A plant thermosensitive sterile gene mutant tms16 and its application

Abstract

The present invention relates to the field of agricultural technology, and more particularly to a plant thermosensitive sterile gene mutant tms16 and its applications. The present invention provides a plant thermosensitive sterile gene mutant tms16, wherein the nucleotide sequence of the plant thermosensitive sterile gene mutant tms16 comprises the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing. The tms16 mutant provided by the present invention can impart improved sterility properties to plants. The fruit set rate of plants containing this mutant is significantly lower than that of previously discovered thermosensitive sterile mutants such as tms5. Application of this mutant in breeding processes will result in higher breeding efficiency and make it easier to obtain high-purity sterile plants.

Description

Technical Field

[0001] The present invention relates to the technical field of agriculture, and in particular to a plant thermosensitive sterile gene mutant tms16 and applications thereof. Background Art

[0002] Utilizing heterosis in self-fertilizing crops like rice requires sterilizing the male gametes of the female parent. Male-sterile plants can avoid self-fertilization during hybrid production, greatly simplifying the hybridization process and improving hybrid yield and germplasm purity. Therefore, controlling male sterility in rice is crucial for hybrid breeding. Male sterility can be categorized as either cytoplasmic male sterility or nuclear male sterility. Cytoplasmic male sterility can be restored to fertility through the use of restorer genes, thereby producing hybrids. Nuclear male sterility is controlled by the nuclear genome. Conflicts among the genes regulating meiosis, tapetum, or male gamete development can easily lead to male sterility. However, these sterile materials lack maintainer lines, making the generation of pure sterile lines difficult and thus unsuitable for hybrid seed production. Nuclear sterile materials, however, which are susceptible to environmental factors, can be restored to fertility under specific conditions, thus serving as both sterile and maintainer lines. Light / temperature-sensitive nuclear male sterile plants will show male sterility under high temperature or long light conditions, and restore fertility to produce self-pollinated seeds under low temperature or short light conditions.

[0003] Nongken 58S is the first photosensitive male sterile plant discovered. Its photosensitive sites are located on PMS1 on chromosome 7 and PMS3 on chromosome 12. Both genes produce long non-coding RNA. The transcription product of PMS1, PMS1T, is cleaved to generate phasiRNA. Under long-day conditions, phasiRNA accumulates in large quantities. The transcription product of PMS3 is named LDMAR. Its mutation produces changes in its secondary structure, resulting in increased methylation of its promoter and decreased transcription levels, ultimately causing male sterility. The two have overlapping effects. The first indica thermosensitive male sterile line, Annong S-1, has its thermosensitive characteristics controlled by the TMS5 site. TMS5 encodes RNaseZ, an RNA enzyme that can specifically cleave Ub L40 mRNA, TMS5 mutation leads to Ub L40 In 2022, the OsMS1 gene that controls Hengnong S-1 was discovered. wenmin1 locus, OsMS1 wenmin1 Both the protein and wild-type OsMS1 encode histidine-binding proteins that can interact with the transcription factor TDR protein and activate the expression of downstream genes. However, the former is located in the nucleus and cytoplasm, while the latter is located entirely in the nucleus. At high temperatures, OsMS1 located in the nucleus wenmin1 The protein decreases, causing the expression of the downstream gene EAT1 to decrease excessively, ultimately leading to male sterility.

[0004] In the two-line hybrid breeding industry, the vast majority of photo- / thermo-sensitive male sterility loci are Nongken 58S or Annong S-1. However, photo- / thermo-sensitive nuclear sterile lines still have problems with impure seed production and limited genetic resources. Therefore, the discovery of new photo- / thermo-sensitive genetic resources is of great significance. Summary of the Invention

[0005] To address the above-mentioned technical problems, the present invention provides a plant thermosensitive sterility gene mutant, tms16, and its application. Its sterility at high temperatures is more stable than that of tms5. High-throughput sequencing shows that the sterility locus of tms16 is different from any reported photothermosensitive sterility locus. This gene encodes an acyl-CoA reductase (FAR), which is involved in the synthesis of the pollen exine. This mutant has a single base substitution in the last exon of the coding region, resulting in a significant decrease in protein activity at high temperatures compared to the wild type. Due to the reduced FAR activity at high temperatures, the sporopollenin precursors produced are insufficient to protect the pollen, resulting in the loss of pollen inner and outer wall structures and plant abortion. However, at low temperatures, due to the slowed development of microspores, the microspores' need for exine protection is reduced, and the residual enzyme activity produces some sporopollenin precursors, which are sufficient to ultimately overcome microspore developmental defects and restore fertility.

[0006] To this end, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a plant thermo-sensitive sterile gene mutant tms16 in an optional embodiment, wherein the nucleotide sequence of the plant thermo-sensitive sterile gene mutant tms16 comprises the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.

[0008] In the present invention, the phenotype of the tms16 mutant is caused by the mutation of G to A in the last exon of its genome, resulting in the mutation of methionine (Met) at position 549 to isoleucine (Ile).

[0009] Preferably, the nucleotide sequence of the plant thermo-sensitive sterile gene mutant tms16 consists of the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.

[0010] In a second aspect, the present invention provides, in an optional embodiment, an expression vector containing the above-mentioned plant thermo-sensitive sterility gene mutant tms16.

[0011] In a third aspect, the present invention provides, in an optional embodiment, a use of the above-mentioned plant thermo-sensitive sterile gene mutant tms16 in preparing recessive male sterile transgenic plants.

[0012] In a fourth aspect, the present invention provides, in an optional embodiment, a use of the plant thermo-sensitive sterile gene mutant tms16 in plant breeding. The use includes any of the following:

[0013] (1) The plant thermosensitive sterility gene mutant tms16 was introduced into the target plant, and its phenotype showed high-temperature sterility and low-temperature fertility recovery;

[0014] (2) The plant thermosensitive sterility gene mutant tms16 was introduced into other plant varieties through hybridization, and the sterile plants obtained in the F2 generation showed a thermosensitive sterility phenotype;

[0015] (3) Using the sterile plants obtained in (1) and (2) as female parents and using different plant varieties as male parents for hybridization, cultivating hybrid target plants, and obtaining corresponding hybrid seeds.

[0016] In a fifth aspect, the present invention provides, in an optional embodiment, a method for cultivating plants whose fertility can be restored in plants whose pollen development is affected by temperature, the method comprising introducing the above-mentioned plant thermosensitive sterility gene mutant tms16 into plant seed cells, and using plant seeds introduced with the plant thermosensitive sterility gene mutant tms16 to cultivate corresponding plants.

[0017] In a sixth aspect, the present invention provides, in an optional embodiment, a method for regulating the temperature-sensitive fertility shape of a plant, the method comprising replacing the TMS16 gene in a wild-type plant with the nucleotide sequence shown in SEQ ID NO.1 in the sequence table; or replacing the corresponding protein of the TMS16 gene with the amino acid sequence shown in SEQ ID NO.2.

[0018] In a seventh aspect, the present invention provides, in an optional embodiment, a use of the above-mentioned plant thermo-sensitive sterility gene mutant tms16 for regulating or providing the thermo-sensitive fertility trait of the plant, or as a selection marker for transgenic plants.

[0019] Preferably, the marker trait of the selection marker is a reversible change of the temperature-sensitive fertility trait, and the reversible change of the temperature-sensitive fertility trait means that under low temperature conditions, the plant shows a trait of fertility recovery; under high temperature conditions, the plant shows a sterility trait.

[0020] In the above technical solution, the plant is a monocot or a dicot, the monocot includes rice, and the dicot includes Arabidopsis thaliana. The high temperature is a temperature greater than 29°C, and the low temperature is a temperature less than 25°C.

[0021] The nucleotide sequence of SEQ ID NO.1 is as follows:

[0022]

[0023] The amino acid sequence of SEQ ID NO.2 is as follows:

[0024] .

[0025] Compared with the prior art, the present invention has one of the following beneficial effects:

[0026] 1. The tms16 mutant provided by the present invention can bring better sterility performance to plants. The fruit set rate of plants containing this mutant is significantly lower than that of the previously discovered temperature-sensitive sterile mutant plants such as tms5. Applying it in the breeding process will achieve higher breeding efficiency and make it easier to obtain high-purity sterile plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Figures 2A and 2B show the cultivation of the tms16 mutant and wild-type Zhonghua 11 plants in Example 2, wherein Figures AC show the rice plants of wild-type Zhonghua 11 and thermosensitive male sterile plants under high and low temperature conditions, Figure D shows the panicle seed setting rate of wild-type Zhonghua 11, tms16, and tms5 under high temperature, Figures E and F show the anther morphology of wild-type Zhonghua 11 and thermosensitive male sterile plants under high and low temperature conditions, and Figure G shows iodine staining of wild-type Zhonghua 11 and tms16 pollen under high and low temperature conditions. Scale bars in the figures are: 5 cm for Figures AC, 2 cm for Figure D, and 100 μm for Figure G.

[0028] Figure 2 Figures 1 and 2 are SEM observation images and fluorescence staining images of anthers and pollen of tms16 mutant plants and wild-type Zhonghua 11 at different temperatures in Example 2, wherein Figures AC are SEM images of pollen and pollen wall of wild-type Zhonghua 11, Figure DF is fluorescence staining image of inner and outer walls of pollen of wild-type Zhonghua 11, Figure GI is SEM images of pollen and pollen wall of tms16 mutant plants at high temperature, Figure JL is fluorescence staining image of inner and outer walls of pollen of tms16 mutant plants at high temperature, Figure MO is SEM image of pollen and pollen wall of tms16 mutant plants at low temperature, and Figure PR is fluorescence staining image of inner and outer walls of pollen of tms16 mutant plants at low temperature. Scale bars in the figures: 50 μm for Figures A, G, and M, 10 μm for Figures B, H, and N, and 1 μm for Figures C, I, and O.

[0029] Figure 3 Figures 1 and 2 are semi-thin section images and transmission electron microscopy (TEM) observation images of anthers of wild-type plants and mutant tms16 plants at different temperatures in Example 3, wherein Figures AD are semi-thin section observation images of anthers of wild-type plants, Figures GJ are semi-thin section observation images of anthers of mutant tms16 plants at high temperature, Figure MP is semi-thin section observation image of anthers of mutant tms16 plants at low temperature, Figures E and F are TEM observation images of anthers of wild-type plants at stage 9-10, and Figures K and L are TEM observation images of anthers of mutant tms16 plants at stage 9-1 TEM observation image at stage 0, Figures Q and R are TEM observation images of anthers of mutant tms16 plants at stages 9-10 under low temperature, E is anther epidermis, En is the inner wall of the anther chamber, T is the tapetum, Msp is the microspore, BP is the binucleate spore, MP is the mature spore, Ex is the pollen exine, AEx is the defective pollen exine, Se is the outer layer of the exine, Ne is the inner layer of the exine, scale bars in the figures: Figures AD, GJ, MP are 20 μm, in Figures E, F, K, L, Q and R, the left figure is 5 μm, and the right figure is 1 μm;

[0030] Figure 4This figure compares the seed set rates of the mutant tms16, tms5, and tms18 plants at different booting temperatures in Example 4. Figure A shows the seed set rate data for each mutant plant in 2020. On the horizontal axis, AD represents the population that germinated in the high-temperature season, and FI represents the population that germinated in the low-temperature season. Figure B shows the seed set rate data for each mutant plant in 2021. This year, the plants were affected by a persistent low-temperature surge during the high-temperature season. On the horizontal axis, AC represents the population that germinated in the high-temperature season, and DF represents the population that germinated in the low-temperature season.

[0031] Figure 5 Schematic diagram of the TMS16 gene encoding acyl carrier protein reductase, wherein Figure A is the TMS16 gene model and the mutation pattern of two allelic mutants, Figure B is the tms16-crp mutation site, Figure C is the tms16 mutation site, Figure D is a diagram of TMS16 genetic complemented plants, Figure E is the phenotype of tms16-crp plants under high temperature, and Figure F is the phenotype of tms16-crp plants under low temperature. Scale bars in Figures DF are 2 cm;

[0032] Figure 6 Schematic diagram of TMS16 protein expression starting from the 9th stage tapetum, where Panel A is a semi-quantitative analysis of TMS16 transcription levels, and Panels BJ are anther localization analysis of TMS16-GFP fusion protein. Scale bars in Panels AJ are 70 μm.

[0033] Figure 7 Figure 1 is a schematic diagram of the recombinant TMS16 protease activity experiment, wherein Figure A is a diagram showing the purification results of TMS16 and mTMS16 (M549I) proteins, Figure B is a diagram showing the mass spectrometry results of the enzyme activity reaction products, and Figures C and D are EIC chromatograms of the enzyme activity reaction results with an extracted mass-to-charge ratio of 69. Figure C is a peak diagram of the enzyme activity reaction products in the reaction system of wild-type TMS16 protein and mutant mTMS16 at 30°C (high temperature); Figure D is a peak diagram of the enzyme activity reaction products in the reaction system of wild-type TMS16 protein and mutant mTMS16 at 20°C (low temperature);

[0034] Figure 8 Figures 1 and 2 show that Arabidopsis ms2 exhibits temperature-sensitive male sterility. Figure A shows the MS2 gene model and the mutation patterns of the two allelic mutants. Figures B, F, and J show the plant phenotypes of wild-type Arabidopsis and the ms2 mutant under high and low temperature conditions. Figures C, G, and K show the Alexander staining results of anthers of wild-type Arabidopsis and the ms2 mutant under high and low temperature conditions. Figures D, E, H, and L show scanning electron microscopic observations of anthers and pollen of wild-type Arabidopsis and the ms2 mutant under high and low temperature conditions.

[0035] Figure 9Figures 1 and 2 show the mutation patterns and phenotypic observations of alleles of ms2. Figure A shows the mutation pattern of ms2-crp, Figures B and E show the phenotypes of ms2-crp plants at different temperatures, Figures C and F show the scanning electron microscopy observations of ms2-crp pollen at different temperatures, Figures D and G show the Alexander staining images of ms2-crp at different temperatures, Figure H shows the plant phenotype of comp-ms2(mut) at 28°C, Figure I shows the scanning electron microscopy observations of comp-ms2(mut) pollen at 28°C, and Figure A shows the Alexander staining image of comp-ms2(mut) at 28°C. Scale bars in the figures: 2 cm for Figures A and D, 2 cm for Figures B, E and H, 5 μm for Figures C, F and J, and 50 μm for Figures G and I. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] Example 1

[0040] Acquisition of tms16 mutants

[0041] The chemical mutagen ethyl methanesulfonate (EMS) was used to randomly induce mutagenesis in the japonica rice variety Zhonghua 11. After the M1 generation was self-pollinated, the M2 generation population was planted under high temperature (>29°C) and long day conditions to screen for sterile plants. After the stumps were cut, the plants that recovered fertility were further screened under low temperature (<25°C) and short day conditions, and finally temperature-sensitive male sterile plants were obtained.

[0042] Sequencing verification revealed that the gene sequence of the thermosensitive male sterile plant contained a tms16 mutant, and the gene sequence of the mutant was shown as SEQ ID No. 1 in the sequence listing.

[0043] Example 2

[0044] Identification of the temperature-sensitive properties of tms16

[0045] Thermosensitive male sterile plants and wild-type Zhonghua 11 plants were cultivated under high temperature. During the vegetative growth stage, the thermosensitive male sterile plants had a slightly longer growth cycle than the wild-type Zhonghua 11 plants. During the reproductive growth stage, the wild-type rice Zhonghua 11 plants had bent and drooping ears and plump fruits. Figure 1A; Thermosensitive male sterile plants have upright panicles and almost no fruiting. Figure 1 B. Under low temperature, some rice ears of thermosensitive male sterile plants bent and drooped, and the seed setting rate recovered to a certain extent. Figure 1 C. In addition, the wild type anthers are plump and yellow, while the anthers of the thermosensitive male sterile plants under high temperature are white and shrunken, and the anther morphology under low temperature is close to that of the wild type, see Figure 1 E and F. Pollen iodine staining experiments showed that Zhonghua 11 produced a large number of mature pollen grains in its anthers. Under high temperature conditions, the pollen of thermosensitive male sterile plants was very small and all aborted, while under low temperature conditions, normal mature pollen grains could be seen. Figure 1 The above data show that the thermosensitive male sterile plants are sterile at high temperatures but fertile at low temperatures, and are typical thermosensitive nuclear male sterile mutants.

[0046] To further understand the phenotype of tms16 anthers at different ambient temperatures, scanning electron microscopy was used to observe the mature anthers of wild type and tms16 at different ambient temperatures. The pollen of wild type and tms16 was co-stained with basic fuchsin and fluorescent whitening agent and observed under a confocal microscope (basic fuchsin is a specific dye for the outer layer of the pollen exine, which can cause the phenylpropanoid substances in the pollen exine to produce red autofluorescence under 561nm excitation light; fluorescent whitening agent 28 is a common cellulose polysaccharide dye that can cause the inner wall of the pollen to produce green fluorescence under 405nm excitation light.). The results are shown in Figure 2 .

[0047] Scanning electron microscopy results showed that under high temperature conditions, the pollen grains of the wild type material were mature and round, with a granular outer wall structure on the pollen surface. Figure 2 AC, while the pollen of tms16 mutant material is small in number, almost all of which is dry and wrinkled, and adheres to the wall of the chamber and is difficult to disperse. Figure 2 GI. It can be seen that compared with the wild type Zhonghua 11, the pollen outer wall of tms16 is not only wrinkled, but also lacks the granular and dense structure of the wild type pollen outer wall. Under low temperature conditions, although tms16 still has a small amount of shriveled pollen, most of the mature pollen has a rounded appearance close to that of the wild type, and the pollen surface also shows a dense outer wall structure, see Figure 2 MO.

[0048] The staining results showed that the outer wall of the wild type was stained red and the inner wall was stained green, see Figure 2 DF. In the tms16 mutant, the phenylpropanoids in the outer wall emit red fluorescence at high temperature, but the outer wall is obviously wrinkled, and the green fluorescence of the inner wall almost disappears. Figure 2JL. At low temperatures, both the outer and inner walls of tms16 return to a state similar to that of the wild type, see Figure 2 Although phenylpropanoids can be detected in tms16 at high temperatures, abnormal metabolism of other substances in sporopollenin may cause defects in the pollen exine, resulting in microspore rupture before the inner wall is formed, leading to abortion.

[0049] Example 3

[0050] Cytological analysis

[0051] In order to determine the specific period of pollen abortion and cytological defects in the mutant tms16 plants, we observed and analyzed the development of anthers at multiple stages in wild-type plants and mutant tms16 plants under high temperature using semi-thin sections. Figure 3 .

[0052] pass Figure 3 The cytological results showed that there was no significant difference between the wild-type plants (ZH11 in the figure) and the mutant tms16 plants in the 9th stage of anther development, which is the period when microspores are released from the tetrad. Figure 3 A, G, and M. At stage 10, the microspores of the wild-type plants swelled normally, while the tapetum cells of the mutant tms16 plants became hypertrophic and the microspores appeared irregularly shrunken. At low temperatures, the microspores swelled normally. Figure 3 B, H, and N. At stage 11, the microspores of wild-type plants shrink to form crescent-shaped binucleate microspores, which eventually develop into mature trinucleate pollen grains at stage 12. Figure 3 C and D: The microspores of the mutant tms16 plant further shrank dramatically and could not develop into a crescent shape. The microspores were completely degraded at the 12th stage, with only some remnants remaining in the spore cavity. Figure 3 I and J. At low temperatures, the mutant tms16 plants formed crescent-shaped microspores, and the contents of the 12th microspores accumulated normally, eventually forming mature trinuclear pollen grains similar to those of the wild type, see Figure 3 O and P.

[0053] Subsequently, anthers of wild-type plants and mutant tms16 plants were resin-embedded and ultrathinly sectioned, and the pollen wall structure was observed using transmission electron microscopy (TEM). The results showed that at the ninth stage of anther development, the wild-type microspores released from the tetrad of wild-type plants were approximately spherical in shape; the electron-dense sporopollenin was orderly deposited around the microspores, forming a complete pollen wall structure. Figure 3 E. At the same time, although the microspores of the mutant tms16 plant are nearly spherical, their outer wall is missing and there is almost no sporopollenin deposition, see Figure 3K. Under low temperature, sporopollenin accumulates on the periphery of microspores in the mutant tms16 plant to form an outer wall structure, but this structure is not as clear and complete as the outer wall in the wild type, see Figure 3 Q. In the 10th issue, the double-layer structure of the pollen wall of the wild-type plant can be clearly observed. Figure 3 F, while the microspores of the mutant tms16 plants are shrunken and lack the pollen outer wall, see Figure 3 L, Under low temperature, a double-layered outer wall structure can be observed in the mutant tms16 plant, but its electron density is not as deep as that in the wild type, see Figure 3 R.

[0054] The above results show that under high temperature, the synthesis and deposition of sporopollenin in the mutant tms16 plants are abnormal, and the defective pollen outer wall leads to microspore abortion; under low temperature, the mutant tms16 plants cannot fully recover to the wild type, but can largely compensate for this developmental defect.

[0055] Example 4

[0056] In order to further study the fertility recovery effect of the mutant tms16 plant, the fruit set rate was statistically analyzed at different temperatures in the summer of 2020 and 2021 and compared with the mutant tms5 plants and mutant tms18 plants under the same genetic background. Starting from May, a batch of seeds were germinated every week and moved into the field one month later. The average temperature within one week of the booting period was taken as the booting temperature. In the summer of 2020, when the booting temperature was above 29°C, the fertility of the mutant tms16 plants and the mutant tms5 plants was close to 0, while when the average temperature during the booting period dropped to 28°C, the fertility of the mutant tms16 plants and the mutant tms5 plants were restored to a certain extent. It is worth noting that the fruit set rate of the mutant tms16 plants is only 50% of that of the mutant tms5 plants, indicating that under the same genetic background, the high temperature sterility of the mutant tms16 plants is better than that of the mutant tms5 plants, see Figure 4 A. In the summer of 2021, Shanghai experienced a cold wave due to the typhoon. This cold wave caused the fruit set rate of the mutant tms5 plants to recover to 13%-15%, while the fruit set rate of the mutant tms16 plants remained at around 0.3%. Figure 4 B. As the temperature gradually dropped, although the seed set rate of mutant tms16 and mutant tms5 plants increased significantly, the seed set rate of mutant tms16 plants (26%) was still significantly lower than that of mutant tms5 plants (49%). At an average booting temperature of 26°C, the seed set rate of mutant tms16 plants was approximately 9%, far lower than the 57% of mutant tms5 plants. It was not until the average booting temperature dropped to 24.8°C that the seed set rate of mutant tms16 plants recovered to 32%, see Figure 4 B. The above results indicate that the mutant tms16 plants have better high-temperature sterility than the mutant tms5 plants and are a good temperature-sensitive male sterility locus.

[0057] TMS16 encodes an acyl-CoA reductase

[0058] When the tms16 mutant was crossed with the wild-type Zhonghua 11, the ratio of fertile to sterile plants in the F2 generation was consistent with 3:1, indicating that the tms16 phenotype is controlled by a single recessive gene. From the F2 generation, 60 fertile and 60 sterile plants were selected, and total DNA was extracted from leaves. The two pools were then pooled for high-throughput sequencing analysis. The analysis revealed that the candidate locus is located on chromosome 3, in the last exon of gene number LOC_Os03g07140, where guanine at position 40 mutates to adenine, resulting in a mutation from methionine to isoleucine at amino acid position 549. Figure 5 A and C. Genetic complementation experiments were subsequently performed to transfer the promoter and genome of LOC_Os03g07140 into the tms16 mutant. The fertility of transgenic plants under high temperature was close to that of the wild type, confirming that the mutation of LOC_Os03g07140 caused the phenotype of tms16. Figure 5 D. This gene encodes an acyl-CoA reductase (FAR), which is responsible for reducing 16-carbon acyl carrier protein (C16:0-ACP) or acyl-CoA (C16:0-CoA) to 16-carbon fatty alcohol (C16:0-OH), which is one of the key components of sporopollenin precursors.

[0059] Related studies have shown that the allelic mutant defective pollen wall (dpw) of tms16 will produce a male sterile phenotype, and the pollen is wrinkled and the outer wall is abnormally developed. This mutant has a single base deletion and leads to premature termination of translation, but the temperature-sensitive effect of tms16 has not been reported so far. In the present invention, it was found that the tms16 mutant produced by a single base mutation showed a phenotype of high-temperature sterility and low-temperature fertility, rather than the complete sterility reported by previous researchers. Therefore, it is speculated that different intensities of mutation types of the TMS16 gene will produce different degrees of male sterility phenotypes. In order to verify this speculation, an allelic mutant tms16-crp was created by CRISPR-Cas9 technology, and sequencing showed that a 4-base deletion occurred in the first exon of the TMS16 gene, which led to premature termination of translation, see. Figure 5 A and B, tms16-crp were grown at different temperatures and found to be male sterile, which was not affected by temperature and could not recover fertility at low temperatures. Figure 5 E and F. In summary, it can be considered that Tms16-Crp is a strong allelic mutant of Tms16.

[0060] To further analyze the spatiotemporal localization of this gene at the transcriptional and protein levels, we extracted RNA from various parts of wild-type rice plants and analyzed it by reverse transcription-PCR. We found that TMS16 transcripts were specifically expressed in the middle stage of anther development. Figure 6 A; In addition, by linking the TMS16 gene sequence to a green fluorescent protein (GFP) tag and constructing it into the pCambia1300 vector, the wild-type was infected and observed under a laser confocal microscope. It was found that the TMS16 protein was localized in the tapetum of the 9th stage and entered the ventricle cavity after the 10th stage. Figure 6 BJ.

[0061] Active residues of the TMS16 protein allow pollen to recover fertility at low temperatures

[0062] The tms16 mutation involves a single base substitution, resulting in a change from methionine (Met) to isoleucine (Ile) at position 549. We hypothesized that this mutation might affect protein activity, leading to thermosensitive male sterility. Therefore, we performed in vitro enzyme activity assays on wild-type TMS16 and the point-mutated mTMS16(M549I) protein.

[0063] We extracted RNA from wild-type spikelets of Zhonghua11 and the tms16 mutant, obtained cDNA templates by reverse transcription, and amplified TMS16 and mTMS16(M549I) fragments by PCR. The fragments were then constructed into the pCold-TF (Takara) vector and expressed and purified in vitro in Rosetta Escherichia coli. Finally, recombinant TMS16 and mTMS16(M549I) proteins were obtained. Figure 7 A. Subsequently, an in vitro enzyme activity reaction was performed using the expression product of an empty pCold as a control and C16:0-CoA as a substrate in a 30°C water bath for 30 minutes. The enzyme activity product was extracted with 1.5 volumes of n-hexane, and 2 μL of the extract was analyzed by gas chromatography-mass spectrometry (GC-MS).

[0064] Before the enzyme activity product is detected, a 100 μg / mL hexadecanol standard is first analyzed by gas chromatography-mass spectrometry to determine the peak position and mass spectrum of the product. Figure 7B. We then extracted the EIC peak at a mass-to-charge ratio of 69 from the TIC peak to eliminate background interference. Detection of the enzymatic reaction products revealed that in both experimental groups containing TMS16 and mTMS16(M549I) proteins, products were detected at the peak corresponding to the C16:0 fatty alcohol, while no product signal was detected in the empty control group. Further analysis revealed that the peak area generated by TMS16 was 10 times that of mTMS16(M549I), indicating that the mutation from methionine at position 549 to isoleucine significantly reduced the enzymatic activity of the TMS16 protein, but retained a small amount of protein activity. Figure 7 C.

[0065] The homologous gene TMS16 in Arabidopsis exhibits a thermosensitive sterile phenotype

[0066] Through protein sequence comparison and relevant literature review, it was found that the homologous gene of TMS16 in Arabidopsis is MaleSterility 2 (MS2, AT3G11980), which was first reported by Aarts et al. in 1993. In 2011, Chen Weiwei et al. found that the pollen outer wall of ms2 mutants developed abnormally and the pollen grains were sensitive to acid hydrolysis treatment. The homologous gene Defective Pollen Wall (TMS16) in rice can successfully complement the outer wall defects of Arabidopsis. However, previous studies have not reported the effect of MS2 mutation on Arabidopsis fertility. To investigate whether the Arabidopsis ms2 mutant exhibits a similar thermosensitive phenotype to the rice tms16 mutant, the ms2 T-DNA mutant SAIL_92_C07 was purchased from the Arabidopsis Germplasm Resource Center. The T-DNA insertion of this mutant is located in the penultimate exon. Simultaneously, using CRISPR-Cas9 technology, a sequence within the first exon was selected as a target, resulting in an allelic mutant with a single-base insertion in the first exon and a premature termination at 60 bp. This mutant was named ms2-crp. Using these two mutants as research subjects, both the ms2 and ms2-crp mutants exhibited normal fertility under normal temperature (24°C) cultivation conditions. Figure 8 J and Figure 9 However, as the planting temperature increased, at high temperature (28°C), both ms2 and ms2-crp mutants showed short siliques and a significant decrease in fertility, see Figure 8 F and Figure 9 B, while the wild type is still fertile at 28℃, see Figure 8 B. Alexander staining shows that the pollen of wild-type anthers is all stained purple and rounded in shape when exposed to high temperatures. Figure 8 C, ms2 and ms2-crp mutants have almost no surviving pollen at 28°C, see Figure 8 G and Figure 9D. In addition, although the pollen of ms2 and ms2-crp is still dark purple at 24℃, it is found that these pollen are more likely to break when pressed. Figure 8 K and Figure 9 G. Scanning electron microscopy further revealed that although the pollen of the ms2 mutant was fertile at 24°C, it lacked the honeycomb-like pollen outer wall structure compared to the wild type. Figure 8 L, M and Figure 9 F, while at 28°C, ms2 anthers were difficult to dehisce and had almost no fertile pollen, see Figure 8 H, I and Figure 9 C, The pollen outer wall of the wild type still develops normally under high temperature, see Figure 8 D and E. Protein sequence alignment showed that the MS2 protein shared 59% similarity with the TMS16 protein sequence. Therefore, we believe that the TMS16 protein is highly conserved, which results in similar temperature-sensitive male sterility phenotypes in rice and Arabidopsis.

[0067] Phenotypic analyses revealed that the T-DNA insertion mutant of ms2, SAIL_92_C07, exhibited the same phenotype as the ms2-crp mutant, which terminates prematurely in the first exon. Both mutants are knockout mutants. Combined with the results of low-temperature fertility restoration in tms16, we hypothesized that the single-base mutation in rice resulted in a degree of residual enzyme activity and restored fertility. To test this hypothesis in Arabidopsis, we sought to obtain a weak allelic mutant of Arabidopsis ms2. Sequence alignment revealed that the amino acid corresponding to the tms16 mutation site, methionine at position 549, is conserved between rice and Arabidopsis. We therefore cloned the MS2 promoter and full-length genome, incorporated the tms16 mutation (methionine to isoleucine) into the MS2 sequence, and ligated the MS2 promoter and mutated MS2 genome into the pCAMBIA1300 vector. The recombinant plasmid was used to infect the ms2(T-DNA) mutant using Agrobacterium, resulting in a transgenic complementary plant with a point mutation, named comp-ms2(mut). When the mutant was grown at 28°C, it was found to produce numerous siliques containing seeds, indicating that fertility had been largely restored. Figure 9 H. We also observed the anthers and pollen of the plant at 28°C, and found that the anthers contained a large amount of mature pollen ( Figure 9 I), scanning electron microscopy revealed that the honeycomb-like pollen wall structure was partially preserved, but still had a certain degree of developmental defects, see Figure 9A comprehensive comparison of the wild-type and ms2 phenotypes at 28°C revealed that the structural integrity of the comp-ms2(mut) pollen exine was intermediate between the wild-type and ms2 mutants. This result suggests that the conserved methionine residue at position 549 of the rice TMS16 protein plays a crucial role in both rice and Arabidopsis, and that mutations at this site result in partial loss of function in both species.

[0068] Although the principles of the present invention have been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely illustrative 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. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent transformations and simple substitutions based on the technical solution of the present invention fall within the scope of protection of the present invention.

Claims

1. A rice thermosensitive male sterility gene mutant tms16 , characterized in that, The rice thermo-sensitive sterile gene mutant tms16 The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing.

2. A rice mutant containing the thermosensitive male sterility gene according to claim 1 tms16 expression vector.

3. A rice thermosensitive male sterile gene mutant according to claim 1 tms16 Application in the preparation of recessive male sterile transgenic rice.

4. A rice thermosensitive male sterile gene mutant according to claim 1 tms16 Application in rice breeding, the application comprising any one of the following: (1) Transform the rice thermosensitive sterile gene mutant tms16 When introduced into the target rice, the phenotype showed high-temperature sterility and low-temperature fertility restoration; (2) Transformation of rice thermosensitive sterile gene mutants tms16 By introducing it into other rice varieties through hybridization, the sterile rice obtained in the F2 generation showed a thermosensitive sterile phenotype; (3) Using the sterile rice obtained in (1) and (2) as the female parent and using different rice varieties as the male parent for hybridization, the target hybrid rice is cultivated to obtain the corresponding hybrid seeds.

5. A method for cultivating rice whose fertility can be restored in which rice pollen development is affected by temperature, characterized in that: The method comprises the steps of: tms16 Introduced into rice seed cells, using the rice thermosensitive sterile gene mutant tms16 The rice seeds are used to cultivate the corresponding rice.

6. A method for regulating the temperature-sensitive fertility trait of rice, characterized in that: The method comprises the steps of: TMS16 The gene is replaced with the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing; or Will TMS16 The corresponding protein of the gene was replaced with the amino acid sequence shown in SEQ ID NO.

2.

7. A rice thermosensitive male sterile gene mutant according to claim 1 tms16 The use is characterized in that The invention is used for regulating or providing the temperature-sensitive fertility trait of the rice, or is used as a selection marker for transgenic rice.

8. The use according to claim 7, characterized in that The marker trait of the selection marker is a reversible change of the temperature-sensitive fertility trait. The reversible change of the temperature-sensitive fertility trait means that under low temperature conditions, the rice shows a trait of fertility restoration; under high temperature conditions, the rice shows a sterility trait.

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