Rice OsNAC4 gene and application thereof in regulation and control of phosphorus nutrient accumulation of leaves

Abstract

The invention discloses a rice OsNAC4 gene and application of the rice OsNAC4 gene in regulation and control of phosphorus nutrient accumulation of leaves. The nucleotide sequence of the OsNAC4 gene is as shown in SEQ ID No: 1. The OsNAC4 gene is overexpressed in the rice body, the tip of the inverted leaf of the obtained OsNAC4-OE-1 and OsNAC4-OE-2 strains has the phenotype of scorched withering and chlorosis, and after the content of inorganic phosphorus in the leaves is measured at the same time, the phosphorus content of the leaves of the NAC4-OE-1 and the NAC4-OE-2 becomes high, and phosphorus accumulation occurs. Compared with a wild type plant, the OsPHT1 in the OsNAC4-OE-1 strain plant and the OsNAC4-OE-2 strain plant has the advantages that the OsPHT1 in the The relative expression level of the gene 1, 2 is obviously increased, which shows that the OsNAC4 improves the phosphorus absorption capacity of the rice so as to realize phosphorus accumulation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a rice OsNAC4 gene and its application in regulating leaf phosphorus accumulation. BACKGROUND

[0002] Seventeen essential elements are required for crops to complete their normal life cycle. Except for C, H and O, which are obtained from air and water, the other fourteen elements are obtained from soil solution. Ca, Mg and S are macronutrients for plants, while Fe, Mn, Zn, Cu, B, Mo, Cl and Ni are micronutrients. N, P and K are the most important nutrients for plants. Phosphorus is one of the most important nutrients for plants. Firstly, phosphorus is an important component of macromolecules in plants, including DNA, RNA, ATP, NADPH, phospholipids and other macromolecules. Secondly, phosphorus is involved in a number of key biochemical processes, such as photosynthesis and respiration. Thirdly, phosphorus plays a key role in improving the adaptability of crops to the external environment.

[0003] NAC (NAM-ATAF-CUC) family transcription factors are plant-specific regulatory proteins that play crucial roles in plant growth and development, stress response, and metabolic processes. NAC family transcription factors have a highly conserved N-terminal domain called the NAC domain. NAC is widely present in various plants, with 117, 151, 559, 157, 93, and 153 NAC genes in Arabidopsis thaliana, rice, wheat, maize, tomato, and soybean, respectively. In terms of nutrient utilization, NAC family transcription factors influence plant growth, development, and final yield by regulating various nutrient signal responses. NAC transcription factors affect plant nitrogen utilization efficiency by regulating genes related to nitrogen absorption, utilization, and transport. In rice, SNAC2 directly binds to the promoters of nitrate transporter genes (OsNRT2.1 / 2.2 and OsNRT1.1A / 1.B), activates their expression, and promotes nitrogen absorption and utilization in rice. In wheat, TaNAC2-5A enhances root growth and nitrate uptake by regulating the expression of nitrate transporter and glutamine synthetase genes. In apple, MhNAC2 promotes nitrogen absorption by regulating the expression of MhNRT2.4 and citrate transporter (MhMATE) genes. In Arabidopsis thaliana, NAC056 promotes lateral root growth and nitrate assimilation by regulating the expression of nitrate assimilation gene NIA1. AtNAC076 helps roots adapt to low-nitrogen environments, and CIPK1 phosphorylates NAC075 to limit its movement in the roots and changes root architecture through WRKY53. NAC transcription factors also play important roles in iron nutrition. ZmNAC78 is specifically expressed in maize kernels and directly regulates the expression of iron transporter genes, increasing iron content in maize kernels. NAC5 in Arabidopsis thaliana is activated under iron deficiency conditions and directly activates the expression of NFYA8, promoting root growth and iron uptake. NAC transcription factor IDEF2 in rice regulates iron stress tolerance by directly regulating the expression of iron transporter genes such as OsYSL2. In addition to nitrogen and iron, NAC transcription factors also regulate plant tolerance to other nutrients such as aluminum and boron. NAC transcription factors play important roles in plant responses to nutrient stress, regulating the expression of genes related to nutrient absorption, transport, and utilization. Currently, NAC transcription factors mainly regulate nitrogen and trace elements, and there is little research on phosphorus regulation. Future exploration of the potential interaction between NAC and phosphorus signaling pathways to improve phosphorus utilization efficiency has great potential. SUMMARY

[0004] The present application aims to provide a rice OsNAC4 gene and its application in regulating leaf phosphorus accumulation.

[0005] The present application aims to provide a rice OsNAC4Genes, the ones mentioned OsNAC4 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating phosphorus accumulation in leaves; (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of regulating phosphorus accumulation in leaves.

[0006] Rice OsNAC4 protein, wherein the amino acid sequence of the OsNAC4 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of regulating phosphorus accumulation in leaves.

[0007] Containing the rice OsNAC4 The carrier of genes.

[0008] Containing the rice OsNAC4 Engineered bacteria with gene vectors.

[0009] Amplification of the rice OsNAC4 Primers for any segment of a gene.

[0010] The rice OsNAC4 Application of genes in regulating phosphorus accumulation in leaves.

[0011] A method to increase phosphorus accumulation in rice leaves by increasing the expression level of OsNAC4 protein in rice.

[0012] The beneficial effects of this invention: This invention allows for overexpression in rice. OsNAC4 Genes, acquired OsNAC4-OE-1 and OsNAC4-OE-2 The tip of the bottom leaf of the strain showed a scorched and chlorotic phenotype, and the inorganic phosphorus content in the leaves was measured at the same time. NAC4-OE-1 and NAC4-OE-2The phosphorus content in the leaves increased, indicating phosphorus accumulation. Compared to wild-type plants, OsNAC4-OE-1 and OsNAC4-OE-2 Among the plant strains OsPHT1;2 The relative expression level of the gene was significantly increased, indicating that OsNAC4 It improves the phosphorus absorption capacity of rice to achieve phosphorus accumulation. Attached Figure Description

[0013] Figure 1 Phosphorus deficiency stress significantly induces OsNAC4 Expression in leaves; A is OsNAC4 Expression levels of B under phosphorus-added (+P) and phosphorus-deficient (-P) conditions, B is OsNAC4 Expression patterns in rice leaves.

[0014] Figure 2 for OsNAC4 Overexpression lines OsNAC4 Expression level detection.

[0015] Figure 3 for OsNAC4 Overexpression lines improved the ability of rice to absorb phosphorus; A represents wild-type (SSBM) and NAC4- OE Phenotype of the top leaf of the strain; B represents SSBM and NAC4-OE Analysis of inorganic phosphorus content in the strains; C is OsNAC4 Overexpression lines OsPHT1;2 The level of expression. Detailed Implementation

[0016] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0017] The experimental materials used in the following examples are: rice material: SSBM; bacterial strain: Escherichia coli strain. DH5α Agrobacterium strains EHA105 Overexpression vector: pCAMBIA1300 .

[0018] Example 1 Rice OsNAC4-GUS Report on transgenic plant preparation and phosphorus-deficient culture conditions OsNAC4 Expression level detection Experimental material treatment: rice SSBM, OsNAC4-OE-1 and OsNAC4-OE-2The strains were subjected to hydroponic treatment. The hydroponic nutrient solution formula was as follows: 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 MnCl2·4H2O, 0.03 mM H3BO3, 0.001 mM (NH4)6Mo7O 24 The nutrient solution contained 0.004 mM ZnSO4·7H2O, 0.002 mM CuSO4·5H2O, and had a pH of 6.2. The hydroponic phosphorus-deficient nutrient solution formula was identical to the complete nutrient solution formula, except for the omission of water and the addition of 0.2 mM NaH2PO4·2H2O. The nutrient solution was changed every 3 days during cultivation. The plant culture conditions were 12 hours light / 12 hours dark, 30℃ / 22℃.

[0019] pCAMBIA1301-NAC4-pro-GUS Vector construction: Using genomic DNA from the aboveground parts of rice SSBM under hydroponic conditions as a template, PCR amplification was performed using the upstream primer 5'-CCGCTCGAGGCTCACGCTTGTTTGGGATG-3' and the downstream primer 5'-CGGGGTACCCCTTCTCCTCTCGTGAAAATGT-3'. Approximately 2600 bp was purified and recovered. OsNAC4 Gene promoter fragments, using Kpn I endonuclease (NEB) and Sal I endonuclease (NEB) for pCAMBIA1301 The vector is digested with enzymes, and the digestion products are recovered. In-Fusion (Novizan) is then used to... OsNAC4 Gene promoter recovery fragments are ligated to linearized pCAMBIA1301 The vector was used to obtain the recombinant vector. pCAMBIA1301-NAC4-pro-GUS .

[0020] Preparation of GUS staining solution (500 ml working solution): Add 50 ml of phosphate-buffered saline (PBS), 0.5 ml of Tritonx-100, 0.33 g of K3[Fe(CN)6], 0.42 g of K4[Fe(CN)6], 10 ml of 0.5 M EDTA, and 0.1 g of 5-bromo-4-chloro-3-indole-β-D to a final volume of 500 ml to prepare the GUS staining working solution.

[0021] OsNAC4-GUS Report on transgenic plant preparation: Using rice SSBM as background material, transgenic plants containing... pCAMBIA1301-NAC4-pro-GUS Agrobacterium vector EHA105 The strain infected rice to obtain T0 generation transgenic positive plants, and the positive plants were identified by GUS staining.

[0022] The top leaf of SSBM rice cultured in normal nutrient solution and phosphorus-deficient nutrient solution was used for testing. OsNAC4 The level of expression, the results are as follows Figure 1 As shown in Figure A, compared to phosphorus-added nutrient solution, rice cultured in phosphorus-deficient nutrient solution droops in the first leaf. OsNAC4 The expression level was significantly increased. Rice samples were taken. OsNAC4-pro-GUS Plant staining, results as follows Figure 1 As shown in B, in the plant's leaves, mesophyll cells, and vascular bundle cells... OsNAC4 High level of expression.

[0023] Example 2 Rice OsNAC4 In the leaves of overexpressing transgenic plants OsNAC4 Expression level detection pCAMBIA1300-NAC4 Overexpression vector construction: Using cDNA from the aboveground parts of rice SSBM grown in hydroponics as a template, PCR amplification was performed using the upstream primer 5'-TCGAGGGGGGGCCCGGTACCATGGAAATGGCGGCGGCG-3' and the downstream primer 5'-TCCTCACCATGTCGACGAATGGTGGCAGGATTGTCTG-3'. Approximately 1000 bp was purified and recovered. OsNAC4 Gene DNA fragments, used Kpn I endonuclease (NEB) and Sal I endonuclease (NEB) for pCAMBIA1300 The vector is digested with enzymes, and the digestion products are recovered. In-Fusion (Novizan) is then used to... OsNAC4 Recycled fragments are connected to linearized pCAMBIA1300 The vector was used to obtain the recombinant vector. pCAMBIA1300-NAC4 .

[0024] OsNAC4 Preparation of transgenic plants by overexpression: Using rice SSBM as background material, transgenic plants containing... pCAMBIA1300- NAC4 Agrobacterium vector EHA105 The strain infected rice to obtain T0 generation transgenic positive plants; the T0 generation transgenic plants were self-crossed twice to obtain homozygous plants. OsNAC4 Rice materials overexpressing the gene were selected, and two homozygous overexpression lines, #1 and #2, were used for real-time quantitative PCR detection using primer pairs consisting of upstream 5'-CTCGTGGTGCACTACCTCTG-3' and downstream 5'-GACGCCTTCTCAGGGA-3'.

[0025] Take rice SSBM and OsNAC4-OE Real-time quantitative PCR was performed on leaves of the two overexpression lines, and the results are as follows:Figure 2 As shown, compared to wild-type plants, in the leaves, OsNAC4-OE-1 and OsNAC4-OE-2 Among the plant strains OsNAC4 The relative expression level of the gene was significantly increased.

[0026] Example 3 Rice OsNAC4 Inorganic phosphorus content, leaf phenotype, and leaf morphology in overexpressed transgenic plants OsPHT1;2 Expression level detection Experimental material treatment: rice SSBM, OsNAC4-OE-1 and OsNAC4-OE-2 The strains were subjected to normal hydroponic cultivation. After 21 days of hydroponics, samples were taken from the top leaf. The samples were then crushed with liquid nitrogen, followed by extraction with 5 M H₂SO₄ for 30 minutes. After extraction, 1 ml of deionized water was added, the mixture was shaken thoroughly, and incubated at 13000 rpm for 10 minutes. The supernatant was then collected and analyzed using the molybdenum antimony colorimetric method. The inorganic phosphorus content was calculated by measuring the color at 695 nm using a UV spectrophotometer. Two [samples] were selected. OsNAC4 Overexpression homozygous lines #1 and #2 were used, employing primers consisting of upstream 5'-GAAAACCCCACAAATCCACAAC-3' and downstream 5'-CACAAACTTCCTCGGTATGCT-3' to target the expression of *Gastroparesis* in plant leaves. OsPHT1;2 Gene expression levels were detected by real-time quantitative PCR.

[0027] The results are as follows Figure 3 As shown in Figure A, under normal phosphorus nutrient solution conditions, the tip of the top leaf of the SSBM rice line did not exhibit the scorching and chlorosis phenotype associated with phosphorus accumulation. OsNAC4-OE-1 and OsNAC4-OE-2 The tip of the bottom leaf of the strain showed a scorched and chlorotic phenotype, and the inorganic phosphorus content in the leaves was measured at the same time. Figure 3 B) Later discovered NAC4-OE-1 and NAC4-OE-2 The phosphorus content in the leaves increases, leading to phosphorus accumulation. For example... Figure 3 As shown in C, compared to wild-type plants... OsNAC4-OE-1 and OsNAC4-OE-2 Among the plant strains OsPHT1;2 The relative expression level of the gene was significantly increased, indicating that OsNAC4 It improves the phosphorus absorption capacity of rice to achieve phosphorus accumulation. The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Rice OsNAC4 Genes, characterized by, The OsNAC4 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating phosphorus accumulation in leaves; (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of regulating phosphorus accumulation in leaves.

2. Rice OsNAC4 protein, characterized in that, The amino acid sequence of the OsNAC4 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of regulating phosphorus accumulation in leaves.

3. Rice containing the rice described in claim 1 OsNAC4 The carrier of genes.

4. Containing the rice described in claim 3 OsNAC4 Engineered bacteria with gene vectors.

5. Expanding the rice according to claim 1 OsNAC4 Primers for any segment of a gene.

6. The rice according to claim 1 OsNAC4 Application of genes in regulating phosphorus accumulation in leaves.

7. A method for increasing phosphorus nutrient accumulation in rice leaves, characterized in that, Increase the expression level of OsNAC4 protein in rice.

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