Application of rice OsPHR2 gene in regulation and control of selenium bioaugmentation

Abstract

The invention discloses an application of a rice OsPHR2 gene in regulation and control of selenium bioaugmentation. The rice OsPHR2 gene is shown as SEQ ID No: 1 in a sequence table, and the amino acid sequence of the protein of the rice OsPHR2 gene is shown as SEQ ID No: 2. According to the invention, the promoter of the rice OsPHR2 is adopted to drive the rice OsPHR2 to increase one to two copy numbers in the rice body, so that the content of phosphorus in the rice body is increased without influencing the growth, development and yield of the rice by means of moderately increasing the transcription expression of the rice OsPHR2, meanwhile, the absorption and accumulation of selenium can be promoted, and the biological enrichment enhancement of rice selenium is realized.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and genetic engineering, and specifically relates to rice. OsPHR2 Application of genes in regulating selenium biofortification. Background Technology

[0002] Selenium is an essential trace element for the human body and a core component of glutathione peroxidase. It plays a vital role in enhancing immunity, delaying aging, reducing cancer incidence, and improving thyroid function. In the body, selenium primarily functions by forming specific compounds through organic binding. Insufficient selenium intake can impair the full expression of organic selenoproteins, preventing them from functioning properly. Approximately one billion people worldwide suffer from selenium deficiency. Therefore, increasing selenium intake is crucial for preventing various diseases and combating hidden hunger. The human body mainly obtains selenium from food, with plant-based foods, especially grains, being the primary source. Rice is the staple food for more than half of the world's population. According to the World Health Organization's recommended daily selenium intake of 50-60 micrograms for adults, approximately 75% of rice globally provides only about 70% of the recommended daily intake. Therefore, increasing the selenium content of rice is essential for improving overall selenium intake.

[0003] Under normal circumstances, the selenium content of rice depends on the selenium level of the soil in which the rice grows. Large areas of the world have selenium-deficient or low-selenium soils, making it difficult for rice to absorb and accumulate significant amounts of selenium from these soils. Therefore, applying selenium is a necessary means to increase the selenium content of rice. Although applying selenium can significantly increase the selenium content of rice, the efficiency of selenium utilization by rice is generally low. This not only leads to increased selenium application in production, wasting selenium resources, but also increases production costs and brings potential local environmental risks. Currently, efforts to improve the selenium level of plants mainly focus on methods such as exogenous foliar application of selenium fertilizers, while research on regulating the selenium content within plants through genetic modification is relatively lacking. Therefore, regulating the ability of rice grains to accumulate selenium through genetic modification is one of the key breeding directions for improving the selenium nutrient level of rice and alleviating the hidden hunger of the world's population. Summary of the Invention

[0004] The purpose of this invention is to provide a rice polypeptide encoding gene. PSIP1 And its application in regulating phosphorus nutrient accumulation.

[0005] rice OsPHR2 Genes, the ones mentioned OsPHR2 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1 of the sequence listing; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of enhancing selenium enrichment in rice. (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of improving selenium enrichment in rice.

[0006] Rice OsPHR2 protein, wherein the amino acid sequence of the OsPHR2 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of improving selenium enrichment in rice.

[0007] Containing the rice OsPHR2 The carrier of genes.

[0008] Containing the rice OsPHR2 Engineered bacteria with gene vectors.

[0009] Amplification of the rice OsPHR2 Primers for any segment of a gene.

[0010] The rice OsPHR2 Application of genes in enhancing selenium enrichment in rice.

[0011] A method to enhance selenium enrichment in rice by increasing the expression level of OsPHR2 protein in rice.

[0012] Beneficial effects of the present invention: The present invention uses rice OsPHR2 The promoter drives the increase of one to two copies in rice to moderately increase its transcriptional expression, thereby increasing the phosphorus content in rice without affecting its growth, development and yield. At the same time, it can also promote the absorption and accumulation of selenium, thus achieving biofortification of rice selenium. Attached Figure Description

[0013] Figure 1 for OsPHR2-Pro-gPHR2 strains OsPHR2 Expression level detection.

[0014] Figure 2 for OsPHR2-Pro-gPHR2 Yield of the strain; In the diagram, A represents NIP and OsPHR2-Pro-gPHR2 Phenotypic results of strains grown outdoors, B represents NIP and OsPHR2-Pro- gPHR2 Yield phenotype of the strain, C for NIP and OsPHR2-Pro-gPHR2 Yield statistics for strains, where D represents NIP and OsPHR2- Pro-gPHR2 Statistical analysis of the effective tiller number of the strain, where E represents NIP and OsPHR2-Pro-gPHR2 Plant height statistics of the strains.

[0015] Figure 3 for OsPHR2-Pro-gPHR2 Nutrient content of the plant; In the diagram, A represents NIP and OsPHR2-Pro-gPHR2 Total phosphorus content of the strain, B is NIP and OsPHR2-Pro-gPHR2 Total selenium content of the strain. Detailed Implementation

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

[0017] The experimental materials used in the following examples are: rice material: NIP; strain: Escherichia coli strain. DH5α Agrobacterium strains EHA105 Plant expression vectors: pBI101 .

[0018] Example 1 Rice OsPHR2 Preparation of plants by increasing gene copy number driven by self-promoter. pBI101-PHR2-Pro-PHR2-genome Vector construction: Using genomic DNA from the aboveground parts of rice NIP grown in hydroponics as a template, PCR amplification was performed using the upstream primer 5'-CCATGATTACGAATTCTGAACTCGGCGGCCTCCA-3' and the downstream primer 5'-GCTTGATATCGAATTCATGGATTCTCATGCCTGTTTGTTTG-3', and the 7800bp vector was purified. OsPHR2 Promoter (SEQ ID No: 3) and gene fragment (SEQ ID No: 4), used Kpn I Endonuclease (NEB) and Sal I Endonuclease (NEB) for pBI101 The vector is digested with enzymes, and the digestion products are recovered. In-Fusion (Novizan) is then used to... OsPHR2Recycled fragments are connected to linearized pBI101 The vector was used to obtain the recombinant vector. pBI101-PHR2-Pro-PHR2-genome (named) OsPHR2-Pro-gPHR2 ).

[0019] OsPHR2 Preparation of transgenic plants with moderately increased gene expression driven by their own promoters: using rice NIP as background material, and employing transgenic plants containing... OsPHR2-Pro-gPHR2 Agrobacterium vector EHA105 The strain infected rice to obtain T0 generation transgenic positive plants. OsPHR2-Pro-gPHR2 The T0 generation transgenic plants were self-crossed twice to obtain homozygous plants. OsPHR2-Pro- gPHR2 For rice materials, two homozygous lines, #1 and #2, were selected, and real-time quantitative PCR was performed using primer pairs consisting of upstream 5'-ACCGATGCCTCAGTGAGAT-3' and downstream 5'-GACAGAATTGTCTGAAGGTTCTT-3'.

[0020] Take rice NIP and OsPHR2-Pro-gPHR2 Real-time quantitative PCR was performed on the leaves of the strain, and the results are as follows: Figure 1 As shown, in the leaves compared to the wild type, OsPHR2-Pro-gPHR2-1 and OsPHR2-Pro-gPHR2-2 Among the plant strains OsPHR2 The relative expression levels of the genes increased by ~1-fold and ~2-fold, respectively, thus achieving... OsPHR2 Transgenic lines with moderately enhanced transcription levels.

[0021] Example 2 OsPHR2-Pro-gPHR2 The plant had no significant effect on major agronomic traits such as plant height and tiller yield. Experimental material treatment: rice NIP lines and OsPHR2-Pro-gPHR2 After the seedlings were grown hydroponically for two weeks, they were transplanted into outdoor pots treated with 10 μM sodium selenite. The rice was harvested after grain filling, and the yield was recorded. Rice yield is as follows: Figure 2 As shown, OsPHR2 The yield of the plant did not change significantly after adding one or two copies of the gene.

[0022] Example 3 OsPHR2-Pro-gPHR2 Increased phosphorus and selenium content in the grains of this strain Experimental material treatment: Harvested rice NIP lines and rice OsPHR2-Pro-gPHR2 After removing the husks from the seeds of the strain, 2.00 g of seeds were weighed, concentrated nitric acid and hydrogen peroxide were added, and the mixture was digested at 220℃ for 2 h. After adjusting the volume and filtering, the phosphorus and selenium content in the seeds was determined by ICP-MS.

[0023] The results are as follows Figure 3 As shown, OsPHR2 The phosphorus and selenium content in the grains of the multi-copy strains both increased.

[0024] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Rice OsPHR2 Genes, characterized by, The OsPHR2 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of enhancing selenium enrichment in rice. (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of improving selenium enrichment in rice.

2. Rice OsPHR2 protein, characterized in that, The amino acid sequence of the OsPHR2 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of improving selenium enrichment in rice.

3. Containing the rice described in claim 1 OsPHR2 The carrier of genes.

4. Containing the rice described in claim 3 OsPHR2 Engineered bacteria with gene vectors.

5. Expanding the rice according to claim 1 OsPHR2 Primers for any segment of a gene.

6. The rice according to claim 1 OsPHR2 Application of genes in enhancing selenium enrichment in rice.

7. A method for improving selenium enrichment in rice, characterized in that, Increase the expression level of OsPHR2 protein in rice.

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