Rice thermo-sensitive dual-purpose sterile line fertility transformation starting point temperature gene Os Vms1 and application thereof

Abstract

The invention relates to a rice thermo-sensitive dual-purpose genic male sterile line fertility transformation starting point temperature regulation gene Os Vms1 containing a tms5 site and application of the gene. Polynucleotide forming the fertility transformation starting point temperature gene Os Vms1 of the thermo-sensitive dual-purpose genic male sterile line has a base sequence as shown in SEQ ID No: 1 in a sequence table, the gene encodes OsVms1 protein, and the protein has an amino acid sequence as shown in SEQ ID No: 2 in the sequence table. According to the present invention, the gene is knocked out on the temperature-sensitive sterile line strain 1S containing the tms5 site by using the CRISPR-Cas9 technology so as to obtain the mutant tms5 osvms1 with the increased fertility transformation starting point temperature, and the fertility transformation starting point temperature of the mutant is accurately identified by using the artificial climate box; in addition, a genetic complementation experiment proves that Os Vms1 participates in regulation and control of the fertility transformation starting point temperature.

Description

Technical Field

[0001] The present invention belongs to the field of rice molecular breeding, and specifically relates to a temperature-regulating gene OsVms1, which is a starting point for fertility conversion in a thermosensitive dual-purpose sterile rice line, and application of the gene in breeding a thermosensitive dual-purpose sterile rice line. Background Art

[0002] Hybrid rice technology is a major technology that my country has independently innovated and is ahead of the world. Its successful application in rice production has made a huge contribution to ensuring my country's food security (Non-Patent Literature 2). Hybrid rice technology utilizes the hybrid vigor between varieties with different genetic compositions. Compared with conventional rice, hybrid rice has great advantages in growth potential, tillering ability, resource utilization rate and stress resistance during the vegetative growth period (Non-Patent Literature 3). The male sterility system is the key to the successful application of hybrid rice technology. According to the genetic differences of sterility genes, rice male sterility can be divided into cytoplasmic male sterility (CMS) controlled by the interaction of cytoplasmic genes and genomic male sterility (GMS) controlled by nuclear genes.

[0003] like Figure 1 As shown in the figure, using sterile lines with different genetic compositions, breeders have established three-line and two-line breeding systems (non-patent document 4). Three-line hybrid rice consists of a cytoplasmic male sterile line, a maintainer line and a restorer line. In breeding applications, the reproduction of the cytoplasmic male sterile line is completed by hybridizing the maintainer line with the cytoplasmic male sterile line; and hybrid F1 seeds for production are obtained by hybridizing the restorer line with the cytoplasmic male sterile line. Two-line hybrid rice consists of a photothermal-sensitive male dual-purpose nuclear sterile line and a restorer line. In breeding applications, the photothermal-sensitive male dual-purpose nuclear sterile line can restore fertility under certain environmental conditions (low temperature, short daylight) and self-pollinate to complete the reproduction of the sterile line, and can also be hybridized with the restorer line under specific environmental conditions (high temperature, short daylight) to obtain F1 seeds for production. The photothermal-sensitive male dual-purpose nuclear sterile line achieves dual-purpose in one line, so it is also called a dual-purpose sterile line. Compared to the three-line method, the two-line method allows for the "one line, two purposes" of the sterile line, simplifying the sterile line breeding process and saving considerable manpower and material resources. Furthermore, because the fertility of the two-line sterile line is controlled by recessive nuclear genes and is not restricted by the parental restorer relationship, the restorer line selection range is wide and the pairing is free, making it more conducive to the selection of strong hybrid combinations. Over the past 20 years, two-line hybrid rice based on the dual-purpose photothermosensitive male sterile nuclear line has occupied an increasingly important position in rice production (Non-Patent Document 5).

[0004] Strain 1S is a typical indica thermosensitive dual-purpose nuclear sterile line and the most widely used two-line sterile line in my country's hybrid early rice. Strain 1S passed the review in 1998, and the critical temperature for fertility conversion is between 22-23°C. It is one of the dual-purpose sterile lines with the lowest starting temperature for conversion in actual breeding (non-patent document 6, non-patent document 7). Strain 1S has stable fertility and can tolerate low temperatures of around 23°C for 6 consecutive days, which greatly reduces the risk of seed production; under conditions above 23°C, Strain 1S is completely sterile, has no pollen at high temperatures, has good broad affinity, and has strong pairing advantages (non-patent document 8). In 2014, the inventor's research group used the map-based cloning method to clone the thermosensitive sterile gene tms5 that controls Strain 1S. This gene encodes a short version of RNase Z S1 . In strain 1S, the tms5 site mutated at bases 70 and 71, causing premature termination of the protein and leading to high-temperature sterility of strain 1S. TMS5 gene sequencing of 27 two-line sterile lines widely used in production found 26 with tms5 mutation sites (non-patent document 9). Statistics on the promotion of major crop varieties across the country show that the planting area of two-line hybrid rice is about 68.85 million mu, and the planting area of two-line hybrid combinations containing tms5 mutation sites is about 65 million mu, accounting for 14.4% of the total rice planting area, indicating that tms5 has become a key gene for the utilization of two-line hybrid rice.

[0005] The fertility conversion of two-line sterile lines, widely used in production, is primarily controlled by temperature. That is, they are sterile under certain high temperatures but can regain fertility under certain low temperatures. The starting temperature for fertility conversion in two-line sterile lines is a key indicator for the selection of thermo-sensitive dual-purpose genic male sterile lines and a key factor in the competitiveness of hybrid rice varieties. A low starting temperature for fertility conversion in thermo-sensitive dual-purpose genic male sterile lines results in low reproductive yields, increasing seed production costs. Conversely, a high starting temperature makes seed production susceptible to weather influences, leading to fertility fluctuations in the sterile lines. This can cause self-fertilization in the sterile lines, reduce the purity of the F1 seed, and lead to seed production failure. In recent years, frequent extreme cold weather has caused significant losses to hybrid rice production in my country. For example, the rice-growing areas of the middle and lower reaches of the Yangtze River experienced abnormally low temperatures for more than three consecutive days. During this period, the sterile lines were in their sensitive period. The low temperatures caused some thermo-sensitive male sterile lines with higher starting temperatures for fertility conversion to self-fertilize, resulting in impure hybrid seeds and reduced yields, significantly impacting agricultural production.

[0006] Although the thermosensitive dual-purpose male sterile lines widely used in breeding all contain the tms5 mutation site, their fertility conversion starting temperatures vary. The fertility conversion starting temperature of Zhui 1S is 23°C (Non-Patent Document 7), meaning that above 23°C, Zhui 1S is completely sterile; the fertility conversion starting temperature of Guangzhan 63S is around 24°C (Non-Patent Document 10); and the fertility conversion starting temperature of Annong S-1 is 26-27°C (Non-Patent Documents 11 and 12). Zhuang Chuxiong's team at South China Agricultural University used CRISPR-Cas9 technology to edit the TMS5 gene in 11 materials (7 maintainer lines, 3 conventional indica rice, and 1 japonica rice). After two generations, they obtained thermosensitive male sterile lines without transgenic components. When these thermosensitive male sterile lines were tested for fertility conversion starting temperatures, it was found that plants generated by gene editing with the same set of guide RNAs (gRNAs) under different genetic backgrounds had different fertility conversion starting temperatures. Among them, the starting temperature for fertility conversion in gene-edited offspring of Zhongzhe B and Yuejing Si Miao was below 24°C, the starting temperature for fertility conversion in gene-edited offspring of Le B, Tianfeng B, and Wushan Si Miao was approximately 24°C, the starting temperature for fertility conversion in gene-edited offspring of japonica rice GAZ was 26°C, and the starting temperature for fertility conversion in gene-edited offspring of Zhenshan 97B was above 26°C (Non-Patent Document 13). These experimental results all indicate that TMS5 is not involved in regulating the starting temperature for fertility conversion, suggesting that other genes may be involved in regulating the starting temperature for fertility conversion, but research in this area is currently lacking.

[0007] References

[0008] Non-patent literature 1: Khush, GS (2005). What it will take to feed 5.0 billion rice consumers in 2030. Plant Mol. Biol. 59, 1-6.

[0009] Non-patent literature 2: Li Lihong, Ni Jianping, Chen Qian, Li Ximing (2013). Development and prospects of hybrid rice seed industry in China. Seed 32, 56-60.

[0010] Non-patent literature 3: Sun, L., Hussain, S., Liu, H., Peng, S., Husng, J., Cui, K., and Nie, L. (2015). Implications of low sowing rate for hybrid rice varieties under drydirect-seeded rice system in Central China. Field Crops Res. 175, 87-95.

[0011] Non-patent literature 4: Fan, Y., and Zhang, Q. (2017). Genetic and molecular characterization of photoperiod and thermo-sensitive male sterility inrice. Plant Reprod. 31, 3-14.

[0012] Non-patent literature 5: Fan Yourong, Cao Xiaofeng, Zhang Qifa (2016). Research progress on photothermosensitive male sterile rice. Science Bulletin 61, 3822-3832.

[0013] Non-patent literature 6: Liu Aimin, Ling Wenbin, Shi Tianbao (2003). Observation of the main characteristics of thermo-sensitive genic male sterile lines 1S and Lu 18S. Hybrid Rice 2.

[0014] Non-patent literature 7: Yang Yuanzhu, Tang Pinglai, Yang Wencai, Liu Aimin, Chen Yunquan, Ling Wenbin, Shi Tianbao (2000). Breeding and application of rice wide-compatibility thermosensitive sterile line 1S. Hybrid Rice 2.

[0015] Non-patent literature 8: Li Bihu, Wu Houxiong, Xu Mengliang, Liang Manzhong, Zhang Zhenhua, Chen Liangbi (2003). Comparative study on the sensitivity of thermo-sensitive genic male sterile rice to low temperature duration. Acta Agronomica Sinica 29, 930-936.

[0016] Non-patent literature 9: Zhou, H., Zhou, M., Yang, Y., Li, J., Zhu, L., Jiang, D., Dong, J., Liu, Q., Gu, L., Zhou, L., et al. (2014). RNase Z S1 processes Ub L40 mRNAs and controlsthermosensitive genic male sterility in rice.Nat.Commun.4884.

[0017] Non-patent literature 10: Yang Zhenyu, Zhang Guoliang, Zhang Conghe, Chen Jinjie, Wang Heqin, Zhang Jinjia, Yan Zhi (2002). Breeding of high-quality photothermosensitive genic male sterile line Guangzhan 63S in medium-grain rice. Hybrid Rice 17, 4-6.

[0018] Non-patent literature 11: Zhou Guangzhi, Li Xunzhen, Tan Zhouyan, Chen Liangbi (1990). Study on the expression conditions and fertility conversion rules of the sterile gene of the indica sterile rice line Annong S-1. Journal of Natural Sciences of Hunan Normal University 13, 365-372.

[0019] Non-patent literature 12: Deng Huafeng, Shu Fubei, Yuan Dingyang (1999). Overview of research and utilization of Annong S-1. Hybrid Rice 14, 1-3.

[0020] Non-patent literature 13: Zhou, H., He, M., Li, J., Chen, L., Huang, Z., Zheng, S., Zhu, L., Ni, E., Jiang, D., Zhao, B., et al. (2016). Development of commercial thermo-sensitivegenic male sterile rice accelerates hybrid rice breeding using the CRISPR / Cas9-mediated TMS5 Editing System.Sci.Rep.6,37395. Summary of the Invention

[0021] As mentioned above, the fertility conversion starting temperature is the core indicator of the temperature-sensitive dual-purpose sterile line and the core competitiveness of the rice variety. However, the research field of the temperature-sensitive dual-purpose nuclear male sterile line fertility conversion starting temperature regulating gene is still blank, so the gene that controls the fertility conversion starting temperature of the rice temperature-sensitive dual-purpose sterile line can provide a technical reserve for creating a dual-purpose sterile line with a low fertility conversion starting temperature, reduce the risk of hybrid rice seed production under the large environment of frequent extreme climatic conditions, and ensure national food production safety. To this end, the object of the present invention is to separate and clone the gene that controls the fertility conversion starting temperature of the temperature-sensitive dual-purpose sterile line from rice, and apply the gene to the rice temperature-sensitive dual-purpose sterile line with a low fertility conversion starting temperature, particularly to the rice temperature-sensitive dual-purpose sterile line containing the tms5 locus that is used to cultivate a low fertility conversion starting temperature.

[0022] Through extensive research, the inventors of this invention, using strain 1S (tms5), performed a gene knockout in the strain using CRISPR-Cas9 technology, resulting in a mutant, tms5 osvms1, that exhibits an elevated threshold temperature for fertility transition. They isolated and cloned a gene that controls the threshold temperature for fertility transition in thermosensitive dual-purpose genic male sterile lines, naming it Os Vms1. Further research confirmed that Os Vms1 is involved in regulating the threshold temperature for fertility transition in thermosensitive dual-purpose genic male sterile lines.

[0023] Specifically, the present invention relates to the following inventions.

[0024] The present invention relates to a DNA molecule, the base sequence of which is shown in SEQ ID No: 1. The DNA molecule is a temperature-regulating gene Os Vms1, which is the starting point for fertility conversion in a thermo-sensitive dual-purpose sterile rice line. The DNA molecule encodes the temperature-regulating protein Os Vms1, which is the starting point for fertility conversion in a thermo-sensitive dual-purpose sterile rice line. The Os Vms1 protein has the amino acid sequence shown in SEQ ID No: 2 in the sequence listing.

[0025] The present invention also relates to a recombinant vector comprising the above DNA molecule and a transgenic cell line or host bacteria comprising the recombinant vector, such as Agrobacterium.

[0026] The present invention also relates to a protein, the amino acid sequence of which is shown in SEQ ID No: 2. The protein is the temperature-regulated protein Os Vms1, which is the starting point for fertility conversion in thermo-sensitive dual-purpose sterile lines of rice. The Os Vms1 protein is encoded by the base sequence shown in SEQ ID No: 1.

[0027] The present invention also relates to a method for regulating the starting temperature of rice thermosensitive dual-purpose genic male sterile line transformation, which comprises the step of introducing the above-mentioned Os Vms1 gene or a recombinant vector containing the gene into a rice thermosensitive dual-purpose genic male sterile line containing the tms5 site.

[0028] The present invention also relates to a method for cultivating a thermosensitive dual-purpose genic male sterile rice line with a low fertility conversion threshold temperature, which comprises the step of introducing the above-mentioned Os Vms1 gene or a recombinant vector containing the gene into a thermosensitive dual-purpose genic male sterile rice line containing a tms5 locus.

[0029] Among them, in the process of cultivating a thermo-sensitive dual-purpose sterile rice line with a low fertility conversion starting temperature, it is necessary to ensure that the amino acid sequence shown in SEQ ID No: 2 of the temperature-regulating gene Os Vms1 for regulating the fertility conversion starting temperature of the thermo-sensitive dual-purpose sterile rice line in the present invention is complete, and one or more amino acid residues must not be substituted, deleted or added.

[0030] The present invention also relates to the use of the aforementioned DNA molecule, recombinant vector, Agrobacterium, or protein in regulating the threshold temperature for fertility conversion in a thermo-sensitive dual-purpose genic male sterile rice line. The thermo-sensitive dual-purpose genic male sterile rice line is preferably a thermo-sensitive dual-purpose genic male sterile rice line containing the tms5 locus.

[0031] Effects of the Invention

[0032] The present invention clones a gene in rice that controls the starting temperature for fertility conversion of a thermosensitive dual-purpose nuclear male sterile line, providing a new gene resource for breeding dual-purpose male sterile lines with a low starting temperature for fertility conversion, which is of great significance to the field of rice molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of two-line and three-line breeding systems for hybrid rice production.

[0034] Figure 2 This is the technical flow chart of the present invention and the technical route for knocking out the Os Vms1 gene.

[0035] Figure 3 Pollen fertility of WT, zhu1S(tms5) and tms5 osvms1 in Beijing field was shown.

[0036] Figure 4 Represents the Os Vms1 gene structure and Os Vms1 protein structure.

[0037] Figure 5 This is a map of the functional vector pCAMBIA1300 used for Os Vms1 transgenic complementation verification in the invention.

[0038] Figure 6 The phenotypes of WT, zhu1S(tms5), tms5osvms1 and complemented transgenic plants grown in Beijing field are shown.

[0039] Figure 7 The pollen fertility of WT, tms5 and tms5 osvms1 was shown under different temperature treatments (22℃, 25℃ and 30℃) in an artificial climate chamber. DETAILED DESCRIPTION

[0040] The present invention isolated and cloned a complete coding region DNA fragment of a gene that controls the starting temperature for fertility conversion in a thermosensitive dual-purpose genic male sterile line from rice. The inventors named this gene Os Vms1. The nucleotide sequence of the thermosensitive dual-purpose genic male sterile line fertility conversion starting temperature gene Os Vms1 has the base sequence shown in SEQ ID No. 1 in the sequence listing (including its promoter sequence, 5' UTR, CDS containing introns, and 3' UTR). The gene encodes the Os Vms1 protein, which has the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0041] Figure 2This is a technical flow chart of the present invention, which shows the technical route for knocking out the Os Vms1 gene. Using strain 1S (tms5) as the research object, the gene of strain 1S (tms5) was knocked out using CRISPR-Cas9 technology to obtain the mutant tms5 osvms1. Phenotypic observations were conducted on the T1 generation in the fields of Beijing. After the rice ears and flowers emerged, 1% I2-KI staining was performed, and the pollen fertility was observed under an optical microscope to determine the phenotype. It was found that the pollen of the mutant tms5 osvms1 was regularly round and stained black with iodine, indicating that the pollen was fertile. Through the artificial climate chamber system with three groups of temperature treatments (22°C, 25°C and 30°C), it was found that the starting temperature for the mutant's fertility conversion was between 25°C and 30°C (the starting temperature for the fertility conversion of strain 1S was 22°C to 25°C).

[0042] To verify the function of the Os Vms1 gene, the pOs Vms1::Os Vms1 genomic-FLAG-3′UTR vector was constructed and transformed into the tms5 osvms1 mutant via Agrobacterium-mediated transfection. Microscopic examination of pollen from strain 1S(tms5), tms5 osvms1, and the complemented transgenic line revealed that strain 1S(tms5) exhibited typical sterility, while tms5 osvms1 was fertile. The phenotype of the complemented transgenic line was consistent with that of strain 1S(tms5). Genetic complementation experiments demonstrated that Os Vms1 could restore the high-temperature sterility phenotype of strain 1S(tms5) under field conditions.

[0043] Example

[0044] Below, the present invention is further described in detail with reference to Examples. It should be understood that the specific examples described below are only used to explain the present invention and are not intended to limit the present invention. In addition, the experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods known in the art.

[0045] Example 1. Obtaining mutants

[0046] The gene was edited using CRISPR-Cas9 gene editing technology, and a specific target site was designed on the first exon of the gene ( Figure 4 ), using the website ( http: / / skl.scau.edu.cn / home / ) designed target primers (Table 1). A gene editing vector was constructed and transferred into the EHA105 strain by electroporation, and the callus induced by the seeds of the infected strain 1S (tms5) was infected. After obtaining the T0 generation transgenic positive seedlings, primers were designed before and after the target site, and the target fragments were amplified and sequenced to identify whether the target site of the positive plant was edited. Two different types of homozygous gene-edited positive seedlings were obtained: -4bp and +1bp. The transgenic T0 generation plants were planted in Beijing, and the T1 generation seeds were harvested and then harvested for southern breeding. In early May 2021, the two T2 generation editing type mutants 1S (tms5) and WT were planted in the field seedling shed in Changping, Beijing, and transplanted to the paddy field after 25 days. In early August, pollen from unshed florets was stained with 1% I2-KI. Light microscopy revealed that WT pollen was regularly round and stained black with iodine, indicating fertility. Pollen from strain 1S (tms5) was triangular and failed to stain with iodine, indicating abortion. Pollen from both mutants, tms5 and osvms1, was regularly round and stained black with iodine, indicating fertility. Therefore, gene editing demonstrated that Os Vms1 could restore the high-temperature sterility phenotype of strain 1S (tms5) under field conditions.

[0047] Table 1 Os Vms1 gene editing target primers in the present invention

[0048]

[0049] Example 2. Identification of the starting temperature for mutant sports development

[0050] We sowed wild-type rice (WT) (a near-isogenic line of TMS5 on the 1S background), the thermosensitive sterile line 1S (tms5), and the mutant tms5 osvms1 in the nursery fields of the China National Rice Research Institute in Hangzhou. Transplantation was performed 25 days after sowing. Thirty days after transplanting, the rice seedlings were transplanted into plastic pots (minimizing root damage), two seedlings per pot. After transplanting, the seedlings were placed under a shade net for three days to allow root damage to recover. After the rice seedlings spitted out water, the shade net was removed, allowing the seedlings to continue growing and monitoring the panicle development progress. After jointing, a climatic chamber (Koithtron S-153W, Japan) was set and the treatment parameters were set. The chamber temperature was monitored using a temperature recorder, and the chamber settings were fine-tuned based on the temperature data. After the rice seedlings reached stage 3 of panicle differentiation, the seedlings were transferred to climatic chambers maintained at uniform temperatures of 22°C, 25°C, and 30°C for 14 days. After treatment, the material was removed from the climate chamber and continued to grow. After heading, florets were removed and stained with 1% I2-KI for microscopic examination to determine phenotype. Previous results from our laboratory's artificial climate chamber indicated that strain 1S (tms5) was a temperature-sensitive dual-purpose genic male sterile line, with photoperiod having little effect on its fertility. Therefore, we selected a photoperiod of 13.5 hours of light and 10.5 hours of darkness for treatment (Table 2). The light and temperature settings for the artificial climate chamber are shown in Table 1. The diurnal temperature variations in Table 1 simulate those observed in natural conditions.

[0051] Figure 7 This figure shows the pollen fertility of WT, tms5 (1S) strain 1S, and tms5 osvms1 treated at different temperatures (22°C, 25°C, and 30°C) in an artificial climate chamber. Pollen from individual florets of WT, tms5 (1S) strain 1S, and tms5 osvms11 that had not shed pollen after treatment at 22°C was stained with 1% I2-KI. Light microscopic observation revealed that the pollen of WT, tms5, and tms5 osvms1 was regularly round and stained black with iodine, indicating fertility. At 25°C, the pollen of WT and tms5 osvms1 was regularly round and stained black with iodine, indicating fertility. However, the pollen of tms5 (1S) strain 1S was triangular and did not stain with iodine, indicating abortion. After treatment at 30℃, the pollen of WT and tms5 osvms1 were regular circles and stained black with iodine, indicating that the pollen was fertile; the pollen of strain 1S (tms5) was pollen-free, indicating that the pollen was aborted.

[0052] Through the identification of artificial climate chamber system, the starting temperature of fertility conversion of zhu1S (tms5) was 22-25℃, and the starting temperature of fertility conversion of tms5osvms1 was increased to above 30℃.

[0053] Table 2 Light and temperature conditions of artificial climate chamber

[0054]

[0055]

[0056] Example 3: Os Vms1 gene function complementation experiment

[0057] To determine the function of the Os Vms1 gene, we used strain 1S (tms5) genomic DNA as a template and amplified the 3′ UTR region (871 bp) of the Os Vms1 gene using primers CP0266 and CP0267. The amplified fragment was then ligated into the XF2635 vector digested with HindIII and PstI using the Gibson Assembly method. The vector was then transformed into a DH5α clone, and positive clones were selected for sequencing. The plasmid was then digested with XbaI and SalI, and the digestion product was recovered for later use. Using strain 1S (tms5) genomic DNA as a template, the promoter region (992 bp) and gene region (3902 bp) of the Os Vms1 gene were amplified with primer pairs CP0262 and CP0265. The amplified amplified amplification fragment was then ligated into the XF2635 vector with the 3′UTR cut by XbaI and SalI using the Gibson Assembly method, completing the construction of the complementary vector pOs Vms1::Os Vms1genomic-FLAG-3′UTR vector.

[0058] A correctly sequenced cloned plasmid (plasmid number: XF4228) was selected and transformed into Agrobacterium tumefaciens EHA105 by electroporation. Calli from the mutant tms5 osvms1 were then infected to obtain transgenic complemented plants. Transgenic T0 plants were transplanted to the fields of the Southern Propagation Base of the Institute of Genetics and Developmental Biology in Lingshui County, Hainan Province, at the end of November 2020, and T1 seeds were harvested in early April. In early May 2021, seeds of T1 transgenic-positive individual plants, tms5 osvms1, and strain 1S (tms5) were planted in Beijing. Field observations revealed that under high temperature conditions, strain 1S (tms5) exhibited phimosis and non-fruiting, while tms5 osvms1 exhibited non-phimosis and fruiting. The phenotype of the transgenic complemented plants was consistent with that of strain 1S (tms5). Pollen from strain 1S (tms5), tms5 osvms1, and a transgenic complemented line (OsVms1-WTC) was examined microscopically. The results showed that the pollen from strain 1S (tms5) was triangular and could not be stained with iodine, indicating that the pollen was sterile. The pollen from tms5 osvms1 was regular round and stained black with iodine, indicating that the pollen was fertile. The pollen from two independent transgenic complemented lines (Os Vms1-WTC1 and Os Vms1-WTC2) was triangular and could not be stained with iodine, showing the same pollen sterility as strain 1S (tms5). Figure 6The results showed that tms5 osvms1, tms5 osvms1, and the complemented transgenic plants showed pollen fertility phenotypes in Beijing field. Therefore, genetic complementation experiments demonstrated that tms5 osvms1 could restore the high-temperature sterility phenotype of tms5 1S (tms5) under field conditions.

[0059] The primers used in the construction of the Os Vms1 gene complementation vector of the present invention are shown in Table 3. The map of the functional vector pCAMBIA1300 used for the Os Vms1 transgenic complementation verification of the present invention is shown in Figure 5.

[0060] Table 3 Primers used in the construction of the Os Vms1 gene complementation vector in the present invention

[0061]

[0062] In summary, the present invention discovered a gene, OsVms1, that controls the starting temperature for fertility conversion in thermosensitive dual-purpose nuclear male sterile lines. Further in-depth research has demonstrated that OsVms1 is involved in regulating the starting temperature for fertility conversion in thermosensitive dual-purpose nuclear male sterile lines. OsVms1 is essential for maintaining the low fertility conversion of strain 1S. Mutation of this gene can increase the fertility conversion of strain 1S (tms5) to above 30°C. This invention provides a new genetic resource for cultivating dual-purpose male sterile lines with a low starting temperature for fertility conversion, which is of great significance to the field of rice molecular breeding.

[0063] After reading the above statements about the present invention, those skilled in the art may make various modifications or changes to the present invention, and these equivalent forms also fall within the scope defined in the claims attached to this application.

Claims

1. A DNA molecule, the base sequence of which is shown in SEQ ID No: 1, wherein the DNA molecule is the temperature-regulated gene OsVms1, which is the starting point for fertility conversion in thermo-sensitive dual-purpose sterile lines of rice and contains the tms5 locus.

2. The DNA molecule according to claim 1, wherein The DNA molecule encodes a temperature-regulating protein OsVms1, which is the starting point for fertility conversion of a thermo-sensitive dual-purpose sterile line of rice. The OsVms1 protein has an amino acid sequence shown in SEQ ID No: 2 in the sequence table. A recombinant vector comprising the DNA molecule according to claim 1 . An Agrobacterium carrying the recombinant vector according to claim 3 .

5. A protein, whose amino acid sequence is shown in SEQ ID No: 2, wherein the protein is the temperature-regulated protein OsVms1, which is the starting point for fertility conversion in thermo-sensitive dual-purpose male sterile lines of rice.

6. A method for regulating the starting temperature for fertility conversion in a thermo-sensitive dual-purpose nuclear male sterile rice line, characterized in that: The method comprises the steps of introducing the DNA molecule according to claim 1 or 2 or the recombinant vector according to claim 3 into a thermosensitive dual-purpose genic male sterile rice line containing the tms5 site.

7. The method according to claim 6, wherein: The method comprises the steps of contacting plant cells or plant tissues of thermo-sensitive dual-purpose nuclear male sterile rice with the Agrobacterium according to claim 4, thereby transferring the polynucleotide sequence of the temperature-regulating gene OsVms1, the starting point of fertility conversion of the thermo-sensitive dual-purpose male sterile rice, into the plant cells and integrating it into the chromosomes of the plant cells.

8. A method for cultivating a thermo-sensitive dual-purpose genic male sterile rice line with a low fertility conversion threshold temperature, characterized in that: The method comprises the steps of introducing the DNA molecule according to claim 1 or 2 or the recombinant vector according to claim 3 into a thermosensitive dual-purpose genic male sterile rice line containing the tms5 site.

9. The method according to claim 8, wherein The method comprises the steps of contacting plant cells or plant tissues of a thermo-sensitive dual-purpose nuclear male sterile rice line with the Agrobacterium according to claim 4, thereby transferring the polynucleotide sequence of the temperature-regulating gene OsVms1, which is the starting point for fertility conversion of the thermo-sensitive dual-purpose male sterile rice line, into the plant cells and integrating it into the chromosomes of the plant cells.

10. The method according to claim 8 or 9, characterized in that In the process of cultivating a thermo-sensitive dual-purpose sterile rice line with a low fertility conversion starting point temperature, the OsVms1 protein expressed by the fertility conversion starting point temperature regulatory gene OsVms1 of the thermo-sensitive dual-purpose sterile rice line has the amino acid sequence shown in SEQ ID No: 2 in the sequence listing, and does not undergo substitution, deletion or addition of one or more amino acid residues.

11. Use of the DNA molecule according to claim 1 or 2, the recombinant vector according to claim 3, the Agrobacterium according to claim 4, or the protein according to claim 5 in regulating the starting temperature for breeding of a thermo-sensitive dual-purpose genic male sterile line of rice containing the tms5 locus.

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