Rice thermo-sensitive male sterile gene mutant tms18 and uses thereof

Abstract

The invention discloses a rice temperature sensitive male sterile gene mutant tms18 and its uses. During the study of TMS18 gene, the inventors found that the changing of fertility was related to the structural integrity of the second layer of pollen outer wall. The inventors accidentally found the thermo sensitive male sterile mutant tms18. The fertility of the mutant was affected by different temperatures and can be restored under low temperature treatment. More importantly, the fertility sensitive period of the mutant was different from that of other thermo sensitive male sterile genes. This unique feature of the gene can provide a new theoretical basis and apply value for two-line hybrid rice breeding.

Claims

1. A transgenic seed for a crop plant, wherein the genome of said transgenic seed comprises a rice thermo-sensitive male sterile gene mutant tms18 of SEQ ID No.1.

2. A method for cultivating transgenic rice, wherein the method includes introducing a rice temperature sensitive male sterile gene mutant tms18 of SEQ ID No.1 into rice seeds, and planting the rice seeds to obtain the rice.

3. The method according to claim 2, wherein the method is used to cultivate fertility recoverable rice, the fertility of which can be recovered by low temperature treatment.

4. The method according to claim 3, wherein the temperature for low temperature treatment is lower than 23° C.

5. The method according to claim 3, wherein the low temperature treatment is performed on the rice after their pollen mother cell meiosis.

6. The method according to claim 3, wherein the method is used to cultivate Recessive Male Sterile transgenic plants.

7. The method according to claim 3, wherein the method comprises: using a recessive homozygous male sterile plant containing the mutant tms18 as a female parent and using a target rice as a male parent, hybridized the female parent with male parent at high temperature, to obtain F1 generation seeds; cultivating the F1 generation seeds to obtain the F1 generation plants; self pollinating the F1 generation plants to obtain the F2 generation seeds; and screening male-sterile plants from F2 generation plants cultivated by the F2 generation seeds.

Description

DESCRIPTION OF FIGURES

[0018] FIG. 1 shows the environmental temperature and humidity during fertility changes of plants containing tms18.

[0019] FIG. 2 shows the phenotypic analysis results of wild-type and temperature sensitive plants containing tms18 at different temperatures, in which A-C portions show the phenotypic of wild-type and tms18 mutant plants after heading; D portion shows anthers of wild type and tms18 mutant plants after flowering; E portion shows anthers of wild type and osacos12 mutant plants after spikelet shelling; H portion shows single anther of wild type and osacos12 mutant; F portion shows Alexander dye solution of wild type and osacos12 mutant anthers; the character “H” stands for high temperature treatment (>28° C.); the character “L” stands for low temperature treatment (<23° C.); The scale length (E-F) is 100 μm; (G) 200 μM.

[0020] FIG. 3 shows the results of seed setting rate of plants containing tms18 under treatments at different time, wherein A portion shows seed setting phenotypes of tms18 mutant after heading; B portion shows the statistical table of seed setting rate of tms18 mutant after heading.

[0021] FIG. 4 shows the cytological observation results of anther development of wild-type and tms18 mutants at different temperatures.

[0022] FIG. 5 shows the SEM observation results of anthers and pollen of wild-type and tms18 mutants at different temperatures, wherein F and K portions show the SEM observation results of anthers of wild-type and containing tms18 materials at high temperature and low temperature respectively; B, G and L portions show the scanning electron microscope observation results of anther amplification structure of wild-type and tms18 materials at high temperature and low temperature, respectively; C, H and M portions show the SEM observation results of the structure of wild-type and tms18 materials after anther cracking at high temperature and low temperature, respectively; D, I and N portions show the SEM observation results of mature pollen of wild-type and tms18-material at high temperature and low temperature, respectively; E, J and O portions show the SEM observation results of mature pollen amplification structure of wild-type and tms18 materials at high temperature and low temperature, respectively; HT stands for high temperature treatment (>28° C.); LT stands for low temperature treatment (<23° C.); A. The scales in F and K are 200 μm, in B, G, L, D, I and N are 10 μm, and in C, H, M, E, 0.1 and 0 are 1 μM.

[0023] FIG. 6 shows the TEM observation results of wild-type and tms18 mutant pollen at different temperatures;

[0024] wherein A-E portions show the pollen transmission electron microscope observation results of wild-type Anthers from stage 7 to stage 10 and stage 12; F-J portions show the pollen transmission electron microscope observation results of tms18 mutant anther from stage 7 to stage 10 and stage 12 under high temperature; K-O portions show the result of TEM observation of pollen in the 7.sup.th to 10.sup.th and 12.sup.th anthers of tms18 mutant under low temperature; the small figure on the right of each figure is the enlarged result of the figure. HT stands for high temperature treatment (>28° C.); LT stands for low temperature treatment (<23° C.). AP represents abnormal pollen; Ba represents rod structure; E represents anther epidermis; EN represents anther endothelium; Ex stands for pollen wall; Msp stands for microspore; Ne represents the inner layer of the outer wall; PE represents the primary outer wall; Se represents the outer layer of the outer wall; T stands for felt layer; TDS stands for tetrad.

[0025] FIG. 7 shows the thickness of pollen outer wall II of wild type and tms18 mutant at different temperatures. In the figure, the thickness of pollen outer wall II of wild type and tms18 mutant at different temperatures at stages 9, 10 and 12, respectively. The data in the figure are the mean±standard deviation, and each group of data is 4 biological replicates. P<0.05;

[0026] FIG. 8 shows the clone locating and sequence analysis results of tms18 gene, wherein A portion shows the initial locating result of Indel molecular marker on rice chromosome 12. B portion represents the verification of genetic complementarity of tms18 mutant. C portion shows the sequencing and location analysis of tms18 gene SNP.

[0027] FIG. 9 shows the analysis results of homologous protein evolutionary tree of tms18 and its conserved sites, wherein A portion shows the result of evolutionary tree analysis of tms18 and its lineal homologous genes among different species by adjacency method; In the process of model calculation, the self-development value of evolutionary tree is 1000; B portion shows the result of conservative site analysis of tms18 homologous protein.

[0028] FIG. 10 shows the semi-quantitative PCR analysis results of tms18 gene in different tissues of rice, showing the semi quantitative analysis of tms18 gene and internal reference gene OsACTIN in different tissues. Le represents lemma; PA represents palea; L2.5 represents glume length is 2.5 mm; L3.0 represents glume length is 3.0 mm; L4.0 represents glume length is 4.0 mm; and L5.0 represents glume length is 5.0 mm.

[0029] FIG. 11 shows the localization analysis results of tms18-gfp fluorescent protein in rice anthers.

[0030] FIG. 12 shows the results of comparative analysis of different site mutant protein of amino acid sequence of tms18 and wild type, in which “1” is the protein amino acid mutation position of thermo-sensitive genic male sterile line in this experiment; “2” is the position of complete male sterility after protein amino acid mutation as described in Chang et al, 2016.

[0031] FIG. 13 shows the results of gene complementarity experiment.

EMBODIMENTS

[0032] The invention is described in detail below in combination with the accompanying drawings and their embodiments, but the protection scope of the invention is not limited to the scope described in the embodiments.

[0033] The inventors took the Oryza sativa ssp. japonica, ZH11 as the material, screened with ethyl methanesulfonate (EMS) to obtain the plant material of thermo-sensitive genic male sterile line TMS18, and obtained the thermo-sensitive male sterile mutant tms18 in the experiment. The gene sequence of the mutant is shown in SEQ ID No.1 (including exons and introns), and the edited amino acid sequence is shown in FIG. 12. The specific experimental process is described in detail below.

[0034] Plant Materials and their Planting Process

[0035] In this embodiment, the rice material used is the japonica rice variety ZH11. Referring to Zhang et al. 2007 (Zhang Z B, Zhu J, Gao J F, et al. Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis. Plant Journal, 2007, 52: 528-538), EMS mutants are induced and screened. During the screening, the mutant plants containing the thermo-sensitive male sterile mutant tms18 of SEQ ID No.1 was accidentally obtained.

[0036] The inventors, using the recessive homozygous male sterile plant containing the mutant tms18 as the female parent (ovule donor) and using the target rice variety as the male parent (pollen donor), hybridized these two kinds of rice at high temperature, and obtained F1 seeds. Then, the inventors cultivated the F1 seeds to obtain the F1 plants. The F1 plants were self pollinated to obtain the F2 seeds. In the F2 plants, those plants with tms18 recessive homozygous sites were screened.

[0037] Identification of temperature sensitive characteristics of tms18 In this example, from May 18, 2018, 24 copies of three kinds of rice are cultivated per week, that is, rice of ZH11 wild type, rice of Annong S-1 male sterile line and rice containing mutant tms18, and obtaining corresponding seedlings. After the seedlings have already been cultivated and reach a certain height, the seedlings were inserted into Fengxian field in Shanghai (a place of China) for planting. From July 26 to August 1, the average temperature in Shanghai reached the highest point, about 30.5° C., and the first batch (germinated on May 18) reached booting stage (pollen development stage). Since the tms18 plants were treated by high temperature, after the tms18 plant materials flowering and selfing, they showed almost complete sterility because there were no pollen (as shown in FIG. 2). In the eighth batch of materials (plants) (Germinating on July 6), since the booting time was about September 10, the average temperature in Shanghai fell to 26.5 degrees, the pollen development partially returned to normal due to low temperature, and the fertility of self bred materials of the plants were partially restored. It is verified that the high temperature sterility of tms18 plant is better than that of Annong S-1 (the name of another kind of rice).

[0038] Cytological Analysis

[0039] The anther slices at different development (growth) stages of rice were photographed with Olympus dx51 digital camera. The fresh stamens and pollen grains were wrapped with 8 nm gold particles for scanning by electron microscopy, and observed by JSM-840 microscope. For the transmission electron microscope experiment, the rice spikelets were fixed on ice in the fixed solution (0.1M phosphate buffer containing 2.5% glutaraldehyde, pH 7.2). Spikelet materials are further embedded in resin (‘hard plus’ embedding resin, Unite Kingdom). Ultrathin sections (50-70 nm) were observed by jem-1230 transmission electron microscope (Jeol, Japan).

[0040] RNA Extraction and Quantitative RT-PCR

[0041] RNA was extracted from spikelet tissue of mature soil cultured rice plants by Trizol reagent (Invitrogen, USA). Using poly DT (12-18) primers; using MMLV reverse transcriptase and corresponding reagents reverse transcribe RNA and obtain the first cDNA strand (60 minutes, 42° C.). The synthesized cDNA strand was used as a template for PCR. SYBR Green I master mix (Toyobo, Japan) was used for quantitative RT-PCR detection through ABI prism 7300 system (Applied Biosystems, USA). The program parameters of quantitative RT-PCR were: 95° C. for 5 minutes, 94° C. for 10 seconds, denaturation for 40 cycles, annealing at 60° C. for 1 minute. β-Actin was used as control.

[0042] It should be noted that the plant materials in the invention are cultured in fertile soil. The high temperature and low temperature treatment are performed in glass greenhouse and artificial intelligence lighting room. The high-temperature treatment temperature is generally in summer or in the environment with an average temperature higher than 28° C. the low-temperature treatment is generally in autumn or under the condition of an average temperature of 22-24.5° C.

[0043] As shown in the figures, as compared with the wild type, the nutrition and development of tms18 mutant was normal without significant difference (FIG. 2A-C). Tms18 plants showed male sterility under high temperature (>28° C.) (FIG. 1), and the fertility of tms18 plants could be restored under low temperature (<23° C.) (FIG. 1). After flowering, the anthers of tms18 mutant plants did not disperse pollen at high temperature, but the pollen in the anthers of tms18 mutant plants was normal at low temperature (FIG. 2D). Through the observation of spikelets of wild-type and tms18 mutant plants, it was found that there were no significant difference in lemma and palea, but the anthers of wild-type plant were plump and yellow, while the anthers of tms18 mutant plants obviously shrunk and became smaller at high temperature and returned to plump state at low temperature (FIG. 2E-F). Alexander staining results of anthers showed that wild-type pollen accumulated rich contents and showed purplish red, while no pollen grains stained purplish red could be seen in the tms18 mutant at high temperature, but returned to normal at low temperature, indicating that the anther development of tms18 mutant was affected by temperature, and low temperature could make up for the development defect of male gametophyte in tms18 mutant.

[0044] In order to verify the response to different temperatures of tms18, the inventors planted the tms18 mutant at different periods of time. The planting time is from July 1 to September 15. Germination planting is carried out every two weeks to observe the fertility recovery. As shown in FIG. 3, all tms18 materials transplanted before the middle of July showed complete sterility phenotype, and some fertility of the materials transplanted at the beginning of August recovered, with a recovery rate of about 10%. With the postponement of transplanting time, the fertility of tms18 material under natural low temperature can be basically similar to that of wild-type material, the spikelet drooping is obvious, and the seed setting rate is about 90%. The results showed that tms18 had better fertility recovery under appropriate temperature.

[0045] Mechanism Analysis:

[0046] The inventors found that low temperature can make up the development defects after release from tetrads of the tms18 mutant (similar expressions in this application refer to the rice plant/material containing the tms18 gene. The tms18 mutant plant cultivated by the inventor is obtained through experiments. Those skilled in the art can introduce the tms18 gene mutant into the rice plant by using existing Agrobacterium introducing methods according to the gene sequence provided by the invention).

[0047] In order to analyze the changes and differences of anther and pollen development of plants containing tms18 at different temperatures, the young spikelets and florets of wild-type ZH11 and tms18 mutants with anther length of 1-5.6 mm were observed by semi-thin sections. The results of semi-thin sections of wild-type and tms18 mutant plants under high temperature showed that from stage 6 to 8, the development of anthers could normally undergo meiosis and form tetrads, and there was no significant difference between the two kinds of plants (FIGS. 4a-c and G-I). The wild-type tapetum cells formed in the early stage and began normal PCD in the 9th stage. The microspore further developed and expanded. The sporopollen deposition on the pollen wall was relatively dense, and the pollen development was normal (FIG. 4D). In the 10th stage, microspores of wild-type development normally and concentrated to form crescent structure, tapetum cells continued to degrade, then developed into mature pollen, and finally formed plump and normal pollen grains (FIG. 4E-F). Under high temperature, the tapetum of tms18 mutant basically did not change significantly in the early stage, but the development of pollen was abnormal in the later mature stage, and finally showed rupture and could not form normal pollen grains (FIG. 4, K-L). Under low temperature, the development of pollen and tapetum cells of tms18 mutant were normal, and normal pollen could be formed (FIG. 4M-R).

[0048] In order to better know the development of anther and pollen of tms18 mutant, the inventor observed the outer surface and inner surface of anther and the surface structure of pollen by scanning electron microscope. Under the scanning electron microscope, the wild-type anthers development normally and showed plump type, while the anthers of tms18 mutant were relatively small and shriveled compared with the wild-type at high temperature, and returned to normal at low temperature (FIGS. 5a, F and K). At the later stage of anther development, reticular waxy and horny structures appeared on the normal development surface of the wild type. There was no significant difference in the anther surface of the tms18 mutant at low and high temperature (FIGS. 5b, G and L). Through the observation of the structure of anther inner wall, it found that there were more dense globular structures in the anther inner wall of wild-type and tms18 mutant no matter at high or low temperature (FIG. 5C, h and m), but the pollen in the anther of tms18 mutant showed a shrinkage phenotype at high temperature. In addition, compared with the dense structure of wild-type pollen outer wall, the structure of tms18 mutant pollen outer wall has defects indicating cracking and obvious shrinkage (FIGS. 5, D-E, I-J, and N-O). This indicates that tms18 gene mutation does not affect the outer epidermis and inner surface of anthers, but will lead to the defective phenotype of pollen outer wall.

[0049] In order to further make sure the condition of pollen outer wall of tms18 mutant at high and low temperature, the inventors observed the microspores of anther stages 7-10 and 12 by transmission electron microscope. The results showed that at the 7th stage of Anther Development (tetrad stage), the microspores of tms18 mutant and wild-type were surrounded by callose at different temperatures, and there were no significant difference there between (FIGS. 6a, F and K). Then microspores were released from callose and entered the 8th stage. According to the observation of pollen outer wall structure in this stage, it was found that sporopollen deposition gradually appeared on the pollen outer wall of wild-type pollen which was relatively dense, while the pollen outer wall sporopollen deposition of tms18 mutant was less at high temperature, while the pollen outer wall sporopollen deposition recovered at low temperature (FIGS. 6B, G and L). In phase 9, the wild-type microspore expanded and enlarged, and the structure of the pollen outer wall basically formed, showing a shape similar to character “”. The microspore pollen outer wall of tms18 mutant can also show an “” shape structure under high temperature treatment, but its internal second layer structure became significantly thinner, and there were some obvious fractures inside (FIGS. 6C, H and M). In the 10th stage, the microspore entered the crescent structure. At high temperature, the defective structure of the second layer of the pollen outer wall can still be observed, while at low temperature, its thickness still did not recover, but its crack disappeared (see FIGS. 6, D, I and N). In the late stage of anther development (growth), the wild type can normally form fertile pollen, while the tms18 mutant has defects in the structure of pollen outer wall at high temperature, and finally cannot form pollen normally, but can form mature pollen grains at low temperature. It shows that the temperature sensitivity of tms18 mutant is related to the structural defect of the second layer of pollen outer wall (FIG. 6, e, J and o). The second layer structure of pollen outer wall in the 9th, 10th and 12th stages of pollen development was statistically analyzed by Image J software. In the 9th-10th stage of pollen development, microspores entered the rapid expansion stage and concentration stage. Under high temperature treatment, the thickness of the second layer of pollen outer wall of tms18 mutant was significantly thinner, which was significantly reduced by 60.7% and 61.2% compared with the wild type, respectively. At the 10th stage of pollen development, the microspore entered the crescent structure. The tms18 mutant showed significant difference under high temperature and low temperature, microspore high temperature was 32.7% lower than that under low temperature. When the pollen development is in the mature stage, the pollen under high temperature treatment is still broken, while the normal pollen can be formed under low temperature treatment, but there is no significant difference in the thickness between the two. The above results show that temperature has a great influence on the pollen development of tms18 mutant, mainly in the 9th stage (microspore expansion stage) and the 10th stage (crescent stage), and after the meiosis stage of microspore mother cell. The genes with this characteristic have not been reported yet.

[0050] Tms18 gene encodes glucose methanol choline (GMC) family oxidoreductases

[0051] Based on the whole genome SNP sequencing, the inventors found that there is a base mutation from G to A on 183 bp of the second exon of tms18 gene, which leads to the mutation of glycine encoded by wild-type Zhonghua 11 into serine (FIG. 8C).

[0052] The inventor further carried out verification analysis by using genetic complementarity experiment.

[0053] The target gene fragments, including upstream promoter region and downstream region, were cloned and transformed into rice seeds including tms18, which are Hybrid, to carry out the cultivation of transgenic plants.

[0054] Specifically, using wild-type plants as templates, the cDNA of light and temperature sensitive gene was amplified with specific primers, then constructed on Blunt through intermediate vector. The constructed results was amplified in large quantities, and then constructed on 1300-eGFP through BamHI and SalI digestion sites. The plasmid containing vector and eGFP was transformed into Agrobacterium EHA105, and then infected the heterozygous seeds of the corresponding mutants to obtain complementary transgenic plants. The complementary plants showed a normal fertile phenotype at high temperature, and the results are shown in FIG. 13.

[0055] The applicant identified the 13 transgenic lines (wherein 6 of them include tms18/tms18, i.e., the double strands of chromosomes both contain tms18). In high temperature environment (greater than 29° C.), these plants restored fertility and formed normal seeds compared with the non-transgenic tms18/tms18 plant. The Figure in scanning electron microscope also showed that the pollen of transgenic plants returned to normal and there was no significant difference between wild-type and transgenic plants (FIG. 8B).

[0056] TMS18 gene encodes a protein with 587 amino acids and about 65 kDa. Structural analysis shows that TMS18 protein belongs to a member of glucose methanol choline (GMC) oxidoreductase family, which family widely exists in animals, plants and microorganisms, including glucose oxidase and pyranose oxidase (Wongnate and Chaiyen, 2013).

[0057] In the tms18 mutant plant, the microspore collapsed after the tetrad stage, indicating that the gene is closely related to the formation of pollen outer wall.

[0058] Molecular Mechanism of Tms18 Thermo Sensitive Genic Male Sterile Line

[0059] Through comparative observation of the plant containing wild-type gene and mutant gene, it is found that the temperature sensitive gene mutant of the invention is specifically expressed in anthers. The single base mutation of the gene caused the thinning of the second layer of pollen outer wall at high temperature, resulting in the thermo sensitive sterility phenotype.

[0060] Potential Application Value of Tms18 Thermo Sensitive Genic Male Sterile Line

[0061] At present, the sensitive period of fertility of most existing photosensitive male sterile lines, such as Photosensitive Genic Male Sterile Lines Nk58s and Ans-1, is before the meiotic tetrad period. However, there is no corresponding report on the materials for which sensitive period of fertility is in the later stage of meiosis, so the gene of the present invention has a good application prospect.

[0062] In the invention, the gene expression period of tms18 thermo sensitive genic male sterile line is mainly in the middle and late stage of anther, and the outer wall of pollen becomes thinner in the 9.sup.th-11.sup.th stage of pollen development. This means that the temperature sensitive genic male sterile line may be affected by temperature, and the sensitive period may be after the meiosis period of pollen mother cell.

[0063] Since tms18 thermo sensitive genic male sterile lines are mainly affected by temperature in the later stage. Therefore, it can be used to solve the problem of easy recovery of low temperature tide in summer encountered in seed production. It can be used to hybridize with temperature sensitive genic male sterile lines and construct double mutants to prolong the period affected by temperature sensitivity, so as to try to solve some safety problems in seed production.

[0064] The amino acid sequence of the tms18 mutant of the invention is changed mainly from glycine to serine at the N-end of the protein, and other positions are not changed.

[0065] In conclusion, the inventor of the present application cloned and obtained the gene tms18 of glucose methanol choline (GMC) oxidoreductase family in rice, and found that the gene mutant showed the phenotype of photo thermo sensitive male sterility, which was not at the same site as the photo thermo sensitive genic male sterile line currently used in production. Cytological analysis showed that the integrity of the second layer of pollen outer wall of tms18 mutant had obvious thinning and fracture defects, which led to the rupture and abortion of pollen in the rapid expansion stage. However, the rupture of microspore outer wall recovered to a certain extent under low temperature treatment. Further analysis showed that tms18 protein was secreted by tapetum cells and specifically located on tapetum, anther and pollen surface. The inventor's research data show that tms18, as a new temperature sensitive genic male sterile line, its fertility sensitive period is mainly after meiosis of pollen mother cell, which is different from the light and temperature sensitive materials reported at present, and has significant application value.