Method for improving the temperature of the starting point of sterility of a rice tms5 two-line sterile line

Abstract

The present invention provides a method for improving rice yield tms5 The method for the sterility threshold temperature of two-line sterile lines comprises the following steps: TMS5 CRISPR / Cas9 gene editing targets T501 and T502 were designed 22bp and 57bp downstream of the gene translation start site ATG, respectively, and gene editing vectors TMS501 and TMS502 were constructed to transform rice varieties to achieve TMS5 Site-directed mutation of genes to obtain different multiple alleles tms5 Gene frameshift mutant material. Experiments have shown that in the same genetic background, the T501 target site editing tms5-1 The sterility starting temperature of the frameshift mutants was significantly higher than that of the T502 target. tm5-2 Mutants. The method provided by the present invention can quickly obtain rice with different sterility threshold temperature phenotypes under the same genetic background. tms5 Mutants can overcome the interference of different genetic backgrounds and provide a basis for studying rice tms5 The molecular mechanism and genetic regulatory network of the starting temperature of two-line sterility provide new ideas.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant transgenic technology and crop genetic breeding, and particularly relates to a method for increasing the sterile starting temperature of a rice tms5 two-line sterile line, which utilizes CRISPR-Cas9 technology to edit a 22-bp site downstream of a translation initiation site ATG of a rice TMS5 gene, to achieve a frame shift mutation, and thus obtain a rice tms5 two-line sterile line with a significantly increased sterile starting temperature. BACKGROUND

[0002] In the breeding process of two-line hybrid rice, breeding a two-line sterile line with a low sterile starting temperature is crucial for producing qualified hybrid rice seeds, and the tms5 sterile lines currently used in production exhibit different sterile starting temperatures, and the genetic regulation mechanism thereof is relatively complex. Previous studies have shown that the interaction between multiple microgenes determines the sterile starting temperature of photoperiod-thermosensitive genic male sterile rice, and the impurity of the genetic basis may be the internal cause of the genetic drift of the sterile starting temperature. In recent years, multiple research teams have used CRISPR / Cas9 technology to edit the TMS5 site of multiple indica and japonica rice varieties, and most of the indica sterile lines obtained have a starting temperature lower than 24°C, which meets the actual needs of two-line sterile line breeding, but all the japonica sterile lines have a starting temperature higher than 26°C, and some materials are completely sterile only at 32°C, which cannot meet the actual production needs of two-line rice breeding. The above results show that different tms5 sterile lines exhibit significantly different thermosensitive sterile starting temperatures due to differences in genetic backgrounds, and the starting temperature of japonica materials is too high to meet the production needs. It is believed by Zhou Hai, Chen Rirong, Huang Zhongming, and others that the sterile starting temperature of different tms5 sterile lines is determined by the genetic background of different rice varieties, and is not related to the tms5 mutant gene itself.

[0003] In previous studies, we analyzed the sterile starting temperatures of a series of tms5 mutants and found that the tms5 mutants in the japonica background had a higher sterile starting temperature than those in the indica background, which is consistent with previous reports. However, we also found that the sterile starting temperatures of different target site mutants were different in the same genetic background. Whether in the japonica Nipponbare or in the indica Minghui 86 background, the sterile starting temperature of the T501 target site mutant (tms5-1) was significantly higher than that of the mutants produced at other target sites, which is a new phenomenon that has not been reported. Further expression analysis showed that in the Minghui 86 and Nipponbare backgrounds, the mRNA expression of the three Ub L40 s was higher in the tm5-2 mutant with a lower sterile starting temperature, and lower in the tms5-1 mutant with a higher sterile starting temperature, which is consistent with the current explanation of the difference in sterile starting temperature.

[0004] From the current existing research on rice tms5 mutant, different researchers have observed that the sterile starting temperature of tms5 mutant is affected by different genetic backgrounds, but the specific mechanism is still lacking in-depth understanding. This may be because the genetic background involved in the related research is too complex, making it difficult to analyze directly, and new discoveries and new materials are needed. In the process of analyzing the sterile starting temperature of tms5 mutant created by gene editing, it is found that the sterile starting temperature of tms5 is not only affected by the genetic background, but also affected by some different target mutants. Systematic analysis and in-depth study of these tms5 mutants can provide new ideas for revealing the molecular mechanism of the sterile starting temperature of rice tms5 mutant. SUMMARY

[0005] The purpose of the present application is to provide a method for cultivating a rice tms5 two-line sterile line with significantly improved sterile starting temperature by using CRISPR-Cas9 technology to edit the 22bp position downstream of the translation initiation site ATG of the rice TMS5 gene.

[0006] Another purpose of the present application is to use tms5 mutants with the same genetic background but different sterile starting temperatures to study the genetic regulation mechanism of the sterile starting temperature of two-line rice.

[0007] The present application is realized by the following technical solutions:

[0008] The method for cultivating a rice tms5 two-line sterile line with significantly improved sterile starting temperature by using CRISPR-Cas9 technology to edit the 22bp position downstream of the translation initiation site ATG of the rice TMS5 gene is particularly suitable for studying the genetic regulation mechanism of the sterile starting temperature of tms5 two-line rice in the same genetic background;

[0009] Comprising the following steps:

[0010] 1) The 23bp sequence 5'-GAACAGCGGCAAGTCATCGCCGG-3' in the first exon of the TMS5 gene is preferably used as the sgRNA target sequence. The sgRNA specifically targets the 22bp position downstream of the translation initiation site ATG of the TMS5 gene. The sequence of the sgRNA is as follows: 5'-GAACAGCGGCAAGTCATCGC-3'.

[0011] 2) Refer to the CRISPR / Cas9 editing technology to construct a CRISPR-TMS501 gene editing vector.

[0012] 3) Use the Agrobacterium-mediated genetic transformation method to transfer the CRISPR-TMS501 gene editing vector into rice to obtain regenerated plants.

[0013] 4) According to the TMS5 gene sequence targeted by the sgRNA, TMS5 gene-specific primers are designed at 100-500bp upstream and downstream of the sgRNA target site according to the principle of PCR primer design, and the primer base sequences are as follows: TMS5F: 5'-CCATCGTGCTTCGTGCCA-3' as shown in SEQ ID NO. 1

[0014] TMS5R: 5'-GAGTTCTTGGTACATGAGTGC-3' as shown in SEQ ID NO. 2

[0015] 5) The genomic fragment of the TMS5 gene is amplified by PCR using the primers and sequenced to identify and screen the frameshift tms5 mutant strain with base insertion or deletion at the TMS5 gene target site.

[0016] 6) The pollen fertility of each mutant strain is investigated and identified under field and artificial climate chamber conditions to obtain a temperature-sensitive male sterile line and breed the mutant strain.

[0017] A tms5 mutant with the same genetic background but different sterile starting temperature is used to study the genetic regulation mechanism of the two-line rice sterile starting temperature, and the application is specifically that, under the same genetic background, the tms5 mutant with a higher sterile starting temperature and the tms5 mutant with a lower sterile starting temperature are used to screen and analyze the differential expression genes during the development of the young ear, and the genes related to the sterile starting temperature are mined, thereby providing a new strategy for studying the molecular mechanism and genetic regulation network of the two-line rice tms5 sterile starting temperature.

[0018] The application has the following beneficial effects:

[0019] 1) The application utilizes the characteristics of TMS5 gene and its protein participating in the development regulation of rice pollen fertility and the genome targeting modification of the CRISPR / Cas9 system, and by site-directed mutagenesis of the gene, a temperature-sensitive nuclear sterile rice can be created, which has a very important application in hybrid rice production.

[0020] 2) The application uses the CRISPR / Cas9 gene editing technology to site-directly mutate the 22bp downstream of the translation initiation site ATG of the rice TM5 gene, and obtains a rice tms5 mutant with significantly improved sterile starting temperature, which is a new genetic research material and has important research and utilization value.

[0021] 3) The application provides a new idea and strategy for studying the molecular mechanism and genetic regulation network of the two-line rice tms5 sterile starting temperature. DETAILED DESCRIPTION

[0022] Figure 1Sequence of two mutant lines of Minghui 86, the left sequence is a frameshift mutation of inserting 1 base A at T501 target site, and the right sequence is a frameshift mutation of inserting 1 base A at T502 target site.

[0023] Figure 2 Flowering characteristics of wild type, tms5-1 mutant and tms5-2 mutant of two genetic backgrounds of Minghui 86 and Nipponbare. The tms5 mutant of two backgrounds is under the natural long-day high-temperature condition in Fuzhou in early August, MH86 is Minghui 86, NIP is Nipponbare, M-# and N-# represent the mutant of Minghui 86 and Nipponbare genetic background respectively, and M-T501-#1 represents fertile and sterile glume flowers on the same spike.

[0024] Figure 3 Pollen fertility of tms5-1 and tms5-2 mutants of Minghui 86 and Nipponbare genetic background under artificial climate chamber 22℃, 24℃, 28℃ and natural high-temperature condition in Fuzhou in early August (NHT). MH86 is Minghui 86, NIP is Nipponbare, M-# and N-# represent the mutant of Minghui 86 and Nipponbare genetic background respectively. DETAILED DESCRIPTION

[0025] The application will be further described in conjunction with an embodiment. The instruments, reagents, materials and the like involved in the following examples, if not specifically stated, are all conventional instruments, reagents, materials and the like in the prior art, which can be obtained through regular commercial channels. The experimental methods, detection methods and the like involved in the following examples, if not specifically stated, are all conventional experimental methods, detection methods and the like in the prior art.

[0026] Example 1

[0027] The application constructs CRISPR-TMS501 and CRISPR-TMS502, two CRISPR / Cas9 gene editing vectors of rice TMS5 gene, and then introduces the two vectors into indica rice variety Minghui 86 and japonica rice variety Nipponbare by Agrobacterium-mediated transformation method, screens TMS5 gene frameshift knockout mutant lines from the transgenic editing materials, investigates and identifies the pollen fertility of each tms5 mutant line, obtains a temperature-sensitive male sterile line, and reproduces the mutant line under natural low-temperature or artificial climate chamber low-temperature condition. The artificial climate chamber is used to set different temperature treatments, and the male sterility starting temperature of different tms5 mutants is identified. Specifically, the following steps are included:

[0028] I. Construction of CRISPR / Cas9 gene editing vector

[0029] 1. Design and synthesis of sgRNA target site primer

[0030] The first exon of rice TMS5 gene was selected to design a 23 bp target site sequence, 5'- GAACAGCGGCAAGTCATCGCCGG-3' and 5'-CCACCGCGCCGCCACCGGGTCGG-3' were used as the sgRNA target sequences of T501 and T502, respectively. The sgRNA specifically targets the 22 bp and 57 bp downstream of the translation initiation site ATG of the TMS5 gene, respectively. Artificial sequences were synthesized, in which the forward primer is consistent with the target sequence, but does not include the PAM sequence NGG three bases, and the 5' end needs to be added with a linker CAG; the reverse primer is complementary to the target sequence excluding NGG, and the 5' end needs to be added with a linker AAC.

[0031] 2. Formation of primer duplex

[0032] The forward and reverse primers were diluted to 10 μM, and each sample was added according to the following system. The primer duplex was formed by denaturing at 95°C for 3 min, slowly reducing the temperature to room temperature at a rate of 3°C per minute, and then maintaining at 16°C for 5 min.

[0033]

[0034] 3. Connection of primer duplex and VK005-1 vector

[0035] The VK005-1 vector was provided by Beijing Weishanglide Biotechnology Co., Ltd. After adding each sample according to the following system, mix well and place in a 16°C water bath for 2h.

[0036]

[0037] 4. Transform the ligation product into E. coli competent cells DH5α using heat shock method, as follows:

[0038] (1) 100 μl of E. coli competent cells were thawed on ice for 5 min.

[0039] (2) Add 6 μl of ligation product and gently mix with a pipette gun. Place on ice for 30 min.

[0040] (3) 42°C water bath for 90 s (heat shock), quickly transfer to ice for 2 min.

[0041] (4) Add 800 μl of antibiotic-free liquid LB and incubate at 37°C on a shaker for 1 h to recover the cells.

[0042] (5) Prepare the screening medium: prepare LB plates containing 50 μg / ml Amp or kan.

[0043] (6) Shake the above culture liquid, and then take 100 μl and spread on the screening medium. Place the medium with the front face upward for half an hour. After the liquid is completely absorbed by the medium, invert the culture dish, and culture at 37°C for 16-24 h. Pick single colonies for culture and sequencing. The sequencing primer sequence is 5'-AGCCATGAATAGGTCTATGAC-3'. The sgRNA sequence of the expression cassette is 5'-GAACAGCGGCAAGTCATCGC-3'. The edited vector correctly verified is named as CRISPR-TMS501, and the sgRNA sequence of the expression cassette is 5'-CCACCGCGCCGCCACCGGGT-3'. The edited vector correctly verified is named as CRISPR-TMS502.

[0044] 5. Small amount of plasmid DNA extraction by alkaline method

[0045] (1) Take 1.5 mL of CRISPR-TMS501 and CRISPR-TMS502 culture liquids into two 1.5 mL eppendorf tubes, and centrifuge at 9000 g at 4°C for 2 min.

[0046] (2) Discard the supernatant, and invert the tube on filter paper for several minutes to make the liquid flow out.

[0047] (3) Resuspend the bacterial pellet in 100 μl of solution I (need to shake vigorously), and place at room temperature for 10 min.

[0048] (4) Add 200 μl of freshly prepared solution II, tightly cap the tube, and mix the contents by rapidly and gently inverting the eppendorf tube several times (do not shake), and ice-bath for 2 min.

[0049] (6) Add 150 μl of pre-cooled solution III, tightly cap the tube, mix by gently shaking for 10 s, ice-bath for 10 min, and centrifuge at 12000 g at 4°C for 5 min.

[0050] (7) Transfer the supernatant into a clean eppendorf tube, add an equal volume of chloroform\isoamyl alcohol (24:1), shake to mix, and extract at room temperature for 5 min. Centrifuge at 12000 rpm at 4°C for 5 min.

[0051] (8) Transfer the aqueous phase into a clean eppendorf tube, add 2 volumes of pre-cooled anhydrous ethanol, shake to mix, and then place at room temperature for 5 min, and then centrifuge at 12000 g at 4°C for 10 min.

[0052] (9) Discard the supernatant, and invert the tube on filter paper to make all the liquid flow out. Add 1 mL of 70% ethanol to wash the pellet once, and centrifuge at 12000 g at 4°C for 5 min.

[0053] (10) Discard the supernatant, and invert the tube to drain the liquid onto filter paper and dry at room temperature. Dissolve the pellet in 20 μl TE buffer (pH 8.0, containing 20 μg / mL RNase A) and store in a refrigerator at -20°C.

[0054] Solution I: Glucose 50 mmol / L, Tris.HCl (pH 8.0) 25 mmol / L, EDTA (pH 8.0 sterilized) 10 mmol / L.

[0055] Solution II: (freshly prepared) 10 mL ratio: 10 N NaOH 200 μl, 10% SDS 1 mL, H2O 8.8 mL.

[0056] Solution III: 5 mol / L potassium acetate 60 mL, glacial acetic acid 11.5 mL, water 28.5 mL.

[0057] TE: NaCl 0.1 mol / L, Tris.HCl (pH 8.0) 10 mmol / L, EDTA (pH 8.0) 1 mmol / L.

[0058] 6. Transformation of Agrobacterium competent cells by electroporation

[0059] (1) Add 2-10 μl (50-100 ng) of the extracted recombinant plasmids CRISPR-TMS501 and CRISPR-TMS502 to 40 μl of Agrobacterium LBA4404 competent cells per tube, and mix on ice.

[0060] (2) Pre-cool the electroporation tank (0.2 cm) at -20°C for 10 min, add the mixture to the electroporation tank, and gently knock it to the bottom of the tank.

[0061] (3) Electroporation parameters: voltage 2.5 KV, capacitance 25 Uf, resistance 200 Ω. Press the two Pulse buttons at the same time, and release after hearing the "click" sound.

[0062] (4) Immediately add 1 mL of YEB medium, mix gently and quickly. Transfer to a sterile eppendof tube, and incubate at 28°C for 2 hours.

[0063] (5) Set three gradients of 50 μl, 250 μl, and 600 μl for plating, and incubate at 28°C for 2 days. Pick single colonies for culture and sequencing. The sequencing primer sequence is 5'-AGCCATGAATAGGTCTATGAC-3' to verify the correctness of the sgRNA sequence of the expression cassettes of the two vectors CRISPR-TMS501 and CRISPR-TMS502.

[0064] II. Obtaining TMS5 gene variants and temperature-sensitive male sterile rice

[0065] 1. Genetic transformation of rice

[0066] The immature embryos of Minghui 86 and Nipponbare were taken respectively, and after sterilization, they were placed on NB medium for 27℃ dark culture to induce rice callus. The callus was subcultured every two weeks, and the callus after subculturing for three times was used for Agrobacterium transformation experiment. The following steps are included: co-culture (the whole operation process needs 8 days)

[0067] The first day (pre-culture): select the naturally dispersed, moderate hardness, bright yellow, about 2-3 mm in diameter granular callus, and place it on NB medium at 27℃ dark culture for 4 days.

[0068] The third day (bacteria division): Agrobacterium LBA4404 containing recombinant CRISPR-TMS501 and CRISPR-TMS502 was inoculated into YEB medium (containing 50 μg / ml kanamycin and 20 μg / ml rifampicin) respectively, and cultured at 28℃ in the dark for 48 hours.

[0069] The fifth day (transformation): the Agrobacterium was suspended in AMM liquid medium added with 100 μM acetyl-syringone (AS) 20ML, and after 1 min of vigorous shaking, it was placed for 1 h to allow the Agrobacterium to form a suspension, and the concentration was adjusted to OD600 = 1-1.5. The bacterial solution was taken into a culture bottle and added with pre-cultured callus, and after slight shaking for 30 min, the callus was dried on sterile paper and placed on NB medium added with 100 μM acetyl-syringone at 27℃ dark culture for 3 days.

[0070] The eighth day (washing bacteria): when the callus grew to visible bacterial plaque, but not full of callus, the callus was picked up in a sterile culture bottle, washed with sterile water until no visible plaque, finally washed with 250 mg / L carbenicillin sterile water for 1 h, dried on sterile filter paper, and then transferred to the selection medium with 30 mg / L hygromycin for selection.

[0071] The callus was transferred to new selection medium every two weeks, and about three weeks, the nodule-shaped resistant callus grew out of the brown and dry callus. When the resistant callus grew on 30 mg / L hygromycin medium for one month, the newly grown resistant callus was transferred to 50 mg / L hygromycin medium, and after one week of growth, the continuously proliferating and dividing resistant callus was transferred to differentiation medium for regeneration, and the callus began to turn green after one week of growth in the light incubator, and after three weeks, young shoots began to grow, and then roots also grew. When the shoots grew to 2-3 cm, they were moved to 1 / 2MS medium, and only one resistant callus induced shoot was placed in each culture bottle. After the seedlings grew on the rooting medium for 10 days, they were transplanted to the field after hardening in water for three days.

[0072] 2. Identification of transgenic seedlings and mutants

[0073] After the regenerated plants were transplanted and survived, total DNA of the regenerated plants was extracted, and PCR amplification was performed with primers CZTF / CZTR based on the recombinant vector and primers HptF / HptR based on the hygromycin, respectively, to screen the positive transformed plants. The identified transgenic positive plants were further subjected to PCR amplification of the genomic fragment of TMS5 gene with primers TMS5F and TMS5R and sequencing identification, and the identification results are shown in Table 1. Figure 1 The sequences of the primers used in the identification are shown as follows:

[0074] CZTF: 5'-GGGAGATCCAGCTAGAGGTC-3',

[0075] CZTR: 5'-GGAAGGAGGAAGACAAGG-3';

[0076] HptF: 5'-TACACAGCCATCGGTCCAGA-3',

[0077] HptR: 5'-TAGGAGGGCGTGGATATGTC-3';

[0078] TMS5F: 5'-CCATCGTGCTTCGTGCCA-3',

[0079] TMS5R: 5'-GAGTTCTTGGTACATGAGTGC-3'.

[0080] 3. Investigation and identification of the mutant sexual phenotype

[0081] The seeds of each transgenic plant were harvested, and the T1 generation was planted in the field under long-day conditions in Fuzhou in summer. The TMS5 gene fragment was amplified with primers TMS5F and TMS5R, and the mutant heterozygotes and homozygotes were screened according to the sequencing results. The tms5 mutant rice plants and the wild-type parent plants were moved into an artificial climate chamber (Canada Conviron Company) for different temperature treatments when the main stems of the plants were at the fourth stage of panicle differentiation. Five pots of each material were treated under the same temperature conditions. The different temperature treatments were set as three temperature conditions of daily average 22°C, 24°C and 28°C, and the light was 14.5h. The specific light and temperature treatment conditions are listed in Table 1. After 7d of treatment, the test materials were removed from the artificial climate chamber, and the rice panicles with a leaf cushion distance of about ±1cm were marked with a marker pen. After the panicles were harvested, 2 glumes of the upper, middle and lower of the flowering spike were collected, and the pollen was stained with 1% I2-KI. The pollen black staining rate was observed under a microscope. After 25d, 10 panicles were randomly selected to investigate the self-seeding rate and the pollen black staining rate. The panicles were bagged, and the seed setting rate was calculated after the seeds matured. The data of the pollen black staining rate and the seed setting rate were converted by inverse sine square root and subjected to variance analysis. The pollen fertility investigation results are shown in Tables 2-1 and 2-2.Figure 2 , Figure 3 The results show that the tms5 mutant of japonica rice Nipponbare has a higher temperature threshold than 28℃, and the temperature threshold of different tms5 mutants of indica rice Minghui 86 is between 22℃ and 28℃. In addition, the temperature threshold of tms5-1 mutants generated by T501 target editing is significantly higher than that of tm5-2 mutants of T502 target in the same genetic background.

[0082] Table 1 Light and temperature conditions of artificial climate chamber treatment

[0083]

[0084] 4、T501 target mutant and pollen fertility phenotype

[0085] In this experiment, the CRISPR-TMS501 gene editing vector generates an insertion-deletion of bases at 22bp downstream of the translation initiation site ATG of the TMS5 gene, causing a frameshift mutation of the TMS5 gene and forming a new tms5 complex allele. After investigating pollen fertility and identifying the temperature threshold of sterility, it is found that the temperature threshold of tms5-1 mutants generated by T501 target editing is significantly higher than that of tm5-2 mutants of T502 target in the same genetic background. The results are shown in Table 2-1, Table 2-2, Figure 2 , Figure 3 The two types of tms5 mutants show significantly different temperature thresholds of sterility, and it can be inferred that there must be different genetic regulatory mechanisms in their pollen development processes. Exploring these mechanisms will help us further understand the response of rice pollen development to environmental changes.

[0086] Table 2-1 Pollen fertility and self-seeding rate of T2 generation tms5 mutants

[0087]

[0088]

[0089] Table 2-2 Pollen fertility and self-seeding rate of T2 generation tms5 mutants

[0090]

[0091] Note: The pollen blackening rate and self-seeding rate are the average of 5 plants. Natural conditions refer to the long-day high-temperature conditions in Fuzhou in August. Letters A, B, C, D and E represent significant differences (P≤0.01) between materials in the same genetic background according to LSD multiple comparisons.

[0092] Therefore, by the method provided by the application, different genetic background interference can be overcome, and the tms5-1 frameshift mutant and the tms5-2 frameshift mutant are used to study the sterile starting point temperature molecular genetic regulation mechanism in the same background, so that a new research material and strategy for the tms5 rice sterile starting point temperature research is provided.

[0093] Although the specific embodiments of the application are described above in combination with the embodiments, the application is not limited to the scope of the application, and those skilled in the art should understand that various modifications or changes made by those skilled in the art on the basis of the technical solutions of the application without creative labor are still within the protection scope of the application.

Claims

1. Obtain two different starting temperatures in the same genetic background tms5 The method of two-line sterile system is characterized by: Using CRISPR / Cas9 gene editing system in rice TMS5 Genes produce new tms5 Multiple alleles can be used to obtain different sterility threshold temperatures tms5-1 Two-line sterile mutants and tms5-2 Two-line sterile mutant; preparation tms5-1 The method is: The CRISPR / Cas9 gene editing system consists of a CRISPR-TMS501 plasmid vector; the CRISPR-TMS501 plasmid vector is specific for rice. TMS5 A site-directed mutagenesis was performed 22 bp downstream of the ATG translation start site of the gene. The sequence of the sgRNA was 5'-GAACAGCGGCAAGTCATCGC-3', and the rice gene sequence targeted by the sgRNA was 5'-GAACAGCGGCAAGTCATCGCCGG-3'. Agrobacterium tumefaciens containing the CRISPR-TMS501 plasmid vector was transformed into rice varieties. TMS5 The gene was edited at the specified site and the regenerated plants were identified to select homozygous frameshift mutants; preparation tms5-2 The method is: The CRISPR / Cas9 gene editing system consists of a CRISPR-TMS502 plasmid vector; the CRISPR-TMS502 plasmid vector is specific for rice. TMS5 A site-directed mutagenesis was performed 57 bp downstream of the gene translation start site ATG, and the sequence of the sgRNA was: 5'-CCACCGCGCCGCCACCGGGT-3', and the rice gene sequence targeted by the sgRNA was: 5'-CCACCGCGCCGCCACCGGGTCGG-3'; Agrobacterium tumefaciens containing the CRISPR-TMS502 plasmid vector was transformed into rice varieties, and the rice was cultured. TMS5 The gene is edited at the specified site and the regenerated plants are identified to select homozygous mutants; Among rice varieties with the same genetic background, tms5-1 The sterility threshold temperature was significantly higher than tms5-2 ; The rice varieties with the same genetic background are Minghui 86 or Nipponbare.

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