Application of rice OsKNOX8 gene in regulation and control of salt tolerance

Abstract

The invention provides an application of a rice OsKNOX8 gene in regulation and control of salt tolerance. The gene OsKNOX8 is a DNA molecule as shown in 1) or 2): 1) a DNA molecule with a genomic sequence as shown in SEQ ID NO.1; 2) a DNA molecule with a CDS sequence as shown in SEQ ID NO.2; and 3) a DNA molecule which is hybridized with the DNA sequence limited by 1) or 2) under strict conditions and is used for coding the protein. According to the gene engineering application of the gene OsKNOX8 in regulation and control of the salt tolerance of the rice, specifically, the gene OsKNOX8 is knocked out, and the salt tolerance of the rice is improved.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a rice salt tolerance related gene OsKNOX8 and its encoded protein and application. Background Art

[0002] With the growth of the world's population and the development of industrialization and urbanization, arable land resources are constantly decreasing. At the same time, soil salinization caused by improper cultivation and utilization has also reduced the productivity of arable land, further exacerbating the problem of food security. The high content of salt and alkali components in saline soil causes changes in the physical and chemical properties of the soil, causing many problems such as soil compaction and decreased fertility, leading to soil degradation. Soil salinization not only threatens the sustainable development of the global agricultural ecosystem, but also causes the reduction of biodiversity and the destruction of regional ecological balance. my country has a total of 100 million hectares of saline-alkali land, ranking third in the world, of which about one-third has development and utilization potential and is an extremely important reserve arable land resource. Rice is the preferred food crop for the improvement of coastal tidal flats and saline-alkali land. However, rice has poor salt tolerance, and high-salt environment seriously harms the growth, development and yield of rice. Therefore, in-depth analysis of the damage caused by salt stress to rice and the physiological and molecular mechanisms of rice in response to salt stress, screening and breeding of salt-tolerant rice varieties, is of great significance for the development and utilization of coastal tidal flats and saline-alkali land and the promotion of sustainable agricultural development.

[0003] Under salt stress, the growth and development of rice will be significantly inhibited, tissue growth and organ differentiation will slow down, and the inhibitory effect will become more obvious with the increase of salt concentration. When rice is subjected to salt stress during the seed germination stage, not only the physiological water absorption of the seeds is restricted, but also the membrane structure of the cells will be destroyed during the water absorption process, resulting in blocked seed germination, reduced germination rate, and uneven germination. The seedling stage is the period when rice is most sensitive to salt stress. Rice seedlings show symptoms such as growth inhibition, death of old leaves, and decreased green leaf area under salt stress. When rice is subjected to salt stress during the tillering stage, its tillering and leaf growth will be inhibited, the tillering ability of individual plants will decrease, the stems will become thicker, the leaves will be small and yellow or even die, and the plant height will decrease. The booting stage is another period in which rice is sensitive to salt stress besides the seedling stage. Salt stress will cause the rice heading period to be delayed, the spikelets to degenerate severely, and the fruiting rate to be reduced, which will seriously affect the yield. In addition, salt stress can also cause changes in grain fatty acids, minerals and nutrients, affecting rice quality.

[0004] The physiological regulation mechanism of rice salt tolerance includes osmotic regulation, antioxidant system regulation, hormone regulation, etc. Under salt stress, the osmotic potential of the external environment decreases, and the rice root system has difficulty absorbing water, resulting in osmotic stress. In order to reduce its own water loss and absorb water from the outside, rice responds to osmotic stress through two osmotic regulation mechanisms: one is to absorb K from the outside + , Ca 2+Second, it synthesizes organic osmotic regulating substances such as proline, betaine, and soluble sugar. Under salt stress, a large amount of reactive oxygen species will be produced and accumulated in rice, which will lead to membrane lipid peroxidation, increased malondialdehyde content, cell damage, and oxidative stress. There is an antioxidant system in rice that removes reactive oxygen species, including enzymatic defense systems and non-enzymatic defense systems. The enzymatic reaction system mainly includes superoxide dismutase, catalase, ascorbate peroxidase, etc., and the non-enzymatic defense system mainly includes glutathione, ascorbic acid, carotenoids, etc. When rice is in a high-salt environment, a variety of plant hormones will be produced in the plant to jointly regulate the response to salt stress in order to cope with the harm of salt stress to rice growth and development. The hormones that help improve rice salt tolerance mainly include abscisic acid, ethylene, brassinolide, etc., which participate in the response of rice to salt stress through complex signal transduction pathways.

[0005] Rice salt tolerance is a quantitative trait controlled by multiple genes, with a complex genetic basis. It is difficult to breed rice salt-tolerant varieties using traditional breeding methods, and progress is slow. Discovering key genes for rice salt tolerance and analyzing their mechanisms of action will help to quickly improve salt tolerance using molecular breeding techniques. However, among the cloned rice salt tolerance genes, few have been effectively applied in rice breeding practice. It is necessary to further discover important key genes for salt tolerance to lay the foundation for breeding new rice salt-tolerant varieties. Summary of the invention

[0006] In order to overcome the above technical problems existing in the prior art, the present invention provides the genetic engineering application of rice OsKNOX8 gene in regulating rice salt tolerance.

[0007] The technical solution of this patent is as follows:

[0008] Genetic engineering application of rice salt tolerance related gene OsKNOX8, or protein encoded by gene OsKNOX8, or knockout vector of gene OsKNOX8, or primers for amplifying gene OsKNOX8 in regulating rice salt tolerance;

[0009] The gene OsKNOX8 is a DNA molecule as described in 1) or 2) below:

[0010] 1) A DNA molecule having a genome sequence as shown in SEQ ID NO.1;

[0011] 2) A DNA molecule whose CDS sequence is shown in SEQ ID NO.2.

[0012] Furthermore, knocking out the aforementioned gene OsKNOX8, or reducing the expression level of the protein encoded by the gene OsKNOX8, or transferring the knockout vector of the gene OsKNOX8 into rice can improve the salt tolerance of rice.

[0013] Furthermore, the amino acid sequence of the protein encoded by the gene OsKNOX8 is shown in SEQ ID NO.3.

[0014] Furthermore, the knockout vector of the gene OsKNOX8 uses CRISPR-Cas9 technology to edit the gene in rice, knocking out the aforementioned gene OsKNOX8 in the rice plant, rendering the gene inoperable;

[0015] Furthermore, the knockout vector of the gene OsKNOX8 is a CRISPR-Cas9 vector targeting the target shown in SEQ ID NO.6.

[0016] Furthermore, the primers for amplifying the gene OsKNOX8 are selected from Primer1 shown in SEQ ID NO.4 and Primer2 shown in SEQ ID NO.5, and the Primer1 / Primer2 are used to amplify the CDS fragment of the OsKNOX8 gene, or Primer5 shown in SEQ ID NO.9 and Primer6 shown in SEQ ID NO.10, and the Primer5 / Primer6 are used to amplify the mutation target region fragment.

[0017] Beneficial effects:

[0018] The present invention is the first to discover the association between the rice gene OsKNOX8 and rice salt tolerance. Inhibiting the expression of the protein encoding gene can lead to improved plant salt tolerance, thereby cultivating salt-tolerant transgenic plants. The protein and its encoding gene can be used for plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The mutation site of the OsKNOX8 gene and its flanking sequences in the OsKNOX8 gene mutants (osknox8-1 and osknox8-2).

[0020] Figure 2 The seedling phenotypes of wild-type Nipponbare (WT) and OsKNOX8 gene mutants (osknox8-1 and osknox8-2) under salt stress.

[0021] Figure 3 Seedling survival rate of wild-type Nipponbare (WT) and OsKNOX8 gene mutants (osknox8-1 and osknox8-2) under salt stress. DETAILED DESCRIPTION

[0022] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents and kits used in the following examples are purchased from conventional biological reagent companies unless otherwise specified.

[0023] Example 1: Cloning of the coding region sequence (CDS) of rice OsKNOX8 gene

[0024] The cDNA of rice variety Nipponbare seedling leaves was used as template and the primer pair consisting of Primer1 and Primer2 was used to amplify the CDS fragment of OsKNOX8 gene by PCR.

[0025] Primer1:5'-ATGGAGAGCTTCGCCAGTCTCGCA-3' (SEQ ID NO.4);

[0026] Primer2: 5'-TCAAGACCCGAGCCGGTACATGCCG-3' (SEQ ID NO. 5).

[0027] The PCR reaction system (50 μl) was as follows: template cDNA (200 ng / μL) 1 μL, Primer1 (10 μM) 1.5 μL, Primer2 (10 μM) 1.5 μL, 10×PCR Buffer for KOD-Plus-Neo 5 μL, 2 mM dNTP 5 μL, 25 mM MgSO4 3 μL, KOD-Plus-Neo (1 U / μL) 1 μL, ddH2O 32 μL. The PCR amplification reaction was carried out in a Bio-Rad T100 PCR amplifier. The PCR reaction program was as follows: 94°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 60°C annealing for 30 s, 68°C extension for 2 min, 35 cycles; 68°C extension for 5 min; 25°C storage.

[0028] The PCR product was purified using a DNA purification kit (Nanjing Novogene Biotech Co., Ltd.), ligated to the pEASY-Blunt expression vector (Beijing Quanshijin Biotechnology Co., Ltd.), and the ligation product was transformed into Escherichia coli DH5α competent cells (Baoriyi Biotechnology (Beijing) Co., Ltd.), and the positive clones were selected for sequencing (Beijing Qingke Biotech Co., Ltd.).

[0029] The sequencing results showed that the CDS fragment of the OsKNOX8 gene obtained by PCR amplification had a nucleotide sequence as shown in SEQ ID NO.2, encoding a protein consisting of 341 amino acid residues as shown in SEQ ID NO.3.

[0030] Example 2: Construction of transgenic rice OsKNOX8 mutant plants

[0031] 1. Construction of OsKNOX8 gene knockout vector

[0032] According to the genome sequence of OsKNOX8 (SEQ ID NO.1), the CRISPR-Cas9 sgRNA target was designed using the CRISPR-P V2.0 website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The target sequence selected for constructing the gene editing vector for OsKNOX8 was: GCTGCCGCGCCGACGACTCC (SEQ ID NO.6).

[0033] According to the target sequence, Primer3 and Primer4 were synthesized, and the primer pair was annealed to obtain a double-stranded DNA molecule with sticky ends; the double-stranded DNA molecule was connected to the pOs-sgRNA linearized vector cut by Bsa I (the vector construction method is recorded in the document "Targeted mutagenesis in rice using CRISPR-Cassystem") using T4 DNA ligase, and the obtained ligation product was transformed into DH5α Escherichia coli; positive clones were identified by colony PCR, plasmids were extracted, and sequencing was performed. The sequencing results showed that a recombinant vector containing the sequence shown in SEQ ID NO.6 was obtained, named pOs-sgRNA-OsKNOX8t.

[0034] Primer3:5'-ggcaGCTGCCGCGCCGACGACTCC-3' (SEQ ID NO.7);

[0035] Primer4:5'-aaacGGAGTCGTCGGCGCGGCAGC-3'(SEQ ID NO.8)

[0036] The pOs-sgRNA-OsKNOX8t plasmid was mixed with the pH-Ubi-cas9-7 vector (the vector construction method is recorded in the document "Targeted mutagenesis in rice using CRISPR-Cas system"); the LR reaction was performed using the Gateway kit (Invirogen) and Escherichia coli DH5α was transformed; the positive clones were identified by colony PCR, the plasmids were extracted and sequenced, and the plasmid pH-Ubi-cas9-7-OsKNOX8t containing the sequence shown in SEQ ID NO.6 was obtained.

[0037] 2. Obtaining recombinant Agrobacterium

[0038] The pH-Ubi-cas9-7-OsKNOX8t was mixed with the Agrobacterium EHA105 competent cells, incubated on ice for 5 min, and the pH-Ubi-cas9-7-OsKNOX8t was transformed into the Agrobacterium EHA105 competent cells by electric shock (1500 V, 5 ms) to obtain the recombinant strain. The recombinant strain identified correctly by colony PCR was named EH-pH-Ubi-cas9-7-OsKNOX8t.

[0039] 3. Obtaining Transgenic Plants

[0040] The above recombinant Agrobacterium strain was transformed into the rice variety Nipponbare, and the specific method was as follows:

[0041] (1) The EH-pH-Ubi-cas9-7-OsKNOX8t strain was cultured overnight at 28°C and 200 rpm until OD 600 Saturation; inoculate the bacterial solution into new YEP liquid medium at a ratio of 1:100, and culture in suspension at 28°C and 200rpm until OD 600 =0.6-0.8, collect the cells; gently mix 30mL AAM liquid medium (containing 30μL 1000×AS (acetosyringone)) with the cells to make OD 600 =0.05-0.1.

[0042] (2) The mature embryonic callus of Nipponbare cultured for one month was mixed with the bacterial solution in step (1), infected for 90 seconds, dried with filter paper, transferred to 2N6-AS solid medium, and cultured in the dark at 28°C for 48-60 hours.

[0043] (3) The callus in step (2) was removed and placed in a 50 mL centrifuge tube. The callus was washed 8-10 times with sterile water, and the tube was shaken continuously until the water was clear. The callus was washed twice with sterile water containing 500 mg / L Car (carbenicillin disodium), each time for 10-20 min. The sterile water was removed and the callus in the centrifuge tube was poured onto sterile filter paper and drained for 1-2 h.

[0044] (4) The callus obtained in step (3) was inoculated on N6D-S solid medium containing 50 mg / L Hyg (hygromycin B) and 250 mg / L Car, and cultured and screened at 28°C for 3-4 weeks.

[0045] (5) The selected resistant calli were transferred to MS-NK solid culture medium and cultured at 28°C for 3-4 weeks to allow the calli to differentiate into adventitious buds.

[0046] (6) When the adventitious buds grow to 3-4 cm, transfer them to MS-HF rooting medium and grow at 28°C for 1 week.

[0047] (7) The differentiated rice seedlings are hardened and transferred to the field for growth, thereby obtaining T0 generation transgenic plants.

[0048] Example 3: Molecular identification of rice OsKNOX8 gene mutant transgenic plants

[0049] The aboveground part of the seedlings of the T0 generation transgenic plants of the OsKNOX8 gene constructed in Example 2 was sampled, genomic DNA was extracted as a template, and the primer pair consisting of Primer5 and Primer6 was used to perform PCR amplification on the editing target site shown in SEQ ID NO.6 and the DNA fragments on both sides thereof.

[0050] Primer5:5'-TTTGCTAGGGTTTGTTTGCC-3' (SEQ ID NO.9);

[0051] Primer6: 5'-ATACTCACCAGCGAGCGAAT-3' (SEQ ID NO. 10).

[0052] The obtained PCR products were detected by 1% agarose gel electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. According to the sequencing results, two strains with specific mutations in the OsKNOX8 gene were identified: osknox8-1 and osknox8-2. The DNA sequences of the editing target sites and their flanking sites in these two strains are shown in Figure 2. Figure 1 shown.

[0053] In the osknox8-1 strain, a nucleotide C was inserted in the first exon of the OsKNOX8 gene;

[0054] In the osknox8-2 strain, a nucleotide G was inserted in the first exon of the OsKNOX8 gene;

[0055] In these two mutant strains, mutations in the CDS sequence of the OsKNOX8 gene caused frameshift mutations and premature termination of the protein it encoded.

[0056] Example 4: Identification of salt tolerance of OsKNOX8 gene mutant strains at the seedling stage

[0057] The T0 transgenic plants of the two OsKNOX8 gene mutants identified in Example 3 were selfed for two generations to obtain homozygous mutant lines osknox8-1 and osknox8-2. These two homozygous mutant lines were used together with the Nipponbare wild type to conduct a salt tolerance identification experiment at the seedling stage.

[0058] (1) Select full rice seeds, soak them in tap water in a 33°C incubator for 2 days, and germinate them for 1 day. Select seeds with consistent germination, sow them on a 96-well PCR plate without a tube bottom, place them in a black plexiglass culture box filled with pure water, and culture seedlings in an artificial climate chamber. The culture conditions are: 14 h light (28°C) / 10 h dark (24°C), light intensity 1000 μmol·m -2 ·s -1 , relative humidity 70%. After culturing in pure water for 1 week, the culture was switched to Kimura B nutrient solution.

[0059] (2) When the rice seedlings grew to the two-leaf and one-heart stage, the nutrient solution was replaced with Kimura B nutrient solution containing 100 mM NaCl for salt stress treatment.

[0060] (3) After 10 days of NaCl treatment, the rice seedlings were rehydrated with Kimura B nutrient solution without NaCl. After 7 days, the survival rate of rice seedlings was counted. Survival rate = number of surviving seedlings / total number of treated seedlings × 100%.

[0061] The results of salt tolerance identification showed that compared with the wild type (WT) of Nipponbare, the two OsKNOX8 gene mutant strains (osknox8-1 and osknox8-2) showed milder salt damage symptoms such as leaf wilting and drying ( Figure 2 ); After 10 days of salt stress and 7 days of rehydration, the survival rate of seedlings of osknox8-1 and osknox8-2 lines was significantly higher than that of the wild type (WT) ( Figure 3 ).

[0062] The above experimental results show that the OsKNOX8 gene has the function of negatively regulating the salt tolerance of rice seedlings, and knocking out this gene can significantly improve the salt tolerance of rice.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

[0064] SEQ ID NO.1 OsKNOX8 genome sequence:

[0065] CACTGTTGCTGCTGCTGCTGCTGCTTGAAACACTGAATAGCCTAACCTCTTCATTAATTT

[0066] CTCGCTCTGATTTTGACGTTAGAACAAAGCTAAGATGGGCCATACGTATACTAGGAGGAG

[0067] TACCATCATATTCATCTTCTTTCTAGCTTCCTCTCCTCTTCACATTCCCTGCATCCATCCATC

[0068] TCAACCACCACCACCTCATTTCAGGAGATCATCTCCTCCTTTTCTTTGCTAGGGTTTGTTT

[0069] GCCATAACTACTATCTATCGCCTCTTCTTCACCTCACCTGTCCTGTCGGCCATGGAGAGCT

[0070] TCGCCAGTCTCGCAGGAGGTGGCAGCAGCAGCACCACAGCTCGGTTGCCGGAGCTGATC

[0071] TCGCCGGAGAACCCGGACCACATCTCGCCGCCCCCGTTGCTGTACCAGCTGCTCGCCGG

[0072] CCCGGAGTCGTCGGCGCGGCAGCATGGGCATGATGGGCATCACCACGGCGGCGGTGGCG

[0073] GCGAAGCAGCAGCAGCAGCTGTGCAGGGACAGGTCTCGCCGGCAGGCGCGGAGGCGGC

[0074] GGTCAAGGCCGAGATCATGTCGCATCCACAGTACTCGGCTCTCCTAGCCGCCTACTTAGG

[0075] CTGCAAAAAGGTGTAGTAGCTAAGCTAGCAGATGCATATATTTAAACCCATGCAGATACAG

[0076] ACGACAACACGCCATGTGCTGACCAAGCTCACGGCCGTGCCGGATGTACTGACCAAGTT

[0077] GATTTTGTTGGTTTTTTTATCAGGTCGGGGCGCCGCCGGATGTACTGACCAAGCTCACGG

[0078] CTGTGCCGGCGGCGCAGCAGCTGGACGAAGCCGACGGCCACCCTCGTCGCCGGCACGA

[0079] GCCACAGCGTGATGACGACCCGGATCAGCTCGATCAGTTCATGGTATCAACATATATAGAT

[0080] CATCAACTCCTTTGCTCGCATGCGTGCATATATATAGACAGACACACACACATATATATCCA

[0081] CTGTTCTTCTGATCGATCGACATGGGATCTGATCTTGTCATCACAGGACGCGTACTGCAGC

[0082] ATGCTGACGAGGTACAGGGAGGAGCTGGAGCGGCCGATCCTGGAAGCCGCGGAGTTCTT

[0083] CAGCAGGGTGGAGACGCAGCTCGATTCGCTCGCTGGTGAGTATTTTATACGCCTCTTCTCT

[0084] ACAAAGATTTGCAGCTTGCCATATTGGGAAATGCCGAAATGGTATCTCACTTTATGGAATT

[0085] GGAGTTAGTTTTTTTTTTGTCACCTCTTTGGACTCATGTGGCTGTTGCAGCAGTATTTCTTA

[0086] AGGCTGCTAATCTCAGGCTTTAATATGTTTATTAGGGTGGGGGATAAGGGTTGCATGCATT

[0087] AGATCAGTTTCGTAATAATTGGAGCTTAATTAAGTATACATGACCAGTATGATAAGCATCAT

[0088] GAGGTCACACGGTGAGGCTGCGAGCTGAGCTCCAGGGTTTCCAAATTGCCATATATGCAT

[0089] GGCTCTCCTACTTTCCAGTCTCCACTTCCTCCTGTGCAATACAACGATGGTGTCCTCCCTC

[0090] TGTCGTACAATCTTAATGAATCATACAAAGGAATAATTAATACTGGAGCACATGCAGTTCT

[0091] TTGAACTTGTACCTTGTACTGACGTTATTTTTTTTCCTCTGATTTTCTTTTATTTTTAGAATT

[0092] AAGACCCAACCTTGCTGACATTATTAGCTTCTAATTAAAGAGTTCAGTGCATGCATCACAA

[0093] GTGATGAACTGACAGGAGAAAGCTCTCAGCACGTTCTGTCCCCAATATCTCTGCAGGTCC

[0094] TTGTCTAACCTACATTTTTGCTCTCTTGTCATGTCTTCCGCTGGTAGTCACAGAGAAATGG

[0095] CTAGACGATCTTTGTTTTCAACTTAGACGATTTATGTTAAGATTAAGGGCCAAGAGGTAAA

[0096] TTAAACGAACCATCAAAGTTTGCAGAACATAGTGGCCAAATTGAGGTGTAAGTAACATCT

[0097] AGCTGGTTGAAAACATGAGTGGCGGTGGATGACAGAATGAGTGGCCGGTGGGCCTATTG

[0098] TCATTGTGCCTCCGTGAGTGTAATTGGTTTACCGACCTCAAATCCTGGATCGAGTCTTGGC

[0099] TGTCGGACATGAATGGGCACTGTTAGTACCCTGTGCACTTTGTCTCATGGCCTGGATATAT

[0100] AGTTACTGGAGTGGGGTAAGGATCCAGCTATATATTAGACAATTGATAAGTGTGTATGACC

[0101] ATCTAGTTGTGTCCTTTCAAGTGCAGTTGTTTCCGAACATTATTGAGCACTTTCCGTAGCC

[0102] ATACGAATAGATTTCCAGATCAGAACTCTTTCTTATATATACAGACATCAATCGAGTGCCAA

[0103] ATAATTGAAAGAATGAAAGCAGCATAGTGACCAGCATGCATATCATATTGTGTTTTGTTGA

[0104] AACATAGTTCAATAATTTTGTCTATATGAACTAGTGTAGTTTTTTACACTGGTAATTGCATAT

[0105] ATACAAGGTGGTGTTTCATAAGACGACTAATATGGCAAAATTATGAAATTCACTATCCGTA

[0106] CAAATCCTGGATAATATATAGGAGTACATATATGGAAGAATTATCTGGTTTCTTATATATTTA

[0107] GTCTGCCAAAATGTCATTCAGCTATTCCCCTACCGATGAGGCATTTTTTTTTGTGTCTACAC

[0108] ATATATTCCTGTCTACAGATGGTCTCTTTTGGAATTGATTTTTGTTTCTAATTTGTTGGACA

[0109] GATGGTCTCTTTTGGAATTGATTTTTTTTCTAATTTGTTGGATATTTATTGGACTGGTGCAA

[0110] CAATTCATCCCCTCACACATGATTTGATCTATTCCATATACTCAGATGAATTGTTGGAATCC

[0111] TACTATTTCACCAGTATGGATCATGTTCAGATATTAGCTATTGACTTTTAGATATGACGTTTG

[0112] AAATATAAATTATTTTTTGTGACTTGTTTATTATTAAAGGTACATCAAACATAACTTATAATT

[0113] TTACATATTTGCACTATTTTTTTTTGTAAGGTGAATGGACAAACGTCATGTTCAAAAGTCA

[0114] GCAATGTCATATATTTAAAAAAGGAGGGAGGATTGCATTAGGAGTTCATAAGAGTGATAA

[0115] ACACGGCTTGTTAATAAGTGAATGATTATCTAACTTAACATATATATCATCCACAACAATGA

[0116] AAATACAGTCTATGCACACATTACTTTAACCAGTGTCTTGTACTAGCATCAATACTAACTA

[0117] GACTTTGCACTCTTTTGATGTCATTTCGTCTCAATGTTGTCATTTCAGAAATATCCATTTAA

[0118] CTGTTCATAAGTAACACTTTTCATTCTGTTAATTACTTTTTGAATCATATTCTGGTTGCATGC

[0119] TGCTCTACTGTCTGGAAATATTCCAATGTCAACAGAATGTGTATGTGCACACATGCAGCAT

[0120] AAATAGCTCCCAGAAAGTTTCAAATAAATTTTTATATTGGATGGAATTTGAACTGCATACTA

[0121] TAAATATTATCCTATGAATTGTAGGAGTTGTGTAATATATTTGTTATTTATATTTTTTTTAATCT

[0122] TCTGAAATCAAGGGTATGAATAATTTTTTTAGTGGAACTAGTGTAGTCTTTGTAAGTATTTG

[0123] GCCTTATTTCATTGTTAAAATCAATAGTTTTGTCTATATGAATTAGTGTAGTTTTTTACACTA

[0124] GTAGTTGCATATATACAAGGTGGTGTTTCAAAAGACGACTAATATGGCAAAATTTATGAGA

[0125] TTCACTATCCGTACAAATCCTGGATCTATATATTACAATCGCTTTGAGATGAATGCAAATTA

[0126] GGATGGGCACAATTTTTTCATAGCATGGTGGAATGGTTTCTCTTGTCACCATCGTTTTGAA

[0127] CTCATCTTTATTATGTGCCCGCTAATTACTCCCTCCTTTCTAAATTGATTATCATGTAATAGA

[0128] AATCAAAATTTCTTAAATTGATCATCATATAACTGCATGGAGACGGATTTCATCGGAATACA

[0129] ATTAATACAACATGGGAGAATGCGTGCATGCTAGGGTGGAGTAATTGGCATGGAGTTTTAA

[0130] TCGATGTATGCATTGTGGGAGAATGTGTGCATTGTGTCTTGATTGATGTGATTTAAATTATT

[0131] TTTTGGTCTTGGTGCGTAAATTTATATGATGATCAATTTGAGAAGGAGACAGTATATTTTAA

[0132] AACCGTTCTGTCTAAGTGTTCACACAAGATCACTGATTTTTTTTTTTGCCTTCGAATAAAC

[0133] AGAACACAAGCAGGCCTTGTAATATTTAGTATATATTCTTGTCATAGCTTTTCCCTACGAAT

[0134] CAAAATGAATATCCTGTCTCAAATTGAACGGTATGGCGGCTGTTTGACTAGTCTAGTTTT

[0135] CTGCTTGGAATTTGGAAATACCGTGGCCTGTTTGGTGGAGCTTTAGATTCTGATAATCAGC

[0136] TGTTTGGTAGCAAGCTTCTGAAAATCTGGAAATGCTCTGAAACCCAGCTTCTCCAGCTTC

[0137] TGGCTTCTTAGTTCAATTTTCAGAATCTGTAACTACAGATTCTCAGAAGCTGTGGACTGTT

[0138] TGGGACAGCTTCTAGCAGAAGTAGCTTTTGGGAAAAGTTGCAGCTGGAAGAAGCTCCTC

[0139] CAAACAGGGCCTGTCTAGCTGAGCAAGGACAATTATTGGGTGCACAGAGTGTACAACAT

[0140] GTTCAGGCCTTCAGGGTTGCACTGTTGCTTACTTCGATACACTCCTACCTAGTAAACACAA

[0141] AGAATTAATTGACTGACTGGTGGCCTGGTGGGGCTGACCGATTGAGTAATACACAGTACT

[0142] ACCATTATACTCTATGCATAATCTATTCGATCTCCCACACCTCCCTTCATATGAGGTTTACCT

[0143] GCTGTGACGCACGCAGTCATGTAATTTTTAATTATTTTGATAGAACATCATGTTGAACAATT

[0144] AACCTTTTTGAGCTGCTGTTTTTAACAGAGAGTAATTGTGAAGGCACGGGATCATCAGAG

[0145] GAAGAGCAAGACCCTAGTGACAAGCAGCTGAAGCACCAGCTTCTGAGGAAGTACGGTG

[0146] GCTCGTTGGGCGATCTCCGGCAGGTGTTCTCCAAGAGGACCAAGAAAGGGAAGCTTCCC

[0147] AAGGAGGCCAGGCAGAAGCTTCTGCACTGGTGGGAGCTGCACTACAAGTGGCCCTATCC

[0148] CTCCGTACGCACGCATCACATCCCGTTTGATCAATTCAACCACATTTTCTGCACCAAATTA

[0149] ACTCGTTTGAAAATGCGTGAAATTTACGGGGTTGTGTATGAGCAGGAGATGGAGAAGATG

[0150] ACGCTGGCGCAGACGACGGGGCTGGACCAGAAGCAGATCAACAACTGGTTCATCAACC

[0151] AGAGGAAGCGGCACTGGAAGCCGACGCCGGTGGCAGGCACGGCCTTCCCGACGATGGA

[0152] AGCTGCCGGAGGCGGCTTCCGACACTCCGGCCACGGCGGCGGCCTTGCGGCGGCGGCG

[0153] GCGCTGCCGCTGTACATGGGCAGGCCGTTCGTTGTGGACGGCATGTACCGGCTCGGGTCT

[0154] TGAAGCCTCTGGGGATCGCATGGTGTTTGCATGGTGTTTTTTGCTTGGTAAATATTTGCTA

[0155] GCAGCTTCCCATGATCAGCCTGTGGGATGGTGAAATGGAAGAACTGATGAATCTTCATCA

[0156] CGATCTTTCCTCTCATTTTTTTTTCTTTGGTGTGTGATAAAAGCTCAGATGTACCACGTACT

[0157] ACTGTCTGCTTCTTTGCAAATGGATATGTAATTGAAAGCTTTAATTCTAAACATGGGACTC

[0158] CTCCGTTTTTATATTATAAGTCGTTTTGATTTTTTTATTAAACTTGGTTAAGTTTAATTAATTT

[0159] TATAAAAAAATATAGCAATAATTTTAACACAAAACAAATACATCATCAAAAATTTTCAATG

[0160] TTACATATAATCAAATTAATTTGATGTTATAGTTGTTGCTGAATTTTCATATAAACTTAATCA

[0161] AACTTAAAGAAAATTGACTAAAAAGATTAAATTAATACATAATATAAAATGGAGGTAGTAT

[0162] ATTATTCTTGGGCGAGATAGCTCTAACGTCTA

[0163] SEQ ID NO.2 CDS sequence of OsKNOX8 gene:

[0164] ATGGAGAGCTTCGCCAGTCTCGCAGGAGGTGGCAGCAGCAGCACCACAGCTCGGTTGCC

[0165] GGAGCTGATCTCGCCGGAGAACCCGGACCACATCTCGCCGCCCCCGTTGCTGTACCAGCT

[0166] GCTCGCCGGCCCGGAGTCGTCGGCGCGGCAGCATGGGCATGATGGGCATCACCACGGCG

[0167] GCGGTGGCGGCGAAGCAGCAGCAGCAGCTGTGCAGGGACAGGTCTCGCCGGCAGGCGC

[0168] GGAGGCGGCGGTCAAGGCCGAGATCATGTCGCATCCACAGTACTCGGCTCTCCTAGCCG

[0169] CCTACTTAGGCTGCAAAAAGGTCGGGGCGCCGCCGGATGTACTGACCAAGCTCACGGCT

[0170] GTGCCGGCGGCGCAGCAGCTGGACGAAGCCGACGGCCACCCTCGTCGCCGGCACGAGC

[0171] CACAGCGTGATGACGACCCGGATCAGCTCGATCAGTTCATGGACGCGTACTGCAGCATGC

[0172] TGACGAGGTACAGGGAGGAGCTGGAGCGGCCGATCCTGGAAGCCGCGGAGTTCTTCAG

[0173] CAGGGTGGAGACGCAGCTCGATTCGCTCGCTGAGAGTAATTGTGAAGGCACGGGATCAT

[0174] CAGAGGAAGAGCAAGACCCTAGTGACAAGCAGCTGAAGCACCAGCTTCTGAGGAAGTA

[0175] CGGTGGCTCGTTGGGCGATCTCCGGCAGGTGTTCTCCAAGAGGACCAAGAAAGGGAAG

[0176] CTTCCCAAGGAGGCCAGGCAGAAGCTTCTGCACTGGTGGGAGCTGCACTACAAGTGGCC

[0177] CTATCCCTCCGAGATGGAGAAGATGACGCTGGCGCAGACGACGGGGCTGGACCAGAAGC

[0178] AGATCAACAACTGGTTCATCAACCAGAGGAAGCGGCACTGGAAGCCGACGCCGGTGGC

[0179] AGGCACGGCCTTCCCGACGATGGAAGCTGCCGGAGGCGGCTTCCGACACTCCGGCCACG

[0180] GCGGCGGCCTTGCGGCGGCGGCGGCGCTGCCGCTGTACATGGGCAGGCCGTTCGTTGTG

[0181] GACGGCATGTACCGGCTCGGGTCTTGA

[0182] Amino acid sequence of the protein encoded by the gene OsKNOX8 of SEQ ID NO.3:

[0183] MESFASLAGGGSSSTTARLPELISPENPDHISPPPLLYQLLAGPESSARQHGHDGHHHGGGGG

[0184] EAAAAAVQGQVSPAGAEAAVKAEIMSHPQYSALLAAYLGCKKVGAPPDVLTKLTAVPAAQQ

[0185] LDEADGHPRRRHEPQRDDDPDQLDQFMDAYCSMLTRYREELERPILEAAEFFSRVETQLDSL

[0186] AESNCEGTGSSEEEQDPSDKQLKHQLLRKYGGSLGDLRQVFSKRTKKGKLPKEARQKLLH

[0187] WWELHYKWPYPSEMEKMTLAQTTGLDQKQINNWFINQRKRHWKPTPVAGTAFPTMEAAG

[0188] GGFRHSGHGGGLAAAAALPLYMGRPFVVDGMYRLGS

Claims

1. A method for genetically engineering the rice salt tolerance-related gene OsKNOX8, or a protein encoded by the gene OsKNOX8, or a knockout vector of the gene OsKNOX8, or a primer for amplifying the gene OsKNOX8 in regulating rice salt tolerance; characterized in that: The gene OsKNOX8 is a DNA molecule as described in 1) or 2) below: 1) A DNA molecule having a genome sequence as shown in SEQ ID NO.1; 2) A DNA molecule whose CDS sequence is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: Knocking out the gene OsKNOX8 described in claim 1, or reducing the expression level of the protein encoded by the gene OsKNOX8, or transferring the knockout vector of the gene OsKNOX8 into rice can improve the salt tolerance of rice.

3. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the gene OsKNOX8 is shown in SEQ ID NO.

3.

4. The use according to claim 1, characterized in that: The knockout vector of the gene OsKNOX8 is a CRISPR-Cas9 vector targeting the target shown in SEQID NO.

6.

5. The use according to claim 1, characterized in that: The primers for amplifying the gene OsKNOX8 are selected from Primer1 shown in SEQ ID NO.4 and Primer2 shown in SEQ ID NO.5, or Primer5 shown in SEQ ID NO.9 and Primer6 shown in SEQ ID NO.10.

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