Rice storage-tolerant and salt-tolerant gene ospskr3, and encoding protein and application thereof

Abstract

The application discloses a rice storage-tolerant and salt-tolerant gene OsPSKR3, and an encoding protein and application thereof. The gene OsPSKR3 is a DNA molecule as described in the following 1), 2), 3) or 4): 1) a DNA molecule shown in SEQ ID NO. 1; 2) a DNA molecule shown in SEQ ID NO. 2; 3) a DNA molecule hybridized with the DNA sequence defined in 1) or 2) under stringent conditions and encoding the protein; and 4) a DNA molecule having more than 90% homology with the DNA sequence defined in 1), 2) or 3) and encoding a plant sulfpeptin receptor. The application further provides a protein encoded by the gene, and the protein affects the storage tolerance of a plant seed. The encoding gene of the protein is overexpressed in a plant with normal storage tolerance, and a transgenic plant with enhanced salt tolerance can be bred. The protein and the encoding gene thereof can be applied to plant genetic improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a rice storage and salt tolerance gene OsPSKR3, its encoded protein, and its applications. Background Technology

[0002] Rice (Oryza sativa L.) is the staple food for over 50% of the world's population and is also a crucial food crop in my country, playing a vital role in the national economy. Every year, billions of kilograms of rice are lost due to mold, pests, and spoilage during storage. Discovering and utilizing storage-resistant genes can effectively improve seed storability and reduce grain losses caused by decreased vigor, which is of great significance to my country's rice agricultural production and food security.

[0003] The PSKR sulfopeptide receptor was first discovered in carrots at the beginning of this century. Overexpression of this enzyme in carrot cells accelerated callus growth in transgenic plants in the presence of PSK-α. In recent years, there has been considerable research on PSKR in Arabidopsis and tomato, but this has mainly focused on growth and development, ion receptors, fruit ripening and quality formation, and disease resistance regulation. In rice, only OsPSKR1 has been found to positively regulate bacterial leaf streak. However, there are no reports on the role of PSKR in regulating seed vigor and thus affecting storability. Therefore, new functional studies on PSKR will not only enrich our understanding of the regulation of plant seed vigor by the PSKR sulfopeptide receptor but also provide excellent candidate genes and breeding strategies for improving rice storability.

[0004] Researchers have found significant changes in seed vigor during rice storage in their long-term studies. However, it remains unclear what factors regulate seed vigor during this storage process, what interactions occur, and how the external environment affects the reduction of seed vigor. This requires further research at the molecular level into the aging mechanism of rice seeds. Summary of the Invention

[0005] The first object of the present invention is to provide an OsPSKR3 gene, said OsPSKR3 gene being selected from one of the DNA molecules shown in 1) or 2) or 3) or 4) below:

[0006] 1) The DNA molecule shown in SEQ ID NO.1 is the genomic sequence of the OsPSKR3 gene;

[0007] 2) The DNA molecule shown in SEQ ID NO.2 is the CDS sequence of the OsPSKR3 gene;

[0008] 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes the protein described in SEQ ID NO. 3;

[0009] 4) A DNA molecule that has more than 90% homology with the DNA sequence defined in 1), 2), or 3) and encodes a sulfopeptide receptor-associated protein.

[0010] A second objective of this invention is to provide the protein encoded by the aforementioned OsPSKR3 gene.

[0011] Furthermore, the protein encoded by the aforementioned OsPSKR3 gene is selected from either (a) or (b):

[0012] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.3;

[0013] (b) Proteins derived from SEQ ID NO.3 with substitution and / or deletion and / or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NO.3 that are related to storage tolerance.

[0014] A third objective of this invention is to provide a knockout vector for the OsPSKR3 gene, a recombinant expression vector containing the aforementioned OsPSKR3 gene, an expression cassette, or a recombinant bacterium.

[0015] Recombinant expression vectors containing the gene can be constructed using existing plant expression vectors.

[0016] The plant expression vectors include CRISPR-Cas9 knockout vectors, etc.

[0017] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by incorporating antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.) or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, for transgenic plants, no selective marker genes may be added, and transformed plants can be directly screened for resistance under adverse conditions.

[0018] Furthermore, the OsPSKR3 gene knockout vector was constructed using CRISPR-Cas9 technology. The OsPSKR3 knockout vector was named CRISPR-OsPSKR3.

[0019] Furthermore, the recombinant expression vector is a recombinant plasmid obtained by inserting the OsPSKR3 gene between the multiple cloning sites SmaI and SacI of the pGluC vector. The pGluc containing OsPSKR3 is named pGluc-OsPSKR3.

[0020] Primer pairs that knock out the gene (OsPSKR3) are also within the scope of protection of this invention, and the preferred primer pairs are Primer1 / Primer2 and Primer3 / Primer4.

[0021] Primer pairs that amplify the full length or any fragment of the gene (OsPSKR3) are also within the scope of protection of this invention, and the preferred primer pairs are Primer5 / Primer6 and Primer7 / Primer8.

[0022] A fourth objective of this invention is to provide the application of the aforementioned OsPSKR3 gene, or the aforementioned protein, or the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria in the cultivation of rice with storage tolerance.

[0023] Furthermore, the application involves overexpressing the OsPSKR3 gene and transferring it into wild-type rice to obtain rice with strong storage tolerance.

[0024] The fifth objective of this invention is to provide a method for cultivating transgenic rice with improved storage tolerance, wherein the OsPSKR3 gene shown in SEQ ID NO.1 or SEQ ID NO.2 in wild-type normal plants is overexpressed to obtain transgenic rice with improved storage tolerance.

[0025] The sixth object of the present invention is to provide the application of the aforementioned OsPSKR3 gene, or the aforementioned protein, or the aforementioned recombinant expression vector, expression cassette, or recombinant bacteria in the cultivation of salt-tolerant rice.

[0026] Furthermore, the application involves overexpressing the OsPSKR3 gene or introducing the aforementioned recombinant expression vector into wild-type rice.

[0027] Using any vector capable of guiding the expression of exogenous genes in plants, the gene encoding the stated protein can be introduced into plant cells to obtain transgenic cell lines and transgenic plants. The expression vector carrying the stated gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants. The plant host being transformed can be either monocotyledonous or dicotyledonous, such as tobacco, birdsfoot, Arabidopsis, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, and alfalfa.

[0028] Beneficial effects:

[0029] This invention is the first to discover that a sulfopeptide receptor OsPSKR3-related protein influences the storage tolerance process in plants. Increasing the expression of the gene encoding this protein leads to enhanced plant storage tolerance, thereby enabling the cultivation of transgenic plants with improved storage tolerance. Overexpression of the gene encoding this protein in plants can cultivate plants with strong salt tolerance. The protein and its encoding gene can be applied to plant genetic improvement. Attached Figure Description

[0030] Figure 1 The figures show the seed germination rate phenotypes of wild-type Ningjing 7 and PSKR3 knockout lines before and after artificial aging. The upper figure shows the seed germination rate phenotypes of wild-type Ningjing 7 and PSKR3 knockout lines before artificial aging, and the lower figure shows the seed germination rate phenotypes of wild-type Ningjing 7 and PSKR3 knockout lines after artificial aging.

[0031] Figure 2 Phenotypic of seed germination rate after natural storage for wild-type Ningjing 7 and pskr3 knockout lines.

[0032] Figure 3 Seed germination rates of wild-type Ningjing 7 and PSKR3 knockout lines before and after artificial aging were calculated. Figure 3 A represents the seed germination rate of wild-type Ningjing 7 and pskr3 knockout lines before artificial aging. Figure 3 B represents the seed germination rate of wild-type Ningjing 7 and pskr3 knockout lines after artificial aging.

[0033] Figure 4 This is the knockout mutation site for pskr3.

[0034] Figure 5 The images show the seed germination rate phenotypes of wild-type Ningjing 7 and PSKR3 overexpression families before and after artificial aging. The upper figure shows the seed germination rate phenotypes of wild-type Ningjing 7 and PSKR3 overexpression families before artificial aging, and the lower figure shows the seed germination rate phenotypes of wild-type Ningjing 7 and PSKR3 overexpression families after artificial aging.

[0035] Figure 6 The seed germination rates of wild-type Ningjing 7 and PSKR3 overexpression families before and after artificial aging were compared. Figure 6 A represents the seed germination rate of wild-type Ningjing 7 and PSKR3 overexpression families before artificial aging. Figure 6 B represents the seed germination rate of wild-type Ningjing 7 and PSKR3 overexpression families after artificial aging.

[0036] Figure 7 The seedling phenotypes of pGluC:OsPSKR3 T1 generation transgenic plants after salt treatment, among which, Figure 7A represents the seedling phenotype of the pGluC:OsPSKR3 T1 generation transgenic plant in the salt-treated control group (treated with 0 mM NaCl for 8 days). Figure 7 B represents the seedling phenotype of pGluC:OsPSKR3 T1 generation transgenic plants after treatment with 150mM NaCl for 8 days.

[0037] Figure 8 The survival rate of pGluC:OsPSKR3 T1 generation transgenic plants after salt treatment was measured. Figure 8 A represents the seedling survival rate of the pGluC:OsPSKR3 T1 generation transgenic plants in the salt-treated control group (treated with 0 mM NaCl for 8 days). Figure 8 B represents the seedling survival rate of pGluC:OsPSKR3 T1 generation transgenic plants after 8 days of treatment with 150mM NaCl. Detailed Implementation

[0038] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0039] Example 1: Obtaining and Identifying Transgenic Plants

[0040] I. Knockout Vector Construction

[0041] 1. Knockout Primer Design

[0042] (1) On the CRISPR-P website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), select Oryza sativa (RAP-DB) in the targetgenome option;

[0043] (2) Enter the gene accession number in the Locus Tag;

[0044] (3) Click submit to run the program and wait for the results to appear;

[0045] (4) Select primers that are located on the CDS near the ATG start site and have a high score to replicate the first 20 bp; perform blast analysis and select primers with high specificity.

[0046] (5) Primer synthesis: The back primer needs to be reverse complementary to the 20bp sequence before adding AAAC at the 5' end, while the front primer directly adds GGCA at the 5' end of the 20bp sequence;

[0047] After primer design and synthesis, the knockout vector CRISPR-OsPSKR3 was constructed.

[0048] The knockout primer sequences are as follows:

[0049] Primer1:

[0050] 5'CGGGGAATCTCTCTGCGGGT 3' (SEQ ID NO.4);

[0051] Primer2:

[0052] 5'AAACACCCGCAGAGAGATTC 3' (SEQ ID NO. 5);

[0053] Primer3:

[0054] 5'CACGCAGACAAGAAA 3' (SEQ ID NO. 6);

[0055] Primer4:

[0056] 5'GCAGGTTGGAGGAGA 3' (SEQ ID NO. 7);

[0057] 2. Knockout vector construction

[0058] (1) Add 1 μL each of Primer 1 and 2 stock solutions (100 μM) to a 200 μL PCR tube, and then add 8 μL of ddH2O; 95℃ for 5 min, and let it cool to room temperature naturally.

[0059] (2) Preparation of reaction system: ATP 1μL, pCAMBIA1305.1 carrier 1μL, buffer 1μL, oligo 0.2μL, AarⅠ 0.2μL, T4ase 0.2μL, ddH2O 5.5μL, (1) mixture 1μL;

[0060] (3) Reaction program: 37℃ for 5 min; 20℃ for 5 min; 4℃ for 30 min (1×10 cycles).

[0061] Transformation of competent *E. coli* cells: Add 10 μL of the ligation vector, gently pipette to mix, and incubate on ice for 30 min; incubate at 42°C for 45 s, then immediately incubate on ice for 2 min; add 700 μL of LB broth (antibiotic-free), and incubate at 37°C for 45 min using a shaker at 220 rpm; spread the bacterial culture evenly on LB broth (containing appropriate antibiotics), place in a clean bench for about 30 min, and after the culture medium has completely dried, invert it in a 37°C incubator for overnight incubation; after obtaining colonies, colony PCR detection is performed. The primers used are sgRNA-F: AAGGAATCTTTAAACATACGAACAGATC (SEQ ID NO.12) paired with Primer 2. Select 1-2 positive colonies for sequencing. After successful sequencing alignment, plasmid extraction is performed to obtain the OsPSKR3 gene knockout vector, named CRISPR-OsPSKR3.

[0062] II. Construction of Recombinant Overexpression Vectors

[0063] Using cDNA from Ninggeng 7 (from the rice germplasm resource bank of Nanjing Agricultural University) as a template, PCR amplification was performed using Primer5 / 6 to obtain the full-length CDS of the OsPSKR3 gene (SEQ ID NO.2).

[0064] The OsPSKR3 gene was obtained by PCR amplification. The PCR primer sequences are as follows:

[0065] Primer5:

[0066] 5'TTACTTCTGCACTAGGTACCATGGTGGTGCCGCCGCC 3" (SEQ ID NO. 8);

[0067] Primer 6:

[0068] 5'GAATTCCCGGGGATCCTACGCTGTCAAGCCAAGCA 3' (SEQ ID NO. 9).

[0069] The PCR product was recovered and purified. The product was cloned into the pGluC vector using the INFUSION recombination kit (Takara, Japan). The INFUSION recombination reaction system (10 μL) consisted of: 2.0 μL PCR product, 5.0 μL pGluC, 2.0 μL 5× infusion buffer, and 1 μL infusion enzyme mix. After brief centrifugation, the mixture was incubated at 37°C for 15 minutes, followed by incubation at 50°C for 15 minutes. 2.5 μL of the reaction mixture was then used to transform *E. coli* DH5α competent cells (Tiangen, Beijing; CB101) using the heat shock method. All transformed cells were evenly spread on LB agar containing 50 mg / L kanamycin. After incubation at 37°C for 16 hours, positive clones were picked and sequenced. Sequencing results showed that a recombinant overexpression vector containing the gene shown in SEQ ID NO.2 was obtained. The pGluC containing OsPSKR3 was named pGluC-OsPSKR3. The OsPSKR3 gene fragment was inserted between the SmaI and SacI restriction sites of the vector using the INFUSION recombination kit (Takara Corporation, Japan).

[0070] III. Obtaining Recombinant Agrobacterium

[0071] Agrobacterium strain EHA105 was transformed using the freeze-thaw transformation method. Commercial Agrobacterium competent (EHA105) was taken out and thawed on ice. 2-3 μL of transformation plasmids CRISPR-OsPSKR3 and pGluc-OsPSKR3 were added, and the mixture was thoroughly mixed by pipetting. The mixture was then rapidly frozen in liquid nitrogen for 5 min. Immediately afterward, it was heat-shocked in a 37℃ water bath for 5 min. The mixture was then removed and placed on ice, and 1 mL of antibiotic-free LB medium was added. The mixture was incubated at 28℃ for 3-4 h using a shaker. The bacterial culture was then evenly spread onto LB medium (containing the corresponding antibiotics), allowed to air dry naturally in a clean bench, and then incubated upside down in a 28℃ incubator for 2-3 days to obtain CRISPR-OsPSKR3 and pGluC-OsPSKR3 strains, respectively.

[0072] IV. Obtaining Transgenic Plants

[0073] The CRISPR-OsPSKR3 and pGluC-OsPSKR3 strains were transformed into wild-type Ningjing No. 7. The specific method was as follows:

[0074] (1) CRISPR-OsPSKR3 and pGluC-OsPSKR3 strains (or empty vector control strains) were cultured at 28℃ for 16 hours, the bacterial cells were collected and diluted in N6 liquid medium (Sigma, C1416) to a concentration of OD600≈0.5 to obtain bacterial solution;

[0075] (2) Mix the mature embryonic callus of rice Ningjing 7, which has been cultured for one month, with the bacterial solution in step (1) and infect for 30 min. After the bacterial solution is dried with filter paper, transfer it to co-culture medium (N6 solid co-culture medium, Sigma) and co-culture at 24℃ for 3 days.

[0076] (3) The callus from step (2) was inoculated onto N6 solid screening medium containing 100 mg / L hygromycin for the first screening (16 days).

[0077] (4) Select healthy callus and transfer it to N6 solid selection medium containing 100 mg / L hygromycin for a second selection. Subculture every 15 days.

[0078] (5) Select healthy callus and transfer them to N6 solid selection medium containing 50 mg / L hygromycin for the third selection, and subculture every 15 days.

[0079] (6) Select resistant callus and transfer it to differentiation medium for differentiation; obtain T0 generation positive plants that differentiated from CRISPR-OsPSKR3(pskr3) and pGluC-OsPSKR3.

[0080] V. Identification of Transgenic Plants

[0081] PCR amplification was performed to identify the transgenic plants. The PCR primer sequences are as follows:

[0082] Primer7:

[0083] 5'TGCCTTCATACGCTATTTATTTGC 3' (SEQ ID NO. 10).

[0084] Primer8:

[0085] 5'AGTTGTTGCTGGCGTTGAAC 3' (SEQ ID NO. 11).

[0086] 1. PCR molecular identification

[0087] Genomic DNA was extracted from the T0 generation plants of CRISPR-OsPSKR3 (pskr3) and pGluC-OsPSKR3 obtained in step four. Genomic DNA extracted from the pskr3 T0 generation plants was amplified using Primer3 and Primer4 as primers, and genomic DNA extracted from the pGluC-OsPSKR3 T0 generation plants was amplified using Primer7 and Primer8 as primers.

[0088] PCR reaction system: DNA (20 ng / μL) 2 μL, Primer 3 (10 pmol / μL) and Primer 4 (10 pmol / μL) 2 μL each or Primer 7 (10 pmol / μL) and Primer 8 (10 pmol / μL) 2 μL, 10x Buffer (MgCl2 free) 2 μL, dNTP (10 mM) 0.4 μL, MgCl2 (25 mM) 1.2 μL, rTaq (5 U / μL) 0.4 μL, ddH2O 10 μL, total volume 20 μL.

[0089] The amplification reaction was performed on a PTC-200 (MJ Research Inc.) PCR instrument: 94℃ for 3 min; 94℃ for 30 sec, 55℃ for 30 sec, 72℃ for 2 min, 35 cycles; 72℃ for 10 min.

[0090] PCR products were separated using 8% non-denaturing PAGE gel and silver-stained. Transgenic positive plants were identified.

[0091] 2. Storage tolerance, salt tolerance, and phenotypic identification

[0092] T2 generation CRISPR-OsPSKR3 positive plants, pGluC:OsPSKR3 positive plants, and Ningjing 7 were planted in transgenic fields at the Tuqiao Rice Breeding Base of Nanjing Agricultural University. After the seeds matured, the seeds of each material were harvested for storage tolerance assessment.

[0093] The identification methods are as follows:

[0094] Artificial aging was performed after dormancy was broken using an intelligent seed aging test chamber (LH-300S) from Nanjing Yan'ao Instrument Equipment Co., Ltd., Jiangsu Province. The temperature was set at 40℃ and the humidity at 80% RH. Ningjing 7 and its corresponding OsPSKR3 knockout and overexpression families were aged for 36 days. Germination rate was assessed after aging. The assessment method is as follows:

[0095] Seed vigor before, after, and naturally aged seeds was assessed using a standard germination test to reflect seed storage tolerance. Fifty plump, healthy seeds were randomly selected and placed in 9cm diameter petri dishes lined with two layers of filter paper. 10mL of water was added, and the dishes were incubated at 30℃, 100% RH, and in darkness. Germination rate was recorded on day 7. Each seedling was replicated in duplicate. Germination was defined as the radicle or plumule exceeding half the length of a seed.

[0096] Salt tolerance was also assessed using the following methods:

[0097] T2 generation pGluC:OsPSKR3 positive plants and Ningjing 7 rice were selected and planted in a transgenic field at the Tuqiao Rice Breeding Base of Nanjing Agricultural University. After seed maturity, seeds from each material were collected for salt tolerance testing.

[0098] 1. After breaking dormancy, a germination experiment was conducted on the salt tolerance test seeds: the seeds were soaked for 2 days, germinated overnight at 30℃, and then sown in 96-well plates with the tube bottoms cut off.

[0099] 2. Cultivate in an artificial climate chamber until the seedlings reach the 2-leaf, 1-heart stage, which takes about 10 days. (After sowing, first cultivate in dd water for 2 days. When the seedlings are about 2cm tall, switch to 1 / 4 Yoshida nutrient solution for 2 days, then 1 / 2 Yoshida nutrient solution for 2 days, and finally cultivate in a complete nutrient solution.)

[0100] 3. Divide the seedlings that have been cultured to the 2-leaf-1-heart stage into two groups. Treat one group with 150mM NaCl (add 150mM sodium chloride to the Yoshida nutrient solution, adjusting the salt concentration as needed). Culture the other group in normal Yoshida nutrient solution. Determine the treatment time based on phenotype (generally 10 days). After treatment, culture the seedlings in normal Yoshida nutrient solution for 2 days and then calculate the survival rate.

[0101] The Yoshida nutrient solution formula is as follows:

[0102] Yoshida Nutrient Solution Mother Solution Preparation

[0103]

[0104] The above solution is the stock solution. When preparing the working solution, dilute it 800 times (4L plus 5ml).

[0105] NaOH: 2M is used to adjust the pH. The working solution is used to adjust the pH to 5.8-6.0.

[0106] VI. Results

[0107] Storage tolerance test results showed that after artificial aging, the seed germination rate of PSKR3 plants was significantly lower than that of wild-type plants. Figure 1 , Figure 3 Its seed germination rate after natural storage is also significantly lower than that of the wild type. Figure 2 Meanwhile, the knockout lines showed slightly abnormal phenotypes compared to the wild type, with different knockout families exhibiting reduced plant height, paler leaf color, and needle-like leaf phenotypes. Furthermore, the seed germination rate of pGluC:OsPSKR3 overexpressing lines after artificial aging was significantly higher than that of the wild-type Ningjing 7. Figure 5 , Figure 6 Salt tolerance test results showed that the overexpression line exhibited significantly higher salt tolerance at the seedling stage than the wild-type Ningjing 7. Figure 7The survival rate after salt treatment was also significantly higher than that of the wild type. Figure 8 The other agronomic phenotypes of the overexpression lines were consistent with those of the wild type. OsPSKR3, as a potential material for regulating seed storage tolerance and seedling salt tolerance, provides a potential gene locus for improving rice seed vigor and increasing agricultural production efficiency.

[0108] Gene sequence of rice sulfopeptide receptor OsPSKR3 (SEQ ID NO.1)

[0109] AGAGTGGACTTTGCAGCTATCGAGCTTGGATTCTAGCGCCATCTCCATCTCCACCTCCAC

[0110] ATCCCTACTAGTATACATCATTGCTGCTGCTCTGCCATCGTCGCGCGCACACACGCAGAC

[0111] AAGAAAGCTGGACCAAACGGGCCCCGCCAGCAATGGTGGTGCCGCCGCCGCCGCCTTC

[0112] CCTTCTCCCCCTCTCCCTACACCCCGCCTCTGCCCGTGAGGCCAAGTCCATGGCGAGACG

[0113] CGCGCCCCTACGCTGTCTCTTCCTCTCCCTCGTCGCGCTCTTCGCTTTGCTGCCATTCCCG

[0114] CCCGCTGCGGCGGCGCCATGCCACCCGGAAGACCTCCTCGCGCTGCGGGCATTTGCGGG

[0115] GAATCTCTCTGCGGGTGGGGGCGGCGCCGGACTCCGCGCCGCGTGGTCGGGTGACGCCT

[0116] GCTGCGCCTGGGACGGCGTCGCCTGCGACGCCGCCGCCCGAGTCACGGCGCTGCGCCTC

[0117] CCCGGGCGAGGTCTCGAGGGGCCATCCCGCCCTCCCTCGCCGCCCTCGCGCGCCTCCA

[0118] GGACCTCGACCTCAGCCACAACGCGCTCACCGGCGGCATCTCCGCCCTCCTCGCCGCCG

[0119] TCTCCCTCCGCACCGCCAACCTCTCCTCCAACCTGCTCAACGACACGCTCCTCGACCTCG

[0120] CCGCGCTGCCGCACCTGTCCGCGTTCAACGCCAGCAACAACTCGCTGTCCGGCGCGCTC

[0121] GCCCCCGACCTCTGCGCCGGCGCGCCGGCGCTGCGGGTGCTCGACCTCTCCGCCAACCT

[0122] CCTCGCCGGGACGCTCTCGCCGTCCCCGTCGCCGCCGCCCTGCGCCGCCACGCTCCAGG

[0123] AGCTCTACCTCGCATCCAATTCCTTCCATGGCGCCCTACCGCCCACGCTCTTCGGCCTCG

[0124] CCGCGCTGCAGAAGCTCTCCCTCGCCTCCAATGGCCTCACCGGCCAGGTCAGCAGCCGC

[0125] CTCCGCGGCCTCACAAACCTCACCTCTCTCGATTTGTCCGTGAACCGCTTCACCGGCCAC

[0126] CTCCCCGACGTGTTCGCCGACCTCACGTCGCTGCAGCATCTCACCGCGCACTCCAATGG

[0127] CTTCTCCGGCTTGCTGCCGCGCTCGCTCTCGTCGCTCTCATCTCTCCGTGACCTCAACCTC

[0128] CGGAACAACTCCTTTTCCGGCCCAATTGCTCGTGTCAACTTCTCCTCCATGCCGTTCCTC

[0129] GTGTCCATTGACCTTGCCACTAACCACTTGAATGGCTCTCTCCCGCTTAGCCTCGCCGAT

[0130] TGCGGCGACCTCAAGTCACTCAGCATTGCCAAGAACAGCCTCACCGGCCAATTGCCCGA

[0131] GGAGTACGGCCGCCTCGGCTCGCTCTCCGTGCTCTCGCTGTCCAACAACACCATGCGCA

[0132] ACATCTCGGGGGCGCTCACCGTGCTCCGCGCCTGCAAGAACCTCACCACACTGATCCTC

[0133] ACCAAGAATTTCGTCGGCGAGGATCTCCCCGACGACGGCATTGCTGGATTCGACAATCT

[0134] TGAGGTGTTGGCCCTTGGTGATTGTGCTCTCAGGGGAAGGGTCCCGGAATGGCTGCATC

[0135] AGTGCAAGAGACTGGAGGTGCTTGATTTGTCCTGGAACCAATTGGTTGGCACCATCCCT

[0136] GAATGGATCGGTCAACTTGATAACCTGACCTACTTGGATCTTTCAAACAATTCCTTAGTC

[0137] GGCGAGATACCGAAGAGTTTGACGCAGCTCAAGAGCCTTGTCACCGCCCGACGTTCGCC

[0138] GGGTATGGCGTTCACTAACATGCCGTTGTATGTGAAGCATAACAAGAGCACAAGTGGCC

[0139] GGCAATACAACCAGCTCTCAAACTTCCCACCGTCGTTGTTCTTGAATGATAATGGTCTGA

[0140] ATGGGACTATCTGGCCCGAGTTTGGGAACTTGAAAGAGCTACATGTGCTGGATTTGAGC

[0141] AACAATGCCATATCTGGGAGCATTCCTGATGTGCTGTCCCGGATGGAGAATTTGGAGGT

[0142] TCTTGATCTGTCATCCAATAATCTCAGTGGATCGATTCCATCGTCGCTGACGGACCTGAC

[0143] CTTCTTGTCAAAGTTCAGTGTGGCTCACAATCACTTGGTGGGGCCGATCCCAAATGGAG

[0144] GACAGTTCTTCACCTTCTCAAACTCAAGTTTTGAAGGTAACCCTGGTCTGTGCAGGTCAA

[0145] GTTCTTGTGACCAGAATCAGCCTGGGGAAACTCCTACCGACAATGACATACAGCGGAGC

[0146] GGCAGAAACAGGAAAAACAAAATTCTTGGAGTGGCCATCTGCATCGGTTTGGTACTTGT

[0147] AGTACTGTTGGCTGTTATCTTGGTTAACATCTCAAAGAGGGAGGTCAGCATTATTGACG

[0148] ACGAAGAAATTAATGGCTCCTGTCATGACTCGTATGATTACTGGAAGCCAGTGCTATTC

[0149] TTTCAGGATTCTGCCAAGGAGCTAACTGTCAGCGATCTCATCAAGTCAACAAACAACTT

[0150] TGATCAGGCCAACATAATAGGATGTGGCGGTTTTGGCCTTGTGTACAAGGCCTACCTCC

[0151] CAGATGGAACAAAGGCGGCGGTGAAGAGGCTATCTGGAGATTGCGGGCAGATGGAGAG

[0152] GGAGTTCCGTGCTGAGGTGGAAGCGCTGTCGCAGGCTCAACACAAAAATCTTGTTTCAC

[0153] TGAGAGGGTACTGCCGCTATGGAAATGATAGGCTGCTAATCTACTCATACATGGAGAAC

[0154] AATAGCCTGGACTACTGGCTGCATGAGAGATCAGATGGAGGCTACATGCTGAAATGGG

[0155] AGTCAAGGCTCAAAATTGCACAGGGTTCCGCAAGGGGACTGGCTTATCTGCACAAGGA

[0156] CTGTGAGCCCAACATCATCCACCGAGATGTGAAGTCCAGCAACATTCTTCTGAACGAAA

[0157] ATTTCGAAGCGCACCTGGCTGATTTTGGGCTAGCAAGACTGATACAGCCATATGACACT

[0158] CATGTCACAACTGATCTGGTTGGTACATTGGGATACATACCGCCGGAGTACAGCCAGTC

[0159] GGTGATCGCCACTCCAAAAGGGGATGTCTACAGCTTCGGCGTTGTCTTGCTGGAACTTC

[0160] TGACAGGCAGACGGCCCATGGATGTGTCCAAAGCCAAGGGATCAAGGGACTTGGTCTC

[0161] CTATGTTCTTCAGATGAAGTCCGAAAAGAAGGAAGAGCAGATCTTTGACACCCTGATAT

[0162] GGAGCAAAACACATGAGAAGCAACTGTTTTCGGTCCTTGAGGCGGCGTGTAGGTGCATC

[0163] AGCACTGACCCTCGGCAGCGCCCATCGATCGAACAAGTTGTTGCTTGGCTTGACAGCGT

[0164] ATAATCATTAATAATTCTGTGTTTTTAAGTTGTCTGCTGTTCATTTTGGATTGCAGGCTAG

[0165] GCCTGACATGAGAAGTAGACAGAGGGTATAGGAGCAATCTGAGTGCTAATAATCTTTAG

[0166] AAGATGCAGTCTATGTGATGTACATAGCTTAGTGTCGTTTTGTTATATTATATCCAGGAA

[0167] TAGACCTGTATCTTTGTTTTGTTGTCTGAAACTTGGCCTTCCAGCAAAATACCAATCAAG

[0168] AATTGAGTACCTAGTACTATGTGGCATTCCAGTGCTATATCTTGGAGATAAGTGGTGGTT

[0169] TGTGATGAAATGTAGTAATGTTCTTGTAATTTGTCCTGCGAAAACTGAAATTCTGGCAGC

[0170] TTATGGTACAGGGGTACATCATCACAGATTTCTTATGC

[0171] CDS sequence of rice phytosulfokine receptor OsPSKR3 of SEQ ID NO.2

[0172] ATGGTGGTGCCGCCGCCGCCGCCTTCCCTTCTCCCCCTCTCCCTACACCCCGCCTCTGCC

[0173] CGTGAGGCCAAGTCCATGGCGAGACGCGCGCCCCTACGCTGTCTCTTCCTCTCCCTCGTC

[0174] GCGCTCTTCGCTTTGCTGCCATTCCCGCCCGCTGCGGCGGCGCCATGCCACCCGGAAGA

[0175] CCTCCTCGCGCTGCGGGCATTTGCGGGGAATCTCTCTGCGGGTGGGGGCGGCGCCGGAC

[0176] TCCGCGCCGCGTGGTCGGGTGACGCCTGCTGCGCCTGGGACGGCGTCGCCTGCGACGCC

[0177] GCCGCCCGAGTCACGGCGCTGCGCCTCCCCGGGCGAGGTCTCGAGGGGCCCATCCCGCC

[0178] CTCCCTCGCCGCCCTCGCGCGCCTCCAGGACCTCGACCTCAGCCACAACGCGCTCACCG

[0179] GCGGCATCTCCGCCCTCCTCGCCGCCGTCTCCCTCCGCACCGCCAACCTCTCCTCCAACC

[0180] TGCTCAACGACACGCTCCTCGACCTCGCCGCGCTGCCGCACCTGTCCGCGTTCAACGCC

[0181] AGCAACAACTCGCTGTCCGGCGCGCTCGCCCCCGACCTCTGCGCCGGCGCGCCGGCGCT

[0182] GCGGGTGCTCGACCTCTCCGCCAACCTCCTCGCCGGGACGCTCTCGCCGTCCCCGTCGCC

[0183] GCCGCCCTGCGCCGCCACGCTCCAGGAGCTCTACCTCGCATCCAATTCCTTCCATGGCGC

[0184] CCTACCGCCCACGCTCTTCGGCCTCGCCGCGCTGCAGAAGCTCTCCCTCGCCTCCAATGG

[0185] CCTCACCGGCCAGGTCAGCAGCCGCCTCCGCGGCCTCACAAACCTCACCTCTCTCGATTT

[0186] GTCCGTGAACCGCTTCACCGGCCACCTCCCCGACGTGTTCGCCGACCTCACGTCGCTGC

[0187] AGCATCTCACCGCGCACTCCAATGGCTTCTCCGGCTTGCTGCCGCGCTCGCTCTCGTCGC

[0188] TCTCATCTCTCCGTGACCTCAACCTCCGGAACAACTCCTTTTCCGGCCCAATTGCTCGTG

[0189] TCAACTTCTCCTCCATGCCGTTCCTCGTGTCCATTGACCTTGCCACTAACCACTTGAATG

[0190] GCTCTCTCCCGCTTAGCCTCGCCGATTGCGGCGACCTCAAGTCACTCAGCATTGCCAAG

[0191] AACAGCCTCACCGGCCAATTGCCCGAGGAGTACGGCCGCCTCGGCTCGCTCTCCGTGCT

[0192] CTCGCTGTCCAACAACACCATGCGCAACATCTCGGGGGCGCTCACCGTGCTCCGCGCCT

[0193] GCAAGAACCTCACCACACTGATCCTCACCAAGAATTTCGTCGGCGAGGATCTCCCCGAC

[0194] GACGGCATTGCTGGATTCGACAATCTTGAGGTGTTGGCCCTTGGTGATTGTGCTCTCAGG

[0195] GGAAGGGTCCCGGAATGGCTGCATCAGTGCAAGAGACTGGAGGTGCTTGATTTGTCCTG

[0196] GAACCAATTGGTTGGCACCATCCCTGAATGGATCGGTCAACTTGATAACCTGACCTACT

[0197] TGGATCTTTCAAACAATTCCTTAGTCGGCGAGATACCGAAGAGTTTGACGCAGCTCAAG

[0198] AGCCTTGTCACCGCCCGACGTTCGCCGGGTATGGCGTTCACTAACATGCCGTTGTATGTG

[0199] AAGCATAACAAGAGCACAAGTGGCCGGCAATACAACCAGCTCTCAAACTTCCCACCGT

[0200] CGTTGTTCTTGAATGATAATGGTCTGAATGGGACTATCTGGCCCGAGTTTGGGAACTTGA

[0201] AAGAGCTACATGTGCTGGATTTGAGCAACAATGCCATATCTGGGAGCATTCCTGATGTG

[0202] CTGTCCCGGATGGAGAATTTGGAGGTTCTTGATCTGTCATCCAATAATCTCAGTGGATCG

[0203] ATTCCATCGTCGCTGACGGACCTGACCTTCTTGTCAAAGTTCAGTGTGGCTCACAATCAC

[0204] TTGGTGGGGCCGATCCCAAATGGAGGACAGTTCTTCACCTTCTCAAACTCAAGTTTTGA

[0205] AGGTAACCCTGGTCTGTGCAGGTCAAGTTCTTGTGACCAGAATCAGCCTGGGGAAACTC

[0206] CTACCGACAATGACATACAGCGGAGCGGCAGAAACAGGAAAAACAAAATTCTTGGAGT

[0207] GGCCATCTGCATCGGTTTGGTACTTGTAGTACTGTTGGCTGTTATCTTGGTTAACATCTC

[0208] AAAGAGGGAGGTCAGCATTATTGACGACGAAGAAATTAATGGCTCCTGTCATGACTCGT

[0209] ATGATTACTGGAAGCCAGTGCTATTCTTTCAGGATTCTGCCAAGGAGCTAACTGTCAGC

[0210] GATCTCATCAAGTCAACAAACAACTTTGATCAGGCCAACATAATAGGATGTGGCGGTTT

[0211] TGGCCTTGTGTACAAGGCCTACCTCCCAGATGGAACAAAGGCGGCGGTGAAGAGGCTA

[0212] TCTGGAGATTGCGGGCAGATGGAGAGGGAGTTCCGTGCTGAGGTGGAAGCGCTGTCGC

[0213] AGGCTCAACACAAAAATCTTGTTTCACTGAGAGGGTACTGCCGCTATGGAAATGATAGG

[0214] CTGCTAATCTACTCATACATGGAGAACAATAGCCTGGACTACTGGCTGCATGAGAGATC

[0215] AGATGGAGGCTACATGCTGAAATGGGAGTCAAGGCTCAAAATTGCACAGGGTTCCGCA

[0216] AGGGGACTGGCTTATCTGCACAAGGACTGTGAGCCCAACATCATCCACCCGAGATGTGAA

[0217] GTCCAGCAACATTCTTCTGAACGAAAATTTCGAAGCGCACCTGGCTGATTTTGGGCTAG

[0218] CAAGACTGATACAGCCATATGACACTCATGTCACAACTGATCTGGTTGGTACATTGGGA

[0219] TACATACCGCCGGAGTACAGCCAGTCGGTGATCGCCACTCCAAAAGGGGATGTCTACAG

[0220] CTTCGGCGTTGTCTTGCTGGAACTTCTGACAGGCAGACGGCCCATGGATGTGTCCAAAG

[0221] CCAAGGGATCAAGGGACTTGGTCTCCTATGTTCTTCAGATGAAGTCCGAAAGAAGGAA

[0222] GAGCAGATCTTTGACACCCTGATATGGAGCAAAACACATGAGAAGCAACTGTTTTCGGT

[0223] CCTTGAGGCGGCGTGTAGGTGCATCAGCACTGACCCTCGGCAGCGCCCATCGATCGAAC

[0224] AAGTTGTTGCTTGGCTTGACAGCGTATAA SEQ ID NO.3 Amino acid sequence of rice phytosulfokine receptor OsPSKR3

[0225] MVVPPPPPSLLPLSLHPASAREAKSMARRAPLRCLFLSLVALFALLPFPPAAAAPCHPEDLLA

[0226] LRAFAGNLSAGGGGAGLRAAWSGDACCAWDGVACDAAARVTALRLPGRGLEGPIPPSLA

[0227] ALARLQDLDLSHNALTGGISALLAAVSLRTANLSSNLLNDTLLDLAALPHLSAFNASNNSLS

[0228] GALAPDLCAGAPALRVLDLSANLLAGTLSPSPSPPPCAATLQELYLASNSFHGALPPTLFGLA

[0229] ALQKLSLASNGLTGQVSSRLRGLTNLTSLDLSVNRFTGHLPDVFADLTSLQHLTAHSNGFSG

[0230] LLPRSLSSLSSLRDLNLRNNSFSGPIARVNFSSMPFLVSIDLATNHLNGSLPLSLADCGDLKSL

[0231] SIAKNSLTGQLPEEYGRLGSLSVLSLSNNTMRNISGALTVLRACKNLTTLILTKNFVGEDLPD

[0232] DGIAGFDNLEVLALGDCALRGRVPEWLHQCKRLEVLDLSWNQLVGTIPEWIGQLDNLTYLD

[0233] LSNNSLVGEIPKSLTQLKSLVTARRSPGMAFTNMPLYVKHNKSTSGRQYNQLSNFPPSLFLN

[0234] DNGLNGTIWPEFGNLKELHVLDLSNNAISGSIPDVLSRMENLEVLDLSSNNLSGSIPSSLTDLT

[0235] FLSKFSVAHNHLVGPIPNGGQFFTFSNSSFEGNPGLCRSSSCDQNQPGETPTDNDIQRSGRNR

[0236] KNKILGVAICIGLVLVVLLAVILVNISKREVSIIDDEEINGSCHDSYDYWKPVLFFQDSAKELT

[0237] VSDLIKSTNNFDQANIIGCGGFGLVYKAYLPDGTKAAVKRLSGDCGQMEREFRAEVEALSQ

[0238] AQHKNLVSLRGYCRYGNDRLLIYSYMENNSLDYWLHERSDGGYMLKWESRLKIAQGSAR

[0239] GLAYLHKDCEPNIIHRDVKSSNILLNENFEAHLADFGLARLIQPYDTHVTTDLVGTLGYIPPE

[0240] YSQSVIATPKGDVYSFGVVLLELLTGRRPMDVSKAKGSRDLVSYVLQMKSEKKEEQIFDTLI

[0241] WSKTHEKQLFSVLEAACRCISTDPRQRPSIEQVVAWLDSV

[0242] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The OsPSKR3 gene, the protein encoded by the OsPSKR3 gene, and the application of recombinant expression vectors, expression cassettes, or recombinant bacteria containing the OsPSKR3 gene in improving the storage tolerance of rice seeds, characterized in that... The OsPSKR3 gene is selected from one of the DNA molecules shown in 1), 2), or 3) below: 1) The DNA molecule shown in SEQ ID NO.1; 2) The DNA molecule shown in SEQ ID NO.2; 3) A DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and encodes the protein described in SEQ ID NO. 3; The amino acid sequence of the protein encoded by the OsPSKR3 gene is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The recombinant expression vector is a recombinant plasmid obtained by inserting the gene of claim 1 between the multiple cloning sites SmaI and SacI of the pGluC vector.

3. A method for cultivating and improving the storage tolerance of rice seeds, characterized in that, Overexpression of the OsPSKR3 gene shown in SEQ ID NO.1 or SEQ ID NO.2 in wild-type normal plants yields transgenic rice with improved seed storage resistance.

4. The OsPSKR3 gene, the protein encoded by the OsPSKR3 gene, and the application of recombinant expression vectors, expression cassettes, or recombinant bacteria containing the OsPSKR3 gene in improving salt tolerance in rice, characterized in that... The OsPSKR3 gene is selected from one of the DNA molecules shown in 1), 2), or 3) below: 1) The DNA molecule shown in SEQ ID NO.1; 2) The DNA molecule shown in SEQ ID NO.2; 3) A DNA molecule that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and encodes the protein described in SEQ ID NO. 3; The amino acid sequence of the protein encoded by the OsPSKR3 gene is shown in SEQ ID NO.

3.

5. The application according to claim 4, characterized in that, The application involves overexpressing the OsPSKR3 gene or introducing the recombinant expression vector into wild-type rice plants.

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