Sporophyte fertile gene OsSTRL3 and application thereof

Abstract

The invention discloses a sporophyte fertile gene OsSTRL3 and application thereof. A protein encoded by the sporophyte fertile gene is an amino acid sequence as shown in SEQ ID NO.1, or a protein which is subjected to substitution, deletion or addition of at least one amino acid and has the same function as the OsSTRL3; the nucleotide sequence of an encoding region of the OsSTRL3 gene is shown as SEQ ID NO.2. The invention also discloses a mutation site of the OsSTRL3. The OsSTRL3 gene disclosed by the invention encodes a protease with an isosessile synthetase / phospholactonase structural domain, and has a function of regulating and controlling normal development of pollen, sporophytic male sterility can be generated by mutation of the gene, and the mutation of the OsSTRL3 gene can be used for regulating and controlling normal development of pollen. The strain can be used as a female parent for producing hybrids in heterosis utilization.

Description

Technical Field

[0001] This invention relates to the field of genetic breeding, specifically to a sporophyte fertility gene OsSTRL3, and also to the application of this gene. Background Technology

[0002] The successful application of heterosis plays a crucial role in improving plant varieties, and the utilization of heterosis in self-pollinating crops relies on the use of male-sterile lines. Currently, heterosis in rice is mainly achieved through cytoplasmic-nuclear interaction male-sterile lines (three-line method) and photoperiod-thermosensitive male-sterile lines (two-line method). However, due to the limited flexibility in pairing between cytoplasmic-nuclear interaction male-sterile lines, the limited number of usable parents, the complexity of seed production compared to the two-line method, the influence of natural light and temperature conditions on the fertility of photoperiod-thermosensitive male-sterile lines, and the high risk of large-scale seed production, the heterosis in rice cannot be fully realized through these two types of male-sterile lines. In contrast, ordinary sporophytic nucleosterile lines possess fertility unaffected by environmental factors such as light and temperature, stable fertility, and the ability to be restored by all normally fertile materials, allowing for extremely flexible pairing of hybrids. Third-generation hybrid rice represents the future direction of hybrid rice development, and sporophytic nucleosterility is an essential genetic element for third-generation hybrid rice. Therefore, vigorous research on the sterility mechanism and regulation of such sterile lines, creation of such sterile mutations, cloning of related genes, research on their reproductive technology and application are of great value to promoting the research and development of third-generation hybrid rice and making full use of the heterosis of rice. Summary of the Invention

[0003] In view of this, one objective of the present invention is to provide a sporophyte fertility gene OsSTRL3; a second objective of the present invention is to provide the coding region CDS of the sporophyte fertility gene OsSTRL3; a third objective of the present invention is to provide a mutant gene of the sporophyte fertility gene OsSTRL3; a fourth objective of the present invention is to provide a recombinant expression vector, expression cassette, recombinant bacteria, transgenic cell line or plant containing the sporophyte fertility gene OsSTRL3; a fifth objective of the present invention is to provide plant material containing the mutant gene; a sixth objective of the present invention is to provide the application of the sporophyte fertility gene OsSTRL3 in restoring pollen fertility; a seventh objective of the present invention is to provide the application of the mutant gene in preparing rice male-sterile lines or producing seeds using rice male-sterile lines; and an eighth objective of the present invention is to provide a method for restoring pollen fertility using the sporophyte fertility gene OsSTRL3.

[0004] To achieve the above objectives, the present invention provides the following technical solution: 1. A sporophyte fertility gene OsSTRL3, wherein the sporophyte fertility gene OsSTRL3 encodes an amino acid sequence as shown in SEQ ID NO.1, or a protein having at least one amino acid substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO.1 and having the same function as the OsSTRL3 protein.

[0005] 2. The coding region CDS of the sporophyte fertility gene OsSTRL3, wherein the nucleotide sequence of the CDS is as shown in SEQ ID NO.2, or the nucleotide sequence shown in SEQ ID NO.2 is substituted, deleted or added at least one nucleotide and has a nucleic acid sequence with the same function as the OsSTRL3 gene.

[0006] 3. The mutant gene of the sporophyte fertility gene OsSTRL3, wherein the mutation site is preferably a 135bp deletion and an 11bp insertion mutation at positions 1287 to 1422 of the nucleotide sequence shown in SEQ ID NO.2, or a 135bp deletion and an 11bp insertion mutation at positions 4592 to 4727 of the nucleotide sequence shown in SEQ ID NO.3.

[0007] 4. Recombinant expression vectors, expression cassettes, recombinant bacteria, transgenic cell lines or plants containing the sporophyte fertility gene OsSTRL3.

[0008] 5. Plant material containing the mutated gene.

[0009] 6. Application of the sporophyte fertility gene OsSTRL3 in restoring pollen fertility.

[0010] 7. Application of the mutant gene in the preparation of male-sterile rice lines or in the production of seeds using male-sterile rice lines.

[0011] 8. A method for restoring pollen fertility using the sporophyte fertility gene OsSTRL3, wherein the sporophyte fertility gene OsSTRL3 is transferred into a male-sterile mutant generated by OsSTRL3 gene mutation, or a positive plant containing the sporophyte fertility gene OsSTRL3 is crossed with a male-sterile mutant generated by OsSTRL3 gene mutation to obtain offspring with restored pollen fertility.

[0012] The beneficial effects of this invention are as follows: This invention discloses a sporophyte fertility gene OsSTRL3 and its application. The sporophyte fertility gene OsSTRL3 mainly affects rice pollen development. Mutations of this gene result in sporophyte male sterility, whose fertility remains unchanged regardless of temperature and light conditions, representing a novel form of sporophyte male sterility. Compared to cytoplasmic male sterility and temperature- and light-sensitive nuclear male sterility, it offers advantages such as free pairing and lower seed production risks. The male-sterile mutant of this gene can be used for efficient breeding and safe production of hybrid seeds, and for the development of third-generation hybrid rice.

[0013] A prerequisite for the development of third-generation hybrid rice is sporophytic nuclear male sterility reproduction, which in turn requires the possession of sporophytic nuclear male sterility-related genes and mutants. The OsSTRL3 gene and its mutant are precisely such a sporophytic nuclear male sterility-related gene and male sterility mutant. The gene, expression vector, and nuclear male sterility reproduction methods provided by this invention have significant value and broad application prospects in fully utilizing heterosis and vigorously promoting the development of third-generation hybrid rice. Attached Figure Description

[0014] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Comparison of fertility between wild-type and mutant OsSTRL3 gene (A: wild-type Zhonghua 11 (fruit-bearing) and mutant OsSTRL3 (non-fruit-bearing) plants; B: wild-type (yellow, plump, fruit-bearing) and mutant (white, small) anthers; C: wild-type (normal staining and size) and D: mutant (pollen-free) pollen). Figure 2 Map-based cloning of the OsSTRL3 gene (A: Chromosomal localization of the OsSTRL3 gene; B: Mutation detection of the OsSTRL3 gene). Figure 3 Figures for identifying propagation effectiveness and purity (A: colorless spikelet of mutant; B: transgenic purple positive spikelet of OsSTRL3 propagation vector; C: sterile anther of mutant; D: transgenic positive fertile anther; E: pollen-free mutant; F: transgenic positive fertile pollen; G: purity identification of propagated sterile line). Detailed Implementation

[0015] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al.'s Laboratory Manual of Molecular Cloning (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0016] The full-length sequence of the relevant nucleotides or fragments thereof can usually be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in this embodiment, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art, or genomic DNA as templates. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. This usually involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods.

[0017] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0018] Example 1: Obtaining and morphologically observing OsSTRL3 mutant plants The OsSTRL3 mutant was discovered by the first inventor in 2018 at the Jinghong rice experimental field of Yunnan University in Xishuangbanna. Its wild type is japonica rice Zhonghua 11: the wild type Zhonghua 11 droops after the panicles are filled. Figure 1 A), while the spikelets of OsSTRL3 are erect and do not produce grains ( Figure 1 B); the anthers of the 11-flowered medium-sized flower are robust and full ( Figure 1 C), the anthers of the OsSTRL3 mutant are thinner ( Figure 1 D); the pollen of the 11th flower was stained black with iodine ( Figure 1 In the OsSTRL3 mutant pollen, only a few crushed anther wall tissues were observed in the field of view after iodine staining; no pollen grains were seen. Figure 1 F).

[0019] Using the normal Zhonghua 11 as the male parent, it is crossed with the OsSTRL3 mutant. The mutant gene is preserved in the heterozygote. When the mutant is needed, the heterozygote is allowed to self-pollinate, and the OsSTRL3 mutant will segregate from the offspring. The OsSTRL3 mutant is then crossed with Zhonghua 11, and the F1 generation... l All F2 plants were fertile. Segregation occurred in the self-crossed F2 generation, with 220 normal plants and 73 mutant plants, a ratio of 3:1 (= 0.001<3.84), indicating that the male sterile mutant phenotype was caused by a single-nucleus gene mutation.

[0020] Example 2: Map-based cloning of the OsSTRL3 gene (1) Targeting the group The OsSTRL3 mutant was crossed with the indica rice Zhongjiu B, and the F2 generation was obtained by self-pollination as the localization population.

[0021] (2) Extraction of rice DNA Parental DNA was extracted using a modified CTAB method, including the following steps: 0.1-0.2 g (approximately half a leaf) of leaf was placed in a small mortar, and an appropriate amount of liquid nitrogen was added. The mixture was immediately ground into powder and transferred to a 2 mL centrifuge tube. 700 μL of preheated 1.5×CTAB solution (100℃) was added to the centrifuge tube, and the mixture was carefully mixed. The tube was then placed in a 65℃ water bath for 20 minutes. After 20 minutes, the centrifuge tube was removed, and an equal volume of chloroform / isoamyl alcohol was added. The mixture was vigorously mixed and centrifuged at 13000 rpm for 10 minutes. The supernatant was transferred to a new tube, and 900 μL of anhydrous ethanol was added. The mixture was then incubated at -20℃ for at least 30 minutes. The precipitated DNA was centrifuged at 14000 rpm for 10 minutes. The supernatant was removed, and the precipitate was washed once with 1 mL of 70% ethanol. The precipitate was centrifuged, dried, and dissolved in 200 μL of TE solution. The DNA from individual plants in the target population was extracted using a modified alkaline boiling method, including the following steps: 1-2 cm of young leaves were cut into small pieces. 2 Place the solution in a 0.5 mL centrifuge tube, add 100 μL of 0.125 M NaOH solution, incubate in a boiling water bath for 30 seconds, then add 50 μL of 1.0 M Tris-HCl (pH 8.0), and finally add 100 μL of 0.125 M HCl. Incubate in a boiling water bath for 2 minutes and store at 4°C for later use.

[0022] (3) Initial and fine localization of population separation analysis 137 pairs of polymorphic markers were designed, including 42 pairs of SSR primers and 95 pairs of InDel molecular marker primers. The SSR primers were synthesized based on published sequences (see http: / / www.gramene.org.microsat / ssr.html for details). Other InDel molecular markers were designed based on a comparison of the published nucleic acid sequences of the Japonica rice Nipponbare and the Indica rice 9311, with primers designed for the differing regions to verify the polymorphism between the two parental lines: the OsSTRL3 mutant in Japonica rice and the ZhongjiuB mutant in Indica rice. The 137 designed InDel primers were used to simultaneously amplify the parents and the gene pool. The PCR amplification program was as follows: in a 10 μL system, 1 μL template, 1 μL 10 pmol / μL upstream primer, 1 μL 10 pmol / μL downstream primer, and 1 μL 10×Buffer (MgCl₂). 2十The PCR products were analyzed by silver staining and electrophoresis using a 10% PAGE gel. The primer R03010 on chromosome 3 showed polymorphism in both the parent and gene pools, and single-plant verification confirmed its linkage to the OsSTRL3 gene. Further fine-mapping was performed to locate the gene between the two primer sites and to narrow down the candidate gene pool. Thirteen pairs of SSR or InDel primers were synthesized within a 5.0 Mb range around primer R03010. Differentiated primers were screened using sterile parents, fertile parents, and F1 cells. A total of eight pairs of primers with significant differences and linkage to the gene were selected, including InDel primers C03D39, C03D40, C03D41, C03D44, and C03D45, and SSR primers RM333, RM334, and C03D46. The primer sequences are shown in Table 1. Genotypic analysis of recessive single plants showed that C03D41 had 9 single-crossover plants with a genetic distance of 2.94 cM; RM334 had 31 single-crossover plants with a genetic distance of 10.13 cM (the single-crossover plants of these two genotypes were mutually exclusive); C03D40 had 4 single-crossover plants with a genetic distance of 1.31 cM; C03D39 had 2 single-crossover plants with a genetic distance of 0.65 cM; R03010 had 30 single-crossover plants with a genetic distance of 9.80 cM; RM333 had 5 single-crossover plants with a genetic distance of 1.63 cM; C03D46 had 3 single-crossover plants with a genetic distance of 0.98 cM; C03D45 had 2 single-crossover plants with a genetic distance of 0.65 cM; and C03D44 had [missing information - likely a single-crossover plant]. Two plants were identified with a genetic distance of 0.65 cM. The crossover of C03D40 was included in the crossover of C03D41, the crossover of C03D39 was included in the crossover of C03D40, the single crossover of R03010 was included in the crossover of RM334, the crossover of RM333 was included in the crossover of R03010, the crossover of C03D46 was included in the crossover of RM333, the crossover of C03D45 was included in the crossover of C03D46, and the crossover of C03D44 was included in the crossover of C03D45. The crossovers of C03D41 and RM334 were not mutually inclusive, indicating that OsSTRL3 exists between the two markers C03D39 and C03D44. Figure 2(A). On chromosome 3 of the rice variety Nipponbare (http: / / www.gramene.org / ), the physical locations of these two markers are 30531048 and 31219822 (version number: Oryza_sativa.IRGSP-1.0), approximately 688 kb apart, indicating a large number of gene loci within this range. Since no closer polymorphic markers could be found to narrow down the search area, resequencing was performed. An F2 population of the mutant OsSTRL3×ZH11 was constructed and sent to Tianjin Novogene Technology Co., Ltd. for resequencing. Then, using the Nipponbare japonica rice genome as a reference, the genomic data analysis software IGV was used to screen for mutations within the 30531048-31219822 location interval on chromosome 3. A large base deletion mutation, Loc_Os03g53950, was found, which is most likely our candidate gene. Sequencing comparisons of the Loc_Os03g53950 gene in Zhonghua 11 and the mutant osstrl3 revealed a 135bp deletion and an 11bp insertion mutation in the third exon of the Loc_Os03g53950 gene in the OsSTRL3 mutant, resulting in a frameshift of the encoded protein. Therefore, we preliminarily identified Loc_Os03g53950 as the target gene. Figure 2 (B). This gene encodes a protease with isogenin synthase and phospholactone domains. The amino acid sequence encoding this protein is shown in SEQ ID NO. 1, the CDS nucleotide sequence of the coding region is shown in SEQ ID NO. 2, the genomic sequence is shown in SEQ ID NO. 3, and the sequence of the mutant gene is shown in SEQ ID NO. 4.

[0023] Table 1. Key molecular markers for gene localization and their primer sequences Primer name Upstream primer (5'→3') Downstream primer (5'→3') C03D41 5'-TGACAAGTTCACGAGCAAAGC-3' (SEQ ID NO.5) 5'-ACGAGCGTGCGTGAAAAATC-3' (SEQ ID NO.6) C03D39 5'-GACCAGGAGTACGAGTTCGC-3' (SEQ ID NO.7) 5'-GAGCAGCAACAAGAAGCCAC-3' (SEQ ID NO.8) C03D44 5'-TGATCCTAGGCAGCAACAGC-3' (SEQ ID NO.9) 5'-CCTCCTAGCTTGTGTCACCG-3' (SEQ ID NO.10) R03010 5'-ACACTGGCTACGGCAAAG-3' (SEQ ID NO.11) 5'-TTTGTTCGGGAATAATGATGC-3' (SEQ ID NO.12) RM334 5'-GGGTGGTGTGAGCTTTTCTC-3' (SEQ ID NO.13) 5'-TTCCACTTCGACAACCCTTC-3' (SEQ ID NO.14) SF / SR 5'-caagggcgcgccAAATCCTGGGATCTGCTATC-3' (SEQ ID NO.15) 5'-cactcctgcaggAGAGCTCCCAGTCTCTCCCAG-3' (SEQ ID NO. 16) Example 3: Functional verification of the OsSTRL3 gene and reproduction of OsSTRL3-induced male sterility (1) Amplification of OsSTRL3: In order to further confirm that the OsSTRL3 gene is the gene that causes the male sterility mutant phenotype of this male sterility mutant and to propagate OsSTRL3 male sterility, the OsSTRL3 gene and expression regulatory region (containing 2180 bp exons and introns, 2568 bp upstream promoter and 1187 bp downstream terminator) were amplified from wild-type japonica rice Zhonghua 11 containing the OsSTRL3 gene. The PCR primers were SF / SR, and the sequences are shown in SEQ ID NO.15 and SEQ ID NO.16, respectively. The amplified nucleotide sequence is shown in SEQ ID NO.3.

[0024] (2) Functional verification and propagation vector construction: The amplification product from the previous step was ligated into the AscI and SbfI of the plant expression vector pOsMYB76RM (the construction process is described in Chinese Patent No. CN112239762A). Sequencing was used to check its correctness, and the intermediate vector pOsSTRL3 was obtained.

[0025] (3) Construction of gametophyte male sterility module: The interference promoter adopted is the OsPTDa promoter PMCOS1 (SEQ ID NO. 17); utilizing the precise self-splicing characteristics of tRNA, the interference elements of OsPTDa and OsPTDb are tandemly linked with tRNA-Gly and placed between the OsPTDa promoter and the rice endogenous terminator. The interference elements of OsPTDa and OsPTDb are shown as I801 and IM1-1, respectively. The sequence of I801 is shown as SEQ ID NO. 18, the sequence of IM1-1 is shown as SEQ ID NO. 19, the tRNA-Gly sequence is shown as SEQ ID NO. 20, and the sequence of the rice endogenous terminator Taom is shown as SEQ ID NO. 21. Using isosigm technology, the sequence is Taom, IM1-1, tRNA-Gly, I801 and PMCOS1 ( Figure 1 The vector pRNAi-PTDab-2 was obtained by sequentially loading the multiple cloning site BglII of the vector pOsMYB76RM (construction process is described in Chinese Patent Publication No. CN112239762A), which contains genes related to anthocyanin synthesis. Then, a gametophyte male sterility module containing Taom, IM1-1, tRNA-Gly, I801, and PMCOS1 was excised from pRNAi-PTDab-2 using SalI+SbfI and loaded into the aforementioned intermediate vector pOsSTRL3 to obtain the reproductive vector pOsSTRL3-RNAi-PTDab-2.

[0026] (3) pOsSTRL3-RNAi-PTDab-2 was transformed into the sterile Zhonghua 11 OsSTRL3 mutant, and a total of 39 positive plants with purple valley tips were screened out. All positive plants showed normal pollen fertility. Figure 3 ,AF).

[0027] The sterile mutant Zhonghua 11-OsSTRL3 was pollinated with positive plants, and a total of 316 F1 seeds were obtained. After planting and flowering, the fertility of F1 was investigated, and all of them showed male sterility. Figure 3 G).

[0028] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A sporophyte fertility gene OsSTRL3, characterized in that: The sporophyte fertility gene OsSTRL3 encodes an amino acid sequence as shown in SEQ ID NO.1, or a protein having at least one amino acid substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO.1 and having the same function as the OsSTRL3 protein.

2. The coding region CDS of the sporophyte fertility gene OsSTRL3, characterized in that: The nucleotide sequence of the CDS is as shown in SEQ ID NO.2, or the nucleotide sequence shown in SEQ ID NO.2 is modified by substitution, deletion or addition of at least one nucleotide and has a nucleic acid sequence with the same function as the OsSTRL3 gene.

3. The mutant gene of the sporophyte fertility gene OsSTRL3 as described in claim 1 or 2, characterized in that: The mutation sites of the mutant gene are either a 135bp deletion and an 11bp insertion mutation at positions 1287 to 1422 of the nucleotide sequence shown in SEQ ID NO.2, or a 135bp deletion and an 11bp insertion mutation at positions 4592 to 4727 of the nucleotide sequence shown in SEQ ID NO.

3.

4. A recombinant expression vector, expression cassette, recombinant bacteria, transgenic cell line or plant containing the sporophyte fertility gene OsSTRL3 as described in claim 1 or 2.

5. Plant material containing the mutant gene as described in claim 3.

6. The application of the sporophyte fertility gene OsSTRL3 as described in any one of claims 1 to 2 in restoring pollen fertility.

7. The application of the mutant gene described in claim 3 in the preparation of male-sterile rice lines or in the production of seeds using male-sterile rice lines.

8. A method for restoring pollen fertility using the sporophyte fertility gene OsSTRL3 as described in claim 1 or 2, characterized in that: The method involves transferring the sporophyte fertility gene OsSTRL3 into a male-sterile mutant resulting from an OsSTRL3 gene mutation, or crossing a positive plant containing the sporophyte fertility gene OsSTRL3 with a male-sterile mutant resulting from an OsSTRL3 gene mutation to obtain offspring with restored pollen fertility.

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