Plant temperature-sensitive genic male sterile gene mutant tms17 and application thereof

Abstract

The present application relates to the field of agricultural technology, in particular to a plant temperature-sensitive male sterility gene mutant tms17 and application thereof.The nucleotide sequence of the plant temperature-sensitive male sterility gene mutant tms17 comprises the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.The present application screens a new temperature-sensitive male sterile line tms17 by chemical mutagen ethyl methyl sulfone (EMS) mutagenesis of japonica rice variety ZH11, which is completely sterile at high temperature and fertility is restored at low temperature, and the mechanism of temperature-sensitive fertility restoration is disclosed, which provides a new germplasm resource with application potential for rice two-line hybrid breeding.

Description

Technical Field

[0001] This invention relates to the field of agricultural technology, specifically to a plant thermosensitive nuclear male sterility gene mutant tms17 and its applications. Background Technology

[0002] Currently, rice (Oryza sativa) is the staple food for more than half of the population and an important source of nutrition for most of the population in Asia, Latin America, and Africa. Increasing the yield per unit area of ​​rice is an important means to address the growing demand for food. Rice male-sterile lines are key to utilizing hybrid vigor in rice. As the female parent, they cannot self-pollinate, avoiding artificial emasculation. Through cross-pollination, they can accept pollen from other lines, thus enabling hybrid rice breeding. In 1987, Yuan Longping proposed three strategic development stages for rice hybrid breeding in my country: from the three-line method to the two-line method and then to the one-line method. The three-line method has been widely used for over 50 years, but the scarcity of restorer genes and the varying restorer efficiency among different varieties limit the selectivity of three-line pairings. The two-line method includes restorer lines and environment-sensitive genic male sterile lines (EGMS). EGMS can restore fertility under specific conditions, thus replacing CMS in two-line hybridization breeding. Since the sterility gene in EGMS is a recessive nuclear mutation, any wild-type rice variety possesses a dominant restorer gene and can therefore be used as a restorer line to pair with EGMS. Furthermore, one-line hybridization breeding remains theoretical and is still far from commercial application.

[0003] Two-line hybridization breeding offers advantages over three-line hybridization, including simplified breeding models, reduced production costs, and increased yields. However, it also has some drawbacks. Firstly, the germplasm resources of the two-line method are too limited; in currently used two-line hybrid rice varieties, the tms5 locus accounts for over 95%. Although tms5 possesses excellent sterility traits, its singular genetic locus poses a potential risk to seed production. Secondly, two-line seed production is highly dependent on weather conditions; fluctuations in light and temperature can lead to unstable sterility in photoperiod- and temperature-sensitive lines, significantly affecting the purity of hybrid seed production. For example, summer typhoons or abnormally low temperatures can cause partial recovery of fertility in photoperiod- and temperature-sensitive male-sterile lines, resulting in seed production failure. Furthermore, fertility transition temperature drift in photoperiod- and temperature-sensitive male-sterile lines has been reported; higher fertility transition temperatures make them more susceptible to the effects of low temperatures. Finally, the fertility transition mechanisms for each photoperiod- and temperature-sensitive male-sterile line, as well as common mechanisms, still require further in-depth research to provide theoretical support and technical guidance for their application. The limitations of the two-line method are currently a bottleneck in the development of hybrid breeding. The discovery and research of novel photoperiod- and temperature-sensitive male-sterile lines can effectively expand germplasm resources for rice two-line hybrid breeding and fully mechanized seed production. Therefore, the discovery of new photoperiod- and temperature-sensitive genetic resources is of great significance. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a plant thermosensitive male sterility gene mutant, tms17, and its applications. This invention uses the chemical mutagen ethyl methanesulfonate (EMS) to mutate the japonica rice variety ZH11, screening out a new thermosensitive male sterility line, tms17, which is completely sterile at high temperatures and recovers fertility at low temperatures.

[0005] Therefore, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a plant thermo-stress gene mutant tms17 in an optional embodiment, wherein the nucleotide sequence of the plant thermo-stress gene mutant tms17 comprises the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.

[0007] In this invention, the phenotype of the tms17 mutant is determined by a C-to-T mutation at the 22nd base before the ATG on the 5'UTR of its genome.

[0008] Secondly, in an optional embodiment, the present invention provides an expression vector containing the above-mentioned plant thermo-sensitive male sterility gene mutant tms17.

[0009] Thirdly, in an optional embodiment, the present invention provides an expression cassette containing the above-mentioned plant thermo-sensitive male sterility gene mutant tms17.

[0010] Fourthly, in an optional embodiment, the present invention provides the application of the above-mentioned plant thermo-sensitive sterility gene mutant tms17 in the preparation of recessive male sterile transgenic plants.

[0011] Fifthly, in optional embodiments, the present invention provides an application of the above-mentioned plant thermo-sensitive sterility gene mutant tms17 in plant breeding, the application including any of the following:

[0012] (1) The plant thermo-sensitive male sterility gene mutant tms17 was introduced into the target plant, and its phenotype showed high temperature sterility and low temperature fertility recovery;

[0013] (2) The plant thermo-sensitive male sterility gene mutant tms17 was introduced into other plant varieties through hybridization. The sterile plants obtained in the F2 generation showed the thermo-sensitive male sterility phenotype.

[0014] (3) Using the sterile plants obtained in (1) and (2) as the female parent, different plant varieties are used as the male parent to carry out hybridization, cultivate the target hybrid plants, and obtain the corresponding hybrid seeds.

[0015] In a sixth aspect, the present invention provides, in an optional embodiment, a method for cultivating plants whose fertility is restored when anther development is affected by temperature. The method includes introducing the above-mentioned plant thermo-sensitive male sterility gene mutant tms17 into plant seed cells, and using the plant seeds infused with the plant thermo-sensitive male sterility gene mutant tms17 for corresponding plant cultivation.

[0016] In a seventh aspect, the present invention provides, in an optional embodiment, a method for regulating the thermo-sensitive fertility trait of a plant, the method comprising replacing the TMS17 / OsUTP6 gene in a wild-type plant with the aforementioned plant thermo-sensitive sterility gene mutant tms17.

[0017] In an eighth aspect, the present invention provides, in optional embodiments, the use of the above-mentioned plant thermo-sterile gene mutant tms17 for regulating or providing the thermo-sensitive fertility trait of the plant, or for use as a selection marker for transgenic plants.

[0018] Preferably, the marker trait selected is a reversible change in thermosensitive fertility traits, whereby the plant exhibits fertility recovery under low temperature conditions and sterility under high temperature conditions.

[0019] In a ninth aspect, the present invention, in optional embodiments, provides the above-described plant thermo-sensitive male sterility gene mutant tms17, the above-described expression vector, the above-described application, and the above-described method or use, wherein the plant is rice.

[0020] The nucleotide sequence of SEQ ID NO.1 is shown below:

[0021]

[0022] Compared with the prior art, the present invention has one of the following beneficial effects:

[0023] 1. This invention uses the chemical mutagen ethyl methanesulfonate (EMS) to mutate the japonica rice variety ZH11, and screens out a new temperature-sensitive male sterile line tms17, which is completely sterile at high temperatures and recovers its fertility at low temperatures. Attached Figure Description

[0024] Figure 1 The figure shows the phenotypic results of tms17 and wild-type ZH11 at different temperatures in Example 2 of the present invention. Figure 1 a, Figure 1 d and Figure 1 g represents the phenotypic diagrams of wild-type ZH11 and tms17 after heading at different temperatures, where HT represents high temperature, LT represents low temperature, and Bars = 10 cm / 2 cm. Figure 1 b、 Figure 1 e and Figure 1 h is a diagram of the anthers after the glumes have been removed, Bars = 2 mm; Figure 1 c. Figure 1 f and Figure 1 i is the pollen iodine staining pattern, Bars = 100μm;

[0025] Figure 2 The figures shown are agronomic trait results of the tms5, tms15, and tms17 mutants in Example 2 of this invention. Figure 2 'a' represents the statistics of agronomic traits, specifically the seed setting rate. The horizontal axis represents the germination date, the left vertical axis represents the seed setting rate, and the right vertical axis represents the average ambient temperature (AT) during the booting stage. The error bars represent the SD of AT. Figure 2 b is a comparison image of planting in the community;

[0026] Figure 3 This is a diagram showing the fertility results of female tms17 and wild-type ZH11 in Example 2 of the present invention. Figure 3 a represents the rice spikelet after hybridization under high temperature, with Bars = 2 cm. Figure 3 b represents the average seed setting rate of the hybrid rice panicle. The statistical analysis was performed using a two-tailed t-test, with a P-value of 0.2913 (ns, no significantly different).

[0027] Figure 4The diagram shows the anther development results of tms17 and wild-type ZH11 at different temperatures in Example 2 of this invention. Arrows indicate abbreviations; a question mark after an abbreviation indicates suspected developmental abnormalities; and an "a" before an abbreviation indicates developmental abnormalities. E, epidermis; En, endothecium; ML, middlelayer; T, tapetum; aT, abnormal tapetum; MMC, microsporemother cell; MC, meiotic cell; Dy, dyad cell; Tds, tetrads; Msp, microspore; aMsp, abnormal microspore; BP, bicellular pollen; aBP, abnormal bicellular pollen; MP, mature pollen (Bar = 50 μm).

[0028] Figure 5 This is a schematic diagram of the mutation site of tms17 in Embodiment 2 of the present invention, wherein, Figure 5 Image a shows the candidate SNP sites on chromosome 2. The vertical axis represents the P-value corresponding to the SNP, and the horizontal axis represents the chromosome location corresponding to the SNP. The arrows indicate the tms17 mutation site. Figure 5 b is the TMS17 sequencing peak diagram, with boxes marking mutation sites and horizontal lines indicating the start codon and subsequent encoded amino acids. Figure 5 c is a schematic diagram of the LOC_Os02g01350 gene structure. The white box represents the 5' / 3' UTR, the black box represents the exon, the vertical line marks the start codon ATG and the stop codon TAA, the arrow indicates the tms17 mutation site on the 5'UTR, and the diagonal line indicates the CAS9 vector gRNA target site;

[0029] Figure 6 This is a schematic diagram of the results of homozygous strong allelic mutations in TMS17-CAS9-gRNA1-C1 and TMS17-CAS9-gRNA-C22 in Example 2 of the present invention. In this diagram, T0 represents the current generation of transgenic plants, and the sequencing peak is a heterozygous peak due to the different editing of the two chromosomes. T1 represents its self-crossed offspring. The horizontal line and ATG mark the position of the start codon. The red marks mark the inserted and deleted bases. The red marks of a2 and a7 represent strong allelic mutations that may affect the function.

[0030] Figure 7The figures show the high-temperature phenotypic and agronomical traits of TMS17, TMS17-COMP, and TMS17-VENUS in Embodiment 2 of the present invention. Figure 7 Image a shows the florets, anthers stained with iodine, and the rice panicle. Float Bars = 2 mm; anther Bars = 1 mm; rice panicle Bars = 2 cm. Figure 7 b is a statistical chart of the seed setting rate of TMS17-COMP. Figure 7 c is a statistical chart of the seed set rate of TMS17-VENUS. One-way ANOVA analysis was used to compare the data. Data with the same letter label showed no significant difference (P > 0.05). Error bars represent the standard deviation (SD) of the seed set rate.

[0031] Figure 8 This is a diagram showing the expression results of TMS17 at different stages of anther development in Example 2 of this invention. The green fluorescent signal represents TMS17-VENUS, the pink fluorescent signal represents chloroplast autofluorescence, and the green fluorescence on the surface of microspores in stages 10 and 11 represents sporophytin autofluorescence. The black arrows indicate the cells represented by the abbreviations: Bars = 20 μm, 2°P, secondary parietal cell layer; Sp, sporogenous cell; T, tapetum; Tds, tetrads; Dy, dyad cell; E, epidermis; En, endothecium; MC, meiotic cell; ML, middle layer; MMC, microspore mother cell; Msp, microspore; BP, bicellular pollen.

[0032] Figure 9 This is a schematic diagram showing the results of TMS17 encoding the OsUTP6 protein in Example 2 of the present invention, wherein, Figure 9 Figure a shows a schematic diagram of the TMS17 domains. Different colors indicate different domains, and numbers indicate the amino acid numbers at the corresponding boundaries. (UTP6) N-L,U3 small nucleolar RNA-associated protein 6, N-terminal-like; NLS, Nuclearlocalization sequence, nuclear localization sequence; HAT, Half a TPR, tetratricopeptide repeats; CC, Coiled-coil, Figure 9 b shows the phylogenetic analysis of proteins from each component of the UTPB complex in rice and Arabidopsis thaliana. Branch length represents genetic distance, nodes represent common ancestor, and colors distinguish different UTPB complex components.

[0033] Figure 10 This is a phylogenetic tree analysis of TMS17 homologous proteins in different plant species and a sequence alignment diagram of TMS17 in maize, rice, and Arabidopsis thaliana in Example 2 of the present invention. Figure 10 a is a phylogenetic tree analysis of TMS17 homologous proteins from different plant species. Figure 10 b is the sequence alignment diagram of TMS17 in maize, rice and Arabidopsis thaliana. The red line marks the UTP6N-L (U3 smallnucleolar RNA-associated protein 6, N-terminal-like) domain, and the purple line marks the HAT domain.

[0034] Figure 11 This is a schematic diagram of the interaction between OsUTP6 and UTPB complex-related proteins in Example 2 of the present invention, where Chlorophyll represents chloroplast autofluorescence. YFP N YFP fluorescent protein N-terminus; YFP C YFP fluorescent protein C-terminus, Bars = 20μm;

[0035] Figure 12 This diagram illustrates the effect of temperature on the interaction efficiency of OsUTP6 and UTPB complex-related proteins in Example 2 of this invention. The top label shows the leaf infection combinations, the top left shows the experimental group of OsUTP6 and the interacting protein (represented by X), and the rest are negative controls to verify self-activation. Luciferase activity is described using colors from low (white) to high (blue). Red boxes represent high-temperature treatments, and blue boxes represent low-temperature treatments.

[0036] Figure 13This diagram illustrates the results of the downregulation of TMS17 transcriptional levels caused by a point mutation on the 5'UTR in Example 2 of this invention. It shows the relative transcriptional expression levels of TMS17 in the anthers of ZH11 and tms17 during mid-developmental stages under high and low temperatures. Two-way ANOVA analysis was used to compare these data (p < 0.0001). Error bars represent the SD of relative expression values.

[0037] Figure 14 This is a schematic diagram illustrating the alteration of the secondary structure of the OsUTP6 transcript caused by a point mutation on the 5'UTR in Example 2 of the present invention. Figure 14 The image on the left is a schematic diagram of the predicted secondary structure of the 5'UTR in the wild-type OsUTP6. Figure 14 The diagram on the right shows the predicted secondary structure of the 5'UTR from the OsUTP6 point mutation. The arrows indicate the location of the tms17 point mutation. The color of each base represents the probability of its current state; paired bases represent the pairing probability, and unpaired bases represent the unpairing probability. Probabilities are described by colors from low (purple) to high (red). (5'UTR) OsUTP6 ,OsUTP6's 5'UTR; m5'UTR OsUTP6 The point mutation 5'UTR of OsUTP6;

[0038] Figure 15 This is a schematic diagram illustrating the partial recovery of OsUTP6 translation defects caused by a point mutation on the 5'UTR of OsUTP6 in Embodiment 2 of the present invention, achieved at low temperatures. Figure 15 a and Figure 15 Image d shows the fluorescence observation of OsUTP6-VENUS in tobacco leaves under high and low temperatures. Chlorophyll represents the autofluorescence of chloroplasts. The red dashed box marks the range of the cell nucleus. Bars = 20 μm. Figure 15 b and Figure 15 e represents the statistical measurements of average fluorescence intensity at high and low temperatures. Figure 15 c and Figure 15 f represents the statistical measurement of the average fluorescence range at high and low temperatures. One-way ANOVA analysis was used to compare the above data (*p < 0.05, ****p < 0.0001). ns, no significantly different, 5'UTR. OsUTP6 ,OsUTP6's 5'UTR; m5'UTR OsUTP6 Point mutation 5'UTR OsUTP6 ;

[0039] Figure 16 This is a schematic diagram of the abnormal results of the tms17 ribosomal small subunit in Example 2 of the present invention, wherein, Figure 16Image a shows the polysome profiling of anthers from ZH11 and tms17 during mid-developmental stages under high and low temperatures. Anther extracts were separated in a 5%–50% sucrose gradient. A254 was detected and statistically analyzed sequentially. The peaks corresponding to free 40S ribosome small subunit, 60S ribosome large subunit, 80S ribosome, and polysome are marked in the image. Biological replication results were similar, and the average value was used to plot the image. Figure 16 b represents the statistical analysis of peak areas for each component in the polysome profiling spectrum. Unpaired t-tests were used to compare the data (*p < 0.05, **p < 0.01, one-tailed). No significant differences were found between samples without statistical significance markers. Colors distinguish different samples. Error bars represent the standard deviation (SD) of the peak areas.

[0040] Figure 17 This is a schematic diagram showing the decrease in the abundance of anther development-related proteins in tms17 under high temperature in Example 2 of the present invention. Red represents high temperature and blue represents low temperature. Two-way ANOVA analysis was used to compare these data (*p < 0.05 and **p < 0.01). The error bars represent the SD of the relative expression values.

[0041] Figure 18 This is a schematic diagram of the TMS17 thermosensitive infertility mechanism in Embodiment 2 of the present invention, wherein, Figure 18 In the wild-type anther, OsUTP6 is normally expressed and participates in ribosome biogenesis under high temperatures, and protein translation within the anther proceeds normally, providing sufficient support for anther development. Figure 18 b is a point mutation on the 5'UTR in tms17 that, under high temperature, causes a decrease in the abundance of OsUTP6, leading to a decrease in the abundance of the small ribosomal subunit and impaired protein translation. As a result, the plant cannot meet the protein abundance required for anther development, ultimately leading to high-temperature sterility. Figure 18 c indicates that the defects caused by point mutations at low temperatures are alleviated, and the partially restored OsUTP6 can synthesize enough ribosomal subunits to translate the proteins required for anther development, thereby restoring plant fertility. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0045] The wild-type ZH11 (Oryza sativa ssp. japonica) and tms17 (LOC_Os02g01350) used in the following examples were grown in the Shanghai Botanical Garden and rice paddies. When grown in the artificial climate chamber of the botanical garden, the normal temperature conditions for rice were 26°C during the day and 22°C at night, while the high-temperature treatment was 34°C during the day and 28°C at night. The light conditions were natural light supplemented by halogen lamps.

[0046] In the following examples, various rice plants and panicles were photographed using a Nikon D7000 digital camera (Nikon, Tokyo, Japan). Anthers and pistils were photographed using an Olympus SZX10 dissecting microscope (Olympus, Tokyo, Japan). Data statistics, analysis, and plotting were performed using software such as Microsoft Excel, GraphPad Prism 9, Origin 2018, and Photoshop CS6.

[0047] In the following embodiments, the experimental methods involved are as follows:

[0048] 1. Observation of semi-thin sections

[0049] Rice anthers were collected at various stages and fixed using FAA fixative. Vacuum was applied for 1 minute to allow the fixative to fully penetrate and settle to the bottom. The mixture was then allowed to stand at 4°C for 2-3 days. Dehydration and embedding were subsequently performed.

[0050]

[0051] Add 8-9 drops of Spurr pure resin with added catalyst to a small EP tube. Use a toothpick to pick up the embedding material and place it into the tube, ensuring the material is properly aligned and removing any air bubbles from the resin. Then transfer the tube to a drying cabinet and incubate at 24 °C for 24 hours. If the material floats to the surface during this time, adjust it with a toothpick. Next, place the tube in a 65 °C oven to polymerize the resin at a high temperature (generally 36 hours). Slice the material using a microtome, stain with toluidine blue, and observe under an Olympus BX51 digital fluorescence microscope (Olympus, Tokyo, Japan).

[0052] 100% ethanol and 100% propylene oxide must be treated with anhydrous sodium sulfate before use; when adding propylene oxide and resin mixture, transfer the material to a 4 mL centrifuge tube, seal with sealing film, and mix overnight on a decolorizing shaker; after adding catalyst, the sample should be placed in a desiccator to avoid moisture.

[0053] 2. Alexander staining and I2-KI staining

[0054] Unopened spikelets were selected for the experiment. Under a dissecting microscope, the anthers were placed in the center of a glass slide using a dissecting needle. An appropriate amount of Alexander's solution was added for staining. After covering the slide, the stain was gently pressed with forceps to ensure full penetration of the anthers. The slide was then treated in the dark and stained overnight. The next day, the anthers were observed using an Olympus BX51 digital fluorescence microscope (Olympus, Tokyo, Japan). After the anthers released pollen onto a glass slide, an appropriate amount of I2-KI staining solution was added for staining, and the anthers were observed using an Olympus BX51 digital fluorescence microscope (Olympus, Tokyo, Japan).

[0055] 3. BSA sequencing analysis

[0056] F2, a representative segregating population of candidate mutants, was planted, with 50 sterile and 50 fertile plants from which leaves were collected. DNA was extracted from the leaves of each population after liquid nitrogen grinding, and sequencing DNA libraries were constructed and sent to Pasenno Biotechnology Co., Ltd. for BSA sequencing. After obtaining sequencing data using BSA sequencing, candidate SNP loci were analyzed and screened. First, SNP loci that did not conform to the single-gene recessive inheritance frequency were excluded; that is, SNP loci where the frequency of gene a was higher than that of gene A in the fertile population and the frequency of gene A was higher than that of gene a in the sterile population were removed. Then, the chi-square values ​​of the fertile and sterile populations were calculated using statistical formulas, and independence tests were performed on each SNP locus. Finally, candidate SNP loci with a total chi-square value less than 3.841 and a p-value greater than 0.5 were selected, and scatter plots were generated and genetic linkage analysis was performed. Since the candidate SNP loci in the sterile population showed genetic linkage during phenotypic segregation, the chromosomes where candidate SNP loci were concentrated in the scatter plot were carefully observed, as SNP loci controlling temperature-sensitive shapes were highly likely to be located on these chromosomes. Based on the location of SNP sites, they were categorized into intergenic, transposon, promoter, amino acid synonymous mutation, intron, and intragenic regions. Among these, SNP sites located within genes and consistent with the characteristics of genetically linked chromosomes were more likely to be candidate genes. Subsequently, DNA was extracted from 30 samples from the sterile population and verified by PCR. Gene cloning and complementation experiments were then used to further verify the functional location and genetic mechanism of the candidate genes.

[0057] 4. Completion and CRISPR-Cas9-mediated rice gene editing mutants

[0058] The genetic transformation of transgenic rice was carried out by Wuhan Boyuan Biotechnology Co., Ltd. The OsUTP6 genome sequence, initiated by its own promoter, was cloned into a vector (pCAMBIA1300) containing a NOS terminator sequence, and this recombinant vector was then used to infect the corresponding mutants. CRISPR-Cas9 targets were designed using the CRISPR-GE website (http: / / skl.scau.edu.cn / ).

[0059] 5. Yeast two-hybrid

[0060] For Y2H single-pair validation, the target sequence was cloned into the vector pGADT7 or pGBKT7. Yeast competent cells AH109 were thawed on ice while simultaneously denaturing the carrier DNA. 1 μg each of BK and AD plasmids were added to the competent cells, along with 10 μL of carrier DNA and 500 μL of PEG solution, and the mixture was thoroughly pipetted and mixed. The cells were incubated at 30°C for 30 min, inverting once during incubation, followed by incubation at 42°C for 15 min. After centrifugation at 5000 rpm, the cells were resuspended in ddH2O, centrifuged again, and the cells were collected and plated. Yeast was screened on SD / -Leu / -Trp (DDO) and SD / -Leu / -Trp / -His / -Ade (QDO) media at 30°C and cultured for 3–5 days.

[0061] 6. Tobacco Conversion Experiment

[0062] The constructed plasmid was transformed into Agrobacterium GV3101 (pSoup-p19). The Agrobacterium containing the recombinant plasmid was cultured overnight in triple-antibiotic LB liquid medium. The bacterial cells were collected by centrifugation and thoroughly resuspended in a transformation solution containing 10 mmol / L MgCl2, 10 mmol / L LMES (pH 5.6), and 150 μmol / L AS. The bacterial concentration was adjusted to OD600 of 1 using a spectrophotometer and incubated at room temperature for 1 h. The bacterial solution was then injected into the underside of tobacco leaves with 5-6 leaves using a syringe without the needle and grown in a 25°C light incubator. After 2-3 days, the cells were observed under an Olympus FV3000 laser confocal microscope (Olympus, Tokyo, Japan).

[0063] 7. Luciferase Complementation Imaging

[0064] The target protein was cloned into the vector JW771-LUC. N The interacting proteins were cloned into the vector JW772-LUC. CThe recombinant vector was then transformed into Agrobacterium GV3101 (pSoup-p19). The two Agrobacterium strains were mixed at a 1:1 ratio and used to transform tobacco leaves. One day after transformation, the tobacco leaves were placed at 30 / 22°C for one day for further observation. Before observation, an equal volume of substrate chromogenic solution was applied to the original injection site, and fluorescence was observed using a detection system. The average grayscale value was quantified using ImageJ software. At least three experiments were conducted using different batches of tobacco.

[0065] Example 1

[0066] Obtaining the tms17 mutant

[0067] The rice variety ZH11 was induced to develop thermosensitive traits through mutagenesis with the chemical mutagen ethyl methanesulfonate (EMS), followed by thermosensitive selection. The screening process and analysis for thermosensitive traits have been described in previous studies (Zhang et al., 2022). M1 generation seeds treated with EMS were sown in the field and cultured normally. After seed setting and maturity, M2 generation seeds were harvested and sown in late spring / early summer, placing the M2 generation plants under the natural high-temperature conditions of summer to screen for sterile lines that could not produce seeds under high-temperature conditions (>30℃). Subsequently, the sterile lines were cut off, allowing them to re-head and develop seeds under the allowable conditions of natural low temperatures (27℃) in autumn. Lines showing observed recovery of seed setting rate under low-temperature conditions were designated as candidate thermosensitive lines. The background was purified by backcrossing with ZH11, and a sequencing population was constructed. After planting, the phenotypic segregation ratio between sterile and fertile plants was observed in the sequencing population. Populations with a segregation ratio close to 3:1 (i.e., single-gene recessive mutants) underwent BSA (Bulked Segregant Analysis) sequencing to analyze mutation site information and were then used for subsequent related experimental studies.

[0068] Sequence analysis began with alignment of relevant protein sequences using the muscle method in Seaview software, followed by evolutionary visualization of the alignment results using GeneDoc software. Phylogenetic analysis was performed using the muscle method in MEGA7, followed by phylogenetic tree construction based on the JTT matrix-based model using the Maximum Likelihood method. The phylogenetic tree was then adjusted using the iTOL website (https: / / itol.embl.de / ). Protein 3D structure prediction was performed using AlphaFold (https: / / alphafold.com / ) and PyMOL software. RNA secondary structure prediction was performed using the MFE method in RNAfold (http: / / rna.tbi.univie.ac.at / ). Polysome profiling results were standardized and statistically measured using the website (https: / / www.umm.uni-heidelberg.de / ) and following the method of Schiller et al. (2024). Proteome data were statistically analyzed using the Meiji BioCloud website (https: / / www.majorbio.com / ).

[0069] Sequencing verification revealed that the temperature-sensitive male sterile plant gene sequence contained the tms17 mutant, and the mutant gene sequence is shown in SEQ ID NO.1 in the sequence listing.

[0070] Example 2

[0071] Thermosensitive characteristics identification of TMS17

[0072] Thermosensitive male-sterile plant tms17 and wild-type Zhonghua 11 were cultured. Under high temperatures, tms17 exhibited complete sterility, with its anthers noticeably shrunken and whitish, and no mature pollen grains were found after the anthers were dissected (see [link]). Figure 1 d- Figure 1 f). During the same period, the wild-type ZH11 produced normal fruit; its anthers were yellow and plump, and I2-KI staining showed that the anthers contained mature pollen (see f). Figure 1 a- Figure 1 c). Conversely, at low temperatures, tms17 fertility was restored and it could set fruit. The anthers became plump and yellow. I2-KI staining showed that there were a large number of morphologically normal and stainable pollen grains in the anthers, but there were also some aborted pollen grains that could not be stained and appeared yellow and shriveled (see c). Figure 1 g- Figure 1 i). Compared with the wild-type ZH11, this line showed normal vegetative growth and normal panicle and instar development. These results indicate that tms17 has a typical temperature-sensitive male sterility phenotype.

[0073] To further investigate the relationship between temperature and fertility in the tms17 mutant, agronomic trait experiments were conducted. tms17 exhibited stable temperature-sensitive traits (see [link to relevant documentation]). Figure 2 a). Furthermore, by comparing tms5 and tms15 under the ZH11 background, it was found that at high temperatures ( Figure 2 All three mutants (5.18-6.8) exhibited complete sterility. During temperature transitions ( Figure 2 The 6.15 and 6.22 mutants were all affected and regained fertility, with tms5 showing the highest fertility recovery, followed by tms17, while tms15 remained sterile in 6.22. Furthermore, through plot planting comparisons, tms5 in 6.15 exhibited drooping panicles due to fertility recovery, while the panicles of tms17 remained upright (see [link to plant comparison]). Figure 2 (b) indicates that tms5 exhibits the highest fertility conversion temperature under the ZH11 background, while tms17 and tms15 show even better fertility conversion temperatures than tms5. At low temperatures ( Figure 2 (6.29-7.20) The fertility recovery performance of tms17 was better than that of tms5 and tms15. The above results indicate that the fertility conversion curve of tms17 is more stable with temperature changes, the fertility conversion temperature is lower, and the fertility recovery effect is better at low temperatures, which shows great application potential in rice two-line hybrid breeding.

[0074] Although the male fertility of tms17 was severely damaged at high temperatures, whether the female fertility of tms17 was normal still needed to be investigated through further experiments. Through high-temperature hybridization verification, the stigma of tms17 could be normally fertilized and produce seeds after pollination with ZH11 pollen (see...). Figure 3 a), and the seed setting rate was not significantly different from that of ZH11 after the same emasculation and pollination operation (see a). Figure 3 (b) This result indicates that female tms17 are normally fertile, and their thermosensitive sterility phenotype is determined by the development of male gametes.

[0075] To investigate the specific stage of male gamete development impairment in tms17, a semi-thin section experiment was conducted on the anthers of this strain. The results showed that, under high temperature and at stage 7 of anther development, the tapetum and microsporocytes of wild-type ZH11 were morphologically normal and dense, while the tapetum and microsporocytes of tms17 showed vacuolation, which appeared to be caused by a lack of contents. In later stages, the defects in the anthers of tms17 gradually worsened: in stage 8b, the microspore tetrads showed abnormal morphology, while the tetrads of ZH11 at this stage were oval-shaped with a cell plate separating the middle; in stage 9, the tapetum of ZH11 was normally colored, and the microspores were spherical, while the tapetum of tms17 showed abnormal morphology, suspected of premature degradation, and the contents of the microspores were missing and abnormal in shape; in stage 10, the microspores of ZH11 became vacuolated and increased in size, while the tapetum of tms17 showed premature degradation, and the microspores showed abnormal vacuolation; in stage 11, the tapetum of tms17 completely disappeared, and the microspores abnormally shrank and collapsed, unable to form a crescent shape similar to that in the wild type; in stage 12, the tapetum of ZH11 completely degraded and disappeared, and the microspores returned to a round shape, while the microspores in the anther chamber of tms17 completely ruptured and degraded. At low temperatures, although anther development in tms17 still exhibits defects, such as vacuolation of some microspore mother cells in stage 7, some microspores can still develop normally into pollen grains in subsequent stages. These results indicate that tapetum / microspore defects appear in anther development of tms17 starting from stage 7, and these defects can be partially recovered under low temperatures, which matches the thermosensitive male sterility phenotype of tms17.

[0076] To verify whether the tms17 sterility phenotype is controlled by a single-gene recessive mutation, the segregation of subsequent phenotypes was statistically analyzed. The tms17 mutant was crossed with the wild-type ZH11 to obtain fertile heterozygous F1 plants. The F2 segregating population produced by self-pollination was then planted, and the segregation of fertile and sterile plants was statistically analyzed. The results showed that tms17 follows the law of single-point inheritance (χ²). 2 = 3.392 < χ 2 0.05,1 = 3.841; see Table 1).

[0077] Table 1 tms17 + / - Chi-square test for F2 generation population segregation

[0078]

[0079] In order to find the temperature-sensitive gene mutation site of tms17, tms17 + / -Fertile and sterile plants from the F2 population were divided into two DNA pools for BSA (bulked segregant analysis). After removing abnormal sites and calculating the chi-square values ​​of the sterile and fertile populations, the P-value of each SNP (Single nucleotide polymorphism) site was calculated and plotted with the corresponding chromosome location information. The results showed a region with high P-values ​​and multi-site linkage on chromosome 2 (see [link to relevant documentation]). Figure 5 a). Primers were designed targeting SNP sites in these regions, and DNA from different single plants in the sterile population was extracted for sequencing verification. The results revealed a single base substitution (C→T, cytosine→thymine) at the 5' UTR of LOC_Os02g01350; see [link to DNA sequence]. Figure 5 b and Figure 5 c). This SNP site was mutated in all infertile samples (n>30; see c). Figure 5 b), while the remaining SNP sites were excluded because the test results were the same as the reference genome or there were multiple overlapping peaks.

[0080] To verify whether a defect in this gene function could lead to anther development defects, a CRISPR-CAS9 vector was constructed and LOC_Os02g01350 was targeted for editing. First, a gRNA targeting the ATG start codon of this gene was designed (see...). Figure 5 c), eight edited positive seedlings were obtained. These T0 generation positive seedlings all showed abnormal vegetative growth and a severe decrease in seed setting rate (see Table 2), and in the T1 progeny, only the a1 and a6 genotypes, which do not affect function, segregated (see Table 2). Figure 6 These results indicate that the deletion of the gene LOC_Os02g01350 may lead to severe impairment of plant growth and reproductive development, and the failure to isolate homozygous loss-of-function mutants in offspring suggests that homozygous mutations of this gene may lead to embryo lethality.

[0081] Table 2 Statistical table of tms17-CAS9-gRNA1

[0082]

[0083] Table Notes: a1 - An extra A before ATG; a2 - 10bp deletion, ATG is disrupted, no frameshift; a3 - 2bp deletion, ATG is disrupted, frameshift; a4 - 3bp deletion, ATG is disrupted, frameshift; a5 - The first 2bp of ATG are mutated from CC to TGG; a6 - The first 2bp of ATG are deleted, and the 7th base is suspected to have mutated from G to A; a7 - 1bp deletion, ATG is disrupted, frameshift. a2, a3, a4, and a7 represent strong allelic mutations that may affect function.

[0084] To verify whether this SNP site caused the tms17 sterility phenotype, LOC_Os02g01350 and its promoter were cloned. Two vectors were then constructed: one without fluorescent protein (TMS17-COMP) and the other fused to express VENUS fluorescent protein (TMS17-VENUS), and these were reintroduced into the tms17 mutant. Observation and statistical analysis revealed that of the 15 positive TMS17-COMP seedlings, 13 of their self-pollinated progeny showed restored fertility. Of the 18 positive TMS17-VENUS seedlings, 12 showed restored fertility (see [link to relevant documentation]). Figure 7 a). Of these, eight TMS17-COMP lines were able to recover to wild-type levels, five lines had a seed setting rate between 15% and 45%, and the remaining two lines had a seed setting rate of less than 5% (see [link to relevant documentation]). Figure 7 (b) while the seed setting rate of TMS17-VENUS partially fertile restored positive seedlings was between 25% and 50% (see [link]). Figure 7 c). Furthermore, all tms17 cells in each group that were simultaneously incubating ears were completely male-sterile. These results indicate that the addition of LOC_Os02g01350 to tms17 can rescue the high-temperature sterility phenotype of tms17 to varying degrees, suggesting that the temperature-sensitive phenotype of tms17 is caused by a mutation at this SNP site.

[0085] To investigate how TMS17 functions during anther development, and to find that TMS17-VENUS can complement the phenotype of TMS17 (see...), Figure 7 a and Figure 7 c) First, fluorescence observation was used to confirm the specific correlation between the expression pattern of TMS17 in anthers and TMS17 fertility. Using laser confocal microscopy with uniform parameter settings, anthers at different stages of the heading stage in TMS17-VENUS positive seedlings were observed. It was found that TMS17 was expressed in the early stages of anther development, and the fluorescence signal was present in the nuclei of all cell types in the anthers (see [link to study]). Figure 8 Fluorescent signals, albeit weak, were still observed within the nuclei of microspore dimers / tetrads during meiosis in stages 8a / 8b. As the anthers grew and developed, the fluorescence signal of TMS17-VENUS gradually diminished, disappearing completely in the tapetum and microspores after stage 10, with only a small amount of signal remaining in the anther epidermis and endothelial cells. These results indicate that TMS17 is expressed in all cell layers during the early and middle stages of anther development, suggesting that TMS17 may participate in some basic biological process in the anther cells, thereby aiding their development.

[0086] No studies have been reported on LOC_Os02g01350 in rice. To investigate its gene function, the protein domains of TMS17 were first predicted using InterPro. The results showed that TMS17 has one nuclear localization signal (NLS), one coiled-coil (CC) domain, five HAT domains (half a TPR, tetratricopeptide repeats), and one N-terminal-like domain of U3 small nucleolar RNA-associated protein 6 (UTP6N-L; see [link to article]). Figure 9 a).

[0087] The CC domain has been reported to potentially mediate protein-protein interactions. The TPR domain is sequence- and structurally similar to the HAT domain; the former primarily mediates protein-protein interactions, but the latter, besides potentially having the same function, appears to be a component of the macromolecular complex required for RNA processing or capable of directly binding RNA. The yeast ScUTP6 protein has been reported to participate in the formation of the UTPB complex, thereby participating in rRNA synthesis and processing. The N-terminal protein sequence of TMS17 is highly similar to ScUTP6 (UTP6N-L; see...). Figure 9 a), its function is also related to the domain analysis of TMS17.

[0088] Therefore, to investigate whether there is a correlation between TMS17 and ScUTP6, homologous proteins of each component of the UTPB complex in rice and Arabidopsis thaliana were searched using NCBI Protein BLAST (https: / / blast.ncbi.nlm.nih.gov / ), and a phylogenetic tree was constructed (see...). Figure 9 (b) The results showed that homologous proteins of each component of UTPB existed in rice and Arabidopsis thaliana, and the homology among these components was high. Meanwhile, it has been reported that root-specific knockout of AtUTP18 in Arabidopsis thaliana leads to growth arrest and abnormal accumulation of 18S pre-rRNA. These analyses indicate that TMS17 encodes OsUTP6, a homolog of ScUTP6, and also suggest that the components of the UTPB complex in plants may play highly similar functions to those in yeast.

[0089] OsUTP6 homologs were searched for in various plant species, and phylogenetic analysis was performed. The results showed that OsUTP6 homologs exist in plants ranging from algae to dicotyledons, except for gymnosperms (see [link to study]). Figure 10a). No homologous proteins were found between OsUTP6 and ScUTP6 sequences compared using the NCBI gymnosperm database. Furthermore, comparisons of OsUTP6 sequences in model plants Arabidopsis thaliana, rice, and maize revealed high homology in these crops in the N-terminal UTP6 N-like domain and HAT domain (see [link to article]). Figure 10 b). These results suggest that the UTP6 protein may be a highly conserved protein, and its function may be extremely conserved in eukaryotes.

[0090] To investigate whether OsUTP6 functions similarly to ScUTP6 in rice, the interaction relationships in rice were first examined to determine if they were consistent with those in yeast. In yeast, ScUTP6 interacts with ScUTP18 and ScUTP21. One model suggests that ScUTP6 first forms a dimer with ScUTP18, then a trimer with ScUTP21, and finally forms the core component of the UTPB complex with ScUTP1, while ScUTP12 and ScUTP13 form additional dissociable dimers involved in complex formation. BiFC analysis revealed that OsUTP1 and OsUTP12 do not interact with OsUTP6 in rice, while OsUTP21, OsUTP13, and OsUTP18 all interact with OsUTP6, and the localization signal is located in the nucleolus (see BiFC). Figure 11 This corresponds to the fact that the pre-rRNA processing site is located in the nucleolus. Furthermore, ScUTP6 has been reported to interact with ScMPP10. In rice, ScMPP10 comparison revealed two highly homologous proteins (named OsMPP10A and OsMPP10B), and BiFC results showed that OsUTP6 can also interact with both OsMPP10A and OsMPP10B (see [link to BiFC]). Figure 11 ).

[0091] These proteins appear to interact differently with OsUTP6 (see [link]). Figure 11OsUTP21 and OsMPP10B interact with OsUTP6 and are localized in the nucleolus. The interaction between OsUTP13 and OsUTP6 is localized in both the nucleolus and nucleoplasm. The nucleolus itself is a membraneless organelle with liquid-liquid phase separation of LLPS (Frottin et al., 2019), while the interaction between OsUTP18 and OsUTP6 is localized at the nucleolar LLPS interface, possibly indicating that the interaction between these two proteins occurs during the transport of ribosome subunits from the nucleolus to the nucleoplasm. Furthermore, the interaction between OsMPP10A and OsUTP6 is characterized by multiple punctate signals similar to LLPS in the nucleoplasm, in addition to a small amount of signal within the nucleolus, suggesting that OsMPP10A may play some other unknown function. These results indicate that the function of OsUTP6 is very similar to that of ScUTP6, and it is highly likely involved in the formation of the UTPB complex in rice.

[0092] Because tms17 exhibits a thermosensitive phenotype, it is necessary to determine whether high and low temperatures affect the interaction between OsUTP6 and UTPB-related proteins. LCI experiments revealed that the interaction between OsUTP6 and OsUTP21 decreased at low temperatures, while the interaction between OsUTP6 and OsUTP18 and MPP102 appeared to increase at low temperatures (see [link to LCI study]). Figure 12 Furthermore, the interaction between OsUTP6 and MPP101 was very low in tobacco leaves. These results suggest that temperature changes do indeed affect the interaction between OsUTP6 and UTPB-related proteins, potentially influencing the assembly of the UTPB complex and the processing of pre-rRNA.

[0093] To investigate how the point mutation on the 5'UTR leads to the thermosensitive male sterility phenotype of tms17, the transcriptional level of OsUTP6 was first examined. RT-qPCR was used to detect the transcriptional level of OsUTP6 in the anthers of ZH11 and tms17 under high and low temperatures. The results showed that the transcriptional level of OsUTP6 in tms17 was significantly lower than that in ZH11, less than half of its original level, while the transcriptional level of OsUTP6 did not change significantly under high and low temperatures (see [link to relevant documentation]). Figure 13 Cis-regulatory elements responsible for post-transcriptional regulation are typically located in the UTR, and their specific sequences and structures significantly influence mRNA stability and translation levels. Therefore, this result indicates that point mutations may lead to decreased stability of the OsUTP6 transcript, thereby reducing transcription levels, while temperature changes did not significantly affect OsUTP6 transcription levels.

[0094] To investigate whether this SNP site also causes abnormalities in the post-transcriptional process of OsUTP6, the secondary structure of the 5'UTR region of OsUTP6 was predicted using RNAfold (http: / / rna.tbi.univie.ac.at / ) via the MFE (Minimum Free Energy) method. The results showed that the 5'UTR of OsUTP6 (5'UTR...) OsUTP6 The predicted free energy of the system in the entire thermodynamic ensemble is -16.85 kcal / mol. A negative value indicates that the system is in a relatively stable state and can release energy. The frequency of MFE structures in the ensemble is 8.15%, and a higher frequency indicates higher stability and reliability. The ensemble diversity value is 18.81, and a higher value indicates that the ensemble contains more different structural forms. Furthermore, the prediction revealed a 5'UTR... OsUTP6 At the site of the point mutation (22 bp before the promoter ATG), a stem-loop structure will form (see [link to relevant documentation]). Figure 14 Left).

[0095] The 5'UTR of point mutations OsUTP6 (m5'UTR) OsUTP6 The predicted system free energy for the entire thermodynamic ensemble is -20.26 kcal / mol, the frequency of MFE structures in the ensemble is 29.28%, the ensemble diversity value is 9.87, and m5'UTR... OsUTP6 The stem-loop structure at the location of the point mutation becomes more stable due to the mutation (see [link]). Figure 14 (Right). The lower the free energy of an mRNA, the more stable its secondary structure, and the less conducive it is to translation. These results indicate that point mutations can lead to... OsUTP6 The alteration of the 5'UTR secondary structure and the more stable stem-loop structure suggest that the translation process of tms17 may be affected.

[0096] To verify the hypothesis that changes in secondary structure affect the OsUTP6 translation process, p35::5'UTR was constructed. OsUTP6 -OsUTP6-VENUS and p35::m5'UTR OsUTP6 -OsUTP6-VENUS and p35::OsUTP6-VENUS without 5'UTR as a blank control were then used to infect tobacco leaves and cultured at high and low temperatures. Finally, fluorescence was observed, and the average fluorescence intensity and range were measured and statistically analyzed. The results showed that at high temperatures, the subcellular localization fluorescence signal of the OsUTP6-VENUS fusion protein (as a control) was observed within the nucleolus of tobacco cells (see [link to original text]). Figure 15 a). 5'UTR OsUTP6 In its presence, the fluorescence intensity and range of OsUTP6-VENUS were twice that of the blank control (see [link]). Figure 15 a- Figure 15 c). The size and shape of the nucleolus are highly dynamic, adjusting ribosome biogenesis according to the needs of protein synthesis. In cells with high translation requirements, the nucleolus becomes significantly larger. Increased fluorescence intensity indicates the 5' UTR. OsUTP6 The translation of OsUTP6 is extremely important, and the enlarged nucleolus suggests that increased OsUTP6 abundance may promote ribosome biogenesis. However, m5'UTR... OsUTP6 The measurement results in the presence of the blank control showed no significant difference from those in the control group (see [link]). Figure 15 Furthermore, the fluorescence intensity of OsUTP6-VENUS was enhanced at low temperatures in all three configurations. High laser power settings, intended for high-temperature results, led to overexposure during imaging; therefore, low laser power was used for imaging and statistical analysis. The results showed that the localization of OsUTP6-VENUS at low temperatures was similar to that at high temperatures, with a 5' UTR... OsUTP6 The presence of this further enhances the average fluorescence intensity, while the average fluorescence area does not change significantly (see [link to relevant documentation]). Figure 15 d- Figure 15 f). The above results indicate that m5'UTR OsUTP6 This leads to translational defects in OsUTP6, and its reduced abundance may affect the ribosomal biogenesis of tms17. At low temperatures, although it did not recover to the 5'UTR... OsUTP6 The level when it exists, but m5'UTR OsUTP6 The abundance of OsUTP6 recovered significantly at higher temperatures when it was present.

[0097] To investigate whether there are any abnormalities in the ribosome biogenesis process of tms17, polysome profiling was used to analyze the anthers of tms17 and wild-type ZH11 in the mid-development stage under high and low temperatures. A polysome is a complex in which multiple ribosomes simultaneously bind to the same mRNA for translational activity, and the number of ribosomes bound results in different sedimentation coefficients. Therefore, polysome profiling is an analytical method that uses sucrose density gradient centrifugation to separate the large and small ribosome subunits and polysomes in cells, and observes the distribution of each component due to their different sedimentation coefficients. Different batches of polysome profiling experiments may show peaks due to variations in elution rates. Figure XSince the X and Y axes were not aligned, the experimental results were processed as follows using the website (https: / / www.umm.uni-heidelberg.de / ) and following the method of Schiller et al. By selecting a baseline, the X and Y axes were aligned and plotted using the lowest point between the 60S and 80S peaks (see...). Figure 16 a), then the peak length and peak area were standardized and measured statistically (see a). Figure 16 (b) According to the experimental results, the 40S peak area of ​​tms17-HT was significantly lower than that of ZH11-HT and ZH11-LT, and the polysome peak area was significantly lower than that of ZH11-HT. This indicates that the assembly of the small ribosomal subunits in tms17 was severely damaged at high temperatures, resulting in the inability of the translation process to meet the normal anther development of tms17, thus causing plant sterility at high temperatures. However, at low temperatures, the 40S peak area of ​​tms17-LT could recover to the level of ZH11-LT, indicating that the assembly defects of the small ribosomal subunits of tms17 could be partially recovered at low temperatures, thus meeting the basic requirements for anther development and restoring fertility.

[0098] To investigate whether abnormal ribosomal biogenesis in tms17 under high temperatures leads to abnormal anther development-related protein abundance, proteomics analysis was performed. Proteomic data showed that some anther development-related proteins were significantly reduced in tms17 anthers under high temperatures, but recovered to wild-type levels under low temperatures (see [link to data]). Figure 17 These results indicate that the abundance of anther development-related proteins in tms17 is insufficient to meet the needs of anther development at high temperatures, leading to plant sterility. However, the restoration of ribosomal biogenesis in tms17 at low temperatures ensures the translation of anther development-related proteins, thereby restoring plant fertility.

[0099] In summary, in plants, the 45S rDNA transcription unit is responsible for transcribing rRNA precursors carrying 18S, 5.8S, and 25S rRNA, and there are hundreds or thousands of copies of this transcription unit in the plant genome. This rRNA precursor requires processing such as folding and cleavage to remove the outer 5' / 3' ETS and the inner ITS1 / 2, thereby forming mature 18S, 5.8S, and 25S rRNAs and assembling them into ribosome subunits. Most genes that exert enzymatic activity in yeast ribosome processors have homologous genes identified in Arabidopsis, but their enzymatic activities are currently unclear. The Arabidopsis mrl-1 (meerling-1) mutant exhibits delayed embryonic development, with promoter recombination from the yeast UTP18 homolog AtUTP18 leading to reduced protein abundance. The mrl-2 loss-of-function heterozygous mutant fails to produce homozygous offspring, and asynchronous development of female gametophytes leading to embryo sac abortion has also been observed. Arabidopsis thaliana SWA1 (Slow Walker1), a homolog of yeast UTP15, has been shown to be involved in the cleavage of the P site on the 5' ETS. Inhibition of SWA1 expression via RNAi significantly suppresses root growth and leads to abnormal accumulation of 18S pre-rRNA. Mutations in the Arabidopsis thaliana TOZ (TORMOZ) gene produce embryos with abnormal cell division planes and potentially arrested embryos that fail to establish a normal cell division pattern. TOZ encodes a nucleolar protein containing a WD40 repeat sequence, and its yeast homolog, UTP13, is predicted to function in 18S rRNA processing. Although the related processing in yeast and animals has been well-studied, only a few related genes in plants have been reported to be functionally conserved, and research on ribosomal biogenesis remains incomplete.

[0100] In this invention, a novel photothermosensitive male sterile TGMS line, tms17, was screened, with its mutation site located on the 5'UTR of the yeast ScUTP6 homolog LOC_Os02g01350 (see [link to original text]). Figure 5 c), which leads to a significant decrease in the abundance of OsUTP6 in anthers under high temperatures (see c). Figure 13 This affects ribosome biosynthesis and leads to abnormal protein abundance in anthers (see...). Figures 16-18 This ultimately leads to abnormal anther development (see...) Figure 4 Plant sterility (see) Figure 1 The sterility phenotype of tms17 can be restored by using the wild-type LOC_Os02g01350 gene fragment (see [link]). Figure 7 This demonstrates that the male fertility defect under high temperature in tms17 is caused by changes in the abundance of OsUTP6.

[0101] In yeast, ScUTP6 interacts with ScUTP18, ScUTP21, and ScMPP10 to participate in 18S pre-rRNA processing. UTP6 and other UTPB proteins are relatively conserved across different species (see [link to relevant documentation]). Figure 9 b and Figure 10 In rice, OsUTP6 can interact with OsUTP13, OsUTP18, OsUTP21, OsMPP101, and OsMPP102 (see [link to relevant documentation]). Figure 11 and Figure 12 These results suggest that OsUTP6 in rice may participate in 18S pre-rRNA processing in a manner very similar to that in yeast.

[0102] Abnormal pre-rRNA processing leads to a variety of phenotypes, resulting in defects ranging from apoptosis and reduced translation to translatome alterations in Homo sapiens. Related mutants in plants also cause defects of varying degrees, including embryonic abnormalities and abnormal vegetative growth. In maize, RCL1 (RNA 3'-terminal phosphate cyclase-like) is involved in the cleavage of 5'ETS sites P' and A1, and ITS1 site A2 during 18S rRNA maturation. Loss of its function leads to low levels of mature 18S rRNA, abnormal accumulation of related precursor substances, and ultimately, abnormal embryonic cell differentiation and death. The rice RPL3B (Ribosome large subunit protein 3B) mutant rml1 (rice minute-like 1) exhibits phenotypes such as slow growth, reduced plant height, narrower leaves, and smaller seeds. Its protein mutation and significantly reduced gene expression result in a significant reduction in the free 60S large subunit and polyribosomes.

[0103] In this embodiment, OsUTP6 is located within the nucleolus (see [link to example]). Figure 11 High expression was observed in all cell layers during the early and middle stages of anther development (see [link]). Figure 8 OsUTP6 may be involved in ribosome biogenesis in various anther cell layers to aid anther development. No homozygous mutants could be isolated from the self-crossed progeny of loss-of-function heterozygous mutants obtained by CRISPR-Cas9 knockout of OsUTP6 (see Table 2 and...). Figure 6 This indicates that OsUTP6 function is also crucial for embryonic development. m5'UTRUTP6 leads to a significant decrease in the transcriptional level of OsUTP6 in the anther (see [link]). Figure 13 This results in fertility defects in the plant; however, the vegetative growth of tms17 is not significantly different from that of the wild type (see [link]). Figure 1This differs from existing mutant phenotypes related to ribosome biogenesis. Studies have reported that the Arabidopsis heterozygous mutant swa1 / + has disrupted the mitotic cycle of female gametophytes, leading to asynchronous development and arrest of different embryo sacs in the same pistil at two-nucleate, four-nucleate, or eight-nucleate stages. Although this defect occurs during reproductive development, it is essentially a gametophyte defect. Therefore, the unique phenotype of tms17 suggests that ribosome biogenesis may be specifically regulated in reproductive development, providing a new research direction for further exploration in this field.

[0104] The influence of environmental factors on ribosomal biogenesis has been reported. Most small ribosomal subunit genes show significantly increased expression under abiotic stress, suggesting that these genes may possess stress-regulated characteristics in addition to their housekeeping function. High temperatures induce atypical pathways in 35S pre-rRNA processing, leading to the production of abnormal pre-rRNA, a phenomenon conserved in rice and tomato.

[0105] Due to decreased OsUTP6 transcription levels (see...) Figure 13 Meanwhile, the translation process of OsUTP6 and its interaction with interacting proteins are impaired by high temperatures (see [link to article]). Figure 12 and Figure 15 This results in impaired ribosome biogenesis in tms17 (see...). Figure 16 This leads to abnormal protein abundance during anther development (see...). Figure 17 During the seventh stage of anther development, the microsporocyte needs to make extensive preparations for meiosis and undergo two mitotic divisions during subsequent development to form mature pollen grains. From the eighth stage onwards, the tapetum (PCD) gradually supplies abundant nutrients, raw materials, and energy for microspore development. Due to insufficient protein abundance to meet the requirements for anther development, tms17 exhibits developmental defects from the seventh stage of anther development (see...). Figure 4 This ultimately leads to the male infertility phenotype under high temperatures (see...). Figure 18 b).

[0106] High-temperature stress exacerbates the burden on microspore development, and the protection provided by photosensitive and thermosensitive lines is insufficient to support normal microspore development. However, the absence of environmental stress at low temperatures reduces the need for protection during microspore development, and the slower development provides ample time for microspore formation, thus allowing photosensitive and thermosensitive lines to regain fertility. Therefore, although the transcriptional level of OsUTP6 does not recover at low temperatures (see...),... Figure 13 However, the partial recovery of the OsUTP6 translation defect alleviated the abnormalities in ribosomal biogenesis (see [link to article]). Figure 15 and Figure 16This restores the abnormal protein abundance in tms17, thereby ensuring the synthesis of anther development-related proteins (see...). Figure 17 ), ultimately restoring tms17's fertility (see Figure 18 c).

[0107] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A plant thermosensitive sterility gene mutant tms17, characterized in that, The nucleotide sequence of the plant thermo-sensitive sterility gene mutant tms17 includes the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.

2. An expression vector containing the plant thermo-sensitive sterility gene mutant tms17 as described in claim 1.

3. An expression cassette containing the plant thermo-sensitive male sterility gene mutant tms17 as described in claim 1.

4. The application of the plant thermo-sensitive sterility gene mutant tms17 as described in claim 1 in the preparation of recessive male sterile transgenic plants.

5. The application of the plant thermosensitive male sterility gene mutant tms17 according to claim 1 in plant breeding, wherein the application includes any one of the following: (1) The plant thermo-sensitive male sterility gene mutant tms17 was introduced into the target plant, and its phenotype showed high temperature sterility and low temperature fertility recovery; (2) The plant thermo-sensitive male sterility gene mutant tms17 was introduced into other plant varieties through hybridization. The sterile plants obtained in the F2 generation showed the thermo-sensitive male sterility phenotype. (3) Using the sterile plants obtained in (1) and (2) as the female parent, different plant varieties are used as the male parent to carry out hybridization, cultivate the target hybrid plants, and obtain the corresponding hybrid seeds.

6. A method for cultivating fertility-recoverable plants whose anther development is affected by temperature, characterized in that, The method includes introducing the plant thermo-sensitive male sterility gene mutant tms17 as described in claim 1 into plant seed cells, and using the plant seeds infused with the plant thermo-sensitive male sterility gene mutant tms17 for corresponding plant cultivation.

7. A method for regulating the thermosensitive fertility morphology of plants, characterized in that, The method includes replacing the TMS17 / OsUTP6 gene in wild-type plants with the plant thermosensitive sterility gene mutant tms17 as described in claim 1.

8. The use of the plant thermosensitive sterility gene mutant tms17 as described in claim 1 or 2, characterized in that, Used to regulate or provide the temperature-sensitive fertility trait of the plant, or used as a selection marker for transgenic plants.

9. The use according to claim 8, characterized in that, The selected marker trait is a reversible change in thermosensitive fertility traits. The reversible change in thermosensitive fertility traits means that under low temperature conditions, the plant exhibits the fertility recovery trait; under high temperature conditions, the plant exhibits the sterility trait.

10. The plant thermosensitive sterility gene mutant tms17 according to claims 1-2, the expression vector according to claim 3, the application according to claims 4-5, the method according to claims 6-7, or the use according to claims 8-9, characterized in that, The plant in question is rice.

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