CN118360329B - Method for rapidly fixing rice heterosis - Google Patents

Abstract

The present invention belongs to the field of biotechnology and plant breeding, and specifically relates to a method for quickly fixing hybrid vigor of rice. The technical scheme of the present invention is mainly divided into vector construction, genetic transformation, ploidy and genotype identification and result detection. The technology provided by the present invention is to directly knock out 5 endogenous genes of rice by gene editing to obtain a rice apomixis system with a high fruiting rate, which provides a new idea for the construction of a rice apomixis system; it also provides a new solution for fixing hybrid vigor of rice apomixis, which can greatly improve the efficiency and quality of agricultural production, and has great economic value and broad application prospects.

Description

A method for quickly fixing heterosis in rice

Technical Field

The present invention belongs to the field of biotechnology and plant breeding, and specifically relates to a method for creating an apomixis system by using gene editing to fix the hybrid vigor of rice.

Background Art

Hybrid vigor is the phenomenon that after hybridization of two varieties or similar species with different genetic backgrounds, the offspring surpasses both parents in growth potential, vitality, adaptability and yield. It is a wonderful manifestation in the biological world, especially in the breeding and production practice of crop varieties. However, hybrid vigor is not eternal. The offspring of the first generation of hybrids often show separation of traits or fertility and cannot maintain their advantages for a long time. Therefore, new seed production is required every year, which undoubtedly consumes a lot of manpower, material resources and land resources.

In contrast, apomixis can firmly fix heterosis. The seeds obtained through this method of reproduction are genetically identical to the mother plant and can continue to have their genotype unchanged and their traits will not separate over generations.

At present, the apomixis system has been initially established, mainly relying on two strategies: one is to completely knock out endogenous genes through gene editing technology; the other is to ectopically express parthenogenetic genes while knocking out endogenous genes. The former induces apomixis by knocking out three key genes (MiMe) involved in reduction and tillering, combined with the knockout of haploid induction genes. This method is simple and stable, and only requires ensuring the homozygous mutation of four genes, but its fruiting rate and cloning efficiency need to be improved, affecting its practical application in breeding. The latter, on the basis of knocking out the MiMe gene, promotes the autonomous development of egg cells by ectopically expressing parthenogenetic genes. However, due to the characteristics of ectopic expression, there may be differences between strains, resulting in fluctuations in the efficiency of heterosis fixation.

Although the first strategy has the advantages of being simple, direct and stable, its insufficient cloning efficiency limits its wide application. The present invention uses gene editing technology to knock out five endogenous genes, successfully constructs an apomixis system with significantly improved cloning efficiency, achieves efficient fixation of hybrid vigor, opens up a new path for the application of apomixis, and provides a practical solution.

Summary of the invention

The main problem solved by the present invention is to overcome the shortcomings of the prior art and provide a method for quickly fixing the heterosis of rice, which can achieve permanent fixation of the heterosis of rice. In order to achieve the above purpose, the present invention includes the following steps:

(1) First, an expression cassette A for CRISPR/Cas9 knockout of three target sites of rice genes OsPAIR1, OsREC8 and OsOSD1 was constructed, and then two target sites of rice genes OsHAP2 and OsGEX2 were connected to the expression cassette A, thereby constructing an expression cassette B for CRISPR/Cas9 knockout of five target sites of rice OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2. The sequences of the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 are shown in SEQ ID NOs. 1-5, and the target site sequences of the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 are shown in SEQ ID NOs. 6-10;

(2) transforming the expression cassette B into hybrid rice by Agrobacterium-mediated method to obtain T0 generation plants;

(3) Screening T0 generation plants with homozygous knockout of the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2, and OsGEX2, and obtaining seeds through self-pollination;

(4) Germinating the seeds obtained from the self-pollination, and screening plants with fixed hybrid vigor using flow cytometry and genome sequencing technology.

Furthermore, in the method for rapidly fixing rice heterosis of the present invention, the specific method for obtaining the expression cassette B in (1) is:

1) Design target sequences based on the coding region sequences of five genes, OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2;

2) Integrate the target sequence into the SK-gRNA vector respectively to obtain five intermediate vectors SG1, SG2, SG3, SG4 and SG5;

3) Use the enzyme ligation method to connect the three intermediate vectors SG1, SG2 and SG3 to the backbone vector pC1300-Cas9 containing the CRISPR/Cas9 expression element to obtain expression cassette A;

4) Then use the enzyme cutting and ligation method to connect the two intermediate vectors SG4 and SG5 to the expression cassette A to obtain the expression cassette B.

Furthermore, in the method for rapidly fixing rice heterosis of the present invention, during the genetic transformation in (2), a genetic transformation method mediated by Agrobacterium EHA105 strain is utilized.

Furthermore, in the method for rapidly fixing rice heterosis of the present invention, the hybrid rice variety transformed in (1) is the indica-japonica hybrid rice variety Chunyou 84.

Furthermore, in the method for rapidly fixing hybrid vigor of rice of the present invention, the specific steps of screening T0 generation plants in which the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 are homozygous knocked out in (3) are as follows: first, according to the gene sequences of OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2, Hi-TOM detection primers PAIR1-Hi-F/R, REC8-Hi-F/R, OSD1-Hi-F/R, HAP2-Hi-F/R, GEX2-Hi-F/R are designed; then, the T0 generation transgenic plants are amplified; finally, the mutation types of the five genes of all plants are detected by using the Hi-TOM system, and the transgenic plants in which the five genes are homozygous knocked out are screened.

Furthermore, the method for rapidly fixing rice heterosis of the present invention comprises the following steps: in said (4), using flow cytometry and genome sequencing technology to detect plants in which the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 are homozygous knocked out, and screening plants with fixed heterosis.

1) The ploidy of T0 plants in which the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 were homozygous knocked out was detected by flow cytometry, and the plants with diploid ploidy were selected;

2) The above diploid plants are tested using genome sequencing technology, and plants with fixed genotypes are selected.

Finally, the present invention also provides the application of the above-mentioned method for rapidly fixing rice heterosis in preparing excellent rice varieties.

Technical Effects

The technology provided by the present invention is to directly knock out five endogenous genes of rice through gene editing to obtain a rice apomixis system with higher cloning efficiency, which provides a new solution for the construction of a rice apomixis system; it also provides a new solution for fixing hybrid advantages in rice apomixis, which can greatly improve the efficiency and quality of agricultural production, has certain economic value, and thus has broad application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a vector map

Figure 2 is the mutation type detection of transgenic plants

Figure 3 shows the fruiting rate and cloning efficiency data of transgenic plants

Figure 4 is the phenotype of transgenic T0 plants

Figure 5 is the screening of diploids by flow cytometry

Figure 6 is the genome sequencing data

Figure 7 is a phenotype diagram of cloned plants

DETAILED DESCRIPTION

This method was applied to the indica-japonica hybrid rice Chunyou 84, and a transgenic line with a high and stable fruiting rate was obtained. In the ploidy identification of the offspring plants, diploid plants with fixed genotypes were successfully identified. It mainly includes vector construction, genetic transformation, ploidy and genotype identification, and result detection.

1. Expression cassette construction

A vector for knocking out the five endogenous genes of rice, OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2, was constructed, and the target sites of the five endogenous genes were connected to the backbone vector pC1300-Cas9 containing CRISPR/Cas9 expression elements to obtain expression cassette B. The specific construction method is as follows:

The target sequences were designed based on the coding region sequences of the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2. The primers are as follows (the nucleotide sequences of the primers are shown in SEQ ID NOs. 11-20):

OsPAIR1++:GGCAAAGCAACCCAGTGCACCGC

OsPAIR1--:AAACGCGGTGCACTGGGTTGCTT

OsREC8++:GGCACGGAGAGCCTTAGTGCCAT

OsREC8--:AAACATGGCACTAAGGCTCTCCG

OsOSD1++:GGCACTGCCGCCGACGAGCAACA

OsOSD1--:AAACTGTTGCTCGTCGGCGGCAG

OsHAP2++:GGCAGCTTCCGGTCGCACTTGAGG

OsHAP2--:AAACCCTCAAGTGCGACCGGAAGC

OsGEX2++:GGCAGTGTCGGTGTCCCGTAGCAA

OsGEX2--:AAACTTGCTACGGGACACCGACAC

Digest the backbone vector SK-gRNA:

COMPONENT 50 µl REACTION

SK-gRNA 2 µg

10 x Buffer Aar I 5 µl

Aar I 1 µl

50 x oligonucleotide 1 µl

Nuclease-free Water to 50 µl

The enzyme was digested at 37°C for 5 h, and the product was purified using a recovery kit to obtain SK-gRNA.

Primer annealing to double strands

The synthesized primers (g++ and g --) were diluted with water to a concentration of 100µM, 20µL each of g++ and --g were mixed together, incubated at 100°C for 5 minutes, and then left to cool naturally at room temperature.

The target sequences were integrated into the SK-gRNA vectors to obtain five intermediate vectors: SG1, SG2, SG3, SG4 and SG5:

COMPONENT 10 µl REACTION

SK-gRNA cut 30ng

10 x T4 ligase Buffer 1 µl

T4 Ligase 0.5 µl

Primer annealing product 7 µl

Nuclease-free Water to 10 µl

Ligate at 25°C for 1 hr to allow transformation.

Transformation of recombinant products:

Thaw the chemical competent cells for cloning on ice; add 10µl of the recombinant product to 500µl of competent cells, flick the tube wall to mix, and place on ice for 30 min; heat shock in a 42℃ water bath for 45 sec, and immediately place on ice to cool for 2 min; add 900µl of LB medium (without antibiotics), shake at 37℃ for 1 h (speed 200 rpm); centrifuge at 5,000 rpm for 1 min, keep 100µl to resuspend, and spread on the corresponding resistance plate; incubate inverted at 37℃ incubator for 12 - 16 h. Positive clones were detected by colony PCR and sent to the company for sequencing. Sequencing primer T3: ATTAACCCTCACTAAAGGGA (nucleotide sequence as shown in SEQ ID NO.21).

Digest the five intermediate vectors SG1, SG2, SG3, SG4 and SG5:

COMPONENT 50 µl REACTION

SG1 1 µg

10X rCutSmart Buffer 5 µl (1X)

KpnI-HF 20 units

SalI-HF 20 units

Nuclease-free Water to 50 µl

The enzyme digestion was carried out at 37°C for 5 h, and the product was purified using a recovery kit to obtain SG1.

COMPONENT 50 µl REACTION

SG2 1 µg

10X rCutSmart Buffer 5 µl (1X)

XhoI 20 units

NheI-HF 20 units

Nuclease-free Water to 50 µl

The enzyme digestion was carried out at 37°C for 5 h, and the product was purified using a recovery kit to obtain SG2.

COMPONENT 50 µl REACTION

SG3 1 µg

10X r3.1 Buffer 5 µl (1X)

XbaI 20 units

BglII 20 units

Nuclease-free Water to 50 µl

The enzyme was digested at 37°C for 5 h, and the product was purified using a recovery kit to obtain SG3.

COMPONENT 50 µl REACTION

SG4 1 µg

10X rCutSmart Buffer 5 µl (1X)

KpnI-HF 20 units

SalI-HF 20 units

Nuclease-free Water to 50 µl

The enzyme was digested at 37°C for 5 h, and the product was purified using a recovery kit to obtain SG4.

COMPONENT 50 µl REACTION

SG5 1 µg

10X r3.1 Buffer 5 µl (1X)

XhoI 20 units

BglII 20 units

Nuclease-free Water to 50 µl

The enzyme was digested at 37°C for 5 h, and the product was purified using a recovery kit to obtain SG5.

Digest the vector backbone pC1300-Cas9:

COMPONENT 50 µl REACTION

pC1300-Cas9 1 µg

10X rCutSmart Buffer 5 µl (1X)

KpnI-HF 20 units

BamHI-HF 20 units

Nuclease-free Water to 50 µl

The enzyme was digested at 37°C for 5 h, and the product was purified using a recovery kit to obtain pC1300-Cas9.

Expression cassette A construction:

COMPONENT 10 µl REACTION

pC1300-Cas9 cut 100ng

SG1 cut 8ng

SG2 cut 8ng

SG3 cut 8ng

10 x T4 ligase Buffer 1 µl

T4 Ligase 0.5 µl

Nuclease-free Water to 50 µl

Ligate at 25°C for 1 hr to allow transformation.

Transformation of recombinant products:

Thaw the chemical competent cells for cloning on ice; take 10µl of the recombinant product and add it to 500µl of competent cells, flick the tube wall to mix, and place it on ice for 30 min; heat shock in a 42℃ water bath for 45 sec, and immediately place it on ice for 2 min; add 900µl LB medium (without antibiotics), shake at 37℃ for 1 h (speed 200 rpm); centrifuge at 5,000 rpm for 1 min, keep 100µl to resuspend, and spread on the corresponding resistance plate; incubate inverted at 37℃ incubator for 12 - 16 h. Positive clones were detected by colony PCR and sent to the company for sequencing. The sequencing primer pC1300-F: acactttatgcttccggctc (nucleotide sequence as shown in SEQ ID NO.22).

Expression cassette A is digested by enzyme:

COMPONENT 50 µl REACTION

Expression Cassette A 1 µg

10X rCutSmart Buffer 5 µl (1X)

KpnI-HF 20 units

BamHI-HF 20 units

Nuclease-free Water to 50 µl

The enzyme digestion was carried out at 37°C for 5 h, and the product was purified using a recovery kit to obtain the expression cassette A.

Expression cassette B construction:

COMPONENT 10 µl REACTION

Expression cassette A cut 100ng

SG4 cut 8ng

SG5 cut 8ng

10 x T4 ligase Buffer 1 µl

T4 Ligase 0.5 µl

Nuclease-free Water to 50 µl

Ligate at 25°C for 1 hr to allow transformation.

Transformation of recombinant products:

Thaw the chemical competent cells for cloning on ice; take 10µl of the recombinant product and add it to 500µl of competent cells, flick the tube wall to mix, and place it on ice for 30 min; heat shock in a 42℃ water bath for 45 sec, and immediately place it on ice for 2 min; add 900µl LB medium (without antibiotics), shake at 37℃ for 1 h (speed 200 rpm); centrifuge at 5,000 rpm for 1 min, keep 100µl to resuspend, and spread on the corresponding resistance plate; incubate inverted at 37℃ incubator for 12 - 16 h. Positive clones were detected by colony PCR and sent to the company for sequencing. The sequencing primer pC1300-F: acactttatgcttccggctc (nucleotide sequence as shown in SEQ ID NO.22).

2. Genetic transformation

The sequencing of the cloned vector was correct, and the next step was to carry out the Agrobacterium transformation experiment. The indica-japonica hybrid rice variety Chunyou 84 (CY84) was transformed using the genetic transformation method mediated by the Agrobacterium EHA105 strain to obtain transgenic materials. The seeds were shelled, disinfected with 75% ethanol for 1 min, the ethanol was poured out, and 2% sodium hypochlorite solution was added for disinfection for 20 min, and placed on a shaker during this period; the sodium hypochlorite solution was poured out in the clean bench, and the seeds were rinsed with sterile water for 4-5 times, and the seeds were placed on sterilized filter paper to absorb the water; then the seeds were inoculated on N6 mature embryo callus induction medium, cultured in the dark at 28℃ for about 1 month, and the embryonic callus in good condition was selected for subculture 2-3 times, and the embryonic callus of the second subculture 3-5 days was selected for transformation.

The embryonic callus was immersed in the activated Agrobacterium EHA105 bacterial solution (containing acetosyringone) with the target plasmid for 30 min, the callus tissue was washed several times with sterile water, the residual liquid was blown dry in the clean bench, and co-cultured at 19°C for 2-3 days, and then transferred to the screening medium with screening marker antibiotics for screening. Each screening process lasted for 2 weeks. After 2-3 rounds of screening, newly grown callus tissue was obtained. Then the newly grown callus tissue was transferred to the pre-differentiation medium for 7 days, and then transferred to the differentiation medium. It was cultured at 25°C and 16 h/d for about 10 days. Green dots appeared, and then regenerated plants were obtained. After the roots of the differentiated transgenic seedlings were cut off, they were placed in the rooting medium for 2-3 weeks, and then the sealing film was opened, water was added to harden the seedlings for 1 week, and transplanted.

3. Mutation detection of transgenic T0 plants

The method for screening T0 generation plants with homozygous mutations in the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 is as follows:

Hi-TOM detection primers were designed based on the gene sequences of OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2. The primer sequences are as follows (the nucleotide sequences are shown in SEQ ID NOs. 23-32):

PAIR1-Hi-F：ggagtgagtacggtgtgccttcttgcgcgcgagaagagtctc

PAIR1-Hi-R：gagttggatgctgagtggggagatgtagtgcgtgggtcttg

REC8-Hi-F:ggagtgagtacggtgtgcttgggttagtgaggagat

REC8-Hi -R：gagttggatgctgagtggtgcgatcggaactatggagac

OSD1-Hi-F：ggagtgagtacggtgtgctatcaggaggacgacgtcgccg

OSD1-Hi-R：gagttggatgctgagtggctcctcctcttgggtgtagc

HAP2-Hi-F:ggagtgagtacggtgtgcATGCGGAACGGAGATCCT

HAP2- Hi-R:gagttggatgctgagtggAAGACGGGACGGAGTAGA

GEX2- Hi-F:ggagtgagtacggtgtgcCGGTCGCTCTCAGGGAC

GEX2- Hi-R:gagttggatgctgagtggCTGATGCGTCCATCTTCC

The above five pairs of primers were used to perform PCR amplification on T0 generation transgenic plants, and the mutation types of the five genes of all plants were detected using the Hi-TOM system to screen transgenic plants with homozygous mutations in all five genes.

4. Plant ploidy and genotype identification:

Flow cytometry and genome sequencing technologies were used to detect the offspring of T0 transgenic plants with homozygous mutations in all five genes to screen plants with fixed heterosis, specifically:

Flow cytometry was used to identify the ploidy of offspring plants of T0 transgenic plants with homozygous mutations in all five genes. The specific experimental operation was as follows:

Cut fresh rice leaves of 4-5 cm in length that have grown for 10 days and place them in a glass dish. Add 1 ml of plant lysis buffer LB01 and chop the tissue vertically and quickly with a blade. Pipette the lysate in the culture dish and filter it into a centrifuge tube with a 50 µm nylon mesh. Mark the sample on the tube cap. Centrifuge at 1,200 rpm for 5 min at 4°C in a desktop refrigerated centrifuge. Gently remove the centrifuge tube, slowly remove the supernatant, and add 450 µl of LB01, 25 µl of pre-cooled PI and 25 µl of RNase A. Stain at 4°C in the dark for 10 min. Detect on BD Accuri C6. If it is a diploid, its peak should be consistent with the wild type peak.

The specific reagent formula is:

Lysis buffer LB01: Tris 363.4 mg, Na2EDTA 148.9 mg, Sperminetetrahydrochloride 34.8 mg, KCl 1.193 g, NaCl 233.8 mg, Triton X-100 200 µl, dilute to 200 mL, adjust pH to 7.5 with 1M HCl, add 220 µl β-mercaptoethanol in a fume hood. Filter sterilize and aliquot using a 0.22 µm filter in a clean bench, and store at -20°C.

Propidium iodide (PI) stock solution (1 mg/ml): Weigh 50 mg of powder and dissolve it in 50 mL of ddH2O; filter and sterilize with a 0.22 μm filter in a clean bench and store at -20°C.

RNase stock solution (1 mg/ml): weigh 25 mg RNase (IIA Sigma) and dissolve in 25 ml ddH2O; sterilize by filtration using a 0.22 µm filter in a clean bench and aliquot; heat at 90°C for 15 min to inactivate the DNase; store at -20°C.

The genotype of diploid plants was detected using genome sequencing technology, specifically:

For the diploid plants detected above, their DNA was extracted and libraries were constructed. Double-end sequencing was performed using the Illumina Hiseq2500 sequencing platform, and the average sequencing depth of each sample was 10-15 times. First, the obtained raw data was filtered using NGSQCtoolkit v2.3.3, and then the filtered data was aligned to the reference genome to obtain SNPs data. Finally, the SNPs data was aligned with the wild-type Chunyou 84 genome to clarify the genotype of the diploid plants. If it is a diploid plant with fixed hybrid vigor, its genome is theoretically a heterozygous genotype consistent with Chunyou 84.

5. Test results:

A total of 18 strains were obtained through genetic transformation. The mutation types of five genes, OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2, were detected using Hi-TOM detection technology. The results showed that only one strain, MGH-14, had homozygous mutations in all five genes (Figure 2). The growth and development of this strain was consistent with the wild type, and the fruit set rate was significantly lower than that of the wild type (3.26%) (Figure 3, Figure 4). When the seeds matured, the seeds of this strain were harvested and the germinated seeds were used for diploid identification. Its ploidy was identified by flow cytometry, and it was found that a high proportion of diploid offspring were produced, with a cloning efficiency of 42.86% (Figure 3, Figure 5). Subsequently, the genotypes of diploid plants were detected using genome sequencing technology, and the results showed that all diploid genotypes were consistent with the genotype of Chunyou 84 (Figure 6), and they were all apomixis clones, and their growth and development were consistent with the wild type (Figure 7).

Claims (6)

1. A method for rapidly fixing rice heterosis, characterized in that it comprises the following steps: (1) First, an expression cassette A for CRISPR/Cas9 knockout of three target sites of rice genes OsPAIR1, OsREC8 and OsOSD1 was constructed, and then two target sites of rice genes OsHAP2 and OsGEX2 were connected to the expression cassette A, thereby constructing an expression cassette B for CRISPR/Cas9 knockout of five target sites of rice OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2. The sequences of the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 are shown in SEQ ID NOs. 1-5; the target sequences of the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 are shown in SEQ ID NOs. 6-10; (2) transforming the expression cassette B into hybrid rice by Agrobacterium-mediated method to obtain T0 generation plants; (3) Screening T0 generation plants with homozygous knockout of the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2, and OsGEX2, and obtaining seeds through self-pollination; (4) Germinating the seeds obtained from the self-pollination, and screening plants with fixed hybrid vigor using flow cytometry and genome sequencing technology. 2. The method for rapidly fixing rice heterosis according to claim 1, characterized in that the specific method for obtaining the expression cassette B in (1) is: 1) Design target sequences based on the coding region sequences of five genes: OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2; 2) Integrate the target sequence into the SK-gRNA vector respectively to obtain five intermediate vectors SG1, SG2, SG3, SG4 and SG5; 3) Use the enzyme ligation method to connect the three intermediate vectors SG1, SG2 and SG3 to the backbone vector pC1300-Cas9 containing the CRISPR/Cas9 expression element to obtain expression cassette A; 4) Then use the enzyme cutting and ligation method to connect the two intermediate vectors SG4 and SG5 to the expression cassette A to obtain the expression cassette B. 3. The method for rapidly fixing rice heterosis according to claim 2, characterized in that, during the genetic transformation in (2), a genetic transformation method mediated by Agrobacterium EHA105 strain is used. 4. The method for rapidly fixing rice heterosis according to claim 3, characterized in that the hybrid rice variety transformed in (2) is the indica-japonica hybrid rice variety Chunyou 84. 5. The method for rapidly fixing rice heterosis according to claim 4, characterized in that the specific steps of screening T0 generation plants in which the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 are homozygous knocked out in said (3) are: firstly designing Hi-TOM detection primers PAIR1-Hi-F/R, REC8-Hi-F/R, OSD1-Hi-F/R, HAP2-Hi-F/R, GEX2-Hi-F/R according to the gene sequences of OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2, then amplifying the T0 generation transgenic plants, and finally using the Hi-TOM system to detect the mutation types of the five genes of all plants to screen the transgenic plants in which the five genes are homozygous knocked out. 6. The method for rapidly fixing heterosis of rice according to claim 5, characterized in that in said (4), flow cytometry and genome sequencing technology are used to detect plants in which the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 are homozygous knocked out to screen plants with fixed heterosis, and the specific steps are as follows: 1) The ploidy of T0 plants in which the five genes OsPAIR1, OsREC8, OsOSD1, OsHAP2 and OsGEX2 were homozygous knocked out was detected by flow cytometry, and the plants with diploid ploidy were selected; 2) The above diploid plants are tested using genome sequencing technology, and plants with fixed genotypes are selected.