Application of RTMS10.1 gene and RTMS10.2 gene in cultivation of anti-temperature-sensitive male sterile line rice

Abstract

The invention discloses an application of an RTMS10.1 gene and an RTMS10.2 gene in cultivation of reverse temperature-sensitive male sterile line rice. According to the invention, based on a CRISPR / Cas9 system, substances for knocking out, changing or inhibiting transcription or expression of RTMS10.1 genes and RTMS10.2 genes in an anti-thermo-sensitive sterile rice material YnS are found, so that a rice anti-thermo-sensitive male sterile line becomes fertile. Furthermore, after the gene RTMS10.1 and the gene RTMS10.2 are introduced into the rice L422 with normal fertility, the L422 generates an anti-thermo-sensitive sterile phenotype. According to the invention, it is found for the first time that the RTMS10.1 gene and the RTMS10.2 gene jointly regulate and control the anti-thermo-sensitive male sterility character of the rice, and the gene can be used for creation of an anti-thermo-sensitive male sterility line of the rice, is applied to breeding of two-line hybrid rice, and has wide development prospects and application values.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to RTMS 10.1 a gene and RTMS 10.2 application of the gene in breeding of photoperiod-thermosensitive male sterile line of rice. BACKGROUND

[0002] Hybrid rice can significantly improve rice yield by using heterosis, which is of great significance to guarantee China's food security. Photoperiod-thermosensitive male sterile line is the core material of two-line hybrid rice breeding in China. Under restrictive conditions (such as long day length and high temperature), pollen abortion occurs, which can be used as a sterile line to cross with a restorer line to produce hybrid varieties. Under suitable conditions (such as short day length and low temperature), fertility is restored, and self-pollination is successful. Therefore, it is also known as a dual-purpose male sterile line. According to the different responses to light and temperature conditions, photoperiod-thermosensitive male sterile line can be divided into two main types: light-sensitive and temperature-sensitive. In production practice, the application of temperature-sensitive male sterile line is more widely used, but there are still problems such as unstable fertility, difficulty in breeding, etc. In addition, the practical male sterile line gene resources are still few, which limits the genetic improvement of male sterile line and the sustainable development and application of two-line hybrid rice. Therefore, cloning new genes that regulate photoperiod-thermosensitive male sterility and analyzing their molecular mechanisms are important ways to solve the problems of two-line hybrid rice breeding, which can provide a new technical path for the sustainable development of two-line hybrid rice, and have important theoretical significance and practical value. In 1973, Mr. Shi Ming Song discovered a photosensitive sterile mutant (named Nongkeng 58S) from the late japonica variety Nongken 58. It showed male sterility under long-day conditions and fertility restoration under short-day conditions. Based on this, he proposed the "one system and two uses" hybrid rice grouping idea and carried out practical research, which opened the curtain of two-line hybrid rice breeding in China. Subsequently, Deng Huafeng and other breeders discovered and bred the indica warm-sensitive male sterile line Annong S1, which is sterile under high temperature conditions and fertile under low temperature conditions. The breeding of Annong S1 opened up a new way for the utilization of rice heterosis and promoted the development process of two-line hybrid breeding. Using Nongkeng 58S and Annong S1 as parents, breeders have bred many two-use nuclear sterile materials (warm-sensitive, light and warm-sensitive, reverse warm-sensitive, and reverse light and warm-sensitive) regulated by different environments. After more than 50 years of research and development, two-line hybrid rice based on warm-sensitive male nuclear sterile lines has become an indispensable part of hybrid rice. It plays an important role in improving rice yield, improving rice quality, and utilizing intersubspecific heterosis and super rice variety breeding. However, the current two-use nuclear sterile line still faces application bottlenecks, such as single sterile gene resources, and the genetic mechanism of fertility conversion critical temperature lags behind. To solve these problems, new two-use nuclear sterile gene resources need to be explored; the critical temperature regulation gene of fertility conversion needs to be studied to improve the efficiency of breeding; and molecular marker-assisted breeding technology needs to be combined to broaden the genetic basis of warm-sensitive nuclear sterile lines.

[0003] Currently, all cloned warm-sensitive male sterile genes are positive warm-sensitive sterile genes, and reverse warm-sensitive sterile genes have not been reported. Among the existing TGMS genes, more than 95% of two-line hybrid rice relies on tms5 derived sterile lines, leading to serious homogeneity of warm-sensitive sterile lines and their grouping hybrid rice. In addition, positive warm-sensitive sterile lines (sterile at high temperature / fertile at low temperature) are only suitable for seed production in midsummer high temperature, and cannot meet the seed production needs in spring and autumn in low latitude rice areas and summer in high latitude rice areas. Reverse warm-sensitive sterile lines, such as YnS, can remain sterile at an average daily temperature of less than 29°C, which can effectively break through these limitations and has important theoretical research and application value. SUMMARY

[0004] In view of the problems existing in the current two-line breeding, the present application discovers a new reverse warm-sensitive male sterile gene, which provides a new gene resource for two-line breeding. In this study, the reverse warm-sensitive male sterile material YnS (sterile at low temperature / fertile at high temperature) was used as the research object, and the high-generation population obtained by backcrossing the reverse warm-sensitive male sterile line rice YnS and the fertile rice material L422 was used to locate the reverse warm-sensitive male sterile gene locus RTMS 10 . Further experiments such as CRISPR / Cas9 knockout and complementation proved that two genes RTMS 10.1 and RTMS 10.2The genes are co-regulated to phenotype of Yns reverse temperature-sensitive male sterility. The present application provides new genetic resources and clues for breeding new temperature-sensitive sterile lines and improved dual-purpose nuclear sterile lines.

[0005] In a first aspect the present application protects RTMS 10.1 a gene and RTMS 10.2 a gene or biological material related thereto for use in at least one of: A1) breeding a rice reverse temperature-sensitive male sterile line or preparing a rice reverse temperature-sensitive male sterile line; A2) rice variety improvement or preparing a product of rice variety improvement A3) preparing a transgenic plant.

[0006] In some embodiments, the gene is introduced into a normal fertile rice material L422 to breed a rice reverse temperature-sensitive male sterile line; the rice reverse temperature-sensitive male sterile line exhibits male sterile line at 20-28°C (daily average temperature) and restores fertility at 29-34°C (daily average temperature). RTMS 10.1 RTMS 10.2 In certain embodiments, the nucleotide of the gene comprises the sequence shown in SEQ ID NO: 1; the nucleotide of the gene comprises the sequence shown in SEQ ID NO: 2.

[0007] In some embodiments, the nucleotide of the gene comprises the sequence shown in SEQ ID NO: 1; the nucleotide of the gene comprises the sequence shown in SEQ ID NO: 2. RTMS 10.1 RTMS 10.2 >RTMS10.1 genome-seq (YnS)

[0008] >RTMS10.1 genome-seq (YnS) ​​ATGGAGGTTGGCATTTCTAGCTCTTATTTTGCCCTTTCTAAATATTCATTATTCGACTACCAAGCGATGATTTTTTATGCTCTATCAATAAGACGATCCTTCCAAGGAGGAGATCCAAGTGTCTCCAGCAAAAGACCAAGCAATGTGGAATCTAACAGGAAAGGTCCCGCAGGCACATCAAAGAAGAAGAAAAGGGGATGTTGTGTTGATATTCCTTCATCTGAATCTGATGAAGAAGACTGGGCACCAACTCCCACAAGGGAGGACAACCAACCAAATAGGAATATGATGGTGGACGAGCATCAGAGATATCCAGATTCAGGAGAAGATATTGAAGATATGATGGTGGACGAGCCTCAGAGATATCCAGATTCAGAAGATACTGAAGTGGCCAAGGATGTTCATCTTCCTACTGCAAAATGTGTAGGAAGAATCACACTTGACATAGAAGACTGGAGACCGGAGGTTCTGACAGTATGCCCAAGCTCTCCTGATACTGATTCCCCGATTTACATGAGCAGTGAGCCAGTGATCATCAACGAAAAAGAGTCTCAGTAG (SEQ ID NO: 1) >RTMS10.2 genome-seq (YnS) In some embodiments, the biological material related thereto is any one of B1) to B3): B1) a recombinant vector containing the RTMS 10.1 gene and / or RTMS 10.2 gene; B2) a recombinant microorganism containing the RTMS 10.1 gene and / or RTMS 10.2 gene, or a recombinant microorganism containing the recombinant vector of B1); B3) a protein encoded by the RTMS 10.1 gene and / or RTMS 10.2 gene.

[0009] In some embodiments RTMS 10.1 the amino acid sequence of the protein encoded by the

[0010] >RTMS10.1 Protein sequence (YnS) MEVGISSSYFALSKYSLFDYQAMIFYALSIRRSFQGGDPSVSSKRPSNVESNRKGPAGTSKKKKRGCCVDIPSSESDEEDWAPTPTREDNQPNRNMMVDEHQRYPDSGEDIEDMMVDEPQRYPDSEDTEVAKDVHLPTAKCVGRITLDIEDWRPEVLTVCPSSPDTDSPIYMSSEPVIINEKESQ* (SEQ ID NO: 17) In some embodiments RTMS 10.2 the amino acid sequence of the protein encoded by the

[0011] >RTMS10.2 protein-seq (YnS) MEPEGGVRLPTLDRVRRGVRSSCDTESWECFQCGSINLPVEKLLFDLPAFHCRGCEAPFLGEMNFCYDLVKANKRSLIGGLDNIKNQYDNPECIAYSIASCLEIADRIKTVLQGKNPDSVKEIDPIAIVDMFDGKCLANCSDGTSGIGKLVTMALAVQTDGIQSADHSRLYTAVAVETIDKYDFEGICATLADGIPLVGAFYCGSRLEKLEYCQIYRVPKLSKFLDRNLIPTGHAAVIIGAGMRCGIQYLYFLNSWGNFFCPRYDKDGNLVKAGVGKLRFYDLLCNPIMFITDSAKRVGLNRQLLPMGTGKLSDHNKSMLMGRKQTDVIPEDLSIGVTSEFVGNQPQISSKRKMANATLDGDGQTQKRNKCRTFGRSSVDLNEANNKRNAKEDEM* (SEQ ID NO: 18) The second aspect of the present application protects a method for creating a rice reverse temperature-sensitive male sterile plant, comprising the following steps: introducing into a normal fertility rice material L422 RTMS 10.1 a gene and RTMS 10.2 a gene or biological material related thereto, to obtain a rice reverse temperature-sensitive male sterile plant.

[0012] In some embodiments, PCR verification is further included for the rice reverse temperature-sensitive male sterile plant, and the primers of the PCR comprise the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14.

[0013] SEQ ID NO: 13: GGCCGAAGTTCTGTCGATCT (SEQ ID NO: 13) SEQ ID NO: 14: AATCATCGCAAGACCGGCA (SEQ ID NO: 14) The third aspect of the present application protects a method for restoring fertility of a male sterile rice plant, characterized in that it comprises the following: knocking out the RTMS 10.1 gene and / or RTMS 10.2 gene in a rice YnS of a reverse temperature-sensitive male sterile line.

[0014] In some embodiments, the knocking out is performed by using a CRISPR Cas9 system, and 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.

[0015] 5'-GATAGCAGACAGGATTAAGACGG-3' (SEQ ID NO: 3) 5'-ATAAGACGATCCTTCCAAGGAGG-3' (SEQ ID NO: 4) Compared with the prior art, the present invention has the following beneficial effects: This invention discovers a method based on the CRISPR / Cas9 system to knock out, alter, or inhibit temperature-sensitive male sterility in rice. RTMS 10.1 Genes and RTMS 10.2 The transcription or expression of genes transforms thermosensitive sterile plants into fertile ones. Furthermore, this invention utilizes primer amplification... RTMS 10.1 Genes and RTMS 10.2 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, thus possessing significant application potential in agricultural production. Attached Figure Description

[0016] 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.

[0017] 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 individuals below them. 'n' represents the population size. Solid black lines and hollow black lines represent L422 and YnS, respectively.

[0018] Figure 2B This is an example of collinearity analysis of Yns and L422 in the candidate interval in Embodiment 2 of the present invention.

[0019] Figure 2C This is an example of collinearity analysis of YnS and Nipponbare in the candidate interval in Embodiment 2 of the present invention.

[0020] Figure 2D This represents the expression abundance of genes within the candidate region in the transcriptome in Example 2 of the present invention.

[0021] Figure 3 In Embodiment 3 of the present invention RTMS 10.2Anther phenotype and pollen staining images of single knockout plants under high and low temperatures.

[0022] Figure 4 In Embodiment 3 of the present invention RTMS 10.1 Anther phenotype and pollen staining images of single knockout plants under high and low temperatures.

[0023] Figure 5 In Embodiment 3 of the present invention RTMS 10.1 - RTMS 10.2 Anther phenotype and pollen staining images of double-knocked plants under high and low temperatures.

[0024] Figure 6 This is a transition from the complementary verification experiment in Embodiment 4 of the present invention. RTMS 10.2 Genes obtained RTMS 10.2 - Com Anther phenotype and pollen grain staining images of the plant under high temperature and low temperature conditions.

[0025] Figure 7 This is a transition from the complementary verification experiment in Embodiment 4 of the present invention. RTMS 10.1 Genes obtained RTMS 10.1 - Com Anther phenotype and pollen grain staining images of the plant under high temperature and low temperature conditions.

[0026] Figure 8 This is a transition from the complementary verification experiment in Embodiment 4 of the present invention. RTMS 10.1 - RTMS 10.2 Genes obtained RTMS 10.1 - RTMS 10.2 - Com Anther phenotype and pollen grain staining images of the plant under high temperature and low temperature conditions. Detailed Implementation

[0027] 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.

[0028] 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.

[0029] 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.

[0030] In the embodiments described below, the pollen scale is 50 μm and the floret scale is 3 mm.

[0031] Phenotype identification of rice reverse temperature-sensitive male sterile line YnS The rice reverse temperature-sensitive male sterile line YnS is male sterile under low temperature conditions (<29℃), and its pollen development is abnormal, which cannot pollinate normally and lead to self-sterility. Under high temperature conditions (≥29℃), YnS is male fertile, and its pollen development is normal, which can pollinate and set seeds normally. Therefore, the fertility of YnS gradually changes from sterile to fertile at the critical temperature of 29-30℃. Under both high and low temperature conditions, the vegetative growth and main agronomic traits of the rice reverse temperature-sensitive male sterile line YnS have no obvious difference.

[0032] The normal fertility rice L422 is male fertile under low temperature conditions (<29℃), and its pollen development is normal, which can pollinate and set seeds normally. Under high temperature conditions (≥29℃), L422 is also male fertile, and its pollen development is normal, which can pollinate and set seeds normally. Therefore, the fertility of L422 remains stable under different temperature conditions and is not affected by temperature changes.

[0033] The phenotypes of the rice reverse temperature-sensitive male sterile line YnS and the normal fertility rice L422 were observed, and the pollen of the two rice lines was respectively subjected to I2-KI staining. 2- KI staining, and the results are shown in Figure 1 .

[0034] I 2- KI staining steps: drop 1.0% (W / V) I2-KI staining solution on a glass slide, take 2-3 anthers from each flower of YnS and L422, and place them in the staining solution. Use tweezers to repeatedly clamp the anthers, and fully crush the pollen from the anthers. Then, place the glass slide under an optical microscope for observation.

[0035] The principle of I2-KI staining method: iodine (I2) and potassium iodide (KI) solution react with starch in pollen, resulting in color change. Starch in iodine solution will appear blue or blue-violet, and the depth of this color is related to the starch content in pollen. Pollen grains appear blue or blue-violet, indicating that the pollen contains a lot of starch, which usually means that the pollen is fertile. Pollen grains appear light blue or colorless, indicating that the pollen contains little or almost no starch. This usually means that the pollen is sterile.

[0036] From Figure 1 the stereomicroscope observation, it was found that compared with high temperature conditions (30℃), the anthers of YnS under low temperature conditions (22℃) became smaller and the color became lighter. Further I2-KI staining of YnS pollen under the two temperature treatment conditions showed that the pollen grains under high temperature conditions were full and could be stained dark, while the pollen grains under low temperature conditions could not be stained and were irregular in shape Figure 1 ).

[0037] 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.

[0038] 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.

[0039] Example 2: Gene Localization 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).

[0040] Based on the initial positioning, BC6F3 was obtained from the backcross of YnS and L422, and selected from it... RTMS 10 The remaining heterozygotes in the interval developed into a segregating population of BC6F4, which consisted of 23,824 plants.

[0041] 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... RTMS 10 The relative position between markers. After verification through encrypted markers, screening of exchanged individual plants and their offspring, the final result will be... RTMS 10 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.

[0042] 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 ).

[0043] 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 ).

[0044] Analysis was performed based on the genome sequence of Nipponbare (RGAP) from the website http: / / rice.plantbiology.msu.edu / . RTMS 10 The gene in the 55.2 kb interval contains 13 annotated genes, most of which are transposons.

[0045] Transcriptome sequencing result analysis showed that only 2 genes in the candidate interval RTMS 10.1 and RTMS 10.2 were specifically expressed in the young spike lemma of rice Figure 2D , and other genes were not expressed.

[0046] Therefore, the RTMS 10.1 and RTMS 10.2 genes were the primary candidate genes.

[0047] Example 3 CRISPR / Cas9 knockout verification test 3.1, obtaining of transgenic knockout plants RTMS 10.2 The sgRNA target sequence for knockout is: 5'-GATAGCAGACAGGATTAAGACGG-3' (SEQ ID NO: 3) RTMS 10.1 The sgRNA target sequence for knockout is: 5'-ATAAGACGATCCTTCCAAGGAGG-3' (SEQ ID NO: 4) RTMS 10.2 - RTMS 10.1 The sgRNA target sequence for double gene knockout is: 5'-GATAGCAGACAGGATTAAGACGG-3' (SEQ ID NO: 3) 5'-ATAAGACGATCCTTCCAAGGAGG-3' (SEQ ID NO: 4) RTMS 10.2 Primers for single gene knockout cr -RTMS10.2#-cas-FTGTTGATAGCAGACAGGATTAAGA (SEQ ID NO: 7) cr -RTMS10.2#-cas-RAAACTCTTAATCCTGTCTGCTATC (SEQ ID NO: 8) RTMS 10.1 Primers for single gene knockout cr -RTMS10.1#-cas-FGTGTATAAGACGATCCTTCCAAGG (SEQ ID NO: 5) cr -RTMS10.1#-cas-RAAACCCTTGGAAGGATCGTCTTAT (SEQ ID NO: 6) RTMS 10.2 - RTMS 10.1 Double gene knockout primers cr -RTMS10.2# / 10.2-cas-F1: TGTT GATAGCAGACAGGATTAAGA (SEQ ID NO: 7) cr -RTMS10.2# / 10.2-cas-R1: AAAC TCTTAATCCTGTCTGCTATC (SEQ ID NO: 8) cr -RTMS10.1# / 10.1-cas-F2: GTGTATAAGACGATCCTTCCAAGG (SEQ ID NO: 5) cr -RTMS10.1# / 10.1-cas-R2: AAACCCTTGGAAGGATCGTCTTAT (SEQ ID NO: 6) Example of double knock vector process Target sgRNA of two genes were obtained by designing website CRISPR-P 2.0 http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / SCORE, see SEQ ID NO: 3 and SEQ ID NO: 4.

[0048] Vector construction primers: RTMS 10.2 Gene target was connected to intermediate vector pEntryA, TGTT was added to the 5' end of F, AAAC was added to the 5' end of R, primers were seen cr -RTMS10.2# / 10.2-cas-F1 / cr -RTMS10.2# / 10.2-cas-R1 (SEQ ID NO: 7 and SEQ ID NO: 8); RTMS 10.1 Gene target was connected to intermediate vector pEntry B, GTGT was added to the 5' end of F, AAAC was added to the 5' end of R, primers were seen cr -RTMS10.1# / 10.1-cas-F2 / cr -RTMS10.1# / 10.1-cas-R2 (SEQ ID NO: 5 and SEQ ID NO: 6).

[0049] The pEntryA / pEntryB vectors were respectively digested with BsaI endonuclease to obtain the digested pEntryA / pEntryB vectors.

[0050] cr -RTMS10.2# / 10.2-cas-F1 / cr-RTMS10.2# / 10.2-cas-R1, cr -RTMS10.1# / 10.1-cas-F2 / cr -RTMS10.1# / 10.1-cas-R2

[0051] Ligation reaction: annealed primers 0.5 μL, T4 ligase 0.5 μL, digested vector 1 μL (about 40 μg), 10x buffer 1 μL, MilliQ water 7 μL (total 10 μL), room temperature ligation for 2 h, to obtain ligation product cr -RTMS10.1# / 10.1-pEntryA, cr -RTMS10.1# / 10.1-pEntryB.

[0052] The ligation product was heat-shocked to transform competent cells according to the procedure, plated, and incubated at 37°C overnight. The next day, single colonies were selected for colony PCR and sent for testing. The correct sequencing plasmid was used as a template for PCR amplification with primers MP542-F / R, and the gel was recovered.

[0053] Primers used for amplification: MP542-F: CCGGGTCACGCTGCACTGCAGTCTAGAGGATCCGTCGACAA (SEQ ID NO: 15) MP542-R: CCCGAATTACTGCATACTAGTAGATCTCTCGAGAATTGCCC (SEQ ID NO: 16) Meanwhile, the pRHCas9 vector was digested with Pstl / SpeI, and the gel was recovered. The recovered product was inserted into the linearized vector pRHCas9 digested with endonuclease using the ClonExpress Multi S One Step Cloning Kit (Cat. No. C113-01) from Novagen, according to the instructions. The recombinant plasmid was transformed into E. coli competent cells and plated on LB solid medium containing 50 mg / L kanamycin, and incubated in a 37°C incubator until uniform single colonies were grown. Positive single colonies were selected for shake culture. After PCR amplification of the bacterial solution, plasmid digestion identification, and sequencing, etc. detection procedures, the homologous recombination plasmid connecting the two gene targets (i.e., the plasmid containing the RTMS10.2-RTMS10.1# CRISPR Cas9 and the RTMS10.2# CRISPR Cas9) was obtained. cr -RTMS10.2-RTMS10.1# CRISPR Cas9 plasmid DNA, cr -RTMS10.2# CRISPR Cas9 plasmid DNA). The plasmids in the construction process were all donated by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0054] Single gene knockout vector construction process with double knockout vector construction process, respectively, to connect a gene target homologous recombination plasmid (ie with cr -RTMS10.1# CRISPR Cas9 plasmid DNA, cr -RTMS10.2# CRISPR Cas9 plasmid DNA).

[0055] The sequencing success of the homologous recombination plasmid and the fresh harvest of L422S seed to Wuhan Aidier company, for subsequent genetic transformation, obtain transgenic positive seedlings.

[0056] The company through the agrobacterium-mediated genetic transformation technology, the homologous recombination plasmid is integrated into the L422S rice material seed embryo induced plant callus cells, through the induction of callus→subculture→agrobacterium infection and co-culture→resistant callus screening→pre-differentiation→differentiation→rooting→refining seedlings→PCR detection→transplanting etc. link, finally obtain the corresponding positive transgenic seedlings (ie cr-RTMS 10.1 single knockout strain, cr-RTMS 10.2 single knockout strain and cr-RTMS 10.2 - RTMS 10.1 double knockout strain).

[0057] Respectively extract cr -RTMS10.1# and cr -RTMS10.2# and cr -RTMS10.2-RTMS10.1# knockout strain seedling leaf DNA, PCR amplification of DNA fragments containing knockout target, product to the company sequencing, and the sequencing results were analyzed, and the edited plants were retained, continue to plant, the next generation of identification and retention of homozygous edited plants. These homozygous lines can be used for subsequent phenotype analysis of transgenic materials.

[0058] At the same time, L422S as a control group.

[0059] 3.2, phenotype observation of transgenic knockout plants Sequencing identification, respectively, cr-RTMS 10.1 single knockout strain, cr-RTMS 10.2 single knockout strain and cr- RTMS 10.2 - RTMS 10.1 double knockout strain. Low temperature, high temperature anther phenotype, and I 2- KI staining.

[0060] The results are shown in Figure 3 - 5 . Anther phenotype was observed by body type mirror.

[0061] From Figure 3 It can be seen that, under low temperature or high temperature conditions, cr-RTMS 10.2The flowers of a single plant are all full and golden yellow, and can be stained dark by I2-KI.

[0062] from Figure 4 It can be seen that, regardless of whether the conditions are low or high temperature, cr-RTMS 10.1 The flowers of a single plant are all full and golden yellow, and can be stained dark by I2-KI.

[0063] from Figure 5 It can be seen that, regardless of whether the conditions are low or high temperature, cr-RTMS 10.2 - RTMS 10.1 Both flowers of the double-knocked plant are full and golden yellow, and can be stained dark by I2-KI.

[0064] Example 4 Complementary Verification Test 4.1 Obtaining complementary transgenic materials Amplification of YnS materials RTMS 10.1 and RTMS 10.2 and RTMS 10.1 - RTMS 10.2 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.

[0065] 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).

[0066] 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.

[0067] 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.

[0068] The company integrates the recombinant plasmid DNA fragment into the plant callus cells induced by the embryo of L422 rice material through the agrobacterium-mediated genetic transformation technology, and finally obtains the corresponding positive transgenic seedlings through the following steps: induction of callus, subculture, agrobacterium infection and co-culture, screening of resistant callus, pre-differentiation, differentiation, rooting, seedling, PCR detection, and transplanting.

[0069] RTMS 10.1 Single gene complementation primer RTMS10.1-G-F: TATCCAGATCCAGTGGGATCCGTATGCGGCTCCTTAAGTGGAC (SEQ ID NO: 9) RTMS10.1-G-R: CGCACTAGTAAGCTTGGTACCCTGAGACTCTTTTTCGTTGATGAT (SEQ ID NO: 10) RTMS 10.2 Single gene complementation primer RTMS10.2-G-F: TATCCAGATCCAGTGGGATCCCTGTACTACGCCCTAAGTTACA (SEQ ID NO: 11) RTMS10.2-G-R: CGCACTAGTAAGCTTGGTACC CATCTCATCTTCCTTTGCATTCCGT (SEQ ID NO: 12) RTMS 10.1 - RTMS 10.2 Double gene complementation primer RTMS10.1-10.2-G-F: ATCCAGATCCAGTGGGATCCCTGTACTACGCCCTAAGTTACA (SEQ ID NO: 13) RTMS10.1-10.2-G-R: GCACTAGTAAGCTTGGTACCTATGCGGCTCCTTAAGTGGAC (SEQ ID NO: 14) 4.2, Phenotype observation of complementation transgenic materials The single gene complementation materials and double gene complementation materials obtained in step 4.1 are subjected to anther phenotype at low temperature and high temperature, and I 2- KI staining.

[0070] The results are shown in Figure 6 - 8 .

[0071] From Figure 6 and 7 it can be seen that RTMS 10.1 and RTMS 10.2Single gene complementary material, under high temperature (30℃) and low temperature (22℃) conditions, floret is full and golden yellow, and can be dyed dark by I2-KI, and is all fertile.

[0072] From Figure 8 It can be known that RTMS 10.1 - RTMS 10.2 Double gene complementary material, under low temperature (22℃) condition, anther of transgenic material becomes small and color becomes light, pollen grain cannot be dyed, and shape is irregular, under high temperature (30℃) condition, floret is full and golden yellow, and can be dyed dark by I2-KI.

[0073] From Figure 6 - 8 It can be known that, under low temperature (22℃) or high temperature (30℃) condition, floret of control material L422 is full and golden yellow, and can be dyed dark by I2-KI, and is completely fertile.

[0074] The above results show that RTMS 10.1 With RTMS 10.2 Gene is necessary to cause reverse temperature sensitive male sterility phenotype.

[0075] The present application is not limited by the above specific literal description, and the present application can be variously changed within the scope outlined by the claims, and the changes are within the scope of the present application.

Claims

1. RTMS 10.1 genes and RTMS 10.2 application of the gene or biological material related thereto in at least one of: A1) cultivating a rice reverse temperature-sensitive male sterile plant or preparing a product of cultivating a rice reverse temperature-sensitive male sterile plant; A2) rice variety improvement or preparing a product of rice variety improvement; A3) preparing a transgenic plant.

2. The use according to claim 1, characterized in that, The RTMS 10.1 genes and RTMS 10.2 genes are introduced into a normal-fertility rice material L422, cultivation is carried out, and a rice reverse temperature-sensitive male sterile plant is obtained; the rice reverse temperature-sensitive male sterile plant exhibits male sterility at 20-28 DEG C and restores fertility at 29-34 DEG C.

3. The use according to claim 2, wherein The nucleotide of the gene comprises the sequence shown in SEQ ID NO: 1; the nucleotide of the gene comprises the sequence shown in SEQ ID NO:

2. RTMS 10.1 The nucleotide of the gene comprises the sequence shown in SEQ ID NO: 1; the nucleotide of the gene comprises the sequence shown in SEQ ID NO:

2. RTMS 10.2 The nucleotide of the gene comprises the sequence shown in SEQ ID NO: 1; the nucleotide of the gene 4. The use according to claim 1, characterized in that, The biological material related thereto is any one of B1) to B3): B1 ) a composition comprising said RTMS 10.1 gene and / or RTMS 10.2 recombinant vector of said gene; B2) a recombinant microorganism comprising said RTMS 10.1 gene and / or RTMS 10.2 gene, or a recombinant microorganism comprising said recombinant vector of B1). B3) the RTMS 10.1 gene and / or RTMS 10.2 protein encoded by the gene.

5. A method for creating a temperature-sensitive male sterile rice plant, characterized by, comprising the step of introducing into a normal fertile rice material L422 RTMS 10.1 the gene and RTMS 10.2 the gene or biological material related thereto, to obtain a rice reverse temperature-sensitive male sterile plant.

6. The method of creating of claim 5, wherein, PCR verification is further included for the rice reverse temperature-sensitive male sterile plant, primers of the PCR comprising sequences shown in SEQ ID NO: 13 and SEQ ID NO:

14.

7. A method for restoring fertility to male sterile rice plants, characterized by, The following are included: Knocking out genes and / or RTMS 10.1 genes in rice YnS, a reverse temperature-sensitive male sterile line RTMS 10.2 line.

8. The method of claim 7, wherein, Knockout is performed using a CRISPR Cas9 system, sgRNA in the CRISPR Cas9 system being selected from sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4.

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