Application of RTMS10.1 and RTMS10.2 genes in breeding temperature-sensitive male-sterile rice lines

Abstract

This invention discloses RTMS10.1 Genes and RTMS10.2 Application of genes in breeding temperature-sensitive male-sterile rice lines. This invention discovers the ability to knock out, alter, or inhibit the gene in the temperature-sensitive male-sterile rice material YnS based on the CRISPR / Cas9 system. RTMS10.1 Genes and RTMS10.2 The transcription or expression of genes transforms temperature-sensitive male-sterile rice lines into fertile ones. Furthermore, by... RTMS10.1 Genes and RTMS10.2 When the gene was introduced into the normally fertile rice variety L422, L422 exhibited a temperature-sensitive male sterility phenotype. This invention is the first to discover this phenomenon. RTMS10.1 Genes and RTMS10.2 Genes jointly regulate the temperature-sensitive male sterility trait in rice, which can be used to create temperature-sensitive male sterile lines in rice and applied to two-line hybrid rice breeding, showing broad development prospects and application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving RTMS10.1 Genes and RTMS10.2 Application of genes in the breeding of temperature-sensitive male sterile rice lines. Background Technology

[0002] Hybrid rice, utilizing heterosis, can significantly increase rice yield, which is of great significance to ensuring my country's food security. Photoperiod- and temperature-sensitive male sterile lines are the core materials for two-line hybrid rice breeding in my country. Under restrictive conditions (such as long days and high temperatures), pollen abortion occurs, allowing them to be used as sterile lines to produce hybrids through crossbreeding with restorer lines. Under suitable conditions (such as short days and low temperatures), fertility is restored, and self-pollination is successful; therefore, they are also called dual-purpose male sterile lines. Based on their different responses to light and temperature conditions, photoperiod- and temperature-sensitive male sterile lines can be divided into two main types: photoperiod-sensitive and temperature-sensitive. In production practice, temperature-sensitive male sterile lines are more widely used, but problems such as unstable fertility and difficulty in propagation still exist. Furthermore, the genetic resources of practical male sterile lines are still relatively limited, restricting the genetic improvement of male sterile lines and the sustainable development and application of two-line hybrid rice. Therefore, cloning a new gene that regulates photoperiod- and temperature-sensitive male sterility and elucidating its molecular mechanism is an important way to solve the breeding problems of two-line hybrid rice. It can provide a new technical path for the sustainable development of two-line hybrid rice and has important theoretical significance and practical value.

[0003] In 1973, Mr. Shi Mingsong discovered a photosensitive male-sterile mutant (named Nongken 58S) in the late-season japonica rice variety Nongken 58. This mutant exhibited male sterility under long-day conditions but recovered fertility under short-day conditions. Based on this, he proposed the concept of "one line, two uses" for hybrid rice pairing and conducted practical research, thus initiating the development of two-line hybrid rice breeding in my country. Subsequently, breeders such as Deng Huafeng discovered and cultivated the indica-type thermosensitive male-sterile line Anong S1 in indica rice. This line exhibited male sterility under high-temperature conditions but recovered fertility under low-temperature conditions. The cultivation of Anong S1 opened up new avenues for utilizing heterosis in rice and promoted the development of two-line hybrid breeding. Using materials such as Nongken 58S and Anong S1 as parents, breeders have cultivated several dual-use nuclear male-sterile materials (thermosensitive, photothermosensitive, antithermosensitive, and antiphotothermosensitive) that are regulated by different environments. After more than 50 years of research and development, two-line hybrid rice based on photoperiod-temperature-sensitive male sterile lines has become an indispensable part of hybrid rice. It plays a crucial role in increasing rice yield, improving rice quality, utilizing heterosis between subspecies, and breeding super rice varieties. However, current two-line hybrids still face application bottlenecks, such as the limited availability of sterility gene resources and lagging research on the genetic mechanisms of fertility transition critical temperatures. To address these issues, it is urgent to explore new two-line hybrid gene resources; strengthen research on genes regulating fertility transition critical temperatures to improve breeding efficiency; and broaden the genetic basis of photoperiod-temperature-sensitive male sterile lines by combining molecular marker-assisted breeding technology.

[0004] Currently, all cloned thermo-sensitive male sterility genes are positive thermo-sensitive male sterility genes; negative thermo-sensitive male sterility genes have not yet been reported. Of the existing TGMS genes, over 95% of two-line hybrid rice varieties rely on... tms5 The development of derivative male-sterile lines has led to severe homogenization of temperature-sensitive male-sterile lines and their hybrid rice varieties. Furthermore, positive temperature-sensitive male-sterile lines (high-temperature sterile / low-temperature fertile) are only suitable for seed production during the hot summer months, failing to meet the seed production needs of mid- and low-latitude rice-growing regions in spring and autumn, and high-latitude rice-growing regions in summer. Inverse temperature-sensitive male-sterile lines, such as YnS, can remain sterile under conditions where the average daily temperature is below 29℃, effectively overcoming these limitations and possessing significant theoretical research and application value. Summary of the Invention

[0005] To address the problems existing in current two-line breeding, this invention has discovered a novel thermosensitive male sterility gene, providing a new genetic resource for two-line breeding. This study used the thermosensitive male sterile line YnS (low-temperature sterile / high-temperature fertile) as the research object, and through a high-generation population of backcrosses between the thermosensitive male sterile rice line YnS and the fertile rice line L422, the thermosensitive male sterility gene locus was located. RTMS10 Further experiments using CRISPR / Cas9 knockout and complementation demonstrated that the two genes within this region... RTMS10.1 and RTMS10.2They jointly regulate the Yns antithermothermic male sterility phenotype. This invention provides new genetic resources and clues for the improvement of new thermo-sensitive male sterile lines and dual-nuclear male sterile lines.

[0006] First aspect of protection of the present invention RTMS10.1 Genes and RTMS10.2 The use of genes or related biological materials in at least one of the following:

[0007] A1) Cultivate or prepare rice plants with temperature-sensitive male sterility;

[0008] A2) Rice variety improvement or preparation of rice variety improvement products

[0009] A3) Preparation of transgenic plants.

[0010] In some implementations, the RTMS10.1 Genes and RTMS10.2 The gene was transferred into the fertile rice material L422 and cultured to obtain a temperature-sensitive male sterile rice plant. The temperature-sensitive male sterile rice plant exhibited male sterility at 20℃-28℃ (daily average temperature) and recovered fertility at 29℃-34℃ (daily average temperature).

[0011] In some embodiments, the RTMS10.1 The gene contains the sequence shown in SEQ ID NO: 1; RTMS10.2 The nucleotides of the gene contain the sequence shown in SEQ ID NO: 2.

[0012] RTMS10.1 genome-seq (YnS)

[0013] ATGGAGGTTGGCATTTCTAGCTCTTATTTTGCCCTTTCTAAATATTCATTATTCGACTACCAAGCGATGATTTTTTATGCTCTATCAATAAGACGATCCTTCCAAGGAGGAGATCCAAGTGTCTCCAGCAAAAGACCAAGCAATGTGGAATCTAACAGGAAAGGTCCCGCAGGCACATCAAAGAAGAAGAAAAGGGGATGTTGTGTTGATATTCCTTCATCTGAATCTGATGAAGAAGACTGGGCACCAACTCCCACAAGGGAGGACAACCAACCAAATAGGAATATGATGGTGGACGAGCATCAGAGATATCCAGATTCAGGAGAAGATATTGAAGATATGATGGTGGACGAGCCTCAGAGATATCCAGATTCAGAAGATACTGAAGTGGCCAAGGATGTTCATCTTCCTACTGCAAAATGTGTAGGAAGAATCACACTTGACATAGAAGACTGGAGACCGGAGGTTCTGACAGTATGCCCAAGCTCTCCTGATACTGATTCCCCGATTTACATGAGCAGTGAGCCAGTGATCATCAACGAAAAAGAGTCTCAGTAG(SEQ ID NO:1)

[0014] >RTMS10.2 genome-seq (YnS)

[0015]

[0016] In some embodiments, the associated biological material is any one of B1) to B3):

[0017] B1) contains the above RTMS10.1 Genes and / or RTMS10.2 Gene recombination vectors;

[0018] B2) contains the above RTMS10.1 Genes and / or RTMS10.2 Recombinant microorganisms containing genes, or recombinant microorganisms containing the recombinant vector described in B1);

[0019] B3) RTMS10.1 Genes and / or RTMS10.2 Proteins encoded by genes.

[0020] In some implementation methods , RTMS10.1 The amino acid sequence of the gene-encoded protein includes the sequence shown in SEQ ID NO: 17.

[0021] >RTMS10.1 Protein sequence (YnS)

[0022] MEVGISSSYFALSKYSLFDYQAMIFYALSIRRSFQGGDPSVSSKRPSNVESNRKGPAGTSKKKKRGCCVDIPSSESDEEDWAPTPTREDNQPNRNMMVDEHQRYPDSGEDIEDMMVDEPQRYPDSEDTEVAKDVHLPTAKCVGRITLDIEDWRPEVLTVCPSSPDTDSPIYMSSEPVIINEKESQ* (SEQ ID NO: 17)

[0023] In some implementation methods , RTMS10.2 The amino acid sequence of the gene-encoded protein includes the sequence shown in SEQ ID NO: 18.

[0024] RTMS10.2 protein-seq (YnS)

[0025] MEPEGGVRLPTLDRVRRGVRSSCDTESWECFQCGSINLPVEKLLFDLPAFHCRGCEAPFLGEMNFCYDLVKANKRSLIGGLDNIKNQYDNPECIAYSIASCLEIADRIKTVLQGKNPDSVKEIDPIAIVDMFDGKCLANCSDGTSGIGKLVTMALAVQTDGIQSADHSRLYTAVAVETIDKYDFEGICATLADGIPLVGA FYCGSRLEKLEYCQIYRVPKLSKFLDRNLIPTGHAAVIIGAGMRCGIQYLYFLNSWGNFFCPRYDKDGNLVKAGVGKLRFYDLLCNPIMFITDSAKRVGL NRQLLPPMTGKLSDHNKSMLMGRKQTDVIPEDLSIGVTSEFVGNQPQISSKRKMANATLDGDGQTQKRNKCRTFGRSSVDLNEANNKRNAKEDEM* (SEQ ID NO: 18)

[0026] The second aspect of this invention protects a method for creating a temperature-sensitive male-sterile rice plant, comprising the following steps: introducing [the following method] into fertile rice material L422. RTMS10.1 Genes and RTMS10.2 Genes or related biological materials were used to obtain temperature-sensitive male-sterile rice plants.

[0027] In some embodiments, the method further includes PCR verification of temperature-sensitive male-sterile rice plants, wherein the primers for the PCR contain the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14.

[0028] SEQ ID NO: 13: GGCCGAAGTTCTGTCGATCT (SEQ ID NO: 13)

[0029] SEQ ID NO: 14: AATCATCGCAAGACCGGCA (SEQ ID NO: 14)

[0030] A third aspect of this invention protects a method for restoring fertility in male-sterile rice, characterized by comprising the following:

[0031] Knockout of the temperature-sensitive male sterile rice line YnS RTMS10.1 Genes and / or RTMS10.2 Gene.

[0032] In some implementations, knockout is performed using a CRISPR Cas9 system, wherein the sgRNA in the CRISPR Cas9 system is selected from one of the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0033] 5'-GATAGCAGACAGGATTAAGACGG-3' (SEQ ID NO: 3)

[0034] 5'-ATAAGACGATCCTTCCAAGGAGG-3' (SEQ ID NO: 4)

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] This invention discovers a method based on the CRISPR / Cas9 system to knock out, alter, or inhibit temperature-sensitive male sterility in rice. RTMS10.1 Genes and RTMS10.2 The transcription or expression of genes transforms thermosensitive sterile plants into fertile ones. Furthermore, this invention utilizes primer amplification... RTMS10.1 Genes and RTMS10.2 By using genetic transformation, the normally fertile rice material L422 can be transformed into a temperature-sensitive male-sterile plant. The temperature-sensitive male-sterile rice plant obtained by this invention shows no obvious abnormalities during the vegetative growth stage, is infertile at low temperatures but fertile at high temperatures, which can improve the heat resistance of rice pollen during the reproductive period and contribute to stable yield. Furthermore, when applied to hybridization breeding, the temperature-sensitive male-sterile line can eliminate the need for emasculation of the female parent, greatly improving production efficiency and reducing labor costs, demonstrating significant application potential in agricultural production. Attached Figure Description

[0037] Figure 1 The images show the plant architecture, anther phenotype, and pollen staining of the thermosensitive male sterile rice line YnS and the fertile rice material L422 under high and low temperatures, respectively, in Example 1 of the present invention.

[0038] Figure 2A This is a high-precision linkage analysis of the RTMS10 interval in Example 2 of the present invention. Vertical lines indicate molecular marker positions, with marker numbers located above the lines and exchanged individual plants below them. 'n' represents the population size. Solid black lines and hollow black lines represent L422 and YnS, respectively.

[0039] Figure 2B This is an example of collinearity analysis of Yns and L422 in the candidate interval in Embodiment 2 of the present invention.

[0040] Figure 2C This is an example of collinearity analysis of YnS and Nipponbare in the candidate interval in Embodiment 2 of the present invention.

[0041] Figure 2D This represents the expression abundance of genes within the candidate region in the transcriptome in Example 2 of the present invention.

[0042] Figure 3 In Embodiment 3 of the present invention RTMS10.2# Anther phenotype and pollen staining images of single knockout plants under high and low temperatures.

[0043] Figure 4 In Embodiment 3 of the present invention RTMS10.1# Anther phenotype and pollen staining images of single knockout plants under high and low temperatures.

[0044] Figure 5 In Embodiment 3 of the present invention RTMS10.1 - RTMS10.2# Anther phenotype and pollen staining images of double-knocked plants under high and low temperatures.

[0045] Figure 6 This is a transition from the complementary verification experiment in Embodiment 4 of the present invention. RTMS10.2 Genes obtained RTMS10.2 - Com Anther phenotype and pollen grain staining images of the plant under high temperature and low temperature conditions.

[0046] Figure 7 This is a transition from the complementary verification experiment in Embodiment 4 of the present invention. RTMS10.1 Genes obtained RTMS10.1 - Com Anther phenotype and pollen grain staining images of the plant under high temperature and low temperature conditions.

[0047] Figure 8 This is a transition from the complementary verification experiment in Embodiment 4 of the present invention. RTMS10.1 - RTMS10.2 Genes obtained RTMS10.1 - RTMS10.2 - Com Anther phenotype and pollen grain staining images of the plant under high temperature and low temperature conditions. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments. The following examples are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0049] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0050] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Experimental methods without detailed specifications are performed according to standard experimental methods or the supplier's recommended operating instructions.

[0051] In the embodiments described below, the pollen scale is 50 μm and the floret scale is 3 mm.

[0052] Example 1: Identification of the temperature-sensitive male sterility phenotype in rice

[0053] The temperature-sensitive male-sterile indica rice line YnS exhibits male sterility under low-temperature conditions (<29℃), with abnormal pollen development, resulting in inability to pollinate normally and thus preventing self-pollination and seed setting. Under high-temperature conditions (≥29℃), it exhibits male fertility, with normal pollen development, enabling normal self-pollination and seed setting. Therefore, its fertility transition critical temperature is 29-30℃. Within this temperature range, YnS gradually transitions from sterility to fertility. Regardless of high or low temperature conditions, there are no significant differences in the vegetative growth and main agronomic traits of the temperature-sensitive male-sterile indica rice line YnS.

[0054] Under low temperature (<29℃) conditions, the fertile rice variety L422 is male-fertile, with normal pollen development, enabling normal pollination and seed setting. Under high temperature (≥29℃) conditions, it is also male-fertile, with normal pollen development, enabling normal pollination and seed setting. Therefore, the fertility of L422 remains stable under different temperature conditions and is not affected by temperature changes.

[0055] The phenotypes of the temperature-sensitive male-sterile indica rice line YnS and the fertile rice line L422 were observed, and their pollen was subjected to I-type irradiation. 2- KI staining, results are shown in [link to results]. Figure 1 .

[0056] I 2- The KI staining procedure is as follows: Place a drop of 1.0% (W / V) I2-KI staining solution on a glass slide, take 2-3 anthers from each flower and place them in the staining solution, repeatedly pinch the anthers with tweezers to fully extract the pollen from the anthers, and then place the glass slide under an optical microscope for observation.

[0057] The principle of the I2-KI staining method: Iodine (I2) and potassium iodide (KI) solutions react with the starch in pollen, producing a color change. Starch in iodine solution will appear blue or bluish-purple, and the intensity of this color is related to the starch content in the pollen. Blue or bluish-purple pollen grains indicate a high starch content, which usually means the pollen is fertile. Pale blue or colorless pollen grains indicate a low or almost no starch content, which usually means the pollen is infertile.

[0058] from Figure 1 Microscopic observation revealed that, compared to high-temperature conditions (30℃), YnS anthers were smaller and paler in color under low-temperature conditions (22℃). Further I2-KI staining of YnS pollen under both temperature treatments showed that pollen grains under high-temperature conditions were plump and stained darkly, while pollen grains under low-temperature conditions could not be stained and had irregular shapes. Figure 1 ).

[0059] from Figure 1 It can be seen that the size and color of the anthers of L422 did not change significantly under high and low temperatures. The results of I2-KI staining of L422 pollen also confirmed that it was not affected by temperature.

[0060] In summary, the fertility of L422 is not affected by high or low temperatures. Therefore, in Example 2, L422 was hybridized with indica rice material YnS to construct a localized population.

[0061] Example 2: Gene Localization

[0062] The near-isogenic line L422S was obtained by crossing and backcrossing the rice variety Yannong S (YnS) with the normal fertile material Lunhui 422 (L422).

[0063] Based on the initial positioning, BC6F3 was obtained from the backcross of YnS and L422, and selected from it... RTMS10 The remaining heterozygotes in the interval developed into a segregating population of BC6F4, which consisted of 23,824 plants.

[0064] Recombinant plants were screened using markers S1305 and S9 located on either side, resulting in 27 recombinant plants. Using markers within this interval, the exchange intervals and individual phenotypes of these 27 plants were analyzed to determine... RTMS10 The relative position between markers. After verification through encrypted markers, screening of exchanged individual plants and their offspring, the final result will be... RTMS10 It is located within a physical region of 8.04 kb between S6833 and SNP14 on chromosome 10. Figure 2A According to predictions from the MBKbase website, there are two candidate genes in this region.

[0065] Genes within the candidate region showed no specificity in expression in florets. Sequencing of YnS using third-generation PacBio technology and collinearity analysis of the assembly results revealed that, compared to YnS, L422 had a large deletion of 55.2 kb within an 8.04 kb physical region. Figure 2B ).

[0066] The collinearity results of YnS and Nipponbare (NIP) in this region showed that the gene structures of the two materials were highly similar in this region.Figure 2C ).

[0067] Analysis was performed based on the genome sequence of Nipponbare (RGAP) from the website http: / / rice.plantbiology.msu.edu / . RTMS10 The gene in the 55.2 kb interval contains 13 annotated genes, most of which are transposons.

[0068] Transcriptome sequencing analysis showed that only two genes were candidate regions ( RTMS10.1 and RTMS10.2 ) specifically expressed in the spikelet of young rice panicle ( Figure 2D ), and no other genes were expressed.

[0069] Therefore RTMS10.1 and RTMS10.2 As the primary candidate gene.

[0070] Example 3: CRISPR / Cas9 Knockout Validation Experiment

[0071] 3.1 Obtaining transgenic knockout plants

[0072] RTMS10.2 The target sequence of the knocked-out sgRNA is:

[0073] 5'-GATAGCAGACAGGATTAAGACGG-3' (SEQ ID NO: 3)

[0074] RTMS10.1 The target sequence of the knocked-out sgRNA is:

[0075] 5'-ATAAGACGATCCTTCCAAGGAGG-3' (SEQ ID NO: 4)

[0076] RTMS10.2 - RTMS10.1 The target sequence of the sgRNA for double gene knockout is:

[0077] 5'-GATAGCAGACAGGATTAAGACGG-3' (SEQ ID NO: 3)

[0078] 5'-ATAAGACGATCCTTCCAAGGAGG-3' (SEQ ID NO: 4)

[0079] RTMS10.2 Primers for single gene knockout

[0080] cr RTMS10.2#-cas-FTGTTGATAGCAGACAGGATTAAGA (SEQ ID NO: 7)

[0081] cr -RTMS10.2#-cas-RAAACTCTTAATCCTGTCTGCTATC (SEQ ID NO: 8)

[0082] RTMS10.1 Primers for single gene knockout

[0083] cr -RTMS10.1#-cas-FGTGTATAAGACGATCCTTCCAAGG (SEQ ID NO: 5)

[0084] cr -RTMS10.1#-cas-RAAACCCTTGGAAGGATCGTCTTAT (SEQ ID NO: 6)

[0085] RTMS10.2 - RTMS10.1 Double gene knockout primers

[0086] cr -RTMS10.2# / 10.2-cas-F1: TGTT GATAGCAGACAGGATTAAGA (SEQ ID NO: 7)

[0087] cr -RTMS10.2# / 10.2-cas-R1: AAAC TCTTAATCCTGTCTGCTATC (SEQ ID NO: 8)

[0088] cr -RTMS10.1# / 10.1-cas-F2:GTGTATAAGACGATCCTTCCAAGG (SEQ ID NO: 5)

[0089] cr -RTMS10.1# / 10.1-cas-R2:AAACCCTTGGAAGGATCGTCTTAT (SEQ ID NO: 6)

[0090] Taking the double-knock carrier process as an example

[0091] The target sgRNAs of two genes were obtained from the design website CRISPR-P 2.0 http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / SCORE, see SEQ ID NO: 3 and SEQ ID NO: 4.

[0092] Vector construction primers: RTMS10.2The gene target was ligated to the intermediate vector pEntryA, with TGTT added to the F-5' end and AAAC added to the R-5' end. See primers below. cr -RTMS10.2# / 10.2-cas-F1 / cr -RTMS10.2# / 10.2-cas-R1 (SEQ ID NO:7 and SEQ ID NO:8); RTMS10.1 The gene target was ligated to the intermediate vector pEntry B, with GTGT added to the F-5' end and AAAC added to the R-5' end. See primers below. cr -RTMS10.1# / 10.1-cas-F2 / cr -RTMS10.1# / 10.1-cas-R2 (SEQ ID NO:5 and SEQ ID NO:6).

[0093] pEntryA / pEntryB vectors were digested with BsaI restriction enzyme to obtain the digested pEntryA / pEntryB vectors.

[0094] cr -RTMS10.2# / 10.2-cas-F1 / cr -RTMS10.2# / 10.2-cas-R1、 cr -RTMS10.1# / 10.1-cas-F2 / cr 5 μL each of RTMS10.1# and 10.1-cas-R2 primers were denatured at 95℃, allowed to cool naturally at room temperature, and then diluted 20 times for later use.

[0095] Ligation reaction: 0.5 μL annealing primer, 0.5 μL T4 ligase, 1 μL digested vector (approximately 40 μg), 1 μL 10x buffer, 7 μL MilliQ water (total 10 μL), ligation at room temperature for 2 h to obtain the ligation product. cr -RTMS10.1# / 10.1-pEntryA、 cr -RTMS10.1# / 10.1-pEntryB.

[0096] The ligation product was heat-shocked and transformed into competent cells according to the procedure, plated, and incubated overnight at 37°C. The next day, single colonies were selected for colony PCR and sent for analysis. Using the correctly sequenced plasmid as a template, PCR amplification was performed with primers MP542-F / R, and the amplified plasmid was recovered from the gel.

[0097] Primers used for amplification:

[0098] MP542-F:CCGGGTCACGCTGCACTGCAGTCTAGAGGATCCGTCGACAA (SEQ ID NO: 15)

[0099] MP542-R: CCCGAATTACTGCATACTAGTAGATCTCTCGAGAATTGCCC (SEQ ID NO: 16)

[0100] Simultaneously, the pRHCas9 vector was digested with PstI / SpeI, recovered via gel electrophoresis, and the amplified and recovered product was inserted into the linearized pRHCas9 vector after restriction enzyme digestion using the Novizan ClonExpressMultiS One Step Cloning Kit (catalog number: C113-01) (refer to the instruction manual for specific usage). The recombinant plasmid was transformed into competent E. coli cells and plated on LB agar containing 50 mg / L kanamycin. The cells were incubated at 37°C until uniformly distributed single colonies appeared. Positive single colonies were selected for shake culture. The cells underwent PCR amplification, plasmid restriction enzyme digestion identification, and sequencing to obtain a homologous recombinant plasmid linking two gene target sites (i.e., plasmids containing...). cr -RTMS10.2-RTMS10.1# CRISPR Cas9 plasmid DNA, cr -RTMS10.2# plasmid DNA from CRISPR Cas9. All plasmids used in the construction process were provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0101] The construction process for single-gene knockout vectors is the same as that for double-knockout vectors, resulting in homologous recombination plasmids linked to a single gene target (i.e., plasmids containing a single gene target). cr -RTMS10.1# CRISPR Cas9 plasmid DNA, cr -RTMS10.2# CRISPR Cas9 plasmid DNA).

[0102] The successfully sequenced homologous recombination plasmid and freshly harvested L422S seeds were sent to Wuhan Aidijing Company for subsequent genetic transformation to obtain transgenic positive seedlings.

[0103] The company utilizes Agrobacterium-mediated genetic transformation technology to integrate homologous recombinant plasmids into plant callus cells induced by L422S rice seed embryos. Through a series of steps including callus induction, subculture, Agrobacterium infection and co-culture, resistant callus screening, pre-differentiation, differentiation, rooting, hardening-off, PCR detection, and transplanting, the company ultimately obtains the corresponding positive transgenic seedlings (i.e.,...). cr - RTMS10.1# Single knock on the plant, cr - RTMS10.2# Single knock plant and cr - RTMS10.2 - RTMS10.1# (Double-knock plant).

[0104] Extract separately cr -RTMS10.1# and cr -RTMS10.2# andcr DNA from the leaves of seedlings from the -RTMS10.2-RTMS10.1# knockout strains was used to amplify the DNA fragment containing the knockout target site using PCR. The product was sent to the company for sequencing, and the sequencing results were analyzed. Plants that underwent editing were retained and continued to be cultivated. The next generation was identified, and homozygous edited plants were retained. These homozygous families can be used for subsequent phenotypic analysis of transgenic materials.

[0105] Meanwhile, L422S was used as a control group.

[0106] 3.2 Phenotypic observation of transgenic knockout plants

[0107] Sequencing and identification yielded... cr - RTMS10.1# Single knock on the plant, cr - RTMS10.2# Single knock plant and cr- RTMS10.2 - RTMS10.1# Double-knocked plants. Anther phenotypes under low and high temperatures, and I... 2- KI staining.

[0108] The results are shown below. Figures 3 - 5 Anther phenotype was observed using a stereomicroscope.

[0109] from Figure 3 It can be seen that, regardless of whether the conditions are low or high temperature, cr - RTMS10.2# The flowers of a single plant are all full and golden yellow, and can be stained dark by I2-KI.

[0110] from Figure 4 It can be seen that, regardless of whether the conditions are low or high temperature, cr - RTMS10.1# The flowers of a single plant are all full and golden yellow, and can be stained dark by I2-KI.

[0111] from Figure 5 It can be seen that, regardless of whether the conditions are low or high temperature, cr - RTMS10.2 - RTMS10.1# Both flowers of the double-knocked plant are full and golden yellow, and can be stained dark by I2-KI.

[0112] Example 4 Complementary Verification Test

[0113] 4.1 Obtaining complementary transgenic materials

[0114] Amplification of YnS materials RTMS10.1 and RTMS10.2 and RTMS10.1 - RTMS10.2 The genome sequence was obtained, a complementary vector was constructed, and it was transformed into the callus tissue of L422 rice material with normal fertility.

[0115] Using genomic DNA from YnS material as a template, and RTMS10.1-GF / RTMS10.1-GR, RTMS10.2-GF / RTMS10.2-GR, and RTMS10.1-10.2-GF / RTMS10.1-10.2-GR as primers (primers below), the target DNA fragment was amplified using Phanta Max Master Mix (catalog number: P515-01) from Novizan. The primers contained restriction enzyme sites and nearby sequences from the pRHE vector (a gift from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences).

[0116] Using the ClonExpress MultiS One Step Cloning Kit (Catalog No.: C113-01) from Novizan, the recovered product was inserted into the linearized vector pRHE (which had been digested with restriction enzymes) to obtain the recombinant plasmid.

[0117] The recombinant plasmid was transformed into competent *E. coli* cells and plated on LB agar containing 50 mg / L kanamycin. The cells were incubated at 37°C until uniformly distributed single colonies appeared. Positive single colonies were selected for shake culture. The colonies underwent PCR amplification, plasmid restriction enzyme digestion identification, and sequencing. The successfully sequenced homologous recombinant plasmid and freshly harvested L422 seeds were sent to Wuhan Aidi Crystal Co., Ltd. for subsequent genetic transformation to obtain transgenic positive seedlings.

[0118] The company uses Agrobacterium-mediated genetic transformation technology to integrate recombinant plasmid DNA fragments into plant callus cells induced by L422 rice material embryos. Through the following steps: callus induction → subculture → Agrobacterium infection and co-culture → resistant callus screening → predifferentiation → differentiation → rooting → hardening → PCR detection → transplanting, the company finally obtains the corresponding positive transgenic seedlings.

[0119] RTMS10.1 Single gene complementation primers

[0120] RTMS10.1-GF: TATCCAGATCCAGTGGGATCCGTATGCGGCTCCTTAAGTGGAC (SEQ ID NO: 9)

[0121] RTMS10.1-GR:CGCACTAGTAAGCTTGGTACCCTGAGACTCTTTTTCGTTGATGAT (SEQ ID NO: 10)

[0122] RTMS10.2 Single gene complementation primers

[0123] RTMS10.2-GF: TATCCAGATCCAGTGGGATCCCTGTACTACGCCCTAAGTTACA (SEQ ID NO: 11)

[0124] RTMS10.2-GR: CGCACTAGTAAGCTTGGTACC CATCTCATCTTCCTTTGCATTCCGT (SEQ IDNO: 12)

[0125] RTMS10.1 - RTMS10.2 Double gene complementation primers

[0126] RTMS10.1-10.2-GF:ATCCAGATCCAGTGGGATCCCTGTACTACGCCCTAAGTTACA (SEQ IDNO: 13)

[0127] RTMS10.1-10.2-GR:GCACTAGTAAGCTTGGTACCTATGCGGCTCCTTAAGTGGAC (SEQ IDNO: 14)

[0128] 4.2 Phenotypic observation of complementary transgenic materials

[0129] The anther phenotypes of the single-gene complementary materials and double-gene complementary materials obtained in step 4.1 were analyzed under low temperature and high temperature conditions, as well as I... 2- KI staining.

[0130] The results are shown below. Figures 6 - 8 .

[0131] from Figure 6 and 7 It can be seen that, RTMS10.1 and RTMS10.2 The single-gene complementary material has fertile florets that are plump and golden yellow under both high temperature (30℃) and low temperature (22℃) conditions and can be stained dark by I2-KI.

[0132] from Figure 8 It can be seen that, RTMS10.1 - RTMS10.2 In the double gene complementary material, under low temperature conditions (22℃), the anthers of the transgenic material become smaller and lighter in color, the pollen grains cannot be stained, and the shape is irregular. Under high temperature conditions (30℃), the florets are full and golden yellow, and can be stained dark by I2-KI.

[0133] from Figures 6 - 8 It can be seen that the florets of the control material L422 are full and golden yellow under both low temperature (22℃) and high temperature (30℃) conditions, and can be stained dark by I2-KI, making it fully fertile.

[0134] The above results indicate that , RTMS10.1 and RTMS10.2 The combined action of genes is necessary to cause the thermosensitive male sterility phenotype.

[0135] This invention is not limited to the specific textual description above. Various changes can be made to this invention within the scope outlined in the claims, and all such changes are within the scope of this invention.

Claims

1. Use of a RTMS10.1 gene and a RTMS10.2 gene or biological material related thereto in at least one of the following: A1) breeding a rice reverse temperature-sensitive male sterile plant or preparing a product for breeding a rice reverse temperature-sensitive male sterile plant; A2) preparing a transgenic rice; the nucleotide sequence of the RTMS10.1 gene is shown as SEQ ID NO: 1; the nucleotide sequence of the RTMS10.2 gene is shown as SEQ ID NO: 2; the biological material related thereto is any one of B1) to B3): B1) a recombinant vector containing the RTMS10.1 gene and the RTMS10.2 gene; B2) a recombinant microorganism containing the RTMS10.1 gene and the RTMS10.2 gene, or a recombinant microorganism containing the recombinant vector of B1); B3) a protein encoded by the RTMS10.1 gene and the RTMS10.2 gene.

2. The use according to claim 1, characterized in that, The RTMS10.1 gene and the RTMS10.2 gene are introduced into a normal fertile rice material L422 to breed a rice reverse temperature-sensitive male sterile plant; the rice reverse temperature-sensitive male sterile plant exhibits male sterility at 20-28°C and restores fertility at 29-34°C.

3. A method for creating a temperature-sensitive male sterile rice plant, characterized by, The method comprises the following steps: introducing the RTMS10.1 gene and the RTMS10.2 gene or biological material related thereto into a normal fertile rice material L422 to obtain a rice reverse temperature-sensitive male sterile plant; the nucleotide sequence of the RTMS10.1 gene is shown as SEQ ID NO: 1; the nucleotide sequence of the RTMS10.2 gene is shown as SEQ ID NO: 2; the biological material related thereto is any one of B1) to B3): B1) a recombinant vector containing the RTMS10.1 gene and the RTMS10.2 gene; B2) a recombinant microorganism containing the RTMS10.1 gene and the RTMS10.2 gene, or a recombinant microorganism containing the recombinant vector of B1); B3) a protein encoded by the RTMS10.1 gene and the RTMS10.2 gene.

4. The method of creating of claim 3, wherein, PCR verification is further included for the rice reverse temperature-sensitive male sterile plant, and the primers of the PCR are sequences shown as SEQ ID NO: 13 and SEQ ID NO:

14.

5. A method for restoring fertility to male sterile rice plants, characterized by, The method comprises the following steps: knocking out the RTMS10.1 gene and / or the RTMS10.2 gene in a reverse temperature-sensitive male sterile rice YnS; the nucleotide sequence of the RTMS10.1 gene is shown as SEQ ID NO: 1; the nucleotide sequence of the RTMS10.2 gene is shown as SEQ ID NO:

2.

6. The method of claim 5, wherein, The knocking out is performed by using a CRISPR Cas9 system, and sgRNA in the CRISPR Cas9 system is selected from sequences shown as SEQ ID NO: 3 and SEQ ID NO: 4.

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