Application of ERS1 gene in regulating thermo-sensitive male sterility in rice

Abstract

The present invention discloses the application of the ERS1 gene in regulating the thermosensitive sterility of rice, including obtaining a sterile mutant by gene editing the wild type of medium indica 3037 and naming it ers1-2 (glutamyl-tRNA synthetase), and further verifying the genetic relationship between the ERS1 gene and the sterile phenotype through complementation experiments. The mutant exhibits a typical thermosensitive sterile phenotype, with a fruit set rate of only 20% at high temperature and a fruit set rate of 70% at low temperature. Further cell biological analysis of the mutant found that the pollen development and meiotic process of the mutant were severely delayed at high temperature, leading to male sterility, and these defects can be restored at low temperature, thereby giving the mutant the characteristics of thermosensitive sterility. The mutation of the ERS1 gene has created a new thermosensitive sterile line in rice, which has great potential in rice hybrid breeding.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and in particular relates to the application of ERS1 gene in regulating the thermosensitive sterility of rice. Background Art

[0002] Food sustains human life and is crucial to a country's economic development and social harmony. As one of the three major staple crops, rice is the staple food for more than half the world's population. my country began research on hybrid rice in the 1960s, leveraging heterosis to increase yields and successfully developing a three-line hybrid rice system in the 1970s. Since then, hybrid rice has been widely adopted, making a significant contribution to increasing grain production. The three-line hybrid rice system consists of sterile lines, maintainer lines, and restorer lines. Sterile lines are cytoplasmic male sterile lines, which cannot produce offspring through self-pollination. Maintainer lines lack the male sterility gene in their cytoplasm, allowing them to maintain their reproduction through self-pollination. The restorer line contains the nuclear restorer gene in its nucleus. Therefore, hybridizing a restorer line with a sterile line as the male parent can produce F1 plants with hybrid vigor for use in production breeding.

[0003] With the widespread application of three-line hybridization in production, inherent problems have gradually emerged, such as limited restorer and maintainer germplasm resources and the negative impact of mitochondrial gene abnormalities on hybrid vigor. These issues have limited the application of three-line hybridization and highlighted the advantages of two-line hybridization. The fertility transformation of photothermosensitive male sterile lines under different environments is key to two-line hybridization. In 1973, Chinese breeder Shi Mingsong discovered a naturally male sterile plant, NK58S, in the Nongken 58 field. It was sterile under long days and suitable for hybrid seed production, but sterile under short days and suitable for selfing. In 1988, Deng Huafeng and others discovered and developed the first thermosensitive genic male sterile line, Annong S-1, in indica rice. It exhibits male sterility under high temperatures but becomes fertile at low temperatures. The dual-purpose nature of photo- and thermo-sensitive male sterile lines in two-line hybrid systems eliminates the need for maintaining a maintainer line and the constraints of a restorer line during seed production, greatly simplifying the breeding process and shortening the breeding cycle. However, the number of photo- and thermo-sensitive male sterile lines reported in rice is very limited, and even fewer have been adapted for large-scale production. Therefore, the development of new thermo-sensitive male sterile lines is of great application value.

[0004] Meiosis is a special type of division in which eukaryotic gametes form, in which the DNA replicates once and the cells divide twice. The smooth progress of meiosis is a prerequisite for the production of fertile gametes. In rice, several genes have been reported to be involved in the regulation of meiosis. Mutations in the LEPTO1 gene cause meiosis in pollen mother cells to arrest at the leptotene stage, followed by pollen mother cell degradation. Mutations in the AM1 gene prevent pollen mother cells from completing the leptotene-zygotene transition. Mutations in the MEL1 gene cause meiosis in pollen mother cells to arrest at prophase I. Studies on these genes have provided new insights into the regulation of meiosis, but few studies have examined whether temperature affects meiosis. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a rice ERS1 gene mutant.

[0008] To solve the above technical problems, the present invention provides the following technical solution: the mutant is obtained by deleting bases 1156 to 1158 in the coding region of the ERS1 gene (LOC_Os10g22380), wherein the deleted base is GAC.

[0009] Another object of the present invention is to overcome the deficiencies in the prior art and provide a gene encoding the rice ERS1 gene mutant according to claim 1.

[0010] Another object of the present invention is to overcome the deficiencies in the prior art and provide a recombinant plasmid characterized by comprising the gene.

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide a host cell characterized by comprising the recombinant plasmid.

[0012] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of an ERS1 gene mutant in regulating thermosensitive male sterility in rice.

[0013] As a preferred embodiment of the present invention, the characteristic of thermosensitive sterility used in regulating thermosensitive sterility of rice comes from the defect of meiotic process.

[0014] As a preferred embodiment of the present invention, the gene mutant is used to regulate the growth temperature of rice to affect the development of male gametes.

[0015] As a preferred embodiment of the present invention, the gene mutant is used to regulate the growth temperature of rice to influence the fruit setting rate.

[0016] Another object of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing transgenic rice.

[0017] As a preferred embodiment of the present invention, the invention comprises causing the ERS1 gene of the recipient rice to undergo the mutation as described in claim 1, thereby deleting the aspartic acid at position 386 in the catalytic center domain of the ERS1 protein, severely delaying the meiotic process of the mutant, and ultimately leading to infertility, thereby obtaining the transgenic rice.

[0018] Beneficial effects of the present invention:

[0019] The present invention edited the ERS1 gene in the wild-type medium-length indica rice 3037 to generate a sterile mutant named ers1-2 (glutamyl-tRNA synthetase). Genetic complementation experiments then further verified the genetic relationship between the ERS1 gene and the sterile phenotype. This mutant exhibited a typical thermosensitive sterile phenotype, with a seed set rate of only 20% at high temperatures and 70% at low temperatures. Further cell biological analysis of the mutant revealed that pollen development and meiotic progression were severely delayed at high temperatures, leading to male sterility. These defects could be reversed at low temperatures, conferring thermosensitive sterility on the mutant. The mutation of the ERS1 gene created a new thermosensitive sterile line in rice, which has great potential in rice hybrid breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0021] Figure 1 Figure 1 is a diagram of the ers1-2 fertility defect mutant in Example 1 of the present invention ( Figure 1 A shows the plant morphology of the wild type and ers1-2 mutant. Figure 1 B is the spike of the wild type and ers1-2 mutant. Figure 1 C shows the fruiting status of the wild type and ers1-2 mutant after saturated pollination. Figure 1 D shows the mature embryo sac structure in the wild type and ers1-2 mutant.

[0022] Figure 2 This is the genetic verification diagram of the ERS1 gene in Example 1 of the present invention ( Figure 2 A is the plant morphology of the wild type and the complemented plant, Figure 2 B is the spike of wild type, ers1-2, and ers1-2-com, Figure 2 C: Anthers of wild type, ers1-2, and ers1-2-com stained with potassium iodide)

[0023] Figure 3 The structure of the ERS1 gene and its encoded protein in Example 1 of the present invention ( Figure 3 A is the gene structure of ERS1: blue rectangles represent exons, black lines represent introns, and gray rectangles and gray triangles represent non-coding regions. Figure 3 B is the domain structure of ERS1 protein. Figure 3 (C is the three-dimensional structure of ERS1 protein in rice).

[0024] Figure 4 This is a temperature sensitivity diagram of the fertility of the ers1-2 mutant in Example 1 of the present invention ( Figure 4 A is the spike of the wild type under high temperature and the ers1-2 mutant under high temperature (HT) and low temperature (LT). Figure 4 B is the fruiting rate of the wild type under high temperature and the ers1-2 mutant under high temperature, low temperature, and when planted in Hainan. Figure 4 C is the pollen fertility statistics of the wild type at high temperature and the ers1-2 mutant at high and low temperatures. Figure 4 D shows potassium iodide staining of anthers of the wild type under high temperature and the ers1-2 mutant under high and low temperatures.

[0025] Figure 5 The pollen development delay defect of the ers1-2 mutant in Example 2 of the present invention can be restored by low temperature ( Figure 5 A is the comparison of fertility of wild type under high and low temperature conditions, Figure 5 B is the pollen development process in the wild type. Figure 5 C is the pollen development process of ers1-2 mutant under high temperature, Figure 5 (D shows the pollen development process of the ers1-2 mutant under low temperature).

[0026] Figure 6 Figure 2 shows that the meiotic delay defect of the ers1-2 mutant in Example 2 of the present invention is restored at low temperature ( Figure 6 A is the meiotic process of the wild type at high temperature, Figure 6B is the meiotic progression of ers1-2 mutant under high temperature. Figure 6 C shows the meiotic progression of the ers1-2 mutant under low temperature). DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0030] The wild-type rice used in the present invention is the cultivated variety Zhongxian 3037, which is preserved in our laboratory. The ers1-2 mutant mentioned is obtained by gene editing Zhongxian 3037.

[0031] Unless otherwise specified, the raw materials used in the examples of the present invention are commercially available.

[0032] Example 1

[0033] Application of the ERS1 gene provided by the present invention in regulating thermosensitive sterility of rice:

[0034] (1) Obtaining the ers1-2 mutant through gene editing and verifying the genetic complementation of the ERS1 gene

[0035] a. Obtaining a male sterile mutant ers1-2

[0036] Using gene editing, the ERS1 gene in the medium-grain rice variety 3037 was specifically edited. The sequence GGACAAGCAAAGTCATAAGT in the fourth exon of the ERS1 gene was selected as the target for gene editing. The following primer sequences were then designed and synthesized:

[0037] Forward primer sequence:GGCAGGACAAGCAAAGTCATAAGT

[0038] Reverse primer sequence:AAACACTTATGACTTTGCTTGTCC

[0039] The synthesized primers were diluted to 100 μM, 20 μl of the forward primer and 20 μl of the reverse primer were annealed at 100 degrees Celsius for 5 minutes to form a double strand, cooled to room temperature, and stored at -20°C. The final vector SgRNA-1300Cas9 (nucleotide sequence as shown in SEQ ID.3) was digested with AarI endonuclease and purified and recovered after digestion at 37 degrees Celsius for 5 hours. Finally, the annealed product and the digested SgRNA-1300Cas9 were connected using T4 ligase, connected at 22 degrees Celsius for 15 minutes, and the product was transformed into DH5α competent cells. After screening the transformants, positive transformants were obtained. After extracting the plasmid, the vector was transformed into the callus formed by the medium indica 3037 variety using Agrobacterium-mediated genetic transformation. After three months of tissue culture, T0-generation transformed plants were obtained. Sequencing of the target site of the ERS1 gene in these plants revealed a heterozygous deletion from bases 1156 to 1158 (i.e., the ERS1 gene genotype was Aa). Planting and harvesting these heterozygous plants revealed fertility segregation in the second generation, resulting in a sterile mutant. This mutant was derived from a deletion of bases 1156 to 1158 in the coding region of the ERS1 gene (LOC_Os10g22380), resulting in a GAC ​​deletion at position 386. The resulting nucleotide sequence is shown in SEQ ID No. 1 (GAC is deleted within "GACTTT"), and the amino acid sequence is shown in SEQ ID No. 2 (D is deleted within "PTYDF"). We named this mutant ers1-2 (glutamyl-tRNA synthetase).

[0040] The mutant had no significant difference from the wild type in the vegetative growth stage, but exhibited severe fertility defects in the reproductive development stage, producing only a small number of seeds ( Figure 1 A and Figure 1 B). We conducted saturation pollination on the mutant using the wild type as the male parent and found that the mutant could bear fruit normally. In addition, by observing the mature embryo sac of the mutant, we found that the mutant had a seven-cell eight-nucleus embryo sac structure similar to that of the wild type ( Figure 1 C and Figure 1 D) This indicates that the female fertility of this mutant is normal, and its fertility defect may be caused by poor male fertility.

[0041] b. Genetic complementation verification of mutant genes

[0042] Genetic complementation experiments are a powerful means to verify the correspondence between genes and phenotypes. In order to verify the genetic correspondence between the ERS1 gene and the sterility phenotype, we performed genetic complementation experiments in the ers1-2 mutant.

[0043] The full-length genomic sequence of the ERS1 gene, a promoter sequence 3.5 kb upstream of the start codon, and a 2 kb downstream sequence after the stop codon, totaling 8.7 kb, were amplified from the genomic DNA of Zhongxian 3037. The binary vector pCambia1300 (for sequence and map, see http: / / www.biofeng.com / zaiti / zhiwu / pcambia1300.html) was digested with SacI and HindIII endonucleases at 37 degrees Celsius for 3 hours, then the product was purified and recovered. The 8.7 kb sequence was cloned into the binary vector pCambia1300 by homologous recombination to form the final complementation vector. Positive recombinants were screened by colony PCR and first-generation sequencing, and then transformed into the ers1-2 mutant via Agrobacterium-mediated rice genetic transformation. The phenotype of the complemented plants was then observed. We took photos of the wild-type and complementary plants (ers1-2-com) during the rice grain-bearing period. The complementary plants (ers1-2-com) could bear fruit, so both their panicles and those of the wild-type showed a drooping phenomenon due to the gravity of the seeds (e.g. Figure 2 (as shown in A).

[0044] In addition, by observing the panicles of the wild type, ers1-2 mutant, and complemented plants (ers1-2-com), we found that the panicles of ers1-2 mutant had fewer seeds, while the panicles of wild type and complemented plants were full of seeds (e.g. Figure 2 B), which indicates that the fertility of the complemented plant has been restored. Further staining of the anthers of the complemented plant (ers1-2-com) with potassium iodide revealed that the anthers of the complemented plant (ers1-2-com) were full of pollen grains that could be stained with potassium iodide, indicating that the pollen fertility had been restored (as shown in Figure 2 (As shown in C). Since the mutation of the ERS1 gene causes sterility, and fertility is restored after the full-length sequence of the ERS1 genome is transferred into the ers1-2 mutant, it is shown that the mutation of the ERS1 gene indeed causes the sterility phenotype.

[0045] c. Obtaining the full-length cDNA of ERS1 gene and analyzing its domain structure

[0046] By using the rapid amplification of cDNA ends (RACE) method, we obtained a 2654 bp cDNA of the gene, the sequence of which is shown in SEQ ID NO: 4, with the start and stop codons underlined.

[0047] The ERS1 gene contains 6 exons and 5 introns ( Figure 3 A), ERS1 encodes a glutamyl-tRNA synthetase with a total length of 713 amino acids, consisting of an N-terminal GST domain, a central catalytic center, and a C-terminal tRNA binding domain ( Figure 3 B and Figure 3 C) The full-length amino acid sequence of ERS1 protein is shown in SEQ ID NO: 5, wherein the stop codon is indicated by *.

[0048] Example 2

[0049] (1) ERS1 gene regulates thermosensitive male sterility in rice

[0050] We counted the seed setting rate of the ers1-2 mutant in the field and found that the seed setting rate of the mutant was only 20%. However, when the mutant was planted in Hainan, its seed setting rate was 80% ( Figure 4 B). Therefore, we speculate that the fertility of the ers1-2 mutant is affected by the environment. After we subjected the mutant to temperature treatment, we found that under high temperature (the minimum daily temperature was 24 degrees, the maximum daily temperature was 34 degrees, and the average daily temperature was 29 degrees. The temperature range of 24-34 degrees mentioned here is a relatively high temperature environment, which does not mean that every temperature in the range of 24-34 degrees is high temperature), the ears of the mutant were light green and had only a small number of seeds ( Figure 4 A), only a small number of pollen grains in its anther can be stained with potassium iodide ( Figure 4 D), the pollen fertility is 30%, and the fruit set rate is 20% ( Figure 4 B and Figure 4 C); Under low temperature (daily minimum temperature 22 degrees, daily maximum temperature 28 degrees, daily average temperature 25 degrees, the temperature range of 22-28 degrees mentioned here is a relatively low temperature environment, not to say that every temperature within the range of 22-28 degrees is low temperature) the mutant ears are light yellow ( Figure 4 A), almost all pollen grains in anthers can be stained with potassium iodide ( Figure 4 D), the pollen fertility is 80%, and the fruit set rate is also 80% ( Figure 4 B and Figure 4 C).

[0051] Taken together, these data indicate that ers1-2 is a thermosensitive sterile mutant.

[0052] (2) Analysis of the intrinsic mechanism of ERS1 thermosensitivity

[0053] Observation on the pollen development process of a.ers1-2 mutant under high and low temperatures

[0054] To determine the reason why the ers1-2 mutant is sterile at high temperatures and fertile at low temperatures, we observed the pollen development of the wild type and ers1-2 mutant at high and low temperatures by semi-thin resin sections of spikelets and toluidine blue staining. Since there is no significant difference in pollen fertility of the wild type at high and low temperatures, only the wild type at high temperature is shown in the following figure ( Figure 5 A). We found that in florets ranging from 3 mm to 5 mm in length, there was no significant difference between the wild type and the ers1-2 mutant under high and low temperature conditions. However, as development progressed, meiosis was completed and microspores were produced in wild-type florets reaching 7 mm in length, and pollen grains of uniform size and regular morphology were formed at the flowering stage ( Figure 5 B). However, in the 7 mm florets of the ers1-2 mutant under high temperature conditions, pollen development is still in the meiotic process, and pollen grains of inconsistent sizes are formed during the flowering stage ( Figure 5 C). Compared with the mutant grown under high temperature, meiosis has been completed in the 7 mm florets of the ers1-2 mutant under low temperature, and pollen grains similar to those of the wild type are formed at the flowering stage ( Figure 5 D) This indicates that pollen development is delayed in the ers1-2 mutant, and low temperature can repair the delayed defect.

[0055] Observation of meiotic progression in b.ers1-2 mutant at high and low temperatures

[0056] To further elucidate the defects in pollen development in the ers1-2 mutant, we observed the meiotic process of pollen mother cells in the wild type, high temperature, and low temperature conditions by DAPI staining. In wild type florets with a length of 3 mm, the meiotic division of pollen mother cells is at the leptotene stage ( Figure 6 A), while the meiosis of ers1-2 mutants under high and low temperature conditions are both in the pre-leptotene stage ( Figure 6 B) In wild-type florets with a length of 4 mm, the meiosis of pollen mother cells is at the zygotene stage ( Figure 6 A), while the meiosis of ers1-2 mutants under high and low temperature conditions are both in the leptotene stage ( Figure 6 B) In wild-type florets with a length of 5 mm, the meiosis of pollen mother cells is at the pachytene stage ( Figure 6A), while the meiosis of ers1-2 mutants under high and low temperature conditions are both in the zygotene stage ( Figure 6 B) In wild-type 6 mm florets, the meiotic division of pollen mother cells is at metaphase I, dyad, tetrad, etc. ( Figure 6 A), at this time, the meiosis of the ers1-2 mutant under high temperature is in the zygotene stage, while the meiosis of the mutant under low temperature conditions is in the pachytene stage ( Figure 6 B) In wild-type florets with a length of 7 mm, meiosis of the pollen mother cells has been completed, forming microspores ( Figure 6 A), at this time, the meiosis of the ers1-2 mutant under high temperature conditions is in the pachytene and diakinesis-telophase I, while the meiosis of the mutant under low temperature conditions is in the metaphase I, tetrad, microspore and other stages, and the meiotic process is also completed ( Figure 6 B) This indicates that meiotic progression is severely delayed in the ers1-2 mutant and is restored at low temperature.

[0057] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

[0058] SEQ ID NO:1 sequence:

[0059] ATG

[0060] SEQ ID NO:2 sequence:

[0061] MEAKLSFSQDSPPISIISAAKVAGVSLSIDPSLAAGSAPVLCFSSGESLRGINPILEYIAQSSPSLHGRDAIESGHVVEWLEYAPTFLLGSEFEVACSFVDGYLMSRTFLVGHVLTIADITVWSNLAGIGQRWESLRKSKKYQNLVRWFNGIDSDYKDTLNEVIAAYVGKRGIGKSPAPNLKEKVNDSKDPSAPEVDLPGAKFGEVCVRFAPEPSGYLHIGHAKAALLNKYFAERYQGRLIVRFDDTNPSKESNEFVENLLKDIETLGIKYDAVTYTSDYFPKLMEMAEKLIKQGKAYVDDTPKEQMRSERMDGVESKCRNNTVEENLSLWKEMINGSERGMQCCVRGKLDMQDPNKSLRDPVYYRCNTDPHHRIGSKYKVYPTYFACPFVDALEGVTHALRSSEYHDRNAQYYRILQDMGMRRVEIYEFSRLNMVYTLLSKRKLLWFVQNKKVEDWTDPRFPTVQGIVRRGLKVEALIQFILQQGASKNLNLMEWDKLWTINKKIIDPVCARHTAVLKDQRVIFRLTNGPEKSFVQILPRHKKFDGAGKKATTFTNRIWLDYADASAISKGEEVTLMDWGNAIIKEIKMENGVITELIGELHLEGSVKTTKLKVTWLPDIDDLVPLSLVEFDYLISKKKLEEDENFLDNLNPCTRKETLALGDANMRNLLRGEVIQLERKGYYRCDAPFVRSSKPVVLFAIPDGRQQASLN*

[0062] SE1 ID NO:3 sequence

[0063]

[0064] SEQ ID NO:4 sequence:

[0065] CGTAGGTGGTAGGAGGGGCCCCATCCGTCATCCTGCTGCTTCCGCCGCG

[0066] TCCATCCTCCCTGCTTAGGTTAGCCCTTCGCACATCCTCCGCCGCCGCCGC

[0067] CGACGATCCCCATCAATCCCGCGAGCGAGCGGGGCCCCGTCGTTCGTCC

[0068] TCCTGCCGCTTCCGCCGCATCCATCCTGTAGGATCG ATG GAGGCGAAGTT

[0069] ATCGTTTTCTCAGGACAGCCCGCCAATTTCCATAATTTCTGCTGCTAAGGT

[0070] TGCCGGTGTCTCCCTGTCCATAGATCCTAGTCTTGCTGCGGGCTCAGCAC

[0071] CCGTTCTATGCTTCAGTTCTGGAGAATCACTCCGTGGTATCAACCCTATTC

[0072] TCGAGTACATTGCCCAATCCTCTCCAAGCCTCCATGGCCGAGATGCTATT

[0073] GAGTCTGGACATGTTGTTGAATGGCTTGAATACGCCCCCACCTTCCTTTT

[0074] AGGATCAGAATTTGAGGTTGCCTGCTCATTTGTTGATGGATACTTGATGT

[0075] CTCGGACCTTTCTGGTTGGTCATGTTTTAACGATTGCTGATATCACTGTA

[0076] TGGTCAAATCTGGCTGGGATTGGTCAACGATGGGAGAGTCTACGGAAAT

[0077] CAAAGAAATACCAAAATCTTGTCCGTTGGTTCAATGGCATAGATTCAGAC

[0078] TACAAAGACACATTGAATGAGGTTATAGCTGCTTATGTTGGGAAACGAG

[0079] GAATTGGGAAATCCCCTGCACCAAACCTTAAGGAAAAGGTTAATGACTCA

[0080] AAGGATCCTTCAGCTCCAGAAGTTGATCTCCCTGGTGCAAAATTTGGAGA

[0081] GGTGTGTGTTCGCTTTGCCCCAGAGCCCAGTGGTTACCTCCATATAGGTC

[0082] ATGCAAAGGCTGCACTCTTGAACAAGTACTTTGCAGAAAGATATCAAGG

[0083] TCGATTAATTGTTCGATTTGATGACACTAACCCTTCAAAAGAAAGCAATG

[0084] AGTTTGTTGAAAACCTCCTAAAAGATATTGAGACCCTGGGAATCAAATAT

[0085] GATGCTGTCACATACACATCTGATTATTTTCCAAAGCTAATGGAAATGGC

[0086] TGAAAAACTGATTAAGCAGGGAAAAGCATATGTTGATGACACACCAAAG

[0087] GAGCAAATGAGAAGTGAGAGAATGGATGGTGTGGAGTCAAAGTGCAGA

[0088] AATAATACTGTGGAGGAAAATCTGTCTCTATGGAAAGAGATGATAAATG

[0089] GTTCTGAAAGGGGTATGCAATGCTGTGTGAGGGGTAAACTTGACATGCA

[0090] GGATCCTAACAAGTCCCTTCGGGATCCAGTTTACTACCGTTGCAACACTG

[0091] ATCCTCACCATCGTATTGGTTCAAAGTACAAGGTCTATCCAACTTAT GAC T

[0092] TTGCTTGTCCATTCGTTGATGCGCTGGAGGGAGTGACCATGCTCTTCGT

[0093] TCCAGTGAATATCATGACAGGAATGCACAATACTATCGAATTCTTCAAGA

[0094] TATGGGGATGAGGAGAGTAGAAATATATGAGTTCAGCAGATTGAATAT

[0095] GGTTTAACTCTTCTTAGCAAGCGGAAGCTGCTTTGGTTTGTGCAGAACA

[0096] AGAAGGTTGAAGATTGGACAGACCCTCGTTTCCCCACTGTTCAAGGCATA

[0097] GTACGCCGTGGCTTGAAAGTTGAGGCTTTGATACAATTTATACTCCAACA

[0098] GGGTGCTTCGAAAAAATTGAATCTTATGGATGGGATAAACTCTGGACA

[0099] ATCAACAAGAAAATAATTGACCCAGTTTGTGCAAGGCATACTGCTGTGTT

[0100] GAAAGATCAGCGTGTGATCTTCAGGCTTACTAATGGTCCAGAGAAATCA

[0101] TTTGTTCAAATTTTGCCAAGGCACAAGAAATTTGATGGTGCAGGAAAGA

[0102] AGGCAACAACCTTCACAAACAGAATTTGGCTAGATTATGCTGATGCATCT

[0103] GCAATTAGCAAGGGTGAGGAAGTTACTCTGATGGACTGGGGAAATGCC

[0104] ATCATTAAAGAAATCAAGATGGAGAATGGAGTAATTACTGAACTGATTG

[0105] GAGAACTACATCTTGAGGGATCGTGTGAAGACAACAAAATTGAAGGTTAC

[0106] ATGGCTTCCGGACATAGATGATCTGGTTCCCTCTCTCATTGGTGGAATTTG

[0107] ATTACCTGATTAGCAAGAAGAAGCTTGAGGAGGATGAGAACTTCCTTGA

[0108] CAACCTGAACCCATGCACTCGGAAAGAAACCCTGGCCCTAGGAGACGCA

[0109] AATATGAGGAACCTCCTGCGTGGGGAGGTCATACAGCTTGAGAGGAAA

[0110] GGCTATTACAGGTGCGATGCCCCTTTTGTCAGATCCTCGAAGCCGGTGG

[0111] TTTTGTTCGCTATCCCTGATGGTCGACAGCAGGCCAGTTTGAAC TAG GCG

[0112] GCATGATTCTGATGAGCTTTCTCAAGTCGTTGAGTTGTCAGAGATTCCTGA

[0113] ATGCTCTACTGAAACACTCCCATCTCATACTGTAATGTCGCTTATGTGTCA

[0114] TGGGTGATTTGCGCTAAGTTTTGGTGCACAGTTTAATGTTTCTCATGCTC

[0115] CATGCTCCAGTTATTAAGAAGACACAACATTTCTTAGATGGCAACGTGTT

[0116] AGCTTGGCGATATCATACTGCCAGATACAAGCATCTGAATTTTTTGAAGA

[0117] CTAATATCTGATATTGCTTGGCATCTGAATTTTTTGAAGACTAATACCGCT

[0118] CTGGGAAACTGAGTTGAGGACA

[0119] SEQ ID NO:5 sequence:

[0120] MEAKLSFSQDSPPISIISAAKVAGVSLSIDPSLAAGSAPVLCFSSGESLRGINPILE

[0121] YIAQSSPSLHGRDAIESGHVVEWLEYAPTFLLGSEFEVACSFVDGYLMSRTFL

[0122] VGHVLTIADITVWSNLAGIGQRWESLRKSKKYQNLVRWFNGIDSDYKDTLN

[0123] EVIAAYVGKRGIGKSPAPNLKEKVNDSKDPSAPEVDLPGAKFGEVCVRFAP

[0124] EPSGYLHIGHAKAALLNKYFAERYQGRLIVRFDDTNPSKESNEFVENLLKDI

[0125] ETLGIKYDAVTYTSDYFPKLMEMAEKLIKQGKAYVDDTPKEQMRSERMDG

[0126] VESKCRNNTVEENLSLWKEMINGSERGMQCCVRGKLDMQDPNKSLRDPVY

[0127] YRCNTDPHHRIGSKYKVYPTY D FACPFVDALEGVTHALRSSEYHDRNAQYY

[0128] RILQDMGMRRVEIYEFSRLNMVYTLLSKRKLLWFVQNKKVEDWTDPRFPTV

[0129] QGIVRRGLKVEALIQFILQQGASKNLNLMEWDKLWTINKKIIDPVCARHTA

[0130] VLKDQRVIFRLTNGPEKSFVQILPRHKKFDGAGKKATTFTNRIWLDYADAS

[0131] AISKGEEVTLMDWGNAIIKEIKMENGVITELIGELHLEGSVKTTKLKVTWLP

[0132] DIDDLVPLSLVEFDYLISKKKLEEDENFLDNLPTCRKETLALGDANMRNLLRGEVIQLERKGYYRCDAPFVRSSKPVVLFAIPDGRQQASLN*.

Claims

1. A method for preparing thermosensitive sterile transgenic rice, characterized by: Including making the recipient rice ERS1 The gene mutates, resulting in a deletion of aspartic acid at position 386 in the catalytic center domain of the ERS1 protein, causing a severe delay in the meiotic process of the mutant, ultimately leading to sterility, thereby obtaining the transgenic rice; The mutation is ERS1 Gene LOC_Os10g22380 The coding region is deleted from base 1156 to base 1158, wherein the deleted base is GAC.

2. The preparation method according to claim 1, wherein: The transgenic rice is sterile at high temperatures but fertile at low temperatures.

3. The preparation method according to claim 1, wherein: The transgenic rice has delayed pollen development under high temperature.

4. The preparation method according to claim 1, wherein: The transgenic rice has a reduced fruit setting rate under high temperature.

5. The preparation method according to claim 1, wherein: The nucleotide sequence after mutation is shown in SEQ ID No. 1, and the amino acid sequence after mutation is shown in SEQ ID No. 2.

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