Rice SPS11 gene and application thereof in regulation and control of phosphorus nutrient accumulation

Abstract

The invention discloses a rice SPS11 gene and application thereof in regulation and control of phosphorus nutrient accumulation. The nucleotide sequence of the rice SPS11 gene is shown as SEQ ID No: 2, and the amino acid sequence of the protein of the rice SPS11 gene is shown as SEQ ID No: 3. The invention provides an application of an OsSPS11 gene and a related biological material in realizing high-efficiency enrichment of phosphorus elements. Specifically, the phosphorus accumulation level of plants can be effectively improved by inhibiting expression of the OsSPS11. By changing the expression level of the OsSPS11, the phosphorus enrichment level of the plant is changed, so that the efficient enrichment of the phosphorus element by the rice is realized.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering technology, and specifically relates to a type of rice. SPS11 Genes and their application in regulating phosphorus accumulation. Background Technology

[0002] Phosphorus is one of the essential macronutrients for plant growth and development. Although soil contains a large amount of phosphorus, the content of available phosphorus is low and cannot meet the nutritional needs of crops. Statistics show that approximately 30% of the world's arable land has insufficient available phosphorus to meet crop nutritional requirements. To promote the Green Revolution and ensure food security, global phosphate fertilizer use increased from approximately 4.6 million tons in 1961 to approximately 21 million tons in 2015, and is projected to reach 22-27 million tons by the middle of this century. my country's long-term phosphate fertilizer use has been almost twice the amount absorbed by crops. After more than forty years of extensive phosphate fertilizer use, the average available phosphorus content in Chinese farmland soil has reached over 20 mg / kg, and the agronomical threshold for available phosphorus in most soil-crop systems is close to this value. This means that my country has largely completed its phased task of improving soil phosphorus fertility through large-scale excessive application of phosphate fertilizer. Future effective methods for improving phosphate fertilizer utilization efficiency should be based on two premises: stabilizing target crop yields and maintaining the available phosphorus content in the soil at 20 mg / kg. Improving the efficiency of crop phosphorus absorption and utilization is a method to effectively increase phosphate fertilizer utilization efficiency and yield while maintaining a stable level of available phosphorus in the soil. Therefore, identifying key genes for efficient phosphorus nutrient absorption and utilization is an effective way to ensure that rice yield remains unchanged or even increases while reducing phosphate fertilizer application, and it is also an important approach in modern agricultural breeding.

[0003] Phosphorus plays a crucial role in numerous processes during plant growth and development, including photosynthesis, signal transduction, and respiration. Plants convert solar energy and carbon dioxide into organic compounds through photosynthesis. Sucrose is a primary carbonate produced during photosynthesis, and phosphorus has a key influence on sucrose synthesis. Although phosphorus is not present in the reaction substrates or final products, its intermediate products and reaction processes are closely related to phosphorus, including fructose hexaphosphate and sucrose hexaphosphate. Phosphorylation and dephosphorylation are important steps controlling the synthesis of sucrose starch in plants. Furthermore, the energy required for sucrose synthesis is provided by the breaking and linking of high-energy phosphate bonds during the conversion of ATP and ADP. Sucrose phosphate synthase (SPS) is a key enzyme controlling sucrose biosynthesis in plants. Higher plants have at least three SPS gene families: A, B, and C, while monocotyledonous grasses have at least five: A, B, C, DIII, and DIV. Different SPS gene families exhibit different expression patterns in different plants. Therefore, different SPS gene families are involved in a variety of biological functions, including sucrose accumulation, plant growth and production, and tolerance to abiotic stresses. The activity of SPS in plants is regulated by exogenous factors through gene expression and reversible protein phosphorylation, and is thus influenced by in vivo phosphorus levels. Improving crop traits through SPS gene transformation is a feasible approach. However, current research lacks details on how SPS affects phosphorus accumulation in plants. Summary of the Invention

[0004] The purpose of this invention is to provide rice SPS11 Genes and their application in regulating phosphorus accumulation.

[0005] rice SPS11 Genes, the ones mentioned SPS11 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 2 of the sequence listing; or (b) A polynucleotide that can hybridize with the complementary sequence of SEQ ID No: 2 under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating phosphorus absorption and accumulation; (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 2; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 2, wherein the protein encoded by the polynucleotide mutant still has the function of regulating phosphorus nutrient absorption and accumulation.

[0006] The rice SPS11The genome sequence of the gene is shown in SEQ ID No: 1.

[0007] Rice SPS11 protein, wherein the amino acid sequence of the SPS11 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 3 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: 3; 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: 3, and the protein still has the function of regulating phosphorus nutrient absorption and accumulation.

[0008] Containing the rice SPS11 The carrier of genes.

[0009] Containing the rice SPS11 Engineered bacteria with gene vectors.

[0010] Amplification of the rice SPS11 Primers for any segment of a gene.

[0011] The rice SPS11 Application of genes in regulating phosphorus nutrient absorption and accumulation.

[0012] A method to improve phosphorus nutrient absorption and accumulation in rice by reducing the expression level of SPS11 protein in rice.

[0013] Beneficial effects of the present invention: The present invention provides OsSPS11 The application of genes and related biomaterials in achieving efficient phosphorus enrichment is specifically manifested in inhibiting... OsSPS11 The expression of this factor can effectively increase the level of phosphorus accumulation in plants. By changing... OsSPS11 By adjusting the expression level of phosphorus, the enrichment level of phosphorus in plants can be altered, thereby achieving efficient enrichment of phosphorus in rice. Attached Figure Description

[0014] Figure 1 A represents phosphorus-deficient conditions. OsSPS11 Relative expression level change; B is OsSPS11-pro-GUS Results of GUS staining observation of transgenic plants under phosphorus-free conditions.

[0015] Figure 2 A in the middle is OsSPS11 Schematic diagram of genome and target sites; B represents Ossps11-1 , Ossps11-2 Sequencing results for identifying mutant transgenic plants.

[0016] Figure 3 A represents both wild-type and mutant strains.Ossps11-1, Ossps11-2 Plant phenotypes under high phosphorus (HP: 200 μM) and low phosphorus (LP: 10 μM) conditions, respectively; B represents wild type and mutant. Ossps11-1, Ossps11-2 Leaf tip phenotypes under high and low phosphorus conditions, respectively; C represents wild type and mutant. Ossps11-1, Ossps11-2 Fresh weight biomass under high and low phosphorus conditions, respectively; D represents wild type and mutant. Ossps11-1, Ossps11-2 Available phosphorus content under high phosphorus and low phosphorus conditions respectively. Detailed Implementation

[0017] 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.

[0018] Experimental materials used in the following examples: wild-type rice (Shishoubaimao: SSBM); bacterial strains: Escherichia coli strain DH5α, Agrobacterium tumefaciens strain EHA105; vector: GUS tag vector. pCAMBIA1300-GUS plus Gene knockout vector pYLCRISPR / Cas9 .

[0019] Experiment Example 1: Quantitative Detection of Phosphorus Deficiency-Induced Phosphorus Effects in Rice OsSPS11 Gene and Observation of GUS Staining 1. Quantitative real-time PCR detection of phosphorus-deficient rice OsSPS11 Gene expression levels: Wild-type rice material SSBM was hydroponically treated with both complete rice nutrient solution and phosphorus-free rice nutrient solution. The complete rice nutrient solution formula was: 1.43 mM NH4NO3, 0.2 mM NaH2PO4•2H2O, 0.19 mM K2SO4, 0.35 mM CaCl2·2H2O, 0.55 mM MgSO4•7H2O, 0.1 mM NaFe(3)-EDTA•3H2O, 0.005 mM MnCl2•4H2O, 0.03 mM H3BO3, 0.001 mM (NH4)6Mo7O 24 • 4H2O, 0.004 mM ZnSO4•7H2O, 0.002 mM CuSO4•5H2O, and phosphorus-free nutrient solution (a complete nutrient solution for rice without the addition of 0.2 mM NaH2PO4•2H2O), pH 6. Rice seedlings were cultured for 18 days in a rice culture chamber (30℃ during the day, 22℃ at night, light intensity 3000 Lux, light duration 12 hours). Aboveground parts and roots of rice seedlings cultured under phosphorus-added and phosphorus-free conditions were sampled. RNA was extracted by liquid nitrogen flash freezing and reverse transcribed to obtain cDNA. Ubi was selected as an internal reference gene for quantitative real-time PCR amplification and determination.OsSPS11 The relative expression levels were determined. Real-time fluorescence quantification was performed using a Light Cycler 480II (Roche, USA), with three replicates per parallel experiment. -ΔΔCT The relative expression level is calculated using this method.

[0020] The results are as follows Figure 1 As shown in Figure A, compared to the phosphorus-added condition, wild-type rice grown for 18 days under phosphorus-free conditions... OsSPS11 Gene expression was induced 5-fold and 29-fold in the aboveground parts and roots, respectively, indicating that... OsSPS11 Genes are significantly induced under phosphorus-free conditions.

[0021] Real-time PCR OsSPS11 The primer sequences are: Forward primer sequence F: 5'-CTGCTCATCAACCCCAACA-3'; Reverse primer sequence R: 5'-GTGAAGAACCGGAGCACCT-3'.

[0022] The primer sequences for the internal control actin in quantitative real-time PCR are as follows: Forward primer sequence F: 5'-CAACACCCCTGCTATGTACG-3'; Reverse primer sequence R: 5'-CAACACCCCTGCTATGTACG-3'.

[0023] 2. OsSPS11-pro-GUS GUS staining results of transgenic plants under phosphorus deficiency conditions pCAMBIA1300-OsSPS11-pro-GUS Vector construction: Using DNA from wild-type rice SSBM grown in hydroponics as a template, PCR amplification was performed using the upstream primer 5'-TCCCCCCGGGTCTCCGTTACAACAAACCAAAT-3' and the downstream primer 5'-ACGCGTCGACCCTCCTCCTCCTTCTTCTCC-3'. Approximately 2250 bp of the vector was purified and recovered. OsSPS11 Gene promoter fragments, amplification products are used SmaI and SalI Double digestion with NEB enzyme, followed by ligation using T4 ligase for use. SmaI and SalI Double digestion with endonuclease (NEB) pCAMBIA-1300-GUS plus In the vector, a recombinant vector was obtained. pCAMBIA1300- OsSPS11-pro-GUS .

[0024] OsSPS11-pro-GUS Preparation of transgenic plants: Using wild-type rice SSBM as background material, transgenic plants containing...pCAMBIA1300-OsSPS11-pro-GUS Agrobacterium EHA105 strain containing the vector was used to infect rice callus tissue to obtain T0 generation transgenic positive plants; the T0 generation positive transgenic plants were self-crossed twice to obtain homozygous transgenic plants. OsSPS11 GUS-tagged genetically modified rice materials OsSPS11-pro-GUS .

[0025] OsSPS11-pro-GUS GUS staining observation of transgenic plants under phosphorus deficiency conditions: Experimental material treatment: wild-type (SSBM) and OsSPS11 GUS-tagged genetically modified rice materials OsSPS11-pro-GUS Hydroponic treatment was carried out. The rice was first cultured in a complete rice nutrient solution for 7 days, and then transferred to a complete rice nutrient solution and a phosphorus-free rice nutrient solution without 0.2 mM NaH2PO4•2H2O for 10 days. The leaves and roots were then subjected to GUS staining for observation. Whole root GUS staining observation: The whole root of the plant was placed in GUS staining solution and left to stand at 37℃ for 8 hours. The staining was observed directly. Then, the mature part of the root after GUS staining was taken and placed on a glass slide about 3 cm from the root tip. The sample was observed and photographed using a Leica CTR6 microscope. Leaf and root section observation: The middle part of the second leaf from the bottom, about 1 cm long, was taken. The mature part of the root was taken and placed about 3 cm from the root tip. The sample was embedded in 4% agarose. The embedded material was fixed on the microtome base and sectioned using a Leica VT 1000s vibrating microtome. The section thickness was 50 μm. The complete section material was picked up and placed in GUS staining solution and left to stand at room temperature for 8 hours. Then, the GUS phosphorus staining solution was aspirated, a clearing agent was added, and the sample was left to stand at room temperature for 5 hours. The complete section material was picked up and placed on a glass slide. The sample was observed and photographed using a Leica CTR6 microscope.

[0026] The results are as follows Figure 1 As shown in B, this is in contrast to rice grown under conditions of complete nutrient nutrition. OsSPS11-pro-GUS Compared to transgenic plants, under phosphorus-free conditions, OsSPS11-pro-GUS The transgenic plant showed darker GUS staining in the entire root, the mature root zone, and the middle of the leaves, indicating that under phosphorus-free conditions, OsSPS11 The protein expression level will increase, consistent with the results of quantitative real-time PCR.

[0027] Experiment Example 2: Rice OsSPS11 Preparation of mutant transgenic plants 1. OsSPS11 Construction of gene knockout vector Based on the target site selection requirements of the plant CRISPR / Cas9 vector system, select OsSPS11 The target sequence of the gene is 5'-GCTGGAGAACCTGTGCTGG-3', and the target site is located at... OsSPS11The second exon in the genome, such as Figure 2 As shown in A, the vector was constructed according to the method provided in the literature: Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A Robust CRISPR / Cas9 System for Convenient, High-Efficiency MultiplexGenome Editing in Monocot and Dicot Plants. Mol Plant 8: 1274-1284. pYLCRISPR / Cas9-OsSPS11 .

[0028] 2. OsSPS11 Obtaining rice mutants Will pYLCRISPR / Cas9-OsSPS11 The recombinant plasmid was introduced into Agrobacterium tumefaciens EHA105, and then the vector was introduced into wild-type SSBM callus of rice using Agrobacterium-mediated genetic transformation to obtain transgenic plants. T0 generation transgenic positive plants were obtained. Genomic DNA was extracted from the T0 generation transgenic positive plants, and then amplified using primers flanking the target site. The PCR products were sequenced to identify the gene. OsSPS11 The location and form of the mutation. Sequencing results show that the mutant... Ossps11-1 Inserting a base T results in OsSPS11 Gene reading frame shift, mutant Ossps11-2 The deletion of 5 bases at the target site, TGTGC, also leads to OsSPS11 Frameshifting of gene reading frames ultimately leads to OsSPS11 The gene function was inactivated, resulting in two OsSPS11 Rice mutant lines Ossps11-1, Ossps11-2 The sequencing results of the homozygous mutant are as follows: Figure 2 As shown.

[0029] The primer sequences used for constructing the CRISPR-Cas9 vector target sites are as follows: U3 forward primer sequence F: 5'-gccGGCTGGAGAACCTGTGCTGG-3'; U3 reverse primer sequence R: 5'-aaacCCAGCACAGGTTCTCCAGC-3'.

[0030] The PCR product sequencing amplification sequence is as follows: Forward primer sequence F: 5'-CGAGTGGATCAACGGGTAC-3'; Reverse primer sequence R: 5'-GCCGGTGAGTTCTTCTTTC-3'.

[0031] Experiment Example 3: Rice OsSPS11 Phenotypic observation of mutant plants under low phosphorus conditions, changes in biomass and available phosphorus content Experimental material processing: Wild-type rice SSBM, OsSPS11 mutant plants Ossps11-1, Ossps11-2 After culturing rice in a complete nutrient solution for 7 days, the rice plants were transferred to a complete nutrient solution and a low-phosphorus nutrient solution containing only 0.01 mM NaH2PO4•2H2O for 4 weeks. The overall plant phenotype and the phenotype of the mature third leaf tip were observed. The fresh weight biomass of the aboveground parts and roots were collected. The aboveground parts and roots were quick-frozen in liquid nitrogen and ground into powder. The available phosphorus was extracted with 5M H2SO4, and then diluted with twice the volume of sulfuric acid in pure water. After centrifugation at 12000 rpm for 15 min, 200 μL of the supernatant was collected. 7 ml of pure water and 20 μL of dinitrophenol indicator were added. The solution was adjusted to a pale yellow color with 4M NaOH solution and 4M H2SO4 solution. The volume was then brought up to 9 ml with pure water. 1 ml of molybdenum antimony available phosphorus assay solution was added. After standing at room temperature for 30 min, the available phosphorus content was measured at a wavelength of 695 nm using a microplate reader (TECANinginite F50, Switzerland).

[0032] The results are as follows ​ As shown, where ​ A represents the overall phenotype of plants under high and low phosphorus culture conditions, regardless of whether the conditions are high or low phosphorus. ​ The mutant plants were generally smaller than the wild type, and their fresh biomass was also significantly lower. Furthermore, under high phosphorus conditions... ​ The mutant exhibited a scorched leaf tip phenotype on the third leaf from the bottom, indicating phosphorus accumulation in the mutant leaves. The results of available phosphorus content determination showed that, under high phosphorus conditions, both mutant lines... ​ and ​ The available phosphorus content in the aboveground parts and roots was significantly increased compared to the wild type. Under low phosphorus conditions, due to the low phosphorus supply level, there was no significant difference between the wild type and the mutant.

[0033] 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. Oryza sativa SPS11 gene characterized in that, The SPS11 polynucleotide of the gene is as set forth in (a), (b), (c), or (d): (a) a polynucleotide as shown in SEQ ID No: 2; or (b) a polynucleotide capable of hybridizing to the complement of SEQ ID No: 2 under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating phosphorus nutrient absorption and accumulation; (c) a polynucleotide having at least 90% or more homology with the polynucleotide as shown in SEQ ID No: 2; or (d) a polynucleotide mutant obtained by deletion, substitution or insertion of one or more bases based on the polynucleotide as shown in SEQ ID No: 2, and the protein encoded by the polynucleotide mutant still has the function of regulating phosphorus nutrient absorption and accumulation.

2. A rice SPS 11 protein characterized in that, The amino acid sequence of the SPS11 protein is as shown in (a), (b) or (c): (a) an amino acid sequence as shown in SEQ ID No: 3; or (b) an amino acid having at least 90% or more homology with the amino acid as shown in SEQ ID No: 3; or (c) a protein mutant obtained by deletion, substitution or insertion of one or more amino acids based on the protein as shown in SEQ ID No: 3, and the protein still has the function of regulating phosphorus nutrient absorption and accumulation.

3. A vector comprising the rice gene of claim 1. SPS11 gene.

4. A rice plant comprising the rice plant cell of claim 3. SPS11 Engineered bacteria of the gene vector.

5. The rice of claim 1 wherein the amplified fragment is any one of the fragments of the SEQ ID NO: 1 gene. SPS11 primers.

6. The rice plant of claim 1 SPS11 application of the gene in regulating phosphorus nutrient uptake and accumulation.

7. A method for improving phosphorus nutrient uptake and accumulation in rice, characterized by, Reducing the expression level of the SPS11 protein in rice.

Images