Gene for improving utilization efficiency and yield of phosphate fertilizer and nutritional quality of seeds and application of gene

Abstract

The invention relates to a gene for improving phosphate fertilizer utilization efficiency, yield and seed nutrition quality and application thereof. Specifically, the invention provides application of ROPD (REEGLAATOR OF PHOSPHORUS UPTAKE AND DISTRIBUTION) or an allele thereof in the aspects of reducing the phytic acid content in seeds and improving the nutritional quality of the seeds while improving the utilization efficiency and the yield of a crop phosphate fertilizer. Methods of improving phosphate fertilizer utilization efficiency, yield and seed nutritional quality of crops such as rice, wheat and corn are also disclosed. The ROPD gene has a wide application prospect in breeding of crops with high phosphorus efficiency and high yield.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. It relates to new functions and applications of known genes. Specifically, it relates to the new functions and applications of the known growth regulator GRF4 encoded by the ROPD gene in improving crop phosphate fertilizer utilization efficiency and yield, reducing seed phytic acid content, and improving seed nutritional quality. More specifically, it relates to the new functions of the growth regulator GRF4 and its applications in crops such as rice, wheat, and corn. Background Art

[0002] Phosphorus is a macronutrient essential for plant growth and development and a key factor influencing crop yield. Plants directly absorb inorganic phosphate from the soil through their roots. However, since most inorganic phosphate in the soil forms complexes with metal ions such as iron, aluminum, and calcium, it is difficult for plants to directly absorb and utilize it. This results in crops in farmland and natural ecosystems often experiencing low-phosphorus stress. In agricultural production, the large-scale application of phosphorus fertilizer is a key measure to increase crop yields. However, the utilization efficiency of phosphorus fertilizer is very low, with typically only 10-25% of the fertilizer being absorbed and utilized by plants. The remaining fertilizer leads to environmental problems such as soil compaction and eutrophication. Therefore, improving crop phosphorus use efficiency (PUE) and reducing P fertilizer input are of great significance for ensuring national food security and sustainable agricultural development.

[0003] Although phosphorus in seeds accounts for 60–85% of total plant phosphorus (Rose et al., 2013), it primarily exists as phytic acid (phytic acid, PA), also known as inositol hexaphosphate (InsP6), which is often excreted in urine and cannot be absorbed or utilized by humans and non-ruminant animals. Therefore, phytic acid is a significant factor in phosphorus loss and nutrient waste. Furthermore, because phytic acid is negatively charged, it can chelate with metal ions such as calcium, magnesium, potassium, iron, and zinc to form insoluble phytates. Therefore, excessive phytic acid intake can interfere with the absorption of dietary elements such as iron, calcium, and zinc. Therefore, efforts should be made to improve phosphorus absorption and utilization by crop roots to increase phosphorus fertilizer use efficiency and yield, and to reduce the phytic acid content in seeds to improve their nutritional quality.

[0004] Phosphorus in rice seeds comes from two sources: newly absorbed phosphorus during flowering and grain filling, and redistributed phosphorus from older leaves (Yamaji and Ma, 2017). Numerous plant hormone signaling pathways, as well as numerous transcription factors and regulatory factors, have been identified as involved in regulating phosphorus use efficiency and seed phosphorus content. For example, PHO1, a member of the SPX-EXS protein family, plays a crucial role in belowground-aboveground phosphate transport (Secco et al., 2010; 2012). OsPHO1;2 directly regulates seed phosphorus content. Mutations in OsPHO1;2 increase phosphorus content in rice nodes, glumes, and brown rice, while decreasing it in leaves. This mutation also interferes with starch synthesis in seeds, thereby affecting yield. Overexpression of OsPHO1;2 significantly reduces seed phosphorus content and increases thousand-grain weight, resulting in synergistic improvements in yield and phosphorus use efficiency (Ma et al., 2021). The PHT1 protein family is involved in the distribution of phosphorus from leaves to seeds. Compared with the wild type, the ospht1;8 mutant showed no change in root phosphorus uptake, but increased phosphorus content in the cob and decreased phosphorus content in unfilled glumes, indicating that OsPHT1;8 is involved in phosphorus transport from the cob to the seed during reproductive growth (Jia et al., 2011). OsPHT1;4 not only regulates phosphorus uptake and aboveground-to-aboveground transport, but also regulates phosphorus transport into the embryo. Mutation of this gene significantly reduces phosphorus concentration in the embryo and phytic acid content in the seed, while overexpression significantly increases phosphorus concentration in the embryo and phytic acid content in the seed (Zhang et al., 2015). The sulfate transporter OsSULTR3;3 regulates phosphorus content in the seed. The seeds of the ossultr3;3 mutant show increased phosphorus accumulation, and the expression of genes involved in phosphorus and phytic acid metabolism in the seed is also altered (Zhao et al., 2016). OsSPDT encodes a plasma membrane-localized protein that is highly expressed in rice stem nodes and positively regulates phosphorus allocation from leaves to seeds (Yamaji et al., 2017a). The osspdt mutant has reduced phosphorus content in seeds and increased phosphorus content in leaves and stems, altering plant phosphorus allocation, reducing seed phosphorus content without affecting yield or seed germination rate (Yamaji et al., 2017a). However, to date, few genes have been identified that can improve crop phosphorus uptake and utilization while reducing seed phosphorus content. Even fewer genes exist that achieve both high phosphorus efficiency, high yield, and low seed phytic acid content and are truly applicable to crop breeding.

[0005] The "Green Revolution" of the 1960s, characterized by semi-dwarf breeding, improved crop tolerance to high fertilizer levels and lodging resistance, significantly increasing rice and wheat yields. However, these semi-dwarf varieties exhibited decreased root phosphorus uptake rates and increased phosphorus accumulation and phytic acid content in their seeds. Enhancing the phosphorus uptake capacity of crops, including these semi-dwarf varieties, to improve their phosphorus fertilizer use efficiency and yield, while also reducing phytic acid accumulation in seeds and enhancing their nutritional quality, are key issues in increasing grain production and improving its nutritional quality.

[0006] References:

[0007] 1. Rose TJ, Liu L, and Wissuwa M (2013) Improving phosphorus efficiency incereal crops: is breeding for reduced grain phosphorus concentration part of the solution.

[0008] Front Plant Sci 4:444.

[0009] 2.Yamaji N, Takemoto Y, Miyaji T, Mitani-Ueno N, Yoshida KT, and Ma JF (2017)

[0010] Reducing phosphorus accumulation in rice grains with an impaired transporter in the node. Nature 541:136-136.

[0011] 3. Secco D, Wang C, Arpat BA, Wang Z, Poirier Y, Tyerman SD, Wu P, Shou H, and Whelan J (2012) The emerging importance of the SPX domain-containing proteins in phosphate homeostasis. New Phytol 193:842-851.

[0012] 4.Secco D,Baumann A,and Poirier Y(2010)Characterization of the RicePHO1

[0013] Gene Family Reveals a Key Role for OsPHO1;2in Phosphate Homeostasisand the Evolution of a Distinct Clade in Dicotyledons.Plant Physiology 152:1693-1704.

[0014] 5.Ma B,Zhang L,Gao Q,Wang J,Li X,Wang H,Liu Y,Lin H,Liu J,Wang X etal.

[0015] (2021)A plasma membrane transporter coordinates phosphatereallocation and grain filling in cereals.Nat Genet 53:906-915.

[0016] 6.Jia HF,Ren HY,Gu M,Zhao JN,Sun SB,Zhang X,Chen JY,Wu P,and Xu GH

[0017] (2011)The Phosphate Transporter Gene OsPht1;8Is Involved in PhosphateHomeostasis in Rice.Plant Physiol 156:1164-1175.

[0018] 7.Zhang F,Sun YF,Pei WX,Jain A,Sun R,Cao Y,Wu XN,Jiang TT,Zhang L,FanXR et al.(2015)Involvement of OsPht1;4in phosphate acquisition andmobilization facilitates embryo development in rice.Plant J 82:556-569.

[0019] 8. Zhao H, Frank T, Tan Y, Zhou C, Jabnoune M, Arpat AB, Cui H, Huang J, He Z, Poirier Y et al. (2016) Disruption of OsSULTR3; 3reduces phytate and phosphorusconcentrations and alters the metabolite profile in rice grains. New Phytol211:926-939. Summary of the Invention

[0020] The present invention relates to new functions and applications of known genes. Specifically, the present invention relates to the new functions and applications of the known growth regulator GRF4 encoded by the ROPD gene in improving crop phosphate fertilizer utilization efficiency and yield while reducing phytic acid content in seeds and improving seed nutritional quality. More specifically, the present invention relates to the new functions of the growth regulator GRF4 and its applications in crops including, but not limited to, rice, wheat, and corn.

[0021] The GRF4 is known to have the function of regulating rice carbon and nitrogen metabolism and growth and development, and has been patented by the inventor, see patent number ZL 2017 1 1498519.7, the invention name is "Genes and applications for improving rice nitrogen fertilizer utilization efficiency and yield", and WO 2019 / 158911, the invention name is "METHODS OF INCREASING NUTRIENT USEEFFICIENCY".

[0022] Based on the research objectives of improving the phosphorus fertilizer utilization efficiency and yield of crops, including "Green Revolution" semi-dwarf varieties, while reducing phytic acid accumulation in seeds, the inventors determined the root phosphorus uptake capacity of a series of rice resource materials carrying the "Green Revolution" gene sd1, and simultaneously detected the phytic acid concentration in the seeds of these materials. Based on the differences in phosphorus uptake capacity and phytic acid concentration in the seeds of different materials, rice materials with higher phosphorus uptake capacity and lower seed phytic acid content, as well as rice materials with lower phosphorus uptake capacity and higher seed phytic acid content, were identified. Genetic populations were constructed, and using QTL analysis and map-based cloning methods, a candidate gene ROPD (REGULATOR OF PHOSPHORUS UPTAKE AND DISTRIBUTION) that regulates phosphorus uptake and distribution in rice was cloned. Sequencing analysis revealed that the gene was the known gene GRF4 (nucleotide sequence see SEQ ID NOs. 1, 2), which encodes a growth regulatory factor (amino acid sequence see SEQ ID NOs. 1, 2). IDNO.5); through phenotypic analysis and transgenic research on near-isogenic lines, it was proved that the ROPD gene has a new function of regulating the phosphorus absorption rate of crops and the distribution of phosphorus to seeds, thereby regulating the phosphorus fertilizer utilization efficiency, yield and seed nutritional quality of crops; further, the inventors of the present invention have identified the ROPD gene and its superior allele ROPD SLC (See SEQ ID NO.3, 4 for nucleotide sequence; SEQ ID NO.6 for amino acid sequence of encoded protein) applied to crops such as rice, wheat and corn, the phosphate fertilizer utilization efficiency and yield were improved while the phytic acid content in seeds was reduced and the nutritional quality of seeds was improved. SLC It is an important genetic resource for crop phosphorus efficiency, high yield and high quality breeding.

[0023] In summary, the present invention provides a new function of the growth regulator GRF4 to improve the phosphorus fertilizer utilization efficiency, yield and seed nutritional quality of crops and its application. Specifically, the present invention relates to the growth regulator GRF4, which synergistically improves the phosphorus fertilizer utilization efficiency and yield of crops without changing the excellent semi-dwarfing and lodging resistance traits of the "Green Revolution" varieties, while reducing the phytic acid content in the seeds and improving the nutritional quality of the seeds, and this new function can also be widely used in other crops such as wheat and corn. The purpose of the present invention is to reveal the molecular basis of plant phosphorus fertilizer utilization efficiency at the molecular level, and to provide a theoretical basis and technical support for efficient phosphorus utilization and high-yield genetic improvement of crops including rice, wheat and corn.

[0024] Specifically, the present invention mainly relates to the following aspects:

[0025] In the first aspect, the inventors cloned the gene ROPD and its alleles that improve crop phosphorus fertilizer utilization efficiency, yield and seed nutritional quality. The gene is known as GRF4, which is known to have the function of regulating plant growth and carbon and nitrogen metabolism. The inventors determined that ROPD can directly regulate the expression of genes related to phosphorus absorption and distribution, thereby playing a new role in regulating crop phosphorus fertilizer utilization efficiency and phytic acid content in seeds. The superior allele ROPD SLC It can promote crop phosphorus absorption, significantly improve crop phosphorus fertilizer utilization efficiency and yield, and reduce the phytic acid content in seeds.

[0026] In one embodiment, the inventors utilized 32 P isotope-labeled phosphate ions ( 32 PO4 3- ), tested the phosphorus uptake rate of roots of 31 rice varieties and the phytic acid concentration in seeds of these varieties. It was found that the phosphorus uptake rate and phytic acid content in seeds of different rice varieties were different. Among them, "Sanlicun" (SLC) had a higher phosphorus uptake rate and lower phytic acid content in its seeds, while the high-yielding indica rice variety 9311 (China National Rice Research Institute, also commercially available) had a lower phosphorus uptake rate and higher phytic acid content in its seeds. Using 9311 as the recipient and "Sanlicun" as the donor, chromosome segment substitution lines were constructed and QTL analysis was performed. A QTL that simultaneously regulates phosphorus uptake rate and phosphate content in seeds was identified and named qROPD (REGULATOR OF BOTH PHOSPHORUS UPTAKE AND DISTRIBUTION) Figure 2 A, B), and further through fine positioning and map-based cloning, the candidate gene ROPD ( Figure 2 C, D, E), which is a known gene encoding growth-regulating factor 4 (GRF4); the ROPD from 9311 was named ROPD, and the ROPD from SLC was named ROPD SLC The nucleotide sequence, amino acid sequence of the encoded protein, and the differences between indica and japonica rice can be found in the reference "Modulating plant growth-metabolism coordination for sustainable agriculture" in Extended Data Fig. 1 for GRF4. ngr2 .

[0027] In one embodiment, the present inventors constructed a pair of near-isogenic lines 9311 and 9311-ROPD in the 9311 background. SLC , respectively detect 9311 and 9311-ROPD SLCThe expression level and protein abundance of ROPD gene in the root tips of seedlings showed that compared with 9311, 9311-ROPD SLC The transcription level of ROPD gene was significantly increased in Figure 2 A) Using ROPD autoantibodies to detect the abundance of ROPD protein in the root tip, the results showed that compared with 9311, 9311-ROPD SLC The abundance of ROPD protein was significantly increased in Figure 2 B) Therefore, the allele ROPD SLC Increased its own transcription level and protein level.

[0028] In one embodiment, the inventors used gene editing methods to create rice materials with ROPD knockout, and verified that ROPD has the function of regulating crop phosphorus absorption and utilization and the phosphorus content in seeds. Specifically, the inventors constructed a CRISPR / Cas9 knockout vector and transformed the indica rice variety 9311 and the japonica rice variety Wuyunjing No. 7 (WYJ7) by Agrobacterium-mediated method, respectively, to obtain homozygous knockout lines 9311-ropd and WYJ7-ropd. Compared with 9311, 9311-ropd plants showed phosphorus-deficient phenotypes such as reduced plant height, short flag leaves, and reduced tillering, and the phytic acid content in its seeds was significantly increased ( Figure 3 A, C); Similarly, the phytic acid content in the seeds of WYJ7-ropd was significantly increased compared with that of WYJ7 ( Figure 7 B); Further, the present inventors conducted 32 The results of the P isotope absorption experiment showed that the root phosphorus absorption rate of 9311-ropd was significantly decreased compared with 9311 ( Figure 3 B); Similarly, the root phosphorus uptake rate of WYJ7-ropd was significantly decreased compared with that of WYJ7 ( Figure 7 C). Therefore, ROPD regulates rice phosphorus absorption and metabolism, as well as the phosphorus content in seeds. Loss of ROPD function leads to a decrease in rice phosphorus uptake rate and inhibition of rice growth and development, while increasing the phytic acid content in rice seeds.

[0029] In another embodiment, the present inventors obtained a pair of near-isogenic lines 9311-ROPD and 9311-ROPD in the 9311 background by crossing SLC with the variety 9311 carrying the "Green Revolution" gene sd1 and backcrossing 9311 multiple times. SLC Compared with 9311-ROPD, 9311-ROPD SLC The expression of ROPD gene in the plant was significantly increased, the phosphorus absorption rate of its root system was significantly increased, and the phytic acid content in the seeds was significantly reduced. Therefore, the allele ROPD SLC It can promote phosphorus absorption in rice and reduce the phytic acid content in seeds ( Figure 4).

[0030] In one embodiment, the present inventors utilized 9311, 9311-ropd and 9311-ROPD SLC RNA-seq analysis and gene expression analysis were performed on the root tips of rice materials. The results of RNA-seq analysis showed that compared with 9311, the expression of phosphorus transporter genes OsPHT1;1 and OsPHT1;8 in 9311-ropd was significantly reduced, and the gene expression of the transcription factor OsWRKY74, which positively regulates phosphorus absorption, was also significantly reduced, while the expression of the gene OsSPDT, which regulates phosphorus distribution, was significantly increased; SLC The expression of OsPHT1;1, OsPHT1;8 and OsWRKY74 genes were significantly increased, while the expression of OsSPDT gene was significantly decreased ( Figure 5 A); To verify the results of RNA-seq analysis, the present inventors used the same batch of rice materials and analyzed the expression levels of OsPHT1;1, OsPHT1;8, OsWRKY74 and OsSPDT genes by fluorescence quantitative PCR. The results showed that compared with 9311, the expression levels of OsPHT1;1, OsPHT1;8 and OsWRKY74 genes in 9311-ropd were significantly decreased, while the expression level of OsSPDT gene was significantly increased; compared with 9311, 9311-ROPD SLC The expression levels of OsPHT1;1, OsPHT1;8 and OsWRKY74 genes were significantly increased, while the expression of OsSPDT gene was significantly decreased ( Figure 5 B) Therefore, the ROPD gene positively regulates the expression of genes related to phosphorus absorption (such as OsPHT1;1, OsPHT1;8, and OsWRKY74), and negatively regulates the expression of genes related to phosphorus distribution (such as OsSPDT). SLC It can significantly increase the expression of genes related to phosphorus absorption in rice, while reducing the expression of genes related to phosphorus distribution.

[0031] In one embodiment, the present inventors created a pActin::ROPD-flag overexpressing transgenic line and used its seedling root tips for ChIP-qPCR experiments. The results showed that ROPD protein can bind to the promoters of OsPHT1;1, OsPHT1;8, and OsWRKY74 ( Figure 6 A). Furthermore, the present inventors amplified the promoter regions of OsWRKY74, OsPHT1;1, OsPHT1;8 and OsSPDT respectively, and performed transcriptional activation experiments using protoplasts of 9311-ropd rice material. The results showed that ROPD and ROPD SLCThe protein has transcriptional activation activity on the OsPHT1;1, OsPHT1;8 and OsWRKY74 gene promoters, and transcriptional repression activity on the OsSPDT gene promoter; ROPD SLC The protein has stronger transcriptional activation activity on OsPHT1;1, OsPHT1;8 and OsWRKY74 genes, and stronger transcriptional repression activity on OsSPDT gene ( Figure 6 B).

[0032] Therefore, the transcription factor ROPD can promote the absorption and utilization of phosphorus by crops by binding to the promoters of phosphorus absorption-related genes (such as OsWRKY74, OsPHT1;1 and OsPHT1;8) and activating their gene expression. At the same time, ROPD can inhibit the expression of phosphorus distribution-related genes (such as OsSPDT) and thus reduce the phosphorus content in seeds. SLC It has stronger transcriptional activation activity on phosphorus absorption-related genes and stronger transcriptional inhibition activity on phosphorus distribution-related genes, thereby significantly improving the crop's phosphorus absorption and utilization capacity while reducing the phosphorus content in seeds.

[0033] The second aspect involves the gene ROPD and its alleles ROPD that improve crop phosphorus fertilizer use efficiency, yield and seed nutritional quality SLC , and the protein it encodes. ROPD (nucleotide sequence preferably SEQ ID NO. 1, 2, amino acid sequence of the encoded protein preferably SEQ ID NO. 5) regulates both the phosphorus absorption and utilization efficiency of crops and the biomass and yield of crops, and also regulates the phytic acid content in crop seeds and the nutritional quality of seeds. Specifically, the ROPD gene and its superior allele ROPD SLC (the nucleotide sequence is preferably SEQ ID NO.3, 4, and the amino acid sequence of the encoded protein is preferably SEQ ID NO.6) The increased expression level can improve the phosphorus absorption and utilization efficiency of crops, increase the biomass and yield of crops, and at the same time reduce the phytic acid content in seeds and improve the nutritional quality of seeds.

[0034] In some embodiments, a gene ROPD and its superior allele ROPD for regulating crop phosphorus fertilizer use efficiency and yield are provided. SLC , wherein the genes ROPD and ROPD SLC The amino acid sequence of the encoded protein comprises one selected from the following amino acid sequences:

[0035] 1) the amino acid sequence shown in SEQ ID NO. 5, 6;

[0036] 2) an amino acid sequence that differs from the amino acid sequence of SEQ ID NOs. 5 and 6 by substitution, deletion, and / or insertion of one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) amino acid residues, but has the same activity as the protein composed of the amino acid sequence of SEQ ID NOs. 5 and 6;

[0037] 3) an amino acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, and especially at least 95%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs. 5 and 6, and has the same activity as the protein consisting of the amino acid sequence of SEQ ID NOs. 5 and 6;

[0038] 4) An active fragment comprising any one of the amino acid sequences described in 1) to 3).

[0039] In some embodiments, the gene ROPD and its superior alleles that regulate crop phosphorus fertilizer utilization efficiency and yield are isolated polynucleotides, and their nucleotide sequences are shown in any one of the following:

[0040] 1) The nucleotide sequences shown in SEQ ID NOs. 1, 2, 3, and 4;

[0041] 2) A nucleotide sequence that differs from the nucleotide sequence shown in SEQ ID NO. 1, 2, 3, or 4 due to substitution, deletion, and / or insertion of one or more nucleotide sequences, but the activity of the protein encoded by it is the same as that of the protein encoded by the nucleotide sequence shown in SEQ ID NO. 1, 2, 3, or 4;

[0042] 3) a nucleotide sequence that is at least 70%, preferably at least 80%, and more preferably at least 90% identical to the nucleotide sequence shown in SEQ ID NO. 1, 2, 3, or 4, and that encodes a protein having the same activity as the protein encoded by the nucleotide sequence shown in SEQ ID NO. 1, 2, 3, or 4;

[0043] 4) Nucleotide sequences that differ from SEQ ID NOs. 1, 2, 3, and 4 due to the degeneracy of the genetic code;

[0044] 5) An active fragment comprising any one of the nucleotide sequences in 1)-4);

[0045] 6) comprising a nucleotide sequence that hybridizes to a complementary sequence of any one of the nucleotide sequences 1) to 5) under moderately stringent conditions, preferably highly stringent hybridization conditions;

[0046] 7) comprising a nucleotide sequence complementary to any one of the nucleotide sequences in 1) to 5).

[0047] Compared with the ROPD protein (SEQ ID NO.5) encoded by the ROPD gene, ROPD SLC Genetically encoded ROPD SLC The protein (SEQ ID NO. 6) differs in two amino acids.

[0048] Table 1. Sequence names and sources of SEQ ID NOs: 1-6

[0049]

[0050] The third aspect relates to a recombinant construct comprising the gene ROPD or its allele ROPD for improving crop phosphate fertilizer utilization efficiency, yield and seed nutritional quality according to the present invention. SLC The vector used in the construct can be a cloning vector or an expression vector for expressing the polynucleotide.

[0051] The fourth aspect relates to a recombinant host cell comprising the recombinant construct of the third aspect of the present invention, or integrating into its genome the gene ROPD or its allele ROPD for improving crop phosphorus fertilizer utilization efficiency, yield and seed nutritional quality of the present invention. SLC The host cell may be selected from plant cells or microbial cells, such as Escherichia coli cells or Agrobacterium cells, preferably plant cells, more preferably rice, wheat, or corn cells, and most preferably rice cells. The cell may be isolated, in vitro, cultured, or part of a plant.

[0052] The fifth aspect involves the gene ROPD and its superior allele ROPD that improve crop phosphorus fertilizer utilization efficiency, yield and seed nutritional quality SLC Application in the improvement of high nitrogen and phosphorus nutrition efficiency and high yield crop varieties. Specifically, the present invention provides a method of polymer breeding, that is, the gene ROPD of the present invention that improves crop phosphorus fertilizer utilization efficiency, yield and seed nutritional quality SLC The poly-breeding method may include over-expressing ROPD or its allele ROPD in rice varieties carrying the dep1-1 gene, which is a key gene for high nitrogen efficiency and high yield, to obtain high-yield rice varieties with high nitrogen and phosphorus efficiency. SLC or other alleles after ROPD is modified (e.g., modified by gene editing).

[0053] In one embodiment, ROPD is overexpressed in rice varieties carrying the dep1-1 gene. SLC Genes that increase the rate of phosphorus absorption in rice varieties ( Figure 8C) and tillering, number of grains per ear and yield under low phosphorus fertilizer conditions ( Figure 8 D, E, F), and significantly reduced the phytic acid concentration in seeds ( Figure 8 B), which ultimately manifests as an improvement in phosphorus utilization efficiency. Phosphorus utilization efficiency refers to the fact that, under the same phosphorus application rate, the higher the crop yield, the higher the phosphorus utilization efficiency of the crop.

[0054] Among them, the dep1-1 gene is an upright and dense ear gene, which has the function of improving nitrogen fertilizer utilization efficiency and yield. The inventor has applied for patents for related technical content, which can be found in 200810111529.5 and 20111002759.9. The above patent documents are fully incorporated herein by reference.

[0055] The sixth aspect relates to a method for improving the phosphate fertilizer utilization efficiency and yield of crops, the method comprising: cultivating a plant containing the gene ROPD or its allele ROPD according to the present invention for improving the phosphate fertilizer utilization efficiency, yield and seed nutritional quality of crops; SLC The method may include: introducing the gene ROPD or its allele ROPD containing the polynucleotide sequence or related construct of the present invention to improve the phosphorus fertilizer utilization efficiency, yield and seed nutritional quality of crops. SLC regeneration of recombinant plant cells to obtain transgenic plants; or regeneration of recombinant plant cells containing the gene ROPD or its allele ROPD containing the gene ROPD or its allele ROPD containing the gene ROPD SLC Or a plant with other forms of alleles after modification (for example, gene editing) is hybridized with another plant to obtain a hybrid plant; or a plant containing the allele ROPD SLC Or other forms of alleles after transformation (for example, gene editing) recombinant Agrobacterium cells are transfected into crop plants to obtain transgenic plants. The ROPD gene or allele ROPD SLC The use of improving the phosphorus fertilizer utilization efficiency, yield and seed nutritional quality of crops includes but is not limited to this. The crops include but are not limited to rice, wheat and corn.

[0056] In one embodiment, the inventors combined 9311 and 9311-ROPD SLC Planted in fields with different phosphorus application rates (0kg / ha, 112kg / ha, 225kg / ha and 450kg / ha), the plant height, tiller number and panicle number were statistically analyzed after the rice grain filling was completed. After the rice was fully mature and harvested, the biomass and yield of each plant were calculated, and the phosphorus assimilation of different parts of the plant aboveground and its percentage in the total phosphorus content of the whole plant were analyzed. The results showed that at lower phosphorus fertilizer inputs, compared with 9311, 9311-ROPD SLCThe plant height becomes higher. When the phosphorus fertilizer input is higher, compared with 9311, 9311-ROPD SLC The plant height remains unchanged ( Figure 9 A, B); Under high and low phosphate fertilizer input, compared with 9311, 9311-ROPD SLC The biomass and yield per plant increased significantly ( Figure 9 E, F); at lower phosphate fertilizer input, compared with 9311, 9311-ROPD SLC The number of grains per ear and the number of tillers increased ( Figure 9 C, D); With the decrease of field phosphorus application, the total phosphorus content of 9311 decreased, and the phosphorus content in seeds increased. However, under different phosphorus application rates, 9311-ROPD SLC The total phosphorus content of the whole plant was significantly higher than that of 9311, and the phosphorus content in the seeds was significantly lower than that of 9311 ( Figure 9 G,H). Therefore, the superior allele ROPD SLC Applied to "Green Revolution" varieties (such as 9311), while maintaining the excellent traits of semi-dwarf plant height, it can, under different phosphorus application conditions, especially under low phosphorus conditions, on the one hand, increase the number of grains per panicle and the number of tillers of rice varieties, thereby increasing rice biomass and yield; on the other hand, it can improve rice's ability to absorb and assimilate phosphorus, while reducing the distribution of phosphorus to seeds, thereby achieving a synergistic improvement in rice phosphorus fertilizer utilization efficiency, yield and seed nutritional quality.

[0057] In one embodiment, the present inventors created transgenic rice pActin::ROPD, pActin::ROPD SLC and proPD SLC ::ROPD SLC , respectively, to detect the root phosphorus absorption rate and seed phytic acid content, the results showed that compared with the control group 9311, the root phosphorus absorption rate of the three transgenic rice materials increased, and the phytic acid content in the seeds decreased. SLC The expression of the gene can increase the phosphorus absorption rate of rice roots and reduce the phytic acid content in seeds ( Figure 5 ).

[0058] In one embodiment, the present inventors provide a ROPD SLC The application of the gene in wheat phosphorus efficient and high yield breeding. Specifically, the inventors first constructed a gene that overexpresses ROPD SLC p35S::ROPD vector SLC -flag and transformed into wheat variety Kenong 199 (KN199) through Agrobacterium-mediated method to obtain p35S::ROPD SLC-flag transgenic wheat. KN199 and p35S::ROPD were grown in the field. SLC -flag, and after maturity, the aboveground parts of the plants were collected and divided into four parts: leaves, stems, shelled seeds, husks, and branches. The phosphorus content was tested and the phosphorus distribution was calculated. The phytic acid content in the seeds was also tested. The results showed that compared with KN199, p35S::ROPD SLC -flag transgenic wheat seeds had a significantly lower phosphorus content and a significantly lower phytic acid content ( Figure 10 A, B). Therefore, ROPD SLC It can also reduce the phosphorus content and phytic acid content in wheat seeds and improve the nutritional quality of seeds.

[0059] In one embodiment, the present inventors provide a ROPD SLC The application of genes in corn phosphorus efficient and high yield breeding. Specifically, the present inventors used pActin::ROPD SLC -flag vector was used to transform the maize inbred line B73 by Agrobacterium-mediated method to obtain transgenic maize, which was then planted in the field. After maturity, B73 and B73 pActin::ROPD were collected. SLC -flag-treated plants were divided into four parts: leaves, stems, shelled seeds, husks, and branches. The phosphorus content was measured to calculate the phosphorus distribution. The phytic acid content in the seeds was also measured. The results showed that compared with B73, the overexpression line B73 pActin::ROPD SLC -flag seeds had a significantly lower phosphorus content ( Figure 10 C), the phytic acid content in seeds was significantly reduced ( Figure 10 D) Therefore, ROPD SLC It can also reduce the phosphorus ratio and phytic acid content in corn seeds and improve the nutritional quality of the seeds.

[0060] The following are definitions of some terms used in the present invention. Unless otherwise specified, the terms used in the present invention have the meanings known to those skilled in the art.

[0061] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably, the RNA is then translated in an organism to produce a protein.

[0062] As used herein, the term "expression cassette" refers to a nucleic acid sequence capable of directing the expression of a specific nucleotide sequence in a suitable host cell, comprising a promoter operably linked to a target nucleotide sequence, which is operably linked to a termination signal.

[0063] A "nucleic acid molecule" or "nucleic acid sequence" is a linear fragment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, preferably, a nucleic acid molecule is a DNA fragment. A "nucleic acid molecule" is also called a polynucleotide molecule.

[0064] A "plant" is any plant at any stage of development, in particular a seed plant.

[0065] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0066] "Plant cell culture" means cultures of plant units at various stages of development such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos.

[0067] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0068] A "plant organ" is a distinct and clearly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0069] As used herein, "plant tissue" means a group of plant cells organized into a structural and functional unit. This includes any tissue of a plant in planta or in culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into a structural and / or functional unit. The use of this term in conjunction with any specific type of plant tissue listed above or encompassed by this definition, or by itself, is not intended to exclude any other type of plant tissue.

[0070] A "promoter" is an untranslated DNA sequence upstream of a coding region that contains a binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region may also contain other elements that act as regulators of gene expression.

[0071] "Protoplasts" are isolated plant cells without or with only a partial cell wall.

[0072] The phrase "substantially identical" in the context of an alignment of two nucleic acid or protein sequences refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90%, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as determined using one of the following sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over at least about 150 residues. In particularly preferred embodiments, the sequences are substantially identical over the entire length of the coding region. Furthermore, substantially identical nucleic acid or protein sequences have substantially the same function.

[0073] To perform a sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When utilizing a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified, if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence, based on the selected program parameters.

[0074] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel et al., infra).

[0075] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.Ncbi.nlm.nih.gov / ). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the search sequence that either match or satisfy some positive threshold score, T, when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits serve as clues to begin searches to find longer HSPs containing them. These word hits are then extended as far as possible in both directions along each sequence until the cumulative alignment score no longer increases. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for pairwise matching residues; always greater than zero) and N (penalty score for mismatching residues; always less than zero). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of word hits in each direction ceases when the cumulative alignment score falls by the number X from the maximum value achieved, when the cumulative score reaches or falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when either sequence reaches an end point. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0076] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability compared to the reference nucleic acid sequence is less than 0.1, more preferably less than 0.01, and most preferably less than 0.001.

[0077] Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specifically hybridizes" refers to the ability of a molecule to bind to a specific nucleotide sequence, forming a duplex or hybridizing under stringent conditions, when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA). "Substantial binding" refers to complementary hybridization between the probe nucleic acid and the target nucleic acid, with minimal mismatches that can be tolerated by reducing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

[0078] In the context of nucleic acid hybridization assays such as Southern and Northern hybridizations, "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and differ under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. A wealth of guidance on nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York. Generally, high stringency hybridization and wash conditions are selected to be approximately 5°C below the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. Typically, under "stringent conditions," a probe will hybridize to its target subsequence but not to other sequences.

[0079] Another indicator that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid immunologically cross-reacts or specifically binds to the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.

[0080] "Transformation" is the process of introducing heterologous nucleic acid into a host cell or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome of the organism of interest.

[0081] "Transformed" refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the host genome or the nucleic acid molecule may exist as an extrachromosomal molecule. Such extrachromosomal molecules may be autonomously replicating. Transformed cells, tissues, or plants are understood to include not only the end product of the transformation process, but also its transgenic progeny.

[0082] The terms "polynucleotide," "polynucleotide molecule," "polynucleotide sequence," "coding sequence," and the like as used herein include single-stranded or double-stranded DNA and RNA molecules, and may contain one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences containing exons and introns, chemically synthesized DNA and RNA sequences, and sense and corresponding antisense strands.

[0083] Methods for producing and manipulating the polynucleotide molecules disclosed herein are known to those skilled in the art and can be accomplished according to the recombinant techniques described (see Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Ausubel et al., 1989, Current Techniques in Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Innis et al. (eds.), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed.), 1992, PCR Techniques, Oxford University Press, New York).

[0084] "Plant transformation" refers to the expression of at least one exogenous gene in a plant for the purpose of conferring one or more desirable phenotypic traits on the transformed plant.

[0085] Plants transformed according to the present invention can be monocots or dicots, including but not limited to corn, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, Brussels sprouts, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, winter squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed oilseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis and woody plants such as conifers and deciduous trees. Particularly preferred are rice, wheat, barley, corn, oats, or rye.

[0086] Once a desired nucleotide sequence has been transformed into a particular plant species, it can be propagated within that species or transferred into other varieties of the same species, particularly including commercial varieties, using conventional breeding techniques.

[0087] Preferably, the nucleotide sequences of the present invention are expressed in transgenic plants, thereby inducing the biosynthesis of proteins that control phosphorus fertilizer use efficiency and yield traits in the transgenic plants. In this way, transgenic plants with improved traits can be generated. To express the nucleotide sequences of the present invention in transgenic plants, the nucleotide sequences of the present invention may require modification and optimization. All organisms have specific codon usage preferences, as is known in the art, and codon usage can be altered to conform to plant preferences while maintaining the amino acids encoded by the nucleotide sequences of the present invention. Furthermore, high levels of expression in plants are best achieved from coding sequences having a GC content of at least about 35%, preferably greater than about 45%, more preferably greater than 50%, and most preferably greater than about 60%. Although preferred gene sequences can be adequately expressed in both monocot and dicot species, sequences can be modified to accommodate the specific codon and GC content preferences of monocot or dicot plants, as these preferences have been shown to differ (Murray et al., Nucl. Acids Res. 17:477-498 (1989)). In addition, the nucleotide sequences may be screened for the presence of unconventional splice sites that result in message truncation. All changes desired in these nucleotide sequences, such as those described above, may be made using methods described in published patent applications EP 0 385 962 (Monsanto), EP 0 359 472 (Lubrizol) and WO 93 / 07278 (Ciba-Geigy) using site-directed mutagenesis techniques, PCR and synthetic gene construction, as are well known in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The above features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0089] Figure 1 , showing that the "Green Revolution" gene sd1 inhibits phosphorus fertilizer uptake in rice and promotes phytic acid accumulation in seeds. (A) Phosphorus allocation in shoots; (B) Comparison of phytic acid concentrations in seeds; (C) Comparison of phosphorus uptake rates in roots.

[0090] Figure 2, showing positional cloning of genes controlling phosphorus uptake and allocation in rice. (A) Screening of 31 rice cultivars carrying the "Green Revolution" gene sd1 revealed cultivar SLC with higher phosphorus uptake rates and lower seed phytic acid concentrations, and cultivar 9311 with lower phosphorus uptake rates and higher seed phytic acid content. (B) QTL analysis of chromosome segment substitution lines constructed by hybridization between 9311 and SLC revealed the major QTL controlling phosphorus uptake and allocation in rice, qROPD. (C) Phytate uptake analysis of ROPD near-isogenic lines. (D) Phytate concentration analysis of ROPD near-isogenic lines. (E) Positional cloning of the gene ROPD controlling phosphorus uptake and allocation in rice was revealed in a BC1F2 population constructed by hybridization between 9311 and SLC.

[0091] Figure 3 , showing that the transcription and protein levels of ROPD are regulated by phosphorus concentration. (A) Analysis of ROPD gene expression under four hydroponic phosphorus concentrations; (B) Analysis of ROPD protein abundance under four hydroponic phosphorus concentrations.

[0092] Figure 4 Figure 1 shows that the ropd loss-of-function mutation affects rice growth, phosphorus uptake, and seed phytic acid concentration. (A) Rice plant phenotype. (B) Comparison of phosphorus uptake in rice. (C) Comparison of phytic acid concentration in rice seeds.

[0093] Figure 5 , indicating that ROPD is a gene that positively regulates phosphorus use efficiency. (A) ROPD expression analysis. (B) Comparison of phosphorus uptake in rice. (C) Comparison of phytic acid concentrations in rice seeds.

[0094] Figure 6 , showing that ROPD regulates the expression of genes related to phosphorus absorption and distribution. (A), Transcriptomic analysis of the expression of genes related to phosphorus absorption and distribution; (B), qPCR analysis of the expression of genes related to phosphorus absorption and distribution.

[0095] Figure 7 Figure 2 shows that ROPD binds to the promoters of genes involved in phosphate uptake and distribution and regulates their transcription. (A) ROPD binds to the promoter of the phosphate uptake gene OsWRKY74. (B) ROPD binds to the promoter of the phosphate uptake gene OsPHT1;1. (C) ROPD binds to the promoter of the phosphate uptake gene OsPHT1;8. (D) ROPD transcriptionally activates phosphate uptake genes and represses the phosphate distribution gene OsSPDT.

[0096] Figure 8, showing that under different phosphorus application levels, WYJ7, WYJ7-ropd knockout material in WYJ7 background and WYJ7 pActin::ROPD overexpression transgenic material SLC Comparative analysis of important agronomic traits, phosphorus uptake rate, and phosphorus percentage of -flag. (A) Plant type; (B) Phytic acid concentration in seeds; (C) Root phosphorus uptake rate; (D) Tiller number; (E) Number of grains per ear; (F) Yield per plant.

[0097] Figure 9 , showing that under different phosphorus application levels, a pair of near-isogenic lines 9311 and 9311-ROPD in the 9311 background SLC Comparative analysis of agronomic traits, phosphorus uptake rate, and phosphorus percentage of different varieties. (A) Plant type; (B) Plant height; (C) Tiller number; (D) Grain number per panicle; (E) Biomass per plant; (F) Yield per plant; (G) Phytic acid concentration in seeds; (H) Analysis of phosphorus percentage in aboveground parts.

[0098] Figure 10 , showing overexpression of ROPD SLC Analysis of phosphorus content and seed phytic acid concentration in transgenic wheat Kenong 199 and maize inbred line B73. (A, C) Analysis of phosphorus content in shoots; (B, D) Analysis of phytic acid concentration in seeds. DETAILED DESCRIPTION

[0099] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further explained below with reference to specific embodiments and accompanying drawings. It should be understood that the embodiments are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.

[0100] The experimental methods in the following examples are all conventional experimental methods unless otherwise specified. The reagents, kits, and experimental instruments used in the experiments can all be purchased from biological instrument and reagent companies unless otherwise specified. Unless otherwise specified, the relevant data involved in the present invention are based on the following methods:

[0101] Phosphorus content analysis method: In field trials, mature plants were cut from the roots, placed individually in mesh bags, and dried at 37°C. Thirty plants were harvested from each sample. After the plants were completely dry, the seeds, husks, branches, stems, and leaves were separated and ground into a powder. The powder was then sent to the Chinese Academy of Agricultural Sciences for determination of phosphorus concentration and content using an Isoprime 100 instrument.

[0102] Phytic acid concentration determination method: Oven-dry the seeds at 80°C for 3 days and then grind them into a powder. Add 0.8 mL of 10% perchloric acid solution and place on ice for 30 minutes, mixing by inversion. Centrifuge at 20,000 rpm for 10 minutes at 4°C, and transfer the supernatant to a fresh centrifuge tube. Centrifuge at 20,000 rpm for 10 minutes at 4°C, and transfer the supernatant to a tube containing 10 mg of titanium dioxide magnetic beads (Lot. No. ZQ5-4172). Incubate at 4°C for 30 minutes. Centrifuge at 8,000 rpm for 1 minute at room temperature, and discard the supernatant. Add 0.8 mL of 10% perchloric acid solution to the centrifuge tube, rinse the beads, and centrifuge at 8,000 rpm for 1 minute. Repeat the rinse once, and discard the supernatant. Add 0.2 mL of 10% ammonia solution to the centrifuge tube, incubate at room temperature for 5 minutes, and then centrifuge at 8,000 rpm for 1 minute. Transfer the supernatant to a fresh centrifuge tube. Repeat the elution once, and collect a total of 0.4 mL of supernatant. Open the cap of the centrifuge tube containing the sample, evacuate and dry at 45°C until all the liquid in the centrifuge tube is evaporated and white crystals appear at the bottom. Dissolve it in 20 μL of single-distilled water, and add 6×Orange G loading buffer. Prepare a 35% PAGE gel and load the sample to be tested and the phytic acid standard for electrophoresis. Stain with toluidine blue dye (20% methanol, 2% glycerol, 0.5% toluidine blue). Decolorize the stained PAGE gel with methanol eluent (20% methanol, 2% glycerol). Calculate the phytic acid concentration by comparing the grayscale value of the band with that of the phytic acid standard.

[0103] Phosphorus absorption determination method: When measuring the phosphorus absorption rate of rice roots, the isotope used 32 The test concentration of P was 8 μCi / L (PerkinElmer, NEX053001MC). 32 Start seedlings 15 days before P arrives. When the seedlings reach 10 cm, start high-low phosphorus (HP: 500μM NaH2PO4, LP: 25μM NaH2PO4) treatment for 7-10 days. Transfer the rice seedlings to the artificial climate chamber in the isotope room. Pre-treatment: Treat the rice seedlings with Solution I (0.5mM CaCl2, 2mM MES pH 5.0) for 10 minutes. Prepare Solution II (rice nutrient solution containing 100μM NaH2PO4) containing 8μCi / L32P and stir in a glass. Transfer the seedlings to a solution containing 8μCi / L 32P solution II was cultured in an artificial climate chamber (28°C, 50% relative humidity, 24 hours of light). Before sampling, the rice seedlings were rinsed with solution III (deionized water) and transferred to pre-cooled solution IV (0.5mM CaCl2, 2mM MES pH5.0). Sampling was performed at 3 hours, 8 hours, and 24 hours, with the sampling sites divided into above-ground and underground parts, with three replicates. The plants were cut at the rhizome junction, weighed, and numbered, and the samples were collected in 10mL centrifuge tubes. 1-2mL of perchloric acid was added to the 10mL centrifuge tube, vortexed vigorously, and left overnight at room temperature. Hydrogen peroxide was added to the centrifuge tube, vortexed to mix thoroughly, then the centrifuge tube cap was opened and placed in a 70°C oven. After 1 hour, the centrifuge tube was removed, the tube cap was fastened, and the tube was inverted to mix thoroughly. Uncover and let stand in a 70°C oven for 4-6 hours until the sample was no longer turbid. The sample was mixed, and an appropriate amount of supernatant was transferred to a 5 mL centrifuge tube. 3.5 mL of scintillation fluid (PerkinElmer Ultima Gold LLT) was added, and the tube was mixed slowly to avoid the generation of bubbles. The tube was placed in a dark place overnight.

[0104] Example 1: Analysis and map-based cloning of major QTLs regulating phosphorus uptake and phytic acid content in rice seeds

[0105] The inventors constructed three pairs of near-isogenic lines NJ6-SD1 and NJ6-sd1, 9311-SD1 and 9311-sd1, HJX74-SD1 and HJX74-sd1 in the background of Nanjing 6 (NJ6), 9311 and Huajingxian 74 (HJX74), and used these materials to systematically analyze the effects of the "Green Revolution" gene sd1 on crop phosphorus absorption and phytic acid content in seeds. These three pairs of materials were planted in the field. After they were fully mature, the aboveground parts were harvested and divided into four parts: leaves, stems, seeds (shelled), and glumes and branches, and the dry weights were measured separately. The samples were ground into powders below 60 mesh using a grinder and sent to the Beijing Academy of Agricultural Sciences to determine the phosphorus content, and the proportion of phosphorus content in the four parts was calculated. The results showed that compared with the control, the phosphorus content in the seeds of the materials NJ6-sd1, 9311-sd1 and HJX74-sd1 carrying sd1 was significantly increased ( Figure 1 A). At the same time, 10 mg of seed powder of different materials was extracted and the phytic acid content in the seed powder was detected using titanium dioxide magnetic beads. The results showed that compared with the control, the phytic acid content in the seeds of NJ6-sd1, 9311-sd1 and HJX74-sd1 was significantly increased ( Figure 1 B) Therefore, the sd1 gene increases the phosphorus content and phytic acid content in rice seeds. In addition, the near-isogenic lines NJ6-SD1 and NJ6-sd1, 9311-SD1 and 9311-sd1, HJX74-SD1 and HJX74-sd1 were used to 32The results of the phosphorus absorption experiment with P isotope labeling showed that the phosphorus absorption capacity of the roots of rice materials carrying sd1 was significantly reduced ( Figure 1 C) Therefore, the sd1 gene reduces the phosphorus uptake capacity of rice roots.

[0106] The present inventors collected and used 31 rice resource materials carrying the sd1 gene, and respectively measured phosphorus absorption and phytic acid concentration in seeds, and screened and obtained a rice line SLC ( Figure 2 A) was crossed with the rice variety 9311, which has a lower root phosphorus uptake rate and higher seed phytic acid concentration. The resulting F1 generation was then backcrossed with 9311 and selfed for one generation to obtain the BC1F2 population. Through QTL analysis, a major QTL regulating crop phosphorus uptake and seed phytic acid content was identified within a 5kb physical range on the long arm of chromosome 2: qROPD ( Figure 2 B); Using the F1 hybrid of SLC and 9311, with 9311 as the recurrent parent, a BC4F2 population was constructed through backcrossing for multiple generations. Fine mapping and positional cloning were performed, and the candidate gene ROPD ( Figure 2 C, D, E), is the known gene GRF4.

[0107] Example 2: ROPD function deficiency leads to decreased phosphorus absorption capacity of crop roots and increased phytic acid concentration in seeds.

[0108] This study used CRISPR / Cas9 to create a ROPD gene knockout rice strain. The CRISPR / Cas9 vector construction and transformation methods are as follows:

[0109] According to the gDNA sequence of the GRF4 gene, primers were designed: ROPD-CRISPR-F1: see SEQ ID NO.7; ROPD-CRISPR-R1: see SEQ ID NO.8.

[0110] Using the gDNA of 9311 as a template, primers ROPD-CRISPR-F1 and ROPD-CRISPR-R1 were used to amplify the target fragment. The specific construction method can be found in the article published by Professor Liu Yaoguang of South China Agricultural University (A robust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. (2015) Molecular Plant, 8 (8): 1274-1284). The vector was introduced into the indica rice variety 9311 and the japonica rice variety WYJ7 by Agrobacterium-mediated transformation, respectively, to obtain the ROPD gene knockout strains 9311-ropd and WYJ7-ropd. The genomic DNA sequence of the editing target site had a C deletion at base 431, the 112th amino acid began to be garbled, and the 300th amino acid was prematurely terminated. The root phosphorus absorption capacity and seed phytic acid concentration were measured and it was found that the root phosphorus absorption capacity of the ROPD knockout plant 9311-ropd was lower than that of the wild type 9311, and the seed phytic acid concentration was higher ( Figure 4 B, C). Similarly, WYJ7-ropd showed a decreased ability to absorb phosphorus fertilizers through its roots and an increased phytic acid concentration in its seeds compared to the wild type WYJ7 ( Figure 8 Therefore, ROPD regulates phosphorus uptake by crop roots and phytic acid concentration in seeds. Loss of ROPD function leads to decreased phosphorus uptake by crop roots and increased phytic acid concentration in seeds.

[0111] Example 3: ROPD and its superior allele ROPD SLC Improves phosphorus absorption capacity of rice roots and reduces phytic acid concentration in seeds

[0112] The inventors created ROPD or ROPD SLC Verification of ROPD and its superior alleles by overexpressing transgenic rice materials SLC It has the function of improving the phosphorus absorption capacity of rice roots and reducing the phytic acid concentration in seeds.

[0113] According to ROPD and ROPD SLC The primers designed based on the cDNA sequence and genomic sequence of the gene are as follows:

[0114] ROPD-XbalI-F: see SEQ ID NO.9; ROPD-SalI-R: see SEQ ID NO.10; ROPD SLC -XbalI-F: see SEQ ID NO.11; ROPD SLC -SalI-R: see SEQ ID NO.12; pROPDSLC ::ROPD SLC -XbalI-F: see SEQ ID NO.13; pROPD SLC ::ROPD SLC -SalI-R: see SEQ ID NO.14.

[0115] The cDNA of 9311 and SLC and the DNA of SLC were used as templates, primers ROPD-XbalI-F and ROPD-SalI-R, primer ROPD SLC -XbalI-F and ROPD SLC -SalI-R and primer proPD SLC ::ROPD SLC -XbalI-F and proPD SLC ::ROPD SLC -SalI-R amplification to obtain ROPD and ROPD SLC CDS fragment and proPD SLC ::ROPD SLC The CDS fragment was ligated into the pCAMBIA2300 vector (purchased from CAMBIA, Australia) using the restriction endonuclease sites of XbalI and SalI, and pActin::ROPD and pActin::ROPD were transformed into pActin::ROPD using Agrobacterium-mediated transformation. SLC and proPD SLC ::ROPD SLC The vector was transferred into 9311 to obtain homozygous overexpression ROPD and ROPD SLC Transgenic rice lines pActin::ROPD, pActin::ROPD SLC and proPD SLC ::ROPD SLC .

[0116] The results of the rice root phosphorus absorption experiment showed that compared with 9311, the transgenic lines pActin::ROPD and pActin::ROPD SLC and proPD SLC ::ROPD SLC The expression of ROPD gene and the phosphorus absorption rate of roots were significantly increased ( Figure 5 A, B); the phytic acid content in the seeds of the above rice materials was detected. The results showed that compared with 9311, the transgenic lines pActin::ROPD and pActin::ROPD SLC and proPD SLC ::ROPD SLC The phytic acid content in the seeds of Figure 5 C) Therefore, ROPD and its superior allele ROPD SLC It has the function of increasing the phosphorus absorption capacity of rice roots and reducing the phytic acid concentration in seeds.

[0117] Example 4: ROPD promotes the expression of genes related to phosphorus absorption and inhibits the expression of genes related to phosphorus distribution. 9311, the knockout line 9311-ropd of Example 2, and 9311-ROPD were used. SLC The seeds were sterilized with 20% sodium hypochlorite solution for 30 minutes, then placed in a 37°C incubator, immersed in water and swollen for 24 hours, drained, and transferred to a 28°C incubator for germination. After turning white, they were transferred to a hollow 96-well plate and cultured for 7 days. The seedlings with uniform growth were selected and transferred to 40L nutrient solution (1.25mM NH4NO3, 0.5mM NaH2PO4·2H2O, 0.75mM K2SO4, 1mM CaCl2, 1.667mM MgSO4·7H2O, 40μM Fe-EDTA(Na), 19μM H3BO3, 9.1μM MnSO4·H2O, 0.15μM ZnSO4·7H2O, 0.16μM CuSO4, and 0.52μM (NH4)3Mo7O 24 4H2O, pH 5.5) for 2 weeks, adjusting the pH every 2 days. RNA was extracted from 3-5 cm root tips and divided into two aliquots: one for RNA-seq (commissioned by BGI) and one for reverse transcription into cDNA. Gene expression levels were determined using quantitative PCR.

[0118] RNA-seq results showed that compared with 9311, the expression of phosphorus transporter genes OsPHT1;1 and OsPHT1;8 in 9311-ropd was significantly reduced, and the gene expression of the transcription factor OsWRKY74, which positively regulates phosphorus absorption, was also significantly reduced, while the expression of the OsSPDT gene, which regulates phosphorus distribution, was significantly increased. SLC The expression of OsPHT1;1, OsPHT1;8 and OsWRKY74 genes were significantly increased, while the expression of OsSPDT gene was significantly decreased ( Figure 6 A).

[0119] Furthermore, the present inventors used cDNA reverse transcribed from RNA of the same batch of rice materials and verified the RNA-seq analysis results using fluorescent quantitative PCR. The results showed that compared with 9311, the expression levels of OsPHT1;1, OsPHT1;8 and OsWRKY74 genes in 9311-ropd were significantly reduced, while the expression level of OsSPDT gene was significantly increased; while 9311-ROPD SLCThe expression levels of OsPHT1;1, OsPHT1;8 and OsWRKY74 genes were significantly increased, while the expression of OsSPDT gene was significantly decreased ( Figure 6 B).

[0120] Therefore, the ROPD gene positively regulates the expression of phosphorus absorption-related genes (such as OsPHT1;1, OsPHT1;8, and OsWRKY74) to promote phosphorus absorption, while negatively regulates the expression of phosphorus distribution-related genes (such as OsSPDT) to reduce phosphorus accumulation in seeds; the superior allele ROPD SLC It can significantly increase the expression of rice phosphorus absorption-related genes, while significantly reducing the expression of phosphorus distribution-related genes.

[0121] Example 5: ROPD and ROPD SLC Can bind to and transcriptionally activate genes related to phosphorus absorption and transcriptionally repress genes related to phosphorus distribution

[0122] ROPD is a transcription factor. The present inventors used ChIP-qPCR experiments and transcriptional activation experiments to further confirm that ROPD can bind to downstream genes and regulate their expression.

[0123] The present inventors constructed pActin::ROPD SLC -flag vector primers are: ROPD SLC -XbalI-F: see SEQ ID NO.11; ROPD SLC -SalI-R: see SEQ ID NO.12.

[0124] Using cDNA of 9311 as template, primers ROPD SLC -XbalI-F and ROPD SLC -SalI-R amplification to obtain ROPD SLC The CDS fragment was ligated into the pCAMBIA2300 vector using the XbalI and SalI restriction endonuclease sites and transformed into 9311 using Agrobacterium-mediated transformation to obtain the homozygous transgenic rice line pActin::ROPD. SLC-flag. The present inventors used the root tips of pActin::ROPD-flag overexpressing transgenic lines to perform ChIP-qPCR experiments (method reference Modulating plant growth–metabolism coordination for sustainable agriculture. Nature. 2018 Aug; 560(7720): 595-600). The results showed that ROPD protein can bind to the promoters of OsPHT1; 1, OsPHT1; 8, and OsWRKY74 genes ( Figure 7 A, B, C).

[0125] The present inventors used the ROPD and ROPD described in Example 4. SLC The full-length cDNA was used as a template and the fragment was ligated into the p35S-GAL4BD vector (provided by Professor Shouyi Chen, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, see Yu-Jun Hao YJ, Wei W, Song QX, Chen HW, Zhang YQ, Wang F, Zou HF, Lei G, Tian AG, Zhang WK, Ma B, Zhang JS and Chen SY. (2011) Soybean NAC transcription factors promote abiotic stress tolerance and lateral root formation in transgenic plants. Plant Journal. 68(2): 302-313) by enzyme digestion and ligation to construct p35S-GAL4BD-ROPD and p35S-GAL4BD-ROPD. SLC carrier.

[0126] The present inventors used the genomic DNA of WYJ7 as a template to amplify the 2.5 kb promoter segments of OsPHT1;1, OsPHT1;8, OsWRKY74 and the 3 kb promoter segment of OsSPDT gene, respectively, and connected them into the reporter gene vector through EcoRV and XbaI to construct pOsPHT1;1::LUC, pOsPHT1;8::LUC, pOsWRKY74::LUC and pOsSPDT::LUC vectors. The primers are: OsPHT1;1-F: see SEQ ID NO.15; OsPHT1;1-R: see SEQ ID NO.16; OsPHT1;8-F: see SEQ ID NO.17; OsPHT1;8-R: see SEQ ID NO.18; OsWRKY74-F: see SEQ ID NO.19; OsWRKY74-R, see SEQ ID NO.20; OsSPDT-F: see SEQ ID NO.21; OsSPDT-R, see SEQ ID NO.22.

[0127] The transcriptional activation assay was briefly described as follows: The coleoptiles of rice seedlings were cut into strips and placed in an enzyme solution. After lysis in the dark with low-speed shaking for 5-6 hours, an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH 5.7) was added. The solution was centrifuged at 200 g for 5 minutes, and the precipitate was collected and resuspended in W5 for washing. After repeated washing twice, the precipitate was resuspended in MMg solution (0.4 M mannitol, 15 mM MgCl2, 4 mM MES pH 5.7) to obtain rice protoplasts. The plasmid DNA of pOsOsPHT1;1::LUC, p35S-REN, and p35S-GAL4BD-ROPD vectors was added to the rice protoplasts at a ratio of REN:LUC:GAL4BD = 1:6:6 (mass ratio). An equal volume of PEG4000 / CaCl2 was used to 2+ After transformation, the cells were allowed to stand for 15 minutes, and then 2 volumes of W5 were added to terminate the reaction and washed. The cells were then placed in a W5 solution and cultured in the dark for 16 hours. The protoplasts were collected and lysate was added. The LUC / REN values ​​were determined using a dual fluorescence detection kit (purchased from PROMEGA).

[0128] The results of transcriptional activation experiments showed that after the addition of ROPD protein, the expression of luciferase gene (LUC) driven by promoters pOsWRKY74, pOsPHT1;1 and pOsPHT1;8 was significantly upregulated, while the expression of luciferase gene (LUC) driven by promoter pOsSPDT was significantly downregulated. SLCAfter the expression of protein, the up-regulation trend of LUC gene expression driven by promoters pOsWRKY74, pOsPHT1;1 and pOsPHT1;8 was more significant, while the down-regulation trend of luciferase gene (LUC) expression driven by promoter pOsSPDT was more significant ( Figure 7 D).

[0129] Therefore, ROPD protein and ROPD SLC The protein can bind to downstream target genes (such as OsWRKY74, OsPHT1;1, OsPHT1;8) and activate the expression of OsWRKY74, OsPHT1;1 and OsPHT1;8 genes, while inhibiting the expression of OsSPDT gene, ultimately promoting the absorption and utilization of phosphorus by crops while inhibiting the distribution of phosphorus to seeds; ROPD SLC The protein has stronger transcriptional activation activity on OsWRKY74, OsPHT1;1 and OsPHT1;8, and stronger transcriptional repression activity on OsSPDT.

[0130] Implementation 6: ROPD SLC It can improve the phosphorus fertilizer use efficiency and yield of indica rice varieties while reducing the phytic acid content in their seeds

[0131] The inventors combined 9311 and 9311-ROPD SLC Planted in the field with different phosphorus application rates (0kg / ha, 112kg / ha, 225kg / ha and 450kg / ha), the aboveground parts of the near-isogenic lines under different phosphorus application rates were harvested after the rice was fully mature. The agronomic traits, biomass and yield were investigated and statistically analyzed. After drying at 80℃, the phosphorus percentage, phosphorus absorption rate and phytic acid content in the seeds were measured and analyzed. The statistical results of agronomic traits showed that the superior allele ROPD SLC Does not affect the plant height and number of grains per ear of 9311 ( Figure 9 A, B, D), but the tiller number, biomass and yield per plant were significantly increased under different phosphorus application rates ( Figure 9 C, E, F). The results of phytic acid test in seeds showed that the concentration of phytic acid in seeds decreased with the decrease of field phosphorus application level. However, compared with 9311, 9311-ROPD SLC The phytic acid concentration decreased more significantly in the seeds of Figure 8 G). The results of the analysis of rice phosphorus distribution showed that as the field phosphorus application level increased, the phosphorus proportion in seeds gradually decreased; under different field phosphorus application levels, compared with 9311, 9311-ROPD SLC The phosphorus content in seeds decreased, and the decrease was more obvious with the increase of field phosphorus application level ( Figure 9 H). Therefore, the superior allele ROPD SLCThe introduction of indica rice varieties can improve the phosphorus fertilizer utilization efficiency and yield of indica rice varieties, while reducing the phytic acid content in their seeds.

[0132] Specific statistical methods: Plant height statistics: After the rice matures, 30 plants are taken in the field to measure the plant height. Ear number statistics: After the rice matures, 30 ears on the main tillers are taken in the field, and the number of grains on each ear is directly counted and recorded. Single plant yield statistics: After the rice is fully mature, a plot (area 15m2) is taken. 2 ) were threshed from 40 individual plants within a certain range. The harvested seeds were dried at 37°C and weighed to obtain the yield per plant. Three replicates were performed. Grain length, width, and 1000-kernel weight were measured: After the seeds were naturally dried, they were rinsed with floating water to remove the empty seeds. After drying at 37°C, they were stored at room temperature for at least three months to ensure that the seeds were fully dried and that the moisture content was relatively consistent across the plant lines.

[0133] Example 7: ROPD SLC It can improve the yield and phosphorus use efficiency of high-yielding japonica rice varieties while reducing the phytic acid content in their seeds

[0134] According to ROPD SLC The cDNA sequence of the gene was sequenced and the following primers were designed:

[0135] ROPD-XbalI-F: see SEQ ID NO.9; ROPD-SalI-R: see SEQ ID NO.10.

[0136] The present inventors used the cDNA of SLC as a template and amplified ROPD with primers ROPD-XbalI-F and ROPD-SalI-R to obtain SLC The CDS fragment was ligated with the pCAMBIA2300 vector using the conventional enzyme digestion and ligation method using the XbalI and SalI restriction sites to obtain pActin::ROPD SLC -flag vector was further transformed into the high-yield japonica rice variety WYJ7 (carrying the high-yield gene dep1-1, WYJ7-dep1) using Agrobacterium-mediated method to obtain the ROPD overexpression in the WYJ7 background. SLC The transgenic rice was planted in fields with different phosphorus application rates (0kg / ha, 112kg / ha, 225kg / ha, and 450kg / ha). Statistical analysis of agronomic traits showed that at a lower phosphorus application rate (112kg / ha), the overexpression of ROPD SLC Significantly increased the number of rice tillers, grains per panicle and yield per plant ( Figure 8 D, E, F). Phytic acid detection results in seeds showed that overexpression of ROPD SLC Significantly reduced the phytic acid content in WYJ7 seeds ( Figure 8 B) Phosphorus absorption experiment results showed that overexpression of ROPD SLC Can increase the phosphorus absorption rate of WYJ7 roots ( Figure 8 C) Therefore, on the one hand, the application of ROPD SLC It can further improve the yield and phosphorus fertilizer utilization efficiency of high-yield japonica rice varieties. On the other hand, ROPD SLC The aggregation of genes with dep1-1 is an effective method to breed high-yielding and nitrogen and phosphorus-efficient rice varieties.

[0137] For details about the dep1-1 gene, please refer to the applicant's authorized patent applications 200810111529.5 and 20111002759.9, which are fully incorporated herein by reference.

[0138] Specific statistical method: Tiller number statistics: After the rice matures, 30 rice plants are taken from the field to measure and count the tiller numbers. The remaining statistical methods are described in Example 8.

[0139] Example 8: ROPD SLC Can reduce the phosphorus content and phytic acid content in wheat seeds

[0140] The present inventors transformed the p35S::ROPD described in Example 4 into SLC -flag vector, and the high-yield wheat variety Kenong 199 (KN199, wheat transformation was completed by Gao Caixia's laboratory at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) was transformed using the Agrobacterium-mediated method to obtain the KN199 background overexpressing ROPD SLC KN199 and transgenic wheat KN199 p35S::ROPD SLC -flag were planted in the field, and the aboveground parts were harvested after they were fully mature. The four parts were divided into leaves, stems, shelled seeds, and hulls and branches. The phosphorus content was tested and the phosphorus percentage was calculated. The phytic acid content in the seeds was also tested. The results showed that compared with KN199, KN199p35S::ROPD SLC The phosphorus percentage and phytic acid concentration in the seeds of the -flag overexpressing strain were significantly reduced ( Figure 10 A, B). The specific detection method is as mentioned above. Therefore, the application of ROPD in wheat SLC Genes can also reduce the phosphorus content and phytic acid content in seeds, thereby improving the nutritional quality of seeds.

[0141] Example 9: ROPD SLC Can reduce the phosphorus content and phytic acid content in corn seeds

[0142] The present inventors transformed the pActin::ROPD described in Example 4 into SLC-flag vector was used to transform maize inbred line B73 (B73, maize transformation was completed by Professor Jiang Caifu's laboratory at China Agricultural University) through Agrobacterium, and the overexpression of ROPD in B73 background was obtained. SLC The transgenic line B73 pActin::ROPD SLC -flag. After corn matures, harvest B73 and B73 pActin::ROPD SLC -flag aboveground part was divided into four parts: leaves, stems, seeds and corn cobs. The phosphorus content was detected and the phosphorus percentage was calculated. The phytic acid content in the seeds was also detected. The results showed that compared with B73, the overexpression strain B73 pActin::ROPD SLC -flag seeds had a significantly lower phosphorus content ( Figure 10 C), and the phytic acid content in seeds was also significantly reduced ( Figure 10 D) Therefore, the application of ROPD in corn SLC Genes can also reduce the phosphorus content and phytic acid content in seeds, thereby improving the nutritional quality of seeds.

[0143] All documents mentioned in this invention are incorporated herein by reference. After reading the above contents of this invention, any changes or modifications made to this invention are deemed equivalent to the present invention and fall within the scope of the appended claims.

[0144] Sequence Listing

[0145] SEQ ID No. 1, gDNA sequence of ROPD (derived from 9311)

[0146] atgacgatgccgtatgcctccctgtctccggcggtggccgaccaccgctcgtccccggcagccgcgaccgcctccctcct

[0147] ccccttctgccgctccaccccgctctccgcgtaagcaacgcgaacccgcggctacaacccattttcttggctccagtggtgc

[0148] atgtgacaacacggtgagacgttgtgtgtgggtgggtgggtgcaggggcggtggtgttgtcgcgatgggggaggacgcg

[0149] ccgatgaccgcgaggtggccgccggcggcggcggcgaggctgccgccgttcaccgcggcgcagtacgaggagctgg

[0150] agcagcaggcgctcatatacaagtacctggtggcaggcgtgcccgtcccgccggatctcgtgctccccatccgccgcgg

[0151] actcgactccctcgccgcccgcttctacaaccatcccgcccgtacgtcgtgttcctatttcttgcctctcctctaccatcgctgc

[0152] attgcttttggatgcttgtttagtgtcggcctctttgtttattccgatcaggcgtactttgcttccatttgttaattggctccgggtcat

[0153] ttgttaatccgggttacgcgattcaagaaacatgcgtgtggtttttatgctatcctccggatttggttataaaaaggcttgtttttaa

[0154] atccaaaactcgtgctcgcttcacgattagcgcatcattttttttttatgggggggggggggggagagtttgcccatcattctgt

[0155] ctctgtttgatctgatagaggacgtgcacacgctcttgtctgaaataaaatcttttgtttatcagtatgcccatgggataagccat

[0156] tttctctgtgaaccaacaccctggcaaactgtttttttgctcgccatttttgagcgattgctaagaacagataactatgccctgca

[0157] tatggatcggatatggacttctcaaatattcaaatgccattctattaggaactcaaaatgcattaccaacaaatgcattcttgtgt

[0158] gtaacacggttgctacgatgtgcctgtttttgtacagttggatatggtccgtacttcggcaagaagctggacccagagccagg

[0159] gcggtgccggcgtacggacggcaagaaatggcggtgctcgaaggaggccgcgccggattccaagtactgcgagcgcc

[0160] acatgcaccgcggccgcaaccgttcaagaaagcctgtggaaacgcagctggtcgcccagtcccaaccgccctcatctgtt

[0161] gtcggttctgcggcggcgccccttgctgctgcctccaatggcagcagcttccaaaaccactctctttaccctgctattgccgg

[0162] cagcaatggcgggggcggggggaggaacatgcccagctcatttggctcggcgttgggttctcagctgcacatggataatg

[0163] ctgccccttatgcagctgttggtggtggaacaggcaaagatctcaggtgattgttcatttctttttttttaatcaaacgccatattta

[0164] cttgtttagcactgtcttgaatcatgatatgtatccttccgttgtctaaaaaaaaggtgtcatgctctaactgattggtgtcaggtg

[0165] gatgcagttatgaatctgtatttttctttgtgatcggttaataactgtgtcccatttgtttgcattggtggcaatcgaaccagctgtc

[0166] catgctcagtagtactacttcgatttggtgctgcaatcactgaaagtctgaaactttactctctgcactgcaaaaatttgtgttatg

[0167] tttaggtttccagagtgctgcctctttgcccttcccatactttctggtatcagttttcagccccagaagccggggacagtctccat

[0168] aagagatttctgctcaggtgaaactggggtgcagggtcttaacatggctttggcccagtagtttgaaacatgtactgtccataa

[0169] agatgatactactacatatttgtgtctgccctcgcagtgcttgtgcctgctggtagctgatcatggcttcccttggcatttactcc

[0170] acttctttattcctccacagaatccagttgtttctgtctctgctcttcaggggcagtcaattatttggcccttgcaaaatactatctct

[0171] gaagatgtctcaccgatcaccactatacctgaaacattttccagtggccagcgtgagctgcatgatgctccaagtcaactcta

[0172] tactcatccaatgttgatgattagattttaacaatgcaactctttgatttatcttccctacaaaaaaaaaggaactctttgatttatct

[0173] tcggtgaatctcagtctgaccttagtacctagcctcattatttacttcaccaaatgtataactctacagtgcttgttcgtgttgattt

[0174] ggtttagtttagttattgaattattcggtcaccttagtctttgattgtttttttctttctgctcttgtcatcaactgtttagggttcagctg

[0175] acttgctgctgcaactaaactgtcttctggttttactgcaaaatagaatgtttcttgggccatgatctgctgctatatatgattagtt

[0176] aaaccatggttctatgttttcttatatgaattcatgacaagaatactaacttttggaaaaggtaattttattttttttgtatgataataat

[0177] gctttggattctttctagtttatctgtcggacttaggttaactacatttcctccggtacatggatttatttcattcttacaattgagccc

[0178] ttatgaatattttcttcctaattctgttctaaaaagttagaattgacatattttcgataggtacatgcctagcacttgcattcgtgtttc

[0179] ctactaattcccaatcactgtatcttctcaaattcaggtatactgcttatggcacaagatctttggcggatgagcagagtcaact

[0180] cattactgaagctatcaacacatctattgaaaatccatggcggctgctgccatctcagaactcgccatttcccctttcaagctat

[0181] tctcagcttggggcactaagtgaccttggtcagaacacccccagctcactttcaaaggttcagaggcagccactttcgttcttt

[0182] gggaacgactatgcggctgtcgattctgtgaagcaagagaaccagacgctgcgtcccttctttgatgagtggccaaaggga

[0183] agggattcatggtcagacctcgctgatgagaatgctaatctttcgtcattctcaggcacccaactgtcgatctccataccaatg

[0184] gcatcctctgacttctcggcggccagttctcgatcaactaatggtacgactacttgatctccccccaattacttcgtgcgtgttta

[0185] tgtctgtatcctgcaatgtctgaagatttcttactgaaaacgtcatctggtctgtgtgcaggtgactga

[0186] SEQ ID No.2, cDNA sequence of ROPD (derived from 9311)

[0187] atgacgatgccgtatgcctccctgtctccggcggtggccgaccaccgctcgtccccggcagccgcgaccgcctccctcct

[0188] ccccttctgccgctccaccccgctctccgcgggcggtggtgttgtcgcgatgggggaggacgcgccgatgaccgcgagg

[0189] tggccgccggcggcggcggcgaggctgccgccgttcaccgcggcgcagtacgaggagctggagcagcaggcgctca

[0190] tatacaagtacctggtggcaggcgtgcccgtcccgccggatctcgtgctccccatccgccgcggactcgactccctcgcc

[0191] gcccgcttctacaaccatcccgcccttggatatggtccgtacttcggcaagaagctggacccagagccagggcggtgccg

[0192] gcgtacggacggcaagaaatggcggtgctcgaaggaggccgcgccggattccaagtactgcgagcgccacatgcacc

[0193] gcggccgcaaccgttcaagaaagcctgtggaaacgcagctggtcgcccagtcccaaccgccctcatctgttgtcggttctg

[0194] cggcggcgccccttgctgctgcctccaatggcagcagcttccaaaaccactctctttaccctgctattgccggcagcaatgg

[0195] cgggggcggggggaggaacatgcccagctcatttggctcggcgttgggttctcagctgcacatggataatgctgcccctta

[0196] tgcagctgttggtggtggaacaggcaaagatctcaggtatactgcttatggcacaagatctttggcggatgagcagagtcaa

[0197] ctcattactgaagctatcaacacatctattgaaaatccatggcggctgctgccatctcagaactcgccatttcccctttcaagct

[0198] attctcagctgtgggcactaagtgaccttggtcagaacacccccagctcactttcaaaggttcagaggcagccactttcgttc

[0199] tttgggaacgactatgcggctgtcgattctgtgaagcaagagaaccagacgctgcgtcccttctttgatgagtggccaaagg

[0200] gaagggattcatggtcagacctcgctgatgagaatgctaatctttcgtcattctcaggcacccaactgtcgatctccataccaa

[0201] tggcatcctctgacttctcggcggccagttctcgatcaactaatggtgactga

[0202] SEQ ID No.3, ROPD SLC gDNA sequence of

[0203] gttttgaataagacgagtggtcaaacgttacaagcaaaaactcaaaatcccttatattatgggacagagtgagtagtacttagt

[0204] agatactccctccattccaaaatataaggcacaaccacccttaatccaaagaccaagaaataattattatcatctggtagtttg

[0205] gatcatcctaataaatactaatgcatgcatccaataggattagataacatgaaagtggaggatttaaaaaaaaataatttaata

[0206] gagaagttgtcatagttaattgcatacttgcatgtatgccttatattatggaacatctaataaaagttattgtgtcttatattatagaa

[0207] tggagggagtatgttgttttggtttgaagacacatcattaccattgctaacatacaatttgaaactttctagattttaatcaaagttt

[0208] cggtttttaactagtagcaaactaaataaactttacccaacgaaagagttggcagtgccactgccaaaacttacctaaattttg

[0209] gcactaccaatatttcgatagagtacgaaatcagacataccctaggatattcctattattcccttcgtcctaatataactgcaacc

[0210] taatacatatgtgatatattctaggatgtccagattcatattcttaggataggtcagtacgtcacacctatactaggttacatttata

[0211] ttggttactttaggggcaagagaactatccgctgccttttctaggaaaatgctagtaatcgggctcccgatcaacgccagaaa

[0212] aatcgggcacccgctgcttcctgcacctccgcgccatccccaccacacgccccaccacatgcgtcgttgcaacaaatcac

[0213] ccacgtattatggaaacgctctattaaggggatcatttcaacttttgtttcaccaacaatatttcatcaagtgtactcgtgatgtttc

[0214] actatgtatagatcaaatgttgcagtgaattaaaatatccttttgctatttgctgaaacattattttatatatggtaaaacaacacct

[0215] gattttttggaaaccgtttgtttcatactttgaagtaaatgttccatgaggtggtttggttgagtttaagctttttaaacaattgaaa

[0216] cgttttcaatctatttaatgaaacaattccggtctacttgatggaacagcgcccgatttttgaaaaaaattcaggtgcctcgcctt

[0217] ttttatcagtttatacgttgattgaaggtaaaacaaatcgaccaaaatggtaaatgcattaatggacatgagtctccatacagttc

[0218] gaccactaataaactagtcaattcgttgaacataggtctaacaatttacttattggcttataaggccttgtttagttcgcgaaaaa

[0219] gaaaattttgggtgtcacatcagacgtttgactggatgtcggaaggggttttcggacatgaatgaaaaaactaatttcataact

[0220] cgactggaaaccgcgagacgaatttattaagcctaataaatccgacattagcacatgtgggttactgtagcacttatggctaa

[0221] tcatcgactaattaggttcgaaaggttcgtctcgcgattttcatacaaactgtgcaattagtttttcattttatctatatttagtgctcc

[0222] atgcatgtgtccaaagattcgatgtaatgtttttgagaaaaaaaattggaaactacgcaaggcctaatttaagttttagaacttaa

[0223] agtgtttattctaagttttcttttcatcgtagtttttcttgcagccggttttcaaaccactagtattatagatataatttttttatttgcaatt

[0224] tattttttacgatttatcaaccgcagtttatccgattagtctttggaaaatgttactggaagaactaaaacccagataacccacac

[0225] caaataatattaataaaaaatctgcatctgcattatagtacgtcaatccagctccacatgttactgatcttgtggtactgtagtagt

[0226] aatagtactccctccgtcctagtatagtggacgtttccattcgttttatttgaaaaattagtgcaaatataaaaaaagataagtcat

[0227] atggaaagtatttgataataaagcaattgacaaacaaaataattaaatattccaaaattttttaaataagacgaatgatcaaaaa

[0228] ttataaacaaaaactcaagaagacaagtaatatgggacagaggtagtaatagatatattagtacactgttttgaggtattccaac

[0229] gtcgaataaaacagagaagtaccacaccattattgtgaactcgaaacctggtagtgattaattgcctcatggcaagcaaact

[0230] gaaacaaactactattactactgctctcccgttttatattgttcatcgtataacccaaaatcagaatttccaaattatatcttgtaatc

[0231] ttgactgcatcgtttgacatacaatactattaaatctatacatataaattgagtctgtatacgtatatacaagcacttgagatggtt

[0232] aagcactttttttagcattctaagtttcttattttgtaggatttttagtacgaggtaagacatacttgaaaaaaattataagaactag

[0233] agtgcatgtgaccacctaactccttgcaatttttattcttataatttgaaaatcctataaaccaaataagcccttcaaaggaaatta

[0234] aatcatgaggtttgaggttaggtttgaattctctaaaaagtggaggaaaggactcaacagaaaaaaaaatcctatagaatttc

[0235] gatcctataaaattttagttaaaaatactttgttccaaaattgccatggataaatgtaatttctatgcatacaactaaattatcgat

[0236] ggcaacagtgcatgagcatatatttatttcattgacctacggttgcatgtcttcgatctctatggagtagtaccgaggctaagttt

[0237] agtttcaaacttttccttcaaacttacagcttttttatcacattaaaactttcctacatataaacttttaacttttccatcacatctttcaa

[0238] tttcaaccaaacttctaattttagcgtgaactaaacacaccctgaattcaaaactctttttatttccttcaagatgtccgatgcaca

[0239] cgctctatgtagacgcaagaagatgttggagcagcagactagatgcaaaaaaaatggcaggtcgaaaagcaactgcg

[0240] acggttgctccgtcatcctctcatcgccttttattgctccggcgttgggaaccgcaacaatggaacagcccaaatcgacagt

[0241] cccctccacccccctcccccatcctctctccccccacgcaatacttgtcactactcgcgctgcccactacagcgtctctgcat

[0242] gtatatccatctatccatccattcccccattttccaaataaaaaatacagcaaaccaaacacaaacgcagcctcgcactgtactc

[0243] gaaaaaatcggtgctgtacgtactacgccacgagataacgagagagagagagagagagagagagagagagagagaaaatgga

[0244] aatgctactgctcgtaccacgccgctacgtccgctaggtcgacaggcccgggggaggcaggtgtttgtcgtctagctcg

[0245] ggtcggagcgcgccttctcgtgtcgggctcgacgtccgcgactcctcgcccctggtcgagagctcgcaggcgcagcggg

[0246] agagagagagagagagagagagagagagccgcgcaataaaagggcgcgcgcgcgagcgagcgaagcaaagc

[0247] accattactaaagaccgcggcgtgtgcttgcgttgcgagcgagcgagagcgagagagagattgagagagagagagg

[0248] agggatggcgatgccgtatgcctccctgtctccggcggtggccgaccaccgctcgtccccggcagccgcgaccgcctcc

[0249] ctcctccccttctgccgctccaccccgctctccgcgtaagcaacgcgaacccgcggctacaacccattttcttggctccagt

[0250] ggtgcatgtgacaacacggtgagacgttgtgtgtgggtgggtgggtgcaggggcggtggtggcgtcgcgatgggggag

[0251] gacgcgccgatgaccgcgaggtggccgccggcggcggcggcgaggctgccgccgttcaccgcggcgcagtacgag

[0252] gagctggagcagcaggcgctcatatacaagtacctggtggcaggcgtgcccgtcccgccggatctcgtgctccccatccg

[0253] ccgcggactcgactccctcgccgcccgcttctacaaccatcccgcccgtacgtcgtgttcctatttcttgcctctcctctacca

[0254] tcgctgcattgcttttggatgcttgtttagtgtcggcttctttgtttattccgatcaggcgtactttgcttccatttgttaattggctcc

[0255] gggtcatttgttaatccgggttacgcgattcaagaaacatgcgtgtgtgtttttatgctatcctccggatttggtaataaaaaggc

[0256] ttgtttttaaatccaaaactcgtgctcgcttcacgattagcgcatcattttttttttttggggggggggggggggaagtttgccca

[0257] tcattctgtctctgtttgatctgatagaggacgtgcacacgctcttgtctgaaataaaatcttttgtttatcagtatgcccatgggat

[0258] aagccattttctctgtgaaccaacaccctggcaaactgtttttttgctcgccatttttgagcgattgctaagaacagataactatg

[0259] ccctgcatatggatcggatatggacttctcaaatattcaaatgccattctattaggaactcaaaatgcattaccaacaaatgcat

[0260] tcttgtgtgtaacacggttgctacgatgtgcctgtttttgtacagttggatatggtccgtacttcggcaagaagctggacccaga

[0261] gccagggcggtgccggcgtacggacggcaagaaatggcggtgctcgaaggaggccgcgccggattccaagtactgcg

[0262] agcgccacatgcaccgcggccgcaaccgtaaaagaaagcctgtggaaacgcagctggtcgcccagtcccaaccgccct

[0263] catctgttgtcggttctgcggcggcgccccttgctgctgcctccaatggcagcagcttccaaaaccactctctttaccctgcta

[0264] ttgccggcagcaatggcgggggcggggggaggaacatgcccagctcatttggctcggcgttgggttctcagctgcacat

[0265] ggataatgctgccccttatgcagctgttggtggtggaacaggcaaagatctcaggtgattgttcatttctttttttttaatcaaac

[0266] gccatatttacttgtttagcactgtcttgaatcatgatatgtatccttccgttgtctaaaaaaaaggtgccatgctctaactgattg

[0267] gtgtcaggtggatgcagttatgaatctgtatttttcattgtgatcggttaataactgtgtcccatttgtttgcattggtggcaatcga

[0268] atcagctgtccatgctcagtagtactacttcgatttggtgctgcaatcactgaaagtctgaaactttactctctgcactgcaaaa

[0269] atttgtgttatgtttaggtttccagagtgctgcctctttgcccttcccatactttctggtatcagttttcagccccagaagccgggg

[0270] acagtctccataagagatttctgctcaggtgaaactggggtgcagggtcttaacatggctttggcccagtagtttgaaacatgt

[0271] actgtccataaagatgatactactacatatttgtgtctgccctcgcagtgcttgtgcctgctggtagctgatcatggcttcccttg

[0272] gcatttactccacttctttattcctccacagaatccagttgtttctgtctctgctcttcaggggcagtcaattatttggcccttgcaa

[0273] aatactgtctctgaagatgtctcaccgatcaccactatacctgaaacattttccagtggccagcgtgagctgcatgatgctcca

[0274] agtcaactctatactcatccaatgttgatgattagattttaacaatgcaactctttgatttatcttccctacaaaaaaaaaggaact

[0275] ctttgatttatcttcggtgaatctcagtctgaccttagtacctagcctcattatttacttcaccaaatgtataactctacagtgcttgt

[0276] tcgtgttgatttggtttagtttagttattgaattattcggtcaccttagtctttgattgtttttttctttctgctcttgtcatcaactgtttag

[0277] ggttcagctgacttgctgctgcaactaaactgtcttctggttttactgcaaatagaatgtttcttgggccatgatctgctgctata

[0278] tatgattagttaaaccatggttctatgttttcttatatgaattcatgacaagaatactaacttttggaaaaggtaattttattttttttgta

[0279] tgataataatgctttggattctttctagtttatctgtcggacttaggttaactacatttcctccggtacatggatttatttcattcttaca

[0280] attgagccccttatgaatattttcttcctaattctgttctaaaaagttagaattgacatattttcgataggtacatgcctagcacttgca

[0281] ttcgtgtttcctactaattcccaatcactgtatcttctcaaattcaggtatactgcttatggcacaagatctttggcggatgagcag

[0282] agtcaactcattactgaagctatcaacacatctattgaaaatccatggcggctgctgccatctcagaactcgccatttccccttt

[0283] caagctattctcagctgggggcactaagtgaccttggtcagaacacccccagctcactttcaaaggttcagaggcagccac

[0284] tttcgttctttgggaacgactatgcggctgtcgattctgtgaagcaagagaaccagacgctgcgtcccttctttgatgagtggc

[0285] caaagggaagggattcatggtcagacctcgctgatgagaatgctaatctttcgtcattctcaggcacccaactgtcgatctcc

[0286] ataccaatggcatcctctgacttctcggcggccagttctcgatcaactaatggtacgactacttgatctccccccaattacttcg

[0287] tgcgtgtttatgtctgtatcctgcaatgtctgaagatttcttactgaaaacgtcatctggtctgtgtgcaggtgactgaatgctgc

[0288] gtggatgatgatcctgctgcccagtgaactcatactggccttgttgctgtcttgctctgcgattttctgcctgctcgcgcccacc

[0289] gtacgatagtagcaaaacattctatgcttctgtaatttaccagtgttcccctgtcagatttgcgtgtgaaatcgatcaaactccgt

[0290] ggtctcctttggacgaagggagatgtcaacgttttccttgatgtttactgctagtaacatcttattacttcccaaatgctgatcag

[0291] cctctgcttgctatgctctcctgtttgtcgactcaacagtgccgtcaacatcagtgcagcaacgtgaggctcatgctttttaagg

[0292] tcattttcccgatgatgacagtgtcctactaaccagttccttttgcaagtggagaggctttgcttgcgatggccgatagatagat

[0293] agacaggtttcatctcttgaacgagccgacgagcgagcagcgcgcaaaacggacgagatggcccccatggctccttttcg

[0294] ccaaaaacacgatcgggcagcaggatccgtttgccttttgtcaactggagcccgtgacaagtaatgttcgttgccccagctc

[0295] cccacggcccctgcatgacatgggctccaccccctcgcacacccgccctccctttttatcattgcgagatagtagtagtgttg

[0296] atggtacgtagtactactaaccgacgacggcgacgtggccatgatgcacactaagggtgccttttgaatagtaggaatgaa

[0297] aaaaaaccaaagtaataagaaaaacataggattttgacagaaatataagtgtaaaacagggaaatacaaaacacaggaaa

[0298] aacataggaatgaccgtttgattgagccgcatgaaaaacacaaaggattcttacatagaaagaggcattctataagaatttca

[0299] taggattatatgggatttattcctttgattcaaagggatatatagaaaaatcctataagaatgaaatcctt

[0300] SEQ ID No.4, ROPD SLC cDNA sequence of

[0301] atggcgatgccgtatgcctccctgtctccggcggtggccgaccaccgctcgtccccggcagccgcgaccgcctccctcct

[0302] ccccttctgccgctccaccccgctctccgcgggcggtggtgttgtcgcgatgggggaggacgcgccgatgaccgcgagg

[0303] tggccgccggcggcggcggcgaggctgccgccgttcaccgcggcgcagtacgaggagctggagcagcaggcgctca

[0304] tatacaagtacctggtggcaggcgtgccccgtccccgccggatctcgtgctccccatccgccgcggactcgactccctcgcc

[0305] gcccgcttctacaaccatcccgcccttggatatggtccgtacttcggcaagaagctggacccagagccagggcggtgccg

[0306] gcgtacggacggcaagaaatggcggtgctcgaaggaggccgcgccggattccaagtactgcgagcgccacatgcacc

[0307] gcggccgcaaccgtaaaagaaagcctgtggaaacgcagctggtcgcccagtcccaaccgccctcatctgttgtcggttct

[0308] gcggcggcgccccttgctgctgcctccaatggcagcagcttccaaaaccactctctttaccctgctattgccggcagcaatg

[0309] gcgggggcggggggaggaacatgcccagctcatttggctcggcgttgggttctcagctgcacatggataatgctgcccct

[0310] tatgcagctgttggtggtggaacaggcaaagatctcaggtatactgcttatggcacaagatctttggcggatgagcagagtc

[0311] aactcattactgaagctatcaacacatctattgaaaatccatggcggctgctgccatctcagaactcgccatttcccctttcaag

[0312] ctattctcagcttggggcactaagtgaccttggtcagaacacccccagctcactttcaaaggttcagaggcagccactttcgt

[0313] tctttgggaacgactatgcggctgtcgattctgtgaagcaagagaaccagacgctgcgtcccttctttgatgagtggccaaa

[0314] gggaagggattcatggtcagacctcgctgatgagaatgctaatctttcgtcattctcaggcacccaactgtcgatctccatac

[0315] caatggcatcctctgacttctcggcggccagttctcgatcaactaatggtgactga

[0316] SEQ ID No.5, Amino acid sequence of ROPD (derived from 9311)

[0317] MAMPYASLSPAVADHRSSPAAATASLLPFCRSTPLSAGGGVVAMGEDAPMTAR

[0318] WPPAAAARLPPFTAAQYEELEQQALIYKYLVAGVPVPPDLVLPIRRGLDSLAA

[0319] RFYNHPALGHGPYFGKKLDPEPGRCRRTDGKKWRCSKEAAPDSKYCERHMH

[0320] RGRNRSRKPVETQLVAQSQPPSSVVGSAAAPLAAASNGSSFQNHSIYTAIAGS

[0321] NGGGGGRNMPSSFGSALGSQLHMDNAAPYAAVGGGTGKDLRYTAYGTRSLA

[0322] DEQSQLITEAINTSIENPWRLLPSQNSPFPLSSYSQLGALSDLGQNTPSSLSKVQ

[0323] RQPLSFFGNDYAAVDSVKQENQTLRPFFDEWPKGRDSWSDLADENANLSSFS

[0324] GTQLSISIPMASSDFSAASSRSTNGD

[0325] SEQ ID No.6, ROPD SLC Amino acid sequence (derived from SLC)

[0326] MAMPYASLSPAVADHRSSPAAATASLLPFCRSTPLSAGGGVVAMGEDAPMTAR

[0327] WPPAAAARLPPFTAAQYEELEQQALIYKYLVAGVPVPPDLVLPIRRGLDSLAA

[0328] RFYNHPALGYGPYFGKKLDPEPGRCRRTDGKKWRCSKEAAPDSKYCERHMH

[0329] RGRNRKRKPVETQLVAQSQPPSSVVGSAAAPLAAASNGSSFQNHSLYPAIAGS

[0330] NGGGGGRNMPSSFGSALGSQLHMDNAAPYAAVGGGTGKDLRYTAYGTRSLA

[0331] DEQSQLITEAINTSIENPWRLLPSQNSPFPLSSYSQLGALSDLGQNTPSSLSKVQ

[0332] RQPLSFFGNDYAAVDSVKQENQTLRPFFDEWPKGRDSWSDLADENANLSSFS

[0333] GTQLSISIPMASSDFSAASSRSTNGD

[0334] SEQ ID NO.7, ROPD - CRISPR - F1

[0335] CTCATATACAAGTACCTGG

[0336] SEQ ID NO.8,ROPD-CRISPR-R1

[0337] CCAGGTACTTGTATATGAG

[0338] SEQ ID NO.9,ROPD-XbalI-F

[0339] GCTCTAGAATGGCGATGCCGTATGCCTCC

[0340] SEQ ID NO.10,ROPD-SalI-R

[0341] ACGCGTCGACTCAGTCACCATTAGTTGATC

[0342] SEQ ID NO.11,ROPD SLC -XbalI-F

[0343] GCTCTAGAATGGCGATGCCGTATGCCTCC

[0344] SEQ ID NO.12,ROPD SLC -SalI-R

[0345] ACGCGTCGACTCAGTCACCATTAGTTGATC

[0346] SEQ ID NO.13,pROPD SLC ::ROPD SLC -XbalI-F

[0347] GCTCTAGAGACGAGTGGTCAAACGTTAC

[0348] SEQ ID NO.14,pROPD SLC ::ROPD SLC -SalI-R

[0349] ACGCGTCGACTCAGTCACCTGCACACAG

[0350] SEQ ID NO.15,OsPHT1;1-F

[0351] ATGTTTGCAATGTGCTGATGGC

[0352] SEQ ID NO.16,OsPHT1;1-R

[0353] GGCTTCCCAACTCTTTGAGC

[0354] SEQ ID NO.17,OsPHT1;8-F

[0355] CCTTCACAGTTGTCTGCACC

[0356] SEQ ID NO.18,OsPHT1;8-R

[0357] GGCTGAACGACTCTGCTCG

[0358] SEQ ID NO.19,OsWRKY74-F

[0359] CTCTCTCCGACGTAAGGGTT

[0360] SEQ ID NO.20,OsWRKY74-R

[0361] CACCCCTCAACCCTTGATCG

[0362] SEQ ID NO.21,OsSPDT-F

[0363] GCCAATGGCCTCTAATATCACAT

[0364] SEQ ID NO.22,OsSPDT-R

[0365] GGCGATCGAGTCGAGAGGGAGGA

Claims

1. The gene ROPD (REGULATOR OF PHOSPHORUS UPTAKE AND DISTRIBUTION) or its allele that improves phosphorus fertilizer utilization efficiency, yield, and seed nutritional quality, and the amino acid sequence of the protein encoded by it is shown as any one of the following: 1) The amino acid sequences shown in SEQ ID NO.5 and 6; 2) An amino acid sequence that is different from the amino acid sequences shown in SEQ ID NO.5 and 6 due to substitution, deletion, and / or insertion of one or more amino acid residues, but has the same activity as the protein composed of the amino acid sequences shown in SEQ ID NO.5 and 6; 3) An amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 90% identity with the amino acid sequences shown in SEQ ID NO.5 and 6, and has the same activity as the protein composed of the amino acid sequences shown in SEQ ID NO.5 and 6; or 4) An active fragment containing any one of the amino acid sequences described in 1)-3).

2. The gene according to claim 1, and its nucleotide sequence is shown as any one of the following: 1) The nucleotide sequences shown in SEQ ID NO.1, 2, 3, and 4; 2) A nucleotide sequence that is different from the nucleotide sequences shown in SEQ ID NO.1, 2, 3, and 4 due to substitution, deletion, and / or insertion of one or more nucleotide sequences, but has the same activity as the protein encoded by the nucleotide sequences shown in SEQ ID NO.1, 2, 3, and 4; 3) A nucleotide sequence that has at least 70%, preferably at least 80%, more preferably at least 90% identity with the nucleotide sequences shown in SEQ ID NO.1, 2, 3, and 4, and has the same activity as the protein encoded by the nucleotide sequence shown in SEQ ID NO.1; 4) A nucleotide sequence that is different from SEQ ID NO.1, 2, 3, and 4 in sequence due to the degeneracy of the genetic code; 5) An active fragment containing any one of the nucleotide sequences described in 1)-4); 6) A nucleotide sequence that contains a complementary sequence of any one of the nucleotide sequences described in 1)-5) and hybridizes under medium stringency conditions, preferably high stringency hybridization conditions; or 7) A nucleotide sequence that contains a complementary sequence of any one of the nucleotide sequences described in 1)-5).

3. A recombinant construct containing the ROPD gene or its allele according to claim 1 or 2.

4. A host cell containing the ROPD gene or its allele according to claim 1 or 2 or the recombinant construct according to claim 3, such as a microbial cell, preferably an Escherichia coli cell or an Agrobacterium cell.

5. A method for cultivating a crop with high phosphorus fertilizer utilization efficiency, high yield, and / or low phytic acid content in seeds, the method comprising increasing the expression level of the gene ROPD or its allele that improves phosphorus fertilizer utilization efficiency, yield, and seed nutritional quality in the crop, preferably the ROPD gene or its allele according to claim 1 or 2.

6. The method according to claim 5, wherein the method comprises transfecting the recombinant construct according to claim 3 or the host cell according to claim 4 into crop cells to obtain a transgenic crop plant, such that the expression level of the ROPD gene or its allele in the transgenic crop is increased, thereby obtaining a crop with high phosphorus fertilizer utilization rate, high yield and / or low phytic acid content in seeds, wherein the crop is a monocotyledonous crop, preferably rice, wheat or maize.

7. The method according to claim 5, wherein the method comprises: Hybridizing a crop plant containing the ROPD gene or its allele according to claim 1 or 2 with another plant of the same crop to obtain a hybrid crop plant, such that the expression level of the ROPD gene or its allele in the hybrid crop is increased, thereby obtaining a crop with high phosphorus fertilizer utilization rate, high yield and / or low phytic acid content in seeds, wherein the crop is a monocotyledonous crop, preferably rice, wheat or maize.

8. A method for cultivating a crop having high phosphorus fertilizer utilization efficiency, high yield and / or low phytic acid content in seeds, wherein the method comprises: Co-expressing the ROPD gene or its allele according to claim 1 or 2 and the dep1-1 gene in a crop plant or over-expressing the ROPD gene or its allele according to claim 1 or 2 in a crop plant carrying the dep1-1 gene to obtain a crop with high phosphorus fertilizer utilization rate, high yield and / or low phytic acid content in seeds, wherein the crop is a monocotyledonous crop, preferably rice.

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