An OsSULTR3 is selected; protein 1 and application of coding gene thereof in cultivation of selenium-rich plants

Abstract

The invention relates to the technical field of gene engineering, in particular to OsSULTR3; 1 protein and the coding gene thereof are applied to cultivation of selenium-rich plants. According to the invention, the OsSULTR3 is disclosed for the first time; 1 gene has the function of increasing the selenium content of rice, and OsSULTR3; 1 overexpression results in increase of the selenium content of rice grains. A new direction is provided for breeding of selenium-rich plants such as rice and the like and preparation of transgenic plants, the selenium content in the plants such as rice and the like can be remarkably improved by constructing and transforming the transgenic plants of the gene, the nutritional quality of the plants can be improved, and the gene has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to the application of OsSULTR3;1 protein and its encoding gene in cultivating selenium-rich plants. Background Art

[0002] Rice ( Oryza sativa ) is an important source of selenium (Se) in the global plant-based diet. Selenium in rice plays a key role in human nutrition and growth and development, especially in developing countries. Studies have shown that human selenium deficiency is closely related to a variety of health problems. Selenium concentrations of 0.05–0.10 ppm in feed or food constitute the minimum threshold required for animal and human nutrition, respectively. When the selenium content in food is lower than 0.05–0.10 ppm, severe selenium deficiency will result; while the intake of 2-5 ppm or higher concentrations of selenium will cause selenium poisoning. The cultivation of selenium-rich crops provides an economical and effective way to fortify selenium. Biofortification of food crops such as rice through the application of selenium fertilizers or genetic improvement is an effective strategy to increase the selenium intake level of specific populations.

[0003] Selenium (Se) and sulfur (S) both belong to Group VIA of the periodic table. They share similar biochemical pathways for plant root uptake, intertissue transport, and metabolism. Selenium available for plant uptake in soil primarily exists as inorganic forms, selenate and selenite, whose abundance is regulated by redox potential and pH. In aerobic, moderately alkaline environments, selenate is chemically similar to sulfate, and plants primarily absorb selenium as selenate via the sulfate transporter (SULTR). Selenium and molybdenum are also absorbed via sulfate transporters, with transport rates dependent on transporter expression levels and sulfate bioavailability. However, under moderate redox potentials (particularly acidic environments such as flooding), selenate can be reduced to selenite, elemental selenium, and even selenides. Selenite exists in aqueous solutions as H2SeO3, SeO3²⁻, and HSeO3⁻, and their relative proportions vary significantly with pH. Rice roots may absorb selenite in the form of neutral H2SeO3 through aquaporins. The absorption of selenite, the main form of selenium in paddy soil, is mediated by the silicon influx transporter Lsi1 (OsNIP2;1), which is permeable to H2SeO3. Another study showed that phosphate transporters OsPT2Knockout and overexpression of significantly reduced and increased the absorption rate of selenite and selenium in rice grains, respectively. Sulfate transporters (SULTRs) belong to the SulP gene family, which contains 12 members in rice and is responsible for regulating transmembrane sulfate transport. SULTRs contain 12 transmembrane domains, and their C-terminus contains a sulfate transport anti-sigma factor domain (STAS). Among them, OsSULTR1;1 、 OsSULTR1;2 and OsSULTR2;1 It is highly expressed in the roots of rice seedlings. Therefore, discovering genes that can regulate selenium in plants has become a technical problem that needs to be solved urgently in this field. Summary of the Invention

[0004] In order to solve the above technical problems, in a first aspect, the present invention provides the use of OsSULTR3;1 protein or a fusion protein of OsSULTR3;1 protein, or a gene encoding the same, or a biological material containing the gene encoding the same, in regulating the selenium content in plants.

[0005] In a second aspect, the present invention provides the use of OsSULTR3;1 protein or a fusion protein of OsSULTR3;1 protein, or a gene encoding the same, or a biological material containing the gene encoding the same, in cultivating new varieties of selenium-enriched plants.

[0006] In a third aspect, the present invention provides the use of OsSULTR3;1 protein or a fusion protein of OsSULTR3;1 protein, or a gene encoding the same, or a biological material containing the gene encoding the same, in plant variety improvement.

[0007] In a fourth aspect, the present invention provides the use of OsSULTR3;1 protein or a fusion protein of OsSULTR3;1 protein, or a gene encoding the same, or a biological material containing the gene encoding the same, in preparing transgenic plants.

[0008] In a specific implementation process, the method of preparing transgenic plants includes but is not limited to transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0009] Preferably, the amino acid sequence of the OsSULTR3;1 protein is at least one of the following: (1) Sequence shown in SEQ ID No. 3; (2) A sequence having the same biological function as the sequence shown in SEQ ID NO. 3 after one or more amino acid residues are substituted, deleted, and / or added.

[0010] Preferably, the nucleotide sequence encoding the gene is at least one of the following: (1) Sequence shown in SEQ ID No. 2; (2) A nucleotide sequence formed by one to several base substitutions and / or one to several base insertions and / or deletions, or insertions / deletions / shifts / inversions of a large fragment of nucleotide sequence based on SEQ ID NO. 2; (3) A nucleotide sequence that can hybridize with the sequence shown in SEQ ID NO. 2 in a 0.1× SSPE (or 0.1× SSC) and 0.1% (w / v) SDS solution at 65°C and the membrane is washed; (4) A nucleotide sequence having more than 85% homology with the sequence shown in SEQ ID NO.2.

[0011] In a specific implementation process, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or non-renewable plant cells or tissues.

[0012] Preferably, the engineered bacteria is Agrobacterium or Escherichia coli.

[0013] In a specific implementation process, the selenium content in plants is increased by increasing the expression level or activity of OsSULTR3;1 protein or its encoding gene.

[0014] In a specific implementation process, the plant is a grass plant; preferably a rice plant; more preferably rice.

[0015] In a fifth aspect, the present invention provides a method for increasing the selenium content in plants, comprising: introducing the OsSULTR3;1 protein or a fusion protein of the OsSULTR3;1 protein, or a gene encoding the OsSULTR3;1 protein, or a biological material containing a gene encoding the OsSULTR3;1 protein, into the plant.

[0016] Preferably, the OsSULTR3;1 protein or a fusion protein of the OsSULTR3;1 protein, or a gene encoding the OsSULTR3;1 protein, or a biological material containing a gene encoding the OsSULTR3;1 protein, is introduced into the plant by genetic engineering.

[0017] Preferably, the OsSULTR3;1 protein or its encoding gene is overexpressed in the plant by genetic engineering means.

[0018] In a specific implementation process, the overexpression method is selected from one or more combinations of (1) to (5): (1) by introducing an expression vector containing the gene; (2) by increasing the copy number of the gene on the plant chromosome; (3) by changing the promoter sequence of the gene on the plant chromosome; (4) by operably linking a strong promoter to the gene; (5) By introducing enhancers.

[0019] In a specific implementation process, the expression vector carrying the gene can be introduced into plant cells by conventional biotechnology methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, etc.

[0020] In a specific implementation process, the fusion protein is an amino acid sequence obtained by connecting a tag, an enzyme cleavage site and / or a connecting peptide sequence to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID No. 3, and the fusion protein has the same biological function as the amino acid sequence shown in SEQ ID No. 3.

[0021] In the art, substitution with amino acids with similar or close properties usually does not change the function of the protein; adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time OsSULTR3;1 The gene has the function of increasing the selenium content of rice. OsSULTR3;1 Overexpression leads to an increase in the selenium content of rice grains. The present invention provides a new direction for the breeding of selenium-rich plants such as rice and the preparation of transgenic plants. By constructing transgenic plants transformed with the gene, the selenium content in plants such as rice can be significantly improved, which is beneficial for improving the nutritional quality of plants and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For rice SULTR Analysis of tissue expression patterns of each gene family member at different developmental stages.

[0024] Figure 2 for OsSULTR3;1 Analysis of gene expression patterns in various tissues.

[0025] Figure 3 for OsSULTR3;1 Gene expression patterns under selenate and sulfate treatments; A represents the expression pattern in roots, and B represents the expression pattern in aerial parts.

[0026] Figure 4 for OsSULTR3;1 and OsLsi1 Yeast transformation vector map.

[0027] Figure 5 for OsSULTR3;1Detection of selenium transport capacity in yeast; A is the result of 0 μM selenate treatment, B is the result of 2 μM selenate treatment, C is the result of 20 μM selenate treatment, D is the result of 0 μM selenite treatment, E is the result of 2 μM selenite treatment, and F is the result of 20 μM selenite treatment.

[0028] Figure 6 For CRISPR / Cas9: OsSULTR3;1 Schematic diagram of the vector structure.

[0029] Figure 7 For pCM1305UPFHN: OsSULTR3;1 Schematic diagram of the vector structure.

[0030] Figure 8 Knockout and overexpression OsSULTR3;1 Results of changes in selenium content in rice after gene modification; where A is a schematic diagram of the gene structure, B is the sequencing result, C is the gene expression level of the three overexpression lines, D is the selenium content in the roots of the different lines treated with 2 μM selenate, E is the selenium content in the aboveground parts of the different lines treated with 2 μM selenate, F is the selenium content in the roots of the different lines treated with 2 μM selenite, G is the selenium content in the aboveground parts of the different lines treated with 2 μM selenite, and H is the selenium content in the grains of the different lines under field growth conditions. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, those without specifying specific techniques or conditions are described in accordance with the techniques or conditions described in the literature in this area, or are carried out according to product specifications. Reagents or instruments used are not specified by manufacturer and are conventional products that can be purchased through regular channels.

[0032] The present invention relates to molecular biology experiments. Unless otherwise noted, reference can be made to the book Molecular Cloning (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used reference books for those skilled in the art when conducting molecular biology experiments. Furthermore, depending on the purpose of the experiment, those skilled in the art can perform the corresponding experiments under the guidance of the operating manuals provided with various commercial kits or outsource them to specialized companies, such as those for gene sequencing, plasmid sequencing, and molecular weight determination.

[0033] Example 1 SULTRs Expression analysis of gene family members at different growth stages The present invention has a great influence on rice SULTR Systematic RT-qPCR analysis was performed on the tissue-specific expression patterns of each gene family member at 3 weeks (seedling stage), 12 weeks (reproductive growth stage), and 16 weeks (grain filling stage) after transplanting (primers are shown in Table 1). SULTR All genes were detected to be expressed, and their spatiotemporal expression patterns showed significant differences. OsSULTR3;1 It showed consistently high expression levels in all tested tissues (roots, leaf sheaths, leaves, and grains) and at all developmental stages, and was a SULTR The only member of the family that is significantly expressed in kernels ( Figure 1 ). Based on its specific high expression characteristics in grains, OsSULTR3;1 ( LOC_Os10g28440 ) can serve as an important candidate gene for the research on selenium biofortification of rice grains. OsSULTR3;1 The gDNA is shown as SEQ ID No. 1, the CDS sequence is shown as SEQ ID No. 2, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 3.

[0034] Table 1 SULTR Gene family RT-qPCR detection primers

[0035] Example 2 OsSULTR 3;1 Analysis of gene tissue expression patterns based on OsSULTR3;1 The high expression characteristics in grains have been listed as key candidate genes for selenium biofortification of rice grains in this paper and further analyzed. RNA was extracted from different tissue samples of wild type (Zhonghua 11) at the seedling, tillering, booting, flowering and filling stages, and the RT-qPCR technology was used to systematically analyze the OsSULTR3;1 Expression characteristics in different tissues. The results showed that the gene was continuously highly expressed in leaves, sheaths and roots throughout the growth period; during the flowering and filling stages, its expression level in nodes, internodes, spikelets and grains was also significantly increased ( Figure 2 ).

[0036] Example 3 OsSULTR3;1 Differential response characteristics of genes to sulfur / selenium treatments The present invention further analyzes rice OsSULTR3;1 Differential expression patterns of genes in roots under low-selenium, high-selenium, low-sulfur and high-sulfur treatments.

[0037] The specific processing methods are as follows: Seeds of the japonica rice variety Zhonghua 11 (ZH11) were soaked in tap water and incubated in the dark at 37°C for 3 days to germinate. Germinated seeds were then transplanted to a sulfur-deficient liquid medium and grown hydroponically for 12 days. Following incubation, seedling roots were treated with medium containing 2 μM Na₂SeO₃ (low selenium), 20 μM Na₂SeO₃ (high selenium), 10 μM Na₂SO₄ (low sulfate), and 1500 μM Na₂SO₄ (high sulfate). Root and aerial samples were collected at 0, 3, 6, 9, 12, and 24 hours after treatment for RNA extraction and reverse transcription and RT-qPCR analysis.

[0038] The results showed that in rice roots, OsSULTR3;1 It is sensitive to selenium and is strongly induced under low selenium (RL-Na2SeO3) and high selenium (RH-Na2SeO3) conditions. OsSULTR3;1 In roots, the response to selenium was significantly higher than that to sulfur ( Figure 3 A). In the aboveground tissues, OsSULTR3;1 It was also significantly activated under low selenium (SL-Na2SeO3) and high selenium (SH-Na2SeO3) conditions. OsSULTR3;1 In the aboveground tissues, a selenium-specific induction pattern was observed, while there was little response to sulfur ( Figure 3 B), which is highly consistent with its characteristic of preferentially responding to selenium in roots. This result confirms the potential core role of this gene in selenium absorption and transport in rice.

[0039] Example 4 Detection of the transport capacity of OsSULTR3;1 for selenate and selenite using a yeast system In order to directly verify the selenium transport activity of SULTR3;1, the present invention heterologously expressed in yeast system OsSULTR3;1 The gene was expressed in 2477 cells, and OsLsi1, which has been reported to have selenite transport ability, was used as a positive control. The empty vector pYES2C-NT, the recombinant vector pYES2C-NT-OsSULTR3;1 and the pYES2C-NT-OsLsi1 vector ( Figure 4 ) were treated with 0 μM, 2 μM and 20 μM selenate or selenite solution for 30 minutes (primers see Table 2), and then the selenium content in yeast cells was measured. The results showed that compared with the empty vector control, OsSULTR3;1 and OsLsi1 The expression of selenate and selenite can significantly promote the absorption of selenate and selenite at concentrations of 2 μM and 20 μM in yeast ( Figure 5 The results confirmed that SULTR3;1 has the dual function of transporting selenate and selenite simultaneously in the yeast system.

[0040] Table 2 Yeast vector construction primers

[0041] Example 5 Overexpression OsSULTR3;1 Can significantly increase the selenium content of rice To further explore how to use OsSULTR3;1 Improve the absorption function of selenium in rice. The present invention uses CRISPR-Cas9 gene editing technology (vector map as shown in Figure 6 Three genomic loci were constructed in the japonica rice variety Zhonghua 11 (ZH11). OsSULTR3;1 Knockout line (B643). In addition, the maize ubiquitin protein 1 ( Ubiquitin 1 ) promoter drive OsSULTR3;1 Gene construction overexpression vector ( Figure 7 ) and obtained three independent overexpression lines (B679, primers see Table 3).

[0042] Table 3 Primers for constructing gene knockout and overexpression vectors

[0043] Sequencing analysis showed that all three knockout strains had base deletions or insertions, resulting in a frameshift mutation in the OsSULTR3;1 protein ( Figure 8 AB); RT-qPCR analysis confirmed that the gene expression levels of the three overexpression lines were significantly higher than those of the wild type ( Figure 8 C). Knockout lines (B643-11, B643-12, B643-15) and overexpression lines (B697-10, B697-13, B697-19) were placed in 2 μM selenate ( Figure 8 DE) or selenite ( Figure 8 After 24 hours of treatment with FG solution, the selenium content in the roots and aerial tissues of the knockout and overexpression lines was measured and compared. The results showed that the selenium content in the roots, aerial tissues and grains of the overexpression line was significantly higher than that of the wild type, regardless of whether it was treated with selenate or selenite, while the knockout line was the opposite ( Figure 8 We further measured the selenium content of rice grains under field growth conditions in Sanya, Hainan (see Table 4 for soil physical and chemical properties) and found that the selenium content in the grains of the overexpression lines was significantly higher than that of the wild type ( Figure 8 H). These results demonstrate that overexpression OsSULTR3;1 It can significantly increase the selenium content in rice grains.

[0044] Table 4 Contents of various elements in the soil of Hainan experimental field

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of OsSULTR3;1 protein or a fusion protein of OsSULTR3;1 protein, or a gene encoding the same, or a biological material containing the gene encoding the same, in regulating the selenium content in plants.

2. Application of OsSULTR3;1 protein or a fusion protein of OsSULTR3;1 protein, or a gene encoding the same, or a biological material containing the gene encoding the same, in the cultivation of new selenium-enriched plant varieties.

3. Use of OsSULTR3;1 protein or a fusion protein of OsSULTR3;1 protein, or a gene encoding the same, or a biological material containing the gene encoding the same, in plant variety improvement.

4. Use of OsSULTR3;1 protein or a fusion protein of OsSULTR3;1 protein, or a gene encoding the same, or a biological material containing the gene encoding the same, in preparing transgenic plants.

5. The use according to any one of claims 1 to 4, characterized in that The amino acid sequence of the OsSULTR3;1 protein is at least one of the following: (1) Sequence shown in SEQ ID No. 3; (2) A sequence having the same biological function as the sequence shown in SEQ ID NO. 3 after one or more amino acid residues are substituted, deleted, and / or added.

6. The use according to any one of claims 1 to 4, characterized in that The nucleotide sequence encoding the gene is at least one of the following: (1) Sequence shown in SEQ ID No. 2; (2) A nucleotide sequence formed by one to several base substitutions and / or one to several base insertions and / or deletions, or insertions / deletions / shifts / inversions of a large fragment of nucleotide sequence based on SEQ ID NO. 2; (3) A nucleotide sequence that can hybridize with the sequence shown in SEQ ID NO. 2 in a 0.1× SSPE (or 0.1× SSC) and 0.1% (w / v) SDS solution at 65°C and the membrane is washed; (4) A nucleotide sequence having more than 85% homology with the sequence shown in SEQ ID NO.

2.

7. The use according to any one of claims 1 to 4, characterized in that The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector, engineered bacteria or non-renewable plant cells or tissues.

8. The use according to any one of claims 1 to 4, characterized in that The selenium content in plants is increased by increasing the expression level or activity of OsSULTR3;1 protein or its encoding gene.

9. The use according to any one of claims 1 to 4, characterized in that The plant is a grass plant; preferably a rice plant; more preferably rice.

10. A method for increasing the selenium content in plants, characterized in that: include: The OsSULTR3;1 protein or a fusion protein of the OsSULTR3;1 protein according to claim 1, or a gene encoding the OsSULTR3;1 protein or a biological material containing a gene encoding the OsSULTR3;1 protein according to claim 1 is introduced into a plant.

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