A gene for improving the salt tolerance of rice, and a method and application for detecting and identifying the same

Abstract

The present invention provides a gene for improving the salt tolerance of rice, a method for detecting and identifying the same, and applications thereof, relating to the field of biotechnology. The gene is the osrst31 gene, and the nucleotide sequence of the osrst31 gene is as shown in SEQ ID NO: 1. The present invention can save breeding time, accelerate the breeding process, and reduce breeding costs. The entire detection process is simple to operate and highly accurate, greatly reducing the working cycle and costs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a gene for improving rice salt tolerance, and a method and application for its detection and identification. Background Art

[0002] Rice is an important staple food crop in China. On the one hand, with the advancement of the modernization process of industry and agriculture, the salinization of paddy fields in China is becoming increasingly serious. On the other hand, there are large areas of inland saline-alkali land and coastal saline-alkali land in China that have not been reclaimed for crop cultivation. Expanding the cultivation of rice to saline-alkali land can increase rice yields and ensure China's food security. Therefore, improving rice salt tolerance is an urgent task in rice breeding. Among the existing breeding methods, traditional cross-breeding takes a long time and the breeding results are unstable. Existing detection methods also have defects such as low sensitivity and excessive time consumption. Summary of the Invention

[0003] In view of the above-mentioned prior art, the present invention provides a gene for improving rice salt tolerance, and a method and application for its detection and identification.

[0004] A gene for improving rice salt tolerance provided by the present invention is the osrst31 gene, and the nucleotide sequence of the osrst31 gene is as shown in SEQ ID NO: 1.

[0005] The present invention also provides an application of the osrst31 gene in cultivating salt-tolerant rice.

[0006] The present invention also provides a primer set for identifying the osrst31 gene. The primer set includes two specific primers, and the nucleotide sequences of the two specific primers are as shown in SEQ ID No: 2 and SEQ ID No: 3 respectively.

[0007] The present invention also provides a method for identifying rice salt tolerance. Using the genomic DNA of a rice sample to be tested as a template, the template is amplified using the primer set, the amplified product is digested with EcoRI, and the digested product is analyzed by acrylamide electrophoresis. If the fragment size is 106 bp, then the individual is tolerant to salt stress.

[0008] The present invention also provides a kit for identifying rice salt tolerance, including the primer set.

[0009] The present invention also provides an application of the kit in identifying rice salt stress tolerance.

[0010] The beneficial effects of the present invention are as follows: In the present invention, a gene from the rice salt tolerance-enhanced mutant osrst31 has a 1-bp deletion in the coding region, resulting in disordered translation of the protein and premature termination. A primer set osrst31dcaps-F and osrst31dcaps-R for identifying this mutation site was developed. By comparing the fragment sizes of the amplification products of this primer, individuals carrying the functional osrst31 gene can be accurately identified at the seedling stage, saving breeding time, accelerating the breeding process, reducing breeding costs. The entire detection process is simple to operate and highly accurate, greatly reducing the working cycle and costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 FIG. is the amplification results of the DNA of the mutant osrst31, Zhonghua 11, and the F2 generation plants obtained by crossing the mutant osrst31 and Zhonghua 11 in the embodiment of the present invention using osrst31dcaps-F and osrst31dcaps-R; among them, lane 1 is the amplification band pattern of Zhonghua 11; lane 2 is the amplification band pattern of the mutant osrst31; lane 3 is the Marker; lanes 4-27 are the amplification bands of the F2 generation obtained by crossing the mutant osrst31 and Zhonghua 11.

[0012] Figure 2 FIG. Figure 1 FIG. is a photograph of the individuals corresponding to lanes 4-29 after being treated with 120 mM NaCl for 7 days. DETAILED DESCRIPTION OF THE INVENTION

[0013] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0014] Example 1: Development of dCAPS markers based on the rice osrst31 gene:

[0015] In the previous work, the present invention screened a salt tolerance-enhanced mutant osrst31. By map-based cloning, the candidate gene OsRST31 was cloned, which encodes an ABCG transporter protein.

[0016] The full-length sequence of the osrst31 gene from the mutant osrst31 is shown in SEQ ID No: 1. Sequencing revealed a 1-bp deletion in the third exon of the osrst31 gene. Based on this deletion, the present invention designed a pair of dCAPS primers, osrst31dcaps-F and osrst31dcaps-R, with nucleotide sequences shown in SEQ ID No: 2 and SEQ ID No: 3 respectively. This primer set was used to amplify in the parent osrst31 and the rice individuals with enhanced salt tolerance in its offspring. After digestion with EcoRI, the fragment length of the amplified product was 106 bp in the parent osrst31 and the rice individuals with enhanced salt tolerance in its offspring, and 84 bp in other rice individuals. The difference in band size could be observed in acrylamide gel electrophoresis, resulting in polymorphism. Through this marker, individuals with enhanced salt tolerance carrying the osrst31 gene were screened out.

[0017] Example 2: Application of the osrst31 molecular marker in the breeding of salt-tolerant rice:

[0018] (1) Population amplification detection and marker analysis: Seeds of the F1 generation were obtained by crossing the rice variety Zhonghua 11 and the salt stress tolerance-enhanced mutant osrst31. After planting, the individual plants of the F1 generation were obtained, and the individual plants of the F1 generation were self-pollinated to obtain seeds of the F2 generation.

[0019] Genomic DNA of some F2 individual plants was extracted using the CTAB method: At the seedling stage, fresh leaves of the recombinant inbred lines were selected, quickly frozen in liquid nitrogen, ground, and the total DNA of the samples was extracted using the CTAB method. The total DNA of the samples was subjected to PCR amplification using the primers osrst31dcaps-F and osrst31dcaps-R (SEQ ID No: 2 and SEQ ID No: 3), and PCR master mix was used for amplification.

[0020] PCR system:

[0021] 1 μL of genomic DNA

[0022] 5 μL of PCR master mix

[0023] 0.5 μL of osrst31-F

[0024] 0.5 μL of osrst31-R

[0025] 3 μL of sterile water

[0026] The PCR reaction program was: pre-denaturation at 94 °C for 5 minutes; denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 30 seconds, for 30 cycles; final extension at 72 °C for 10 minutes, and the amplified product was stored at 15 °C.

[0027] The amplified product was digested with EcoRI, and the digestion system was as follows:

[0028] 5 μL of the amplified product

[0029] 5 μL of Buffer

[0030] 1 μL of EcoRI

[0031] 39 μL of sterile water

[0032] The digestion program was: 37 °C for 2 hours

[0033] The amplified product was electrophoresed on a 10% non-denaturing polyacrylamide gel, and the gel formulation was as follows (50 ml):

[0034] 12.5 ml of 19:1 solution;

[0035] 5 ml of 10×TBE solution;

[0036] 32.5 ml of ddH2O;

[0037] 50 μL of TEMED;

[0038] 500 μL of 10% ammonium persulfate solution.

[0039] Among them, 10×TBE was prepared by dissolving 108 g of Tris base, 55 g of boric acid and 7.44 g of EDTA in distilled water, and then adding ddH2O to a final volume of 1000 ml; the 19:1 (100 ml) solution was prepared by dissolving 38 g of acrylamide and 2 g of N',N'-methylenebisacrylamide in pure water, diluting to 100 ml, filtering, and storing at 4 °C.

[0040] The electrophoresis was carried out at a constant voltage. The DYY-6E type electrophoresis power supply (Beijing Liuyi) and JY-CX2B type electrophoresis tank (Beijing Junyi) were used. Note to be slow and patient during the gel casting process to avoid the generation of bubbles. After the gel was completely solidified, a 2.5 μL range pipette (Eppendorf) was used for sample loading. The sample loading volume was 1.5 μL, the voltage was 220 V, the current was 100 mA, and the electrophoresis duration was 120 min. After electrophoresis, the gel was taken out for silver staining and color development.

[0041] Silver staining: The gel was washed twice with ultrapure water and then added to 1 L of silver staining solution (0.1% aqueous silver nitrate solution); gently shaken on a shaker for 15 minutes, the silver staining solution was poured out, and the gel was rinsed twice with ultrapure water.

[0042] Color development: 1 L of color development solution (1.5% aqueous sodium hydroxide solution) was added, and then 1 ml of formaldehyde was added. Gently shake until the bands were clear; take out the gel plate, wash it 3 times with tap water, read the band pattern on a light box, and analyze the bands.

[0043] (2) Results and analysis: The partial detection results of the genotypes of the F2 population of osrst31 and Zhonghua 11 are as follows Figure 1 shown. Lane 1 is the amplification band pattern of Zhonghua 11, with a fragment size of 84 bp; Lane 2 is the amplification band pattern of the mutant osrst31, with a fragment size of 106 bp; Lane 3 is the Marker, with the lowest band size of 50 bp, the second-to-last band being 100 bp, and the third-to-last band being 400 bp. The following are the amplification bands of some F2 individuals. Among them, it was found that the amplification band sizes of 22 individual plants were 84 bp, the amplification band sizes of 7 individual plants were 106 bp, and the amplification bands of 2 individual plants were heterozygous, that is, they contained two bands with sizes of 84 bp and 106 bp at the same time.

[0044] After Figure 1 the F2 individuals subjected to detection were treated with 120 mM NaCl for 7 days (as Figure 2 shown), the individuals with an amplification band of 106 bp ( Figure 2 the 7th individual in the 3rd row in

[0045] showed enhanced phenotypic tolerance to salt stress. This indicates that the osrst31dcaps primer set can be used as a material for accurately identifying salt stress tolerance. The above is only the implementation mode of the present invention, and does not limit the patent scope of the present invention accordingly. Any equivalent structure made by using the content of the specification of the present invention, directly or indirectly applied in other related technical fields, is similarly within the patent protection scope of the present invention.

Claims

1. A gene for improving the salt tolerance of rice, characterized in that, It is the osrst31 gene, and the nucleotide sequence of the osrst31 gene is shown as SEQ ID NO:

1.

2. Use of the osrst31 gene according to claim 1 in cultivating salt-tolerant rice.

3. A primer set for identifying the osrst31 gene according to claim 1, characterized in that, The nucleotide sequences of the two specific primers of the primer group are shown as SEQ ID No: 2 and SEQ ID No: 3 respectively.

4. A kit for identifying the salt tolerance of rice, characterized in that, It includes the primer group according to claim 3.

5. Use of the kit according to claim 4 in identifying the salt stress tolerance of rice.

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