinant vector containing the gene recited in claim 1; (2) A recombinant microorganism containing the gene recited in claim 1.

4. The application according to claim 3, characterized in that The vector is a plasmid vector, and / or the microorganism is Agrobacterium.

5. A method for cultivating rice with high-efficiency inorganic phosphorus absorption, characterized in that, Overexpressing the gene recited in claim 1 and / or the protein recited in claim 2 in a recipient plant to obtain a target plant; compared with the recipient plant, the inorganic phosphorus content of the target plant is increased.

6. The method according to claim 5, characterized in that, The method of overexpressing the gene recited in claim 1 and / or the protein recited in claim 2 in a recipient plant is: inserting the gene recited in claim 1 into a vector to obtain a recombinant vector; transferring the recombinant vector into the recipient plant.

7. The method according to claim 5, characterized in that, Compared with the recipient plant, the inorganic phosphorus content of the target plant is increased by at least 20%.

8. The method according to claim 5, wherein The method further includes: Compared with the recipient plant, the expression level of the phosphate transporter gene of the target plant is increased; The phosphate transporter gene includes Os PT2 , Os PT4 , Os PT8 , Os PT10 or at least one of them.

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